D2-dopaminergic receptor and 5-HT3 serotoninergic receptor

Editorial boardEditor-in-Chief

Aida El-Azzouny

[email protected]+202 33371362/33371433+2 0100 52 54 161+202 33370931/3601877Complete professional affiliations:

Pharmaceutical and Drug Industries Research Division,National Research Centre (NRC), Dokki-Cairo,12622-EgyptSpecialization: Medicinal and Pharmaceutical Chemistry

Deputy-Editors

Abdel-Hamid Zaki Abdel-Hamid Amer

[email protected].+201002020747Complete professional affiliation:

Pharmaceutical and Drug Industries Research Division,National Research Centre (NRC), Dokki-Cairo,12622-EgyptSpecialization: Applied Biochemistry

Mohamed Ahmed Abdel-Naby

Egypt+202 24708049+201149921388+202 3370931/[email protected] professional affiliations:

Pharmaceutical and Drug Industries Research Division,National Research Centre (NRC), Dokki-Cairo,12622-EgyptSpecialization: Professor of Biochemistry

Editorial Assistants

Hassan Abdel Zaher Mohamed Mohamed AMER

[email protected] professional affiliations:National Research CentreCenter of Excellence for Advanced Sciences,Dept of Natural and Microbial Products ChemistryDivision of Pharmaceutical and Drug IndustriesDokki, El-behoos StreetCairo, EgyptTel: +201227341899Specialization:Associate professor of bioorganic Chemistry

Mohammad H. A. Ibrahim

[email protected]+201150935326Complete professional affiliations:

Chemistry of Natural and Microbial Products Dept.,Pharmaceutical & Drugs Industries Research Division,National Research Centre, Al-Bohoos st., Dokki,12622 Cairo, EgyptSpecialization: Microbial Biotechnology, FermentationTechnology, Bioplastics

Mona E. Aboutabl

[email protected]+2011155 330 72Complete professional affiliations:

Researcher of Pharmacology, Room # 374, Medicinal andPharmaceutical Chemistry Department, Pharmaceutical andDrug Industries Research Division, National Research Center,El-bohous St., Dokki, Cairo, 12311 EgyptSpecialization: Pharmacology and Toxicology

EGYPTIAN PHARMACEUTICAL JOURNAL

Vol 12 No 1 June 2013

Table of contents

Review article1 D2-dopaminergic receptor and 5-HT3 serotoninergic receptor antagonists having antiemetic profile

Mohamed N. Aboul-Enein, Aida A. EL-Azzouny, Yousreya A. Maklad, Mohamed I. Attia, Mohamed Abd EL-Hamid Ismail andWalaa H.A. Abd EL-Hamid

Original articles11 Synthesis and DPPH radical-scavenging activity of some new 5-(N-substituted-1H-indol-3-yl)-5H-thiazolo[4,3-b]-1,3,

4-thiadiazole derivatives

Heba M. Abo-Salem, Manal Sh. Ebaid, Eslam R. El-Sawy, Abd El-Nasser El-Gendy and Adel H. Mandour

20 Synthesis and antihypertensive activity of certain substituted dihydropyridines and pyrimidinones

Wageeh S. El-Hamouly, Kamelia M. Amine, Hanaa A. Tawfik and Dina H. Dawood

28 Immobilization of Mucor racemosus NRRL 3631 lipase and characterization of silica-coated magnetite (Fe3O4)

nanoparticles

Abeer A. El-Hadi, Hesham I. Saleh, Samia A. Moustafa and Hanan M. Ahmed

36 Extracellular polysaccharides produced by the newly discovered source Scopularis spp.

Siham A. Ismail

40 Biotransformation of soybean saponin to soyasapogenol B by Aspergillus parasiticus

Hala A. Amin, Yousseria M. Hassan and Soad M. Yehia

46 Characterization of ternary solid dispersions of nimesulide with Inutec SP1 and b-cyclodextrin and evaluation of

anti-inflammatory efficiency in rats

Rawia M. Khalil, Mamdouh M. Ghorab, Noha Abd El Rahman and Silvia Kocova El-Arini

57 DNA fingerprinting and profile of phenolics in root and root calli of Arctium lappa L. grown in Egypt

Elsayed A. Aboutabl, Mona El-Tantawy, Nadia Sokkar and Manal M. Shams

63 Influence of formulation parameters on the physicochemical properties of meloxicam-loaded solid lipid nanoparticles

Rawia M. Khalil, Ahmed Abd El-Bary, Mahfoz A. Kassem, Mamdouh M. Ghorab and Mona Basha

73 Effect of pollution on the chemical content and secondary metabolites of Zygophyllum coccineum and Tamarix nilotica

Hanan E. Osman and Reham K. Badawy

83 Optimization of growth conditions and continuous production of inulinase using immobilized Aspergillus niger cells

Nagwa A. Atwa and Enas N. Danial

Short communication90 Chemical constituents from the aerial parts of Salsola inermis

Fatma S. Elsharabasy and Ahlam M. Hosney



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D2-dopaminergic receptor and 5-HT3 serotoninergic receptor

antagonists having antiemetic profileMohamed N. Aboul-Eneina, Aida A. EL-Azzounya, Yousreya A. Maklada,Mohamed I. Attiaa,b, Mohamed Abd EL-Hamid Ismailc

and Walaa H.A. Abd EL-Hamidd

aDepartment of Medicinal and PharmaceuticalChemistry, Pharmaceutical and Drug IndustriesResearch Division, National Research Centre, Giza,bDepartment of Pharmaceutical Chemistry, College ofPharmacy, King Saud University, Riyadh, Saudi Arabia,cDepartment of Pharmaceutical Chemistry, Facultyof Pharmacy, Ain Shams University, Cairo anddDepartment of Pharmaceutical Chemistry, Faculty ofPharmacy, Misr University for Science &Technology,6th of October City, Egypt

Correspondence to Mohamed N. Aboul-Enein,Department of Medicinal and PharmaceuticalChemistry, Pharmaceutical and Drug IndustriesResearch Division, National Research Centre,12622 Dokki, Giza, EgyptTel: + 20 012 216 8624; fax: + 20 233370931;e-mail: [email protected]

Received 15 January 2013Accepted 11 March 2013

Egyptian Pharmaceutical Journal

2013, 12:1–10

Metoclopramide is the prototype of the orthopramide family and is used clinically as a

stimulant of upper gastrointestinal motility and as an antiemetic. Its antiemetic potential

is attributed mainly to the antagonist activity at D2-dopaminergic receptors in the

chemoreceptor trigger zone of the central nervous system. Besides, ondansetron was

the first selective 5-HT3 serotoninergic receptor antagonist used in clinics as an

antiemetic. Herein, the antiemetic profile of different chemical classes of D2-

dopaminergic receptor and 5-HT3 serotoninergic receptor antagonists will be

discussed, which may be helpful in the development of potent antiemetic agents.

Keywords:

antagonists, antiemetic, D2-dopaminergic receptor, 5-HT3 serotoninergic receptor

Egypt Pharm J 12:1–10& 2013 Division of Pharmaceutical and Drug Industries Research,National Research Centre1687-4315

IntroductionVomiting is the forceful repulsion of the contents of one’s

stomach through the mouth and sometimes the nose and

it may result from many causes, ranging from gastritis or

poisoning to brain tumor or elevated intracranial pressure.

It is generally considered to be a protective mechanism by

which undesirable substances are evacuated quickly from

the gastrointestinal tract. Vomiting is different from

regurgitation, although the two terms are often used

interchangeably. Regurgitation is the return of undigested

food back up the esophagus to the mouth, without the

force and displeasure associated with vomiting. Nausea,

which also has an impact on the gastrointestinal tract, is

the sensation of discomfort in the upper stomach with the

urge to vomit [1]. Persistent nausea may lead to loss of

appetite and reduction of food uptake until the point of

malnutrition and debilitation. Gastrointestinal infections

(37%) and food poisoning are the most common causes of

nausea and vomiting besides side effects from medications

(3%) and pregnancy [1,2]. In 10% of people the cause

remains unknown [2]. Prolonged and severe vomiting leads

to hypochloremia, hypokalemia, alkalosis, and dehydration;

it can even cause death, especially in children. Therefore,

treatment should be directed mainly toward elimi-

nating the causes of illness. This review focuses on the

antiemetic agents that potentially act as antagonists to the

D2-dopaminergic and 5-HT3 serotoninergic receptors.

Mechanism of emesisThe act of emesis is very complicated and involves a

series of coordinated activities and changes in the

respiratory and gastrointestinal musculature. It is usually

preceded by salivation, nausea, malaise, lassitude, weak-

ness, retching movements, and characteristic postures

of the head and body adopted to final expulsion of

vomitus [3,4]. This order of events indicates the

existence of at least two central areas concerned with

the vomiting act, namely, the chemoreceptor trigger zone

(CTZ), which can be stimulated by chemical agents such

as the dopaminergic apomorphine and transmits impulses

to the vomiting centre itself, which is located in the

reticular core of the medulla [2].

The latter center lies in proximity to the other centers

such as inspiratory and expiratory centers, the vasomotor

center, salivary nuclei, and vestibular nuclei. The action

of all these centers may manifest as the act of

vomiting [2]. Impulses from all these centers pass

through the CTZ to the vomiting center, resulting in

emesis [5–7].

Sites other than the CTZ may be effective in the

stimulation of emesis. Thus, visceral afferent impulses

mediated through the parasympathetic and sympathetic

routes transmit to the vomiting center impulses that

result in the genesis of vomiting [8,9].

Antiemetic agentsAntiemetic agents are drugs that are used for the

prophylaxis, control, and prevention of nausea and

vomiting. Emesis is the main symptom for motion

sickness, during the first trimester in pregnancy, in the

case of hyperemesis gravidarum, and of radiation sickness

Review article 1

1687-4315 & 2013 Division of Pharmaceutical and Drug Industries Research,National Research Centre

DOI: 10.7123/01.EPJ.0000428875.20180.de

resulting during the treatment of tumors by irradiation

and using cytotoxic drugs. In addition, postoperative

vomiting can also occur, which may be due to the use of

general anesthetics and opiate analgesics after surgical

operations. Gastrointestinal irritation because of peptic

ulcer and ulcerative colitis also leads to nausea and

vomiting. Antiemetics include various classes and groups

of drugs with versatile pharmacological mechanisms.

Dopaminergic antagonists

The neurotransmitter dopamine plays an important role

in neural functions involving reward processes, approach

behavior, economic decision making, adaptive behavior,

motion, and cognition [10]. Dopamine receptors belong

to two subclasses, with D1 and D5 receptors sharing

homology and coupling with Gs and D2, D3, and D4

receptors coupling with Gi.

Selective D1-receptor antagonists have been studied as

potential therapeutics for Parkinson’s disease, psychotic

behavior, substance abuse, and obesity [11] in animal

models and in human clinical trials [12].

D2-receptor antagonists were the first antiemetics used;

these drugs are currently primarily used as rescue

antiemetics. The primary reason for the ignorance of

these drugs is frequent induction of extrapyramidal side

effects such as akinesia, akathisia, and acute dystonic

reactions. Domperidone 1 and metopimazine 2 are

examples of D2-receptor antagonists that are effective

against nausea, which is a more troublesome chemother-

apy-induced side effect than vomiting; these do not cause

extrapyramidal side effects (Fig. 1) [13].

Phenothiazines

Phenothiazine was first synthesized by Bernthsen in

1883. In 1934 [14], it was found to possess insecticidal

properties. Later, Hardwood et al. [15] discovered the

anthelmintic activity of this compound in swine ascar-

idosis. In 1946, Halpern and Ducrot [16] screened various

phenothiazine compounds for antihistaminic properties.

The first compound with definite therapeutic value was

promethazine 3, which has proven antihistaminic as well

as sedative and hypnotic properties [17].

In 1950, the first neuroleptic phenothiazine prototype,

chlorpromazine 4, was introduced. It possesses a large

number of pharmacological activities such as adrenolytic,

antidopaminergic, antihistaminic, antiserotonin, antimus-

carinic, and antiemetic properties [18–22]. Subsequently,

numerous phenothiazines have been introduced such as

antiemetics with increased antiemetic potency, reduced

cardiovascular effects, milder tranquilizing action, and

decreased extrapyramidal side effects [23–25].

The difference in antiemetic activities between the

neuroleptic phenothiazines is because of differences in

the site of their sequestration in the central nervous

system; however, they act predominantly on the CTZ.

Phenothiazines are classified into three groups according

to the substituents on nitrogen: (i) aliphatic analogues,

which bear acyclic groups; (ii) piperidines, which contain

piperidine-derived groups; (iii) piperazine, which incor-

porate piperazine-derived substituents. The most rele-

vant antiemetic phenothiazines (3–15) are illustrated

in Table 1.

Butyrophenones

Neuroleptic drugs like droperidol 16 and haloperidol 17

are major tranquillizing drugs, which possess significant

antiemetic activity as a result of their D2-receptor

antagonist properties, especially when administered

through the intravenous and intramuscular routes [43].

However, side effects [44,45] such as drowsiness,

dysphoria, delayed discharge, extrapyramidal reactions,

restlessness, and anxiety after discharge have led to the

current reluctance to use these agents in the outpatient

setting. The structures of the commonly used antiemetic

butyrophenones are depicted in Table 2.

Benzamides

Metoclopramide hydrochloride monohydrate (Primperan

21; Table 3) is a benzamide derivative related to

orthopramides class that belongs to the neuropsycho-

tropic, antipsychotic neuroleptics (Table 3). It is a

derivative of procainamide but it is virtually devoid of

antiarrhythmic or local anesthetic activity in clinical

doses [67]. It shows both central and peripheral

Figure 1

Dompridone(Motillum ®) Metopimazine

21

Cl

NHN

O

NNHN

O

N

S

O

H2N

SO

O

Examples of D2-receptor antagonist.

2 Egyptian Pharmaceutical Journal

antiemetic activities. It is rapidly absorbed orally but has

a wide range of oral bioavailability. It has a plasma half life

of 4–6 h. In addition to its ability to block dopaminergic

receptors at the CTZ, metoclopramide increases lower

esophageal sphincter tone and enhances gastric and small

bowel motility, thereby preventing delayed gastric

emptying caused by opioid analgesics [68]. Although it

is not effective in controlling motion sickness, metoclo-

pramide has some peripheral cholinergic actions.

High doses of metoclopramide (1–2 mg/kg) are effective

in managing chemotherapy-induced emesis [69]; how-

ever, this is associated with high incidence of dystonic

reactions and extrapyramidal side effects.

It has been successfully used to treat dyspepsia,

gastrointestinal disorders, including irritable colon syn-

drome, and spastic constipation [67].

This gastroprokinetic activity is attributed to the release

of acetylcholine upon stimulation of 5-HT4 receptors,

whereas the antiemetic activity is attributed to the

antagonistic activity at both 5-HT3 serotoninergic and

D2-dopaminergic receptors in the CTZ of the central

nervous system. In addition, it stimulates orthograde

peristalsis, leading to suppression of the bile reflux, with

subsequent promotion of healing of gastric ulcers and

prevention of relapse. However, metoclopramide does not

accelerate healing of duodenal ulcers [70].

The antiemetic activity of metoclopramide as an

antiapomorphine drug is 35 times greater and more

selective than that of chlorpromazine [71]. Further, it

shows no sedative action at its antiemetic doses [71].

However, at large doses it produces extrapyramidal

side effects. It exerts its antiemetic activity through

Table 1 Antiemetic phenothiazines (3–15)

N

S

R1

R2

Name R1 R2 References

Aliphatic analogues Promethazine (3, Phenergan) –CH2-CH(CH3)N(CH3)2 H Couvoisier and colleagues [26,27]Chlorpromazine (4, Largactil) –(CH2)3N(CH3)2 Cl Delay and colleagues [28,29]Promazine (5, Sparine) –(CH2)3N(CH3)2 CF3 Wirth [30]Triflupromazine (6, Vesprin) –(CH2)3N(CH3)2 H Yale and colleagues [31,32]

Piperidines Pipamazine (7, Mornidine)

N(H2C)3 C

O

NH2

Cl Dobkin and Purkin [33]

Mepazine (8, Pacatal)

N

CH3

H2C

H Bowes [34]

Metopimazine (9, Compazine)

N(H2C)3 CONH2

SO2CH3 Jacob and colleagues [35,36]

Piperazines Trifluoperazine (10, Stelazine)

(H2C)3 CH3N N

CF3 Tedeschi et al. [37]

Thiethylperazine (11, Torecan)

(H2C)3 CH3N N

SCH2CH3 Bourquin et al. [38]

Perphenazine (12, Trilafon)

(H2C)3 CH2 CH2 OHN N

Cl Hotovy and Kapff-Walter [39]

Prochlorperazine (13, Campizine )

(H2C)3 CH3N N

Cl Gralla et al. [40]

Fluphenazine (14, Prolixin)

(H2C)3 CH3N N

CF3 Kline and Simpson [41]

Thiopropazate (15, Dartal)

N N(H2C)3 CH2 CH2 COCH3

Cl Toldy et al. [42]

Receptors antagonists as antiemetics Aboul-Enein et al. 3

central [72,73] and peripheral [74] dopamine receptor

antagonism. Moreover, metoclopramide is ineffective

against motion sickness and emesis occurring in labyr-

inthine episodes [71].

Orthopramides possess three common structural elements

required for binding to the receptor site: an aromatic

moiety, a carbonyl group or carbonyl group bioisosteres,

and a basic nitrogen atom. The weak affinity and lack of

selectivity of metoclopramide for dopaminergic and

serotoninergic receptors can be explained by the large

number of permissible conformers because of the

flexibility of its amino chain. Accordingly, Aboul-Enein

and colleagues [75,76] studied certain molecular modifica-

tions of metoclopramide, which imply (i) a change in the

substituents of the aromatic ring, (ii) structural variations

in the amine moiety, and (iii) an increase in the

lipophilicity through a change in the vicinal carbon atom

of the basic nitrogen to a cyclohexane ring (22–24; Fig. 2).

These compounds were evaluated for their dopamine

D2-receptor antagonistic activity in vivo by measuring

their ability to inhibit apomorphine-induced chewing

‘‘Zwansgnagen’’ in rats. Among these compound, 24

possessed an ED50 of 5.94 mmol/kg, being nearly two-fold

more potent than the previously reported cyclohexane-

based dopamine D2-receptor antagonist 23 (ED50 =

11.66 mmol/kg). Molecular simulation study of 24,

including fitting to the three-dimensional model of

dopamine D2-receptor antagonists using Discovery Stu-

dio 2.5 programs showed high-fit values [75]. The

experimental dopamine D2-receptor antagonistic activity

was consistent with the findings of the molecular

modeling study. Other substituted benzamides (Table 3)

that have been evaluated as antiemetics include

trimethobenzamide (25, Tigan), clebopride (27), cisa-

pride (30), and alizapride (39) [77]. Trimethobenzamide

is an antiemetic having some structural similarities to

both reserpine and antihistamines [78] as well as to

orthopramides. It possesses one-tenth to one-twentieth

of the antiemetic activity of chlorpromazine. Its antiemetic

action is primarily on the CTZ. Trimethobenzamide does

not cause depression at very high doses. It has no sedative,

hypotensive, or extrapyramidal effects; moreover, it shows

no antihistaminic activity, and it proved effective against

vomiting from various causes [79,80].

Cisapride 30 has a greater ability than metoclopramide to

reverse morphine-induced gastric stasis and is not

associated with extrapyramidal side effects. However,

cisapride does not prevent the decrease in lower

esophageal tone following antagonism by neostigmine in

the form of neuromuscular blockade and has lesser

antiemetic activity than metoclopramide.

It is worth mentioning that metoclopramide and its

congeners, besides being potent antiemetics, show

neuroleptic, antidyskinetic, and antiulcer effects, also

Table 2 The commonly used antiemetic butyrophenones (16–20)

Name Structure References

Benperidol 16

NH

N

N

O

O

F

Bobon et al. [46]

Droperidol 17

F

ON

N

HN O

Domino et al. [47]

Haloperidol 18F

ON

OH

ClGranger and Albu [48]

Lenperone 19

F

ON

F

O Nakra et al. [49]

Melperone 20

F

NO

Grozinger et al. [50]


Table 3 Antiemetic benzamides 25–39 (orthopramides)


Trimethobenzamide 25

C

OCH3OCH3

H3CO

ONH

O NCH3

CH3

Report of the Workgroup on Vaccines [51]

Bromopride 23

C NH

NC2H5

C2H5

O

NH2

Br

OCH3

Fontaine and Reuse [52]

Clebopride 27

Cl

H2N

NH

N

OCH3

O Cuena Boy and Macia Martinez [53]

Tiapide 28

C

S

NH

O

OH3C

NC2H5

C2H5

O

NH2

OCH3

Fontaine and colleagues [52,54]

Dazopride 29

Cl

H2N

NH

OCH3

ON

N

C2H5

C2H5

Lunsford and Cale [55]

Cisapride 30

OCH3

NH2

Cl

CO NH

N(CH2)3H3CO O

F

Van Daele and colleagues [56,57]

Troxipide 31

OCH3

OCH3H3CO

CONHNH

Burnton and colleagues [58,59]

Sulpiride 32

COOCH3

SO

OH2N

NH CH2N

C2H5 Laville and Margarit [60]

Sultopride 33

COOCH3

SC2H5

NH CH2N

C2H5

O

O

Bruguerolle et al. [61]

Amisulpride 34

COOCH3

SO

OC2H5

NH CH2N

C2H5

NH2

Florvall and Oegren [62]


being useful as nonhormonal therapeutic agents in severe

cases of menopausal disorders [67,81].

5-HT3 serotoninergic receptor antagonists

5-HT3 antagonists are a class of medications that act as

receptor antagonists at the 5-HT3 receptor, a subtype of

the serotonin receptors found at the terminal ends of the

vagus nerve and in certain areas of the brain. They are

used as antiemetics in the prevention and treatment of

nausea and vomiting. They are particularly effective in

controlling nausea and vomiting caused by cancer

chemotherapy and are considered the gold standard for

this purpose [82].

5-HT3 receptors are present at several critical sites

involved in emesis, including vagal afferents, the solitary


Itopride 35

NH

O

ON

OCH3

OCH3

Florvall et al. [63]

Raclopride 36

COOCH3

NH CH2

N

C2H5

Cl

HO

Cl

Florvall and colleagues [63,64]

Remoxipride 37

COOCH3

NH CH2

N

C2H5

Br

H3CO

Florvall et al. [63]

Veralipride 38

COOCH3

NH CH2N

SO

OH2N

OCH3

Thominet et al. [65]

Alizapride 39

COOCH3N

NNH

NH CH2

NBleiberg et al. [66]

Figure 2

NNH

H2N

O

C2H5 C2H5

C2H5C2H5

OMe

Cl

H2N

O

OMe

NH

NCl

Metoclopramide21 22

24

23

NNH

NO

Cl

OMe

OMe

OMe

NNH

N RO

OCH3H2N

Cl

R = C2H5

R = CH2C6H5

R = CH2C6H2(OCH3)3R = CH(C6H5)2

Metoclopramide and structurally related compounds.


tract nucleus, and the area postrema itself. Serotonin is

released by the enterochromaffin cells of the small

intestine in response to chemotherapeutic agents and

may stimulate the vagal afferents (through the 5-HT3

receptor) to initiate the vomiting reflux. 5-HT3 receptor

antagonists suppress vomiting and nausea by preventing

serotonin from binding to the 5-HT3 receptors. The

highest concentration of 5-HT3 receptors in the central

nervous system is found in the solitary tract nucleus and

CTZ, and 5-HT3 antagonists may also suppress vomiting

and nausea by acting at these sites [59].

5-HT3 serotoninergic receptor

5-HT3 antagonists are most effective in prevention and

treatment of chemotherapy-induced nausea and vomiting

(CINV), especially that caused by highly emetogenic

drugs such as cisplatin. When used for prevention and

treatment of CINV, they may be administered alone or,

more frequently, in combination with a glucocorticoid,

usually dexamethasone. They are usually administrated

intravenously, shortly before administration of the che-

motherapeutic agent [60], although some authors have

argued that oral administration may be preferred [83].

The concomitant administration of an NK1 receptor

antagonist, such as aprepitant, significantly increases the

efficacy of 5-HT3 antagonists in preventing both acute

and delayed CINV [84].

5-HT3 antagonists are also indicated in the prevention

and treatment of radiation-induced nausea and vomiting,

when needed, and postoperative nausea and vomiting.

Although they are highly effective at controlling CINV –

they stop symptoms altogether in up to 70% of people

and reduce them in the remaining 30% – they are only as

effective as other agents in controlling postoperative

nausea and vomiting.

Current evidence suggests that 5-HT3 antagonists are

ineffective in controlling motion sickness [85]. A ran-

domized, placebo-controlled trial of ondansetron 40 to

treat motion sickness in air ambulance personnel showed

subjective improvement, but it was statistically insignificant.

Chemical structures of the first generation 5-HT3

receptor antagonists [86] can be categorized into three

main classes (Table 4).

Carbazole derivatives

Ondansetron 40 was the first 5-HT3 antagonist; it was

developed by Glaxo around 1984. Its efficacy was first

established in 1987 in animal models [90]. Several

studies have demonstrated that ondansetron produces

an antiemetic effect equal to or superior to that of high

doses of metoclopramide; however, ondansetron has a

superior toxicity profile compared with dopaminergic

antagonist agents [88,91]. Ondansetron (0.15 mg/kg) is

administered intravenously 15–30 min before chemother-

apy, and this dose is repeated every 4 h for two additional

doses.

Ondansetron is not approved for use in children younger

than 4 years. Its clearance is diminished in patients

with severe hepatic insufficiency; therefore, such

patients should receive a single injectable or oral dose

no higher than 8 mg. The major adverse effects of

ondansetron include headache, constipation or diarrhea,

fatigue, dry mouth, and transient asymptomatic elevation

Table 4 The 5-HT3 receptor antagonists commonly used as antiemetics


Ondansetron (40, Zofran)

N

N N

O Gan [86]

Granisetron (41, Kytril)

NCH3 NH

O NN CH3 Gebbia et al. [87]

Dolasetron (42, Anzemet)

NH

O

ONO

Hainsworth et al. [88]

Ramosetron (43, Nasea)

N

N

HN

O Rabasseda [89]

Palonosetron (44, Aloxi)

N

O

H

N Gebbia et al. [87]


in liver function tests (alanine and aspartate transami-

nases), which may be related to concurrent cisplatin

administration.

Indole derivatives

Dolasetron 42 was first mentioned in the literature in

1989 [92]. Both oral and injectable formulations of dolasetron

are administered for the prevention of nausea and vomiting

associated with moderately emetogenic cancer chemother-

apy, including initial and repeat courses. Dolasetron should

be administered intravenously or orally at 1.8 mg/kg as a

single dose B30 min before chemotherapy [87].

Indazole derivatives

Granisetron 41 was developed around 1988 [93]. It has

demonstrated the same efficacy and safety margin as

ondansetron in preventing and controlling nausea and

vomiting at broad-range doses (e.g. 10–80 mg/kg and

empirically 3 mg/dose) especially in patients receiving

emetogenic chemotherapy, including a high dose of

cisplatin [94].

Ramosetron 43 is only available in Japan and certain

Southeast Asian countries as of 2008. It has a higher affinity

for the 5-HT3 receptors than do the older 5-HT3

antagonists, and it maintains its effects over 2 days. It is

therefore significantly more effective against delayed

CINV [89]. In animal studies, ramosetron was also effective

against irritable bowel syndrome-like symptoms [95].

Palonosetron 44 is the newest 5-HT3 receptor antago-

nist. It shows antiemetic activity at both central and

gastrointestinal sites. In comparison with the older

5-HT3 antagonists, it has a higher binding affinity to

the 5-HT3 receptors, a higher potency, a significantly

longer half life (B40 h; four to five times longer than that

of dolasetron, granisetron, or ondansetron), and an

excellent safety profile. A dose finding study demon-

strated that the effective dose was 0.25 mg or slightly

higher [87].

ConclusionAntiemetics include various classes and groups having

versatile pharmacological mechanisms. This review deals

with D2-dopaminergic receptor and 5-HT3 serotoninergic

receptor antagonists possessing antiemetic potential,

which could be considered as biocandidates in the

development of new antiemetics or targets for extensive

molecular modifications in order to accentuate some

of their effects and attenuate or abolish side effects.

AcknowledgementsConflicts of interestThere are no conflicts of interest.

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Synthesis and DPPH radical-scavenging activity of some new

5-(N-substituted-1H-indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazole

derivativesHeba M. Abo-Salema, Manal Sh. Ebaida, Eslam R. El-Sawya,Abd El-Nasser El-Gendyb and Adel H. Mandoura

aChemistry Department of Natural Compoundsand bMedicinal and Aromatic Plants Department,

National Research Centre, Dokki, Giza, Egypt

Correspondence to Eslam R. El-Sawy, ChemistryDepartment of Natural Compounds, National ResearchCentre, Dokki 12311, Giza, EgyptTel: + 20 23 833 939 4; fax: + 20 33 370 931;e-mail: [email protected]

Received 7 October 2012Accepted 3 January 2013


2013,12:11–19

Background and objectives

Heterocyclic systems with thiadiazole nucleus show a wide spectrum of biological

activities such as antioxidant, analgesic, antitumor, and anti-inflammatory activities.

The aim of this study is to describe the synthesis of some new 5-(N-substituted-1H-indol-

3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazole derivatives and to evaluate their antioxidant

activity using 2,20-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity.

Materials and methods

A one-pot reaction of N-substituted-1H-indol-3-carboxaldehyde 1a,b with thioglycolic

acid and thiosemicarbazide in concentrated sulfuric acid yielded novel 2-amino-5-(N-

substituted-1H-indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazoles 2a,b. The reaction of

2a,b with some benzenesulfonyl chlorides and/or benzoyl chlorides yielded sulfonamides

3a,b and 4a,b and benzamide 5a,b and 6a,b derivatives, respectively, whereas, the

reaction of 2a,b with chloroacetyl chloride yielded chloroacetamide derivatives 7a,b,

which, on cyclization with potassium thiocyanate, yielded thiazolidinone derivatives 8a,b.

The reaction of 2a,b with sodium azide yielded tetrazole derivatives 9a,b. However, the

reaction of 2a,b with benzaldehyde yielded Schiff bases 10a,b, which cyclized with

chloroacetyl chloride and/or phenacyl bromide to yield azetidinone derivatives 11a,b and

12a,b, respectively. However, the reaction of 10a,b with sodium cyanide, followed by

acid hydrolysis yielded the a-amino acid derivatives 14a,b. Diazotization of 2a,b yielded

diazonium salt A, which, on coupling with sodium azide, yielded the azido derivatives

15a,b. Cyclization of 15a,b with ethylacetoacetate yielded tetrazole derivatives 16a,b,

whereas the coupling reaction of A with malononitrile yielded dicyano derivatives 17a,b,

which, on cyclization with hydrazine hydrate, yielded 3,5-diaminopyrazole derivatives

18a,b. The newly synthesized compounds were screened for their antioxidant activity

using 2,20-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity.

Results and conclusion

4-{5-[(1H-Indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl]diazo}-1H-pyrazole-3,5-

diamine (18a) was highly active with radical-scavenging activity (IC50 of 69.14mg/ml)

compared with ascorbic acid (IC50 of 6.50 mg/ml).

Keywords:

DPPH radical-scavenging activity, indole-3-carboxaldehyde, synthesis, tetrazole,

thiazolo[4,3-b]-1,3,4-thiadiazole

Egypt Pharm J 12:11–19& 2013 Division of Pharmaceutical and Drug Industries Research, National Research Centre1687-4315

IntroductionThiadiazole is a versatile moiety that shows a wide variety

of biological activities, viz, antioxidant, analgesic, antic-

onvulsant, anti-hepatitis B, antitubercular, antitumor, anti-

depressant, anti-inflammatory, antimicrobial, and anti-

Helicobacter pylori [1–6]. Besides these, fused 5H-thiazo-

lo[4,3-b]-1,3,4-thiadiazoles have been prepared and be-

come a substance among 1,3,4-thiadiazoles that has drawn

the attention of researchers [7–9]. Moreover, indole, which

is the potent basic pharmacodynamic nucleus, has been

reported to have a wide variety of biological properties, viz,

antioxidant [10], anti-inflammatory [11,12], anti-cancer

[13], and antimicrobial activities [12,14]. On the basis of

the above observations and as a part of our continuous work

on the preparation of new poly-heterocycles with pharma-

ceutical values [11–16], the present study focuses on the

synthesis of some new N-substituted-3-indolyl-5H-thiazo-

lor-1,3,4-thiadiazoles for the evaluation of their antioxidant

activity using 2,20-diphenyl-1-picrylhydrazyl (DPPH) radi-

cal-scavenging activity starting from N-substituted indole-

3-carboxaldehyde.

Materials and methodsChemistry

Melting points were determined in open capillary tubes on

an Electrothermal 9100 digital melting point apparatus

(Electrothermal Engineering Ltd, Serial No. 8694, Rochford,

Original article 11


DOI: 10.7123/01.EPJ.0000426585.93667.87

United Kingdom) and were uncorrected. Elemental analyses

were carried out on a Perkin-Elmer 2400 analyzer (940

Winter Street, Waltham, Massachusetts, USA) and were

found to be within ± 0.4% of the theoretical values

(Table 1). IR spectra were recorded by Perkin-Elmer 1600

Fourier transform infrared spectroscopy against KBr discs.

The 1H NMR spectra were measured using a mass spectro-

meter (JEOL Ltd. 1-2, Musashino 3-chome Akishima,

Tokyo, Japan) 500 MHz in DMSO-d6, and chemical shifts

were recorded in d ppm relative to TMS as an internal

standard. Mass spectra (EI) were run at 70 eV using a

JEOL-JMS-AX500 mass spectrometer (Japan). All reagents

and solvents were of commercial grade. 1H-indole-3-

carboxaldehyde (1a) [17] and N-benzyl-1H-indole-3-car-

boxaldehyde (1b) have been prepared as reported [18].

2-Amino-5-(1H-indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazole

(2a) and 2-amino-5-(N-benzyl-1H-indol-3-yl)-5H-thiazolor-

1,3,4-thiadiazole (2b)

N-substituted-1H-indole-3-carboxaldehydes 1a or 1b

(0.02 mol) and thioglycolic acid (1.84 ml, 0.02 mol) were

mixed for 10–15 min. To the reaction mixture, thiosemi-

carbazide (1.82 g, 0.02 mol) was added with stirring and

then concentrated sulfuric acid (10 ml) was added in

portions upon cooling. The reaction mixture was homo-

genized and left for 24 h in a deep freezer (– 201C). The

reaction mixture was then treated with crushed ice (50 g)

and the suspension obtained was neutralized with an

aqueous sodium hydroxide solution (40%) to pHC7–8.

The precipitate that formed was filtered off, air dried, and

crystallized from aqueous dioxane (Scheme 1 and Table 1).

N-[5-(1H-Indol-3-yl1)-5H-thiazolor-1,3,4-thiadiazol-2-yl]

benzenesulfonamide (3a), N-[5-(N-benzyl-1H-indol-3-yl)-5H-

thiazolo [4,3-b]-1,3,4-thiadiazol-2-yl]benzenesulfonamide

(3b), 4-chloro-N-[5-(1H-indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-

thiadiazol-2-yl]benzene-sulfonamide (4a), and 4-chloro-N-

[5-(N-benzyl-1H-indol-3-yl)-5H-thiazolokr-1,3,4-thiadiazol-2-

yl]benzenesulfonamide (4b)

A mixture of compounds 2a or 2b (0.001 mol) and ben-

zenesulfonyl chloride, or 4-chlorobenzenesulfonyl chlor-

ide (0.001 mol) in dry dioxane (10 ml) containing a few

drops of triethylamine was heated at reflux for 6 h. After

cooling, the reaction mixture was poured onto cold water

(10 ml). The solid that formed was filtered off, air dried,

and crystallized from dioxane (Scheme 1 and Table 1).

N-[5-(1H-Indol-3-yl)-5H-thiazolor-1,3,4-thiadiazol-2-yl]

benzamide (5a), N-[5-(N-benzyl-1H-indol-3-yl)-5H-

thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl]benzamide (5b), 2-chloro-

N-[5-(1H-indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazol-2-

yl]benzamide (6a), and 2-chloro-N-[5-(N-benzyl-1H-indol-3-

yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl]benzamide (6b)

A mixture of compounds 2a or 2b (0.001 mol) and

benzoyl chloride or 2-chlorobenzoyl chloride (0.001 mol)

in dry dioxane (10 ml) containing a few drops of

triethylamine was heated at reflux for 8 h. After cooling,

Table 1 Physical and analytical data of the newly synthesized compounds

Analysis (%; calculated/found)

Compound number Formula (MW) MP (1C) Yield (%) C H N

2a C12H10N4S2 (274.36) 146–148 94 52.53/52.33 3.67/3.58 20.42/20.312b C19H16N4S2 (364.49) 86–88 88 62.61/62.44 4.42/4.26 15.37/15.203a C18H14N4O2S3 (414.52) 111–113 70 52.15/52.01 3.40/3.27 13.52/13.413b C25H20N4O2S3 (504.65) 76–78 65 59.50/59.36 3.99/3.81 11.10/10.994a C18H13ClN4O2S3 (448.97) 212–214 83 48.15/48.01 2.92/2.76 12.48/12.324b C25H19ClN4O2S3 (539.09) 187–189 76 55.70/55.54 3.55/3.41 10.39/10.225a C19H14N4OS2 (378.47) 165–168 84 60.30/60.16 3.73/3.61 14.80/14.665b C26H20N4OS2 (468.59) 136–138 77 66.64/66.48 4.30/4.21 11.96/11.776a C19H13ClN4OS2 (412.92) 300–302 84 55.27/55.04 3.17/3.06 13.57/13.416b C26H19ClN4OS2 (503.04) 300 81 62.08/62.16 3.81/3.66 11.14/11.027a C14H11ClN4OS2 (350.85) 135–137 91 47.93/47.98 3.16/3.20 15.97/15.997b C21H17ClN4OS2 (440.97) 127–129 95 57.20/57.28 3.89/3.76 12.71/12.688a C15H11N5OS3 (373.48) 175–177 90 48.24/48.11 2.97/3.00 18.75/18.808b C22H17N5OS3 (463.60) 162–164 91 57.00/57.23 3.70/3.55 15.11/15.229a C13H9N7S2 (327.39) 252–254 81 47.69/47.73 2.77/2.64 29.95/29.809b C20H15N7S2 (417.51) 175–177 82 57.53/57.40 3.62/3.58 23.48/23.4010a C19H14N4S2 (362.47) 170–172 86 62.96/62.76 3.89/3.99 15.46/15.5410b C26H20N4S2 (452.59) 158–160 83 69.00/69.11 4.45/4.33 12.38/12.5511a C21H15ClN4OS2(438.95) 100–102 78 57.46/57.66 3.44/3.68 12.76/12.5611b C28H21ClN4OS2 (529.08) 70–72 65 63.56/63.44 4.00/4.28 10.59/10.7212a C27H20N4OS2 (480.6) 159–161 75 67.48/67.50 4.19/4.32 11.66/11.4512b C34H26N4OS2 (570.73) 172–174 71 71.55/71.60 4.56/4.44 9.82/9.7813a C20H15N5S2 (389.50) 234–236 79 61.67/61.62 3.88/3.90 17.98/17.9313b C27H21N5S2 (479.62) 106–108 80 67.61/67.69 4.41/4.46 14.60/14.5814a C20H16N4O2S2 (408.5) 196–208 76 58.80/58.77 3.95/3.90 13.72/13.6914b C27H22N4O2S2 (498.62) 130 70 65.04/65.00 4.45/4.35 11.24/11.4415a C12H8N6S2 (300.36) 86–88 40 – – –15b C19H14N6S2 (390.48) 61–3 30 – – –16a C16H12N6O2S2 (384.44) 126–128 46 49.99/49.75 3.15/3.00 21.86/21.6616b C23H18N6O2S2 (474.56) 92–94 36 58.21/58.00 3.82/3.78 17.71/17.6917a C15H9N7S2 (351.41) 144–146 67 51.27/51.33 2.56/2.35 27.90/27.9317b C22H15N7S2 (441.53) 123–125 55 59.85/59.92 3.42/3.33 22.21/22.3018a C15H13N9S2 (383.45) 201–202 83 46.98/47.01 3.42/3.37 32.87/32.6618b C22H19N9S2 (473.58) 130–132 85 55.80/55.71 4.04/4.15 26.61/26.45

Compounds 15a,b was decomposed slowly during the preparation of the samples analyzed.


the reaction mixture was poured onto cold water (20 ml).

The solid that formed was filtered off, air dried, and

crystallized from dioxane (Scheme 1 and Table 1).

N-[5-(1H-Indol-3-yl)-5H-thiazolor-1,3,4-thiadiazol-2-yl]-2-

chloroacetamide (7a) and N-[5-(N-benzyl-1H-indol-3-yl)-5H-

thiazolo [4,3-b]-1,3,4-thiadiazol-2-yl]-2-chloroacetamide (7b)

To a solution of compounds 2a or 2b (0.02 mol) in dry

benzene (60 ml), a solution of chloroacetyl chloride (5 ml,

0.04 mol) in dry benzene (20 ml) was added dropwise

under vigorous stirring at 0–51C. After complete addition,

the reaction mixture was heated at reflux for 3 h. The

solvent was evaporated in vacuo and the solid that formed

was washed with sodium hydrogen carbonate (20 ml, 5%)

and then with water, air dried, and crystallized from

chloroform (Scheme 1 and Table 1).

3-[5-(1H-Indol-3-yl)-5H-thiazolor-1,3,4-thiadiazol-2-yl]-2-

iminothiazolidin-4-one (8a) and 3-[5-(N-benzyl-1H-indol-3-yl)-

5H-thiazolor-1,3,4-thiadiazol-2-yl]-2-iminothiazolidin-4-one (8b)


potassium thiocyanate (0.58 g, 0.006 mol) in dry acetone

(10 ml) was heated at reflux for 3 h. The solid that formed

was filtered off, air dried and crystallized from chloroform

(Scheme 1 and Table 1).

5-(1H-Indol-3-yl)-2-(1H-tetrazol-1-yl)-5H-thiazolor-1,3,4-

thiadiazole (9a) and 5-(N-benzyl-1H-indol-3-yl)-2-(1H-

tetrazol-1-yl)-5H-thiazolor-1,3,4-thiadiazole (9b)

A mixture of compounds 2a or 2b (0.001 mol), triethyl

orthoformate (0.15 ml, 0.001 mol), and sodium azide

(0.065 g, 0.001 mol) in glacial acetic acid (10 ml) was

stirred under reflux for 2 h. After cooling, the reaction

mixture was neutralized with concentrated hydrochloric

acid (10 ml). The solid that formed was filtered off,

washed with water, air dried, and crystallized from

absolute ethanol (Scheme 1 and Table 1).

N-Benzylidene-(5-(1H-indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-

thiadiazol-2-yl)-2-amine (10a) and N-benzylidene-[(5-(N-

benzyl-1H-indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl]-

2-amine (10b)


benzaldehyde (1.06 g, 0.01 mol) in glacial acetic acid

Scheme 1

N

N

S

N

S

NHSO2

R

3a,b, X= H4a,b, X= Cl

TEA

HC(OC2H5)3

NaN3

N

N

S

N

S

N

RN

NN

N

N

S

N

S

NH2

R

N

CHO

R

+H2N

NH

S NH2

H2SO 4

HSOH

O

+

1a,b

X

TEA

N

N

S

N

S

NHCO

RY

5a,b , Y= H6a,b , Y= Cl

N

N

S

N

S

NH

R

O

Cl

N

N

S

N

S

N

RS

O

HN

ClCOCH 2

Cl

KSCN

2a,b

7a,b

8a,b

9a,b

SO 2ClX

COClY

1- 9, R, a=H , b=CH2Ph

Synthesis of compounds 1a,b to 9a,b.

3-Indolylthiazolothiadiazole Abo-Salem et al. 13

(20 ml) was heated at reflux for 6 and 8 h. After cooling,

the reaction mixture was poured onto ice water (50 ml).

The solid that formed was filtered off, air dried, and

crystallized from benzene (Scheme 2 and Table 1).

1-[5-(1H-Indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl]-

3-chloro-4-phenylazetidin-2-one (11a), 1-[5-(N-benzyl-1H-

indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl]-3-chloro-4-

phenylazetidin-2-one (11b), 1-(5-(1H-indol-3-yl)-5H-

thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl)-3,4-diphenylazetidin-2-

one (12a), and 1-[5-(N-benzyl-1H-indol-3-yl)-5H-thiazolo[4,3-

b]-1,3,4-thiadiazol-2-yl]-3,4-diphenylazetidin-2-one (12b)

To a solution of Schiff bases 10a or 10b (0.01 mol) in dry

dioxane (5 ml), a solution of chloroacetyl chloride and/or

phenacyl bromide (0.01 mol) in dry dioxane (5 ml) and

triethylamine (0.59 ml, 0.01 mol) was added. The reac-

tion mixture was heated at reflux for 12–14 h. The

reaction mixture was filtered off while hot and the

Scheme 2

2a,b

N

N

S

N

S

N

R10a,b

N

N

S

N

S

N

R

O

12a,b

PhCH 2COBrTEA

dry dioxaneN

N

S

N

S

NH

RCN

13a,b

N

N

S

N

S

NH

RCOOH

14a,b

50% H 2SO 4

NaCNgl.AcOH

NaNO2/HCl

N

N

S

N

S

N

R A

N Cl

N

N

S

N

S

N3

R

N

N

S

N

S

N

R

N N

CH3

COOH

17a,bN

N

S

N

S

NH

R

N

NC

CN 15a,b

16a,b18a,b

N

N

S

N

S

N

R

N

NHN

NH2

H2N

CHO

gl.AcOH

CH2(CN)2

NH2NH2

NaN3

CH3COCH2COOC2H5

2-18, R, a=H , b=CH2Ph

N

N

S

N

S

NH2

R

N

N

S

N

S

N

R

O

ClClCH2COCl

TEAdry dioxane

11a,b

Synthesis of compounds 10a,b to 18a,b.


solvent was removed in vacuo. The residue solid was

treated with water and filtered, air dried, and crystallized

from absolute ethanol (Scheme 2 and Table 1).

2-[5-(1H-Indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl

amino]phenylacetonitrile (13a) and 2-[5-(N-benzyl-1H-indol-

3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl

amino]phenylacetonitrile (13b)

To a solution of Schiff bases 10a or 10b (0.01 mol) in

glacial acetic acid (20 ml) sodium cyanide (0.49 g,

0.01 mol) was added and the reaction mixture was heated

at reflux for 6 h. After cooling, the reaction mixture

was poured onto cold water (10 ml) and the solid that

formed was filtered off, washed with water, air dried,

and crystallized from acetic acid–water (Scheme 2

and Table 1).

2-[5-(1H-Indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl

amino] phenyl acetic acid (14a) and 2-[5-(N-benzyl-1H-indol-

3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl amino]phenyl

acetic acid (14b)

A solution of compounds 13a or 13e (0.01 mol) in sulfuric

acid (30 ml, 50%) was heated at reflux for 10 h. After

cooling, the dark reaction mixture was poured onto cold

water (20 ml) and then neutralized with ammonia

solution (25%). The precipitate that formed was filtered

off, washed with water, air dried, and crystallized from

aqueous acetic acid (Scheme 2 and Table 1).

2-Azido-5-(1H-indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazole

(15a) and 2-azido-5-(N-benzyl-1H-indol-3-yl)-5H-

thiazolo[4,3-b]-1,3,4-thiadiazole (15b)

To a cold solution of compounds 2a or 2b (0.02 mol) in a

mixture of concentrated hydrochloric acid (5 ml) and ice

water (5 ml), a cold aqueous solution of sodium nitrite

(1.73 g, 0.025 mol) in ice water (5 ml) was added dropwise

under stirring at 0–51C. After 10 min, the reaction

mixture was decanted. To the decanted solution of the

diazonium salt thus formed (A), sodium azide (1.3 g,

0.02 mol) in water (5 ml) was added dropwise. The

reaction mixture was left for 15 min at room temperature

and the azide was extracted by chloroform (3–10 ml) and

dried over anhydrous sodium sulfate. The solvent was

evaporated in vacuo and the residue was used without

subsequent purification, and used in the reaction

immediately after its formation because of its instability


1-[5-(1H-Indol-3-yl)-5H-thiazolor-1,3,4-thiadiazol-2-yl]-5-

methyl-1H-1,2,3-triazole-4-carboxylic acid (16a) and 1-[5-(N-

benzyl-1H-indol-3-yl)-5H-thiazolor-1,3,4-thiadiazol-2-yl]-5-

methyl-1H-1,2,3-triazole-4-carboxylic acid (16b)

To a solution of sodium (0.23 g, 0.01 mol) in absolute

methanol (20 ml) ethylacetoacetate (1.34 g, 0.01 mol) and

compounds 15a or 15b (0.01 mol) were added dropwise

under cooling in an ice bath. The reaction mixture was

kept in an ice water bath for 30 min and then gradually

heated under reflux for 1 h. After cooling, the reaction

mixture was neutralized by diluted hydrochloric acid

(1 : 1). The solid that formed was filtered off, washed

with water, air dried, and crystallized from methanol


2-{5-[(1H-Indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl]

hydrazono}malononitrile (17a) and 2-{5-[(N-benzyl-1H-indol-

3-yl)-5H-thiazolor-1,3,4-thiadiazol-2-yl]hydrazono}-

malononitrile (17b)

To a cold solution of compounds 2a or 2b (0.02 mol) in a

mixture of concentrated hydrochloric acid (5 ml) and ice

water (5 ml), a cold aqueous solution of sodium nitrite

(1.73 g, 0.025 mol) in ice water (5 ml) was added dropwise

under stirring at 0–51C. After 10 min, the reaction

mixture was decanted. To the decanted solution of the

diazonium salt thus formed (A), a cold solution of malo-

nonitrile (1.3 g, 0.02 mol) and sodium acetate trihydrate

(5.4 g, 0.04 mol) in ethanol (10 ml) was added under

stirring at 0–51C. The stirring was continued for an

additional 3 h at 0–51C, and then left overnight in the

refrigerator. The reaction mixture was poured onto water

(250 ml) and the solid that formed was filtered off, air

dried, and crystallized from absolute ethanol (Scheme 2

and Table 1).

4-{5-[(1H-Indol-3-yl)-5H-thiazolor-1,3,4-thiadiazol-2-yl]diazo}-

1H-pyrazole-3,5-diamine (18a) and 4-{5-[(N-benzyl-1H-indol-

3-yl)-5H-thiazolor-1,3,4-thiadiazol-2-yl]diazo}-1H-pyrazole-

3,5-diamine (18b)


hydrazine hydrate (0.75 ml, 0.015 mol) in absolute

ethanol (20 ml) was heated at reflux for 6 h. The solvent

was evaporated in vacuo to half of its volume and the solid

that formed was filtered off, washed with water, air dried,

and crystallized from absolute ethanol (Scheme 2

and Table 1).

Biological assay

DPPH radical-scavenging activity

The antioxidant activity of the test compounds was

measured in terms of hydrogen-donating or radical-

scavenging ability using the stable radical 2,20-diphenyl-1-

picrylhydrazyl (DPPH) (Sigma Chemical Co., Steinheim,

Germany) [19]. A volume of 50 ml of a DMSO stock

solution of tested compounds at four different concen-

trations (50, 100, 200, and 300 mg/ml) was added to 2 ml

of 6� 10–5 mol/l dimethylsulfoxide solution of DPPH

(2.3659 mg from DPPH/100 ml DMSO). The mixtures

were shacked in a vortex (2500 rpm) for 1 min and then

placed in a dark room. Ascorbic acid (Sigma-Aldrich

Chemie GmbH, Taufkirchen, Germany) was used as a

reference. The decrease in absorbance at 517 nm was

determined using a JENWAY 6315 spectrophotometer

(Keison Products, Chelmsford, England) after 1 h for all

samples. Dimethylsulfoxide was used to zero the spectro-

photometer. The absorbance of the radical without a

sample was used as a negative control. The amount of

sample necessary to decrease the absorbance of DPPH

(IC50) by 50% was calculated graphically. The inhibition

percentage of the DPPH radical (scavenging activity) was

calculated according to the following formula:

% I¼ AB�Asð Þ/AB½ ��100;


Table 2 Spectral characterization of the newly synthesized compounds

Compoundnumber IR (gmax/cm) 1H NMR (d, ppm) Mass (m/z, %)

2a 3410 (NH2), 3169 (NH), 1635(C = N), 1575 (C = C)

12.11 (s, 1H, NH), 11.11 (s, 1H, thiazolyl 5-H), 9.90 (s, 1H,thiazolyl 7-H), 8.25 (s, 1H, indolyl 2-H), 8.06 (d, 1H, indolyl7-H), 7.48 (d, 1H, indolyl 4-H), 7.23-7.16 (m, 2H, indolyl6-H and 5-H), 3.73 (s, 2H, NH2)

274 (M + , 1), 256 (16), 192 (5),144 (34), 128 (14), 116 (16),83 (47), 18 (100)

2b 3336 (NH2), 1628 (C = N), 1543(C = C)

12.10 (s, 1H, thiazolyl 5-H), 9.93 (s, 1H, thiazolyl 7-H), 8.25(s, 1H, indolyl 2-H), 8.27-7.18 (m, 9H, Ar-H), 5.57 (s, 2H,CH2-N), 3.85 (s, 2H, NH2)

–

3a 3156 and 3111 (NH), 1636 (C = N),1602 (C = C), 1354 and 1163(SO2-N)

12.14 (s, 1H, thiazolyl 5-H), 9.97 (s, 1H, thiazolyl 7-H), 8.95(s, 1H, NH), 8.31 (s, 1H, indolyl 2-H), 8.11-7.20 (m, 9H,Ar-H), 5.08 (s, 1H, NH)

–

3b 3125 (NH), 1631 (C = N), 1574(C = C), 1363 and 1148 (SO2-N)

– 504 (M + , 21), 430 (12), 353(10), 91 (100)

4a 3232 and 3126 (NH), 1624 (C = N),1575 (C = C), 1368 and 1136(SO2-N), 745 (C-Cl)

– 448/450 (M + /M + + 2, 33/11),330 (2), 191 (20), 113 (37),111 (100)

4b 3168 (NH), 1618 (C = N), 1610(C = C), 1366 and 1134 (SO2-N),747 (C-Cl)

12.01 (s, 1H, thiazolyl 5-H), 9.92 (s, 1H, thiazolyl 7-H), 8.69(s, 1H, NH), 8.42 (s, 1H, indolyl 2-H), 8.21-7.18 (m, 13H,Ar-H), 5.51 (s, 2H, CH2-N)

–

5a 3327 and 3120 (NH), 1695 (C = O),1640 (C = N), 1585 (C = C)


378 (M + , 23), 350 (10), 274(20), 258 (1), 105 (100)

5b 3154 (NH), 1710 (C = O),1638 (C = N), 1563 (C = C)

12.24 (s, 1H, thiazolyl 5-H), 9.94 (s, 1H, thiazolyl 7-H), 8.26(s, 1H, indolyl 2-H), 8.01-7.07 (m, 14H, Ar-H), 5.42 (s, 2H,CH2-N), 3.75 (s, 1H, NH)

–

6a 3260 and 3112 (NH), 1688 (C = O),1644 (C = N), 1585 (C = C), 775(C-Cl)


–

6b 3212 (NH), 1759 (C = O),1643 (C = N), 1578 (C = C),773 (C-Cl)

– 503/505 (M + /M + + 2, 19/6),391 (10), 113 (27), 111 (75),91 (100)

7a 3240 and 3163 (NH), 1722 (C = O),1618 (C = N), 1521 (C = C),747 (C-Cl)

11.85 (s, 1H, thiazolyl 5-H), 9.91 (s, 1H, thiazolyl 7-H), 8.26(s, 1H, indolyl 2-H), 7.94-7.26 (m, 4H, Ar-H), 6.76 (s, 1H,NH), 4.75 (s, 2H, CH2), 4.11 (s, 1H, NH)

–

7b 3265 (NH), 1710 (C = O), 1588(C = N), 1529 (C = C), 734 (C-Cl)

– 440/442 (M + /M + + 2, 30/10),349 (20), 318 (14), 91 (100)

8a 3186 and 3121 (NH), 1753 (C = O),1616 (C = N), 1521 (C = C)

12.12 (s, 1H, thiazolyl 5-H), 9.93 (s, 1H, thiazolyl 7-H), 9.15(s, 1H, NH), 8.29 (s, 1H, indolyl 2-H), 8.10-7.23 (m, 4H,Ar-H), 6.08 (s, 1H, NH), 4.13 (s, 2H, CH2)

373 (M + , 34), 345 (10), 317(20), 142 (100), 117 (15)

8b 3265 (NH), 1725 (C = O),1612 (C = N), 1522 (C = C)

12.15 (s, 1H, thiazolyl 5-H), 9.93 (s, 1H, thiazolyl 7-H), 8.72(s, 1H, NH), 8.31 (s, 1H, indolyl 2-H), 8.26-7.36 (m, 9H,Ar-H), 5.21 (s, 2H, CH2-N), 4.20 (s, 2H, CH2)

–

9a 3160 (NH), 1643 (C = N),1594 (C = C)

12.10 (s, 1H, thiazolyl 5-H), 9.92 (s, 1H, thiazolyl 7-H), 8.86(s, 1H, tetrazolyl 5-H), 8.23 (s, 1H, indolyl 2-H), 7.76-7.24(m, 4H, Ar-H), 6.90 (s, 1H, NH)

–

9b 1635 (C = N), 1572 (C = C) 11.65 (s, 1H, thiazolyl 5-H), 9.92 (s, 1H, thiazolyl 7-H), 8.82(s, 1H, tetrazolyl 5-H), 8.42 (s, 1H, indolyl 2-H), 8.06-7.15(m, 9H, Ar-H), 5.92 (s, 2H, CH2-N)

417 (M + , 17), 385 (2), 353 (21),117 (12), 91 (100)

10a 3157 (NH), 1624 (C = N),1565 (C = C)

12.03 (s, 1H, thiazolyl 5-H), 10.11 (s, 1H, thiazolyl 7-H),9.90 (s, 1H, NH), 8.91 (s, 1H, CH = N), 8.50 (s, 1H,indolyl 2-H), 8.34-7.40 (m, 9H, Ar-H)

–

10b 1628 (C = N), 1571 (C = C) 12.11 (s, 1H, thiazolyl 5-H), 9.95 (s, 1H, thiazolyl 7-H), 9.01(s, 1H, CH = N), 8.54 (s, 1H, indolyl 2-H), 8.23-7.11 (m,14H, Ar-H), 5.66 (s, 2H, CH2-N)

–

11a 3154 (NH), 1702 (C = O),1633 (C = N), 1601 (C = C),736 (C-Cl)

– 438/440 (M + /M + + 2, 12/4),410 (1), 402 (3), 326 (10), 77(100)

11b 1724 (C = O), 1637 (C = N),1563 (C = C), 745 (C-Cl)

12.22 (s, 1H, thiazolyl 5-H), 9.93 (s, 1H, thiazolyl 7-H), 8.33(s, 1H, indolyl 2-H), 8.10-7.07 (m, 14H, Ar-H), 5.20 (d, 2H,CH), 5.07 (d, 2H, CH)

–

12a 3201 (NH), 1739 (C = O),1640 (C = N), 1568 (C = C)

11.53 (s, 1H, thiazolyl 5-H), 9.81 (s, 1H, thiazolyl 7-H), 8.57(s, 1H, NH), 8.25 (s, 1H, indolyl 2-H), 8.12-7.11 (m, 14H,Ar-H), 5.20 and 4.81 (2d, 2H, 2CH)

480 (M + , 2), 328 (10), 115 (14),103 (100)

12b 1737 (C = O), 1635 (C = N),1570 (C = C)

12.23 (s, 1H, thiazolyl 5-H), 9.91 (s, 1H, thiazolyl 7-H), 8.65(s, 1H, indolyl 2-H), 8.32-7.01 (m, 19H, Ar-H), 5.51 (s, 2H,CH2-N), 5.21 and 4.99 (2d, 2H, 2CH)

–

13a 3240 and 3141 (NH), 2211 (CN),1636 (C = N), 1583 (C = C)


389 (M + , 18), 349 (100), 333(10), 103 (6)

13b 3118 (NH), 2216 (CN),1625 (C = N) 1587 (C = C)

– 479 (M + , 31), 388 (7), 312 (2),91 (100)

14a 3418 (OH), 3265 and 3152 (NH),1700 (C = O), 1641 (C = N),1573 (C = C)

– 408 (M + , 46), 392 (1), 315 (20),287 (2), 117 (50), 116 (100)


where I is the DPPH inhibition %, AB the absorbance of

control (t = 0 h), and AS the absorbance of a tested

sample at the end of the reaction (t = 1 h). Each assay was

carried out in triplicate and the results were averaged.

Results and discussionChemistry

The reaction route for the synthesis of the newly

synthesized compounds has been described in Schemes

1 and 2. New 2-amino-5-(N-substituted-1H-indol-3-yl)-

5H-thiazolo[4,3-b]-1,3,4-thiadiazoles (2a,b) were prepared

by a one-pot reaction of N-substituted-1H-indole-3-

carboxaldehyde with thioglycolic acid and thiosemicarba-

zide in concentrated sulfuric acid according to the

procedure of Shukurov et al. [7] (Scheme 1). The IR

spectra of compounds 2a,b showed characteristic absorp-

tion bands at B3241–3410/cm for (NH2) and showed no

absorption band characteristic for C = O (Table 2). Their1H NMR (DMSO-d6) spectra showed two singlet signals

at d 12.12–9.90 ppm attributed to 5-H and 7-H of

thiazolo[4,3-b]-1,3,4-thiadiazole moiety, besides the other

aromatic protons located at their positions (Table 2).

The reaction of compounds 2a or 2b with benzenesulfo-

nyl chloride and 4-chlorobenzenesulfonyl chloride in dry

dioxane and in the presence of triethylamine led to the

formation of N-[5-(N-substituted-1H-indol-3-yl)-5H-

thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl]benzenesulfonamide

derivatives 3a,b and 4a,b, respectively (Scheme 1).

However, the reaction of 2a,b with benzoyl chloride

and 2-chlorobenzoyl chloride yielded N-[5-(N-substi-

tuted-1H-indol-3-yl)-5H-thiazolor-1,3,4-thiadiazol-2-yl]-

benzamide derivatives 5a,b and 6a,b, respectively

(Scheme 1).

In contrast, the reaction of 2a or 2b with chloroacetyl

chloride in dry benzene yielded N-[5-(N-substituted-1-

H-indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl]-2-

chloroacetamides (7a,b). Cyclization of the latter compounds

through their reactions with potassium thiocyanate in

dry acetone yielded 3-[5-(N-substituted-1H-indol-3-yl)-

5H-thiazolor-1,3,4-thiadiazol-2-yl]-2-iminothiazolidin-4-ones

(8a,b) (Scheme 1).

The treatment of 2a or 2b with triethyl orthoformate and

sodium azide according to Abu-Hashem et al. [20] yielded

the new 5-(N-substituted-1H-indol-3-yl)-2-(1H-tetrazol-1-

yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazols (9a,b) (Scheme 1).

The acid-catalyzed reaction of 2a,b with benzaldehyde in

glacial acetic acid under reflux yielded the corresponding

Schiff bases, N-benzylidene-[5-(N-substituted-1H-indol-

3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl]-2-amines

(10a,b) (Scheme 2). Cyclocondensation of the latter

Schiff bases with chloroacetyl chloride and/or phenacyl

bromide under reflux in dry dioxane and in the presence

of triethylamine yielded 3-chloro-4-phenylazetidin-2-one

derivatives 11a,b and 3,4-diphenylazetidin-2-one deriva-

tives 12a,b, respectively (Scheme 2).

However, the reaction of Schiff bases 10a or 10b with

sodium cyanide in glacial acetic acid yielded 2-[5-(N-

substituted-1H-indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-thia-

diazol-2-yl amino]phenylacetonitriles (13a,b) (Scheme 2).

Acid hydrolysis of the latter compounds 13a or 13b

yielded the corresponding a-amino acid 14a,b (Scheme 2).

Diazotization of compounds 2a or 2b with concentrated

hydrochloric acid and sodium nitrite at 0–51C yielded the

corresponding diazonium salts (A), which, under coupling

with sodium azide, yielded the corresponding azides,

namely, 2-azido-5-(N-substituted-1H-indol-3-yl)-5H-thia-

zolo[4,3-b]-1,3,4-thiadiazols (15a,b). The freshly prepared

azides 15a,b reacted with ethylacetoacetate in dry

methanol and in the presence of freshly prepared sodium

methoxide and yielded 1-[5-(N-substituted-1H-indol-3-yl)-

5H-thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl]-5-methyl-1H-1,2,3-

triazole-4-carboxylic acids (16a,b) (Scheme 2).

14b 3400 (OH), 1715 (C = O),1638 (C = N), 1524 (C = C)

13.45 (s, 1H, OH), 11.70 (s, 1H, thiazolyl 5-H), 9.65 (s, 1H,thiazolyl 7-H), 8.81 (s, 1H, NH), 8.40 (s, 1H, indolyl 2-H),8.19-7.01 (m, 14H, Ar-H), 5.51 (s, 2H, CH2-N), 2.3 (s, 1H,CH)

–

16a 3408 (OH), 3135 (NH),1692 (C = O), 1631 (C = N),1563 (C = C)

13.23 (s, 1H, OH), 12.12 (s, 1H, thiazolyl 5-H), 9.93 (s, 1H,thiazolyl 7-H), 8.40 (s, 1H, NH), 8.65 (s, 1H, indolyl 2-H),8.22-7.12 (m, 4H, Ar-H), 1.25 (s, 3H, CH3)

–

16b 3368 (OH), 1707 (C = O),1639 (C = N), 1585 (C = C)

– 474 (M + , 26), 460 (11), 431(10), 389 (8), 91 (100)

17a 3159 and 3112 (NH), 2195 (CN),1628 (C = N), 1560 (C = C)


–

17b 3160 (NH2), 2205 (CN),1644 (C = N), 1615 (C = C)

– 441 (M + , 45), 413 (3), 391 (2),381 (1), 244 (10), 91 (100)

18a 3420 (NH2), 3192 and 3101 (NH),1635 (C = N), 1620 (N = N), 1564(C = C)

11.65 (s, 1H, thiazolyl 5-H), 8.90 (s, 1H, thiazolyl 7-H), 8.53(s, 1H, NH), 8.32 (s, 1H, indolyl 2-H), 7.83-7.20 (m, 4H,Ar-H), 6.50 (s, 1H, NH), 5.21 (s, 2H, NH2), 2.95 (s, 2H, NH2)

–

18b 3363 and 3246 (NH2), 3133 (NH),1638 (C = N), 1616 (N = N),1583 (C = C)

12.01 (s, 1H, thiazolyl 5-H), 9.91 (s, 1H, thiazolyl 7-H), 9.46(s, 2H, NH2), 8.42 (s, 1H, indolyl 2-H), 8.05-7.17 (m, 9H,Ar-H), 6.95 (s, 1H, NH), 5.37 (s, 2H, CH2-N), 3.91 (s, 2H,NH2)

473 (M + , 66), 445 (2), 397 (21),115 (15), 91 (100)

Table 2 (Continued)

Compoundnumber IR (gmax/cm) 1H NMR (d, ppm) Mass (m/z, %)


However, coupling of diazonium salts (A) with mal-

ononitrile in the presence of sodium acetate trihydrate

yielded 2-[(5-(N-substituted-1H-indol-3-yl)-5H-thiazo-

lo[4,3-b]-1,3,4-thiadiazol-2-yl hydrazono] malononitriles

(17a,b). The reaction of the latter compounds with

hydrazine hydrate in absolute ethanol under reflux

yielded the corresponding pyrazoles (18a,b) (Scheme 2).

DPPH radical-scavenging activity

The preliminary DPPH radical-scavenging activity of the

newly synthesized compounds was determined using

ascorbic acid as a reference and IC50 of the most active

compounds were calculated (Table 3 and Fig. 1). From

the data obtained, compounds 14a and 18a showed free

radical-scavenging effects of 84.61 and 80.83% compared

with that of ascorbic acid of 91.25% at a concentration of

300 mg/ml, whereas at a concentration of 200 mg/ml, only

18a showed a radical-scavenging effect of 79.56%

compared with that of ascorbic acid of 85.41%. The

amount of sample necessary to decrease the absorbance

of DPPH by 50% (IC50) was calculated and it was found

that 4-{5-[(1H-indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-thia-

diazol-2-yl]diazo}-1H-pyrazole-3,5-diamine (18a) was

highly active with radical-scavenging activity (IC50 of

69.14 mg/ml) compared with ascorbic acid (IC50 of

6.50 mg/ml); this may be because of the presence of the

N–H moieties of the two primary aromatic amino groups

and secondary amine, which act as good hydrogen bond

donors (Table 3 and Fig. 1).

ConclusionSome new heterocycles derived from novel 2-amino-5-(N-

substituted-1H-indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-thi-

diazoles (2a,b) were prepared and screened for their

antioxidant activity using 2,20-diphenyl-1-picrylhydrazyl

(DPPH) radical-scavenging activity. 4-{5-[(1H-indol-3-

yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl]diazo}-1H-

pyrazole-3,5-diamine (18a) was found to be highly active

with radical-scavenging activity (IC50 of 69.14mg/ml)

compared with ascorbic acid (IC50 of 6.50 mg/ml); this

may be because of the presence of the N–H moieties of

the two primary aromatic amino groups and secondary

amine, which act as good hydrogen bond donors.


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antioxidant studies of some 1,3,4-thiadiazole derivatives. Int J Res PharmBiomed Sci 2011; 2:1590–1592.

2 Mishra G, Singh AK, Jyoti K. Review article on 1, 3, 4-thiadiazolederivatives and its pharmacological activities. Int J ChemTech Res 2011;3:1380–1393.

3 Bhuvaa H, Sahua D, Shaha BN, Modia DC, Patelb MB. Biological profile ofthiadiazole. Pharmacologyonline 2011; 1:528–543.

4 Nelson JA, Rose LM, Bennett LL Jr.. Effects of 2 amino 1,3,4 thiadiazole onribonucleotide pools of leukemia L1210 cells. Cancer Res 1976; 36:1375–1378.

5 Gupta JK, Dudhey R, Sharma PK. Synthesis and pharmacological activity ofsubstituted 1,3,4-thiadiazole derivatives. Medichemonline 2010; 1:1–10.

6 Kushwaha N, Kushwaha SKS, Rai AK. Biological activities of thiadiazolederivatives: a review. Int J ChemTech Res 2012; 4:517–531.

7 Shukurov SSh, Kukaniev MA, Alibaeva AM. One-pot synthesis of2-amino-5-aryl-5H-thiazolo[4,3-b]-1,3,4-thiadiazoles. Russ Chem Bull 1996;45:724–725.

8 Karigar AA, Himaja M, Mali SV, Jagadeesh KP, Sikarwar MS. One-pot synthesisand antitubercular activity of 2-amino-5-aryl-5H-thiazolo [4,3-b]-1,3,4-thiadia-zoles. Int Res J Pharm 2011; 2:153–158.

9 Malipeddi H, Karigar AA, Malipeddi VR, Sikarwar MS. Synthesis and anti-tubercular activity of some novel thiazolidinone derivatives. Trop J Pharm Res2012; 11:611–620.

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11 Mandour AH, El-Sawy ER, Zahran MA, Ebaid MS, Mustafa MA. Anti-in-flammatory analgesic, anticonvulsant and antimicrobial activities of somenew synthesized N-alkyl-3-indolyl pyrimidines and benzimidazolo(1,2-a)pyrimidines. Biohealth SciBull (Malaysia) 2009; 1:57–67.

Table 3 Scavenging activity % on DPPH radicals of the most

active synthesized compounds and IC50 values

Scavenging activity (%)a

Compound number 50 100 200 300 IC50 (mg/ml)

2a 11.39 18.62 30.19 44.42 368.596b 8.49 13.74 18.67 22.24 1254.028a 6.15 8.67 9.40 17.00 2243.399a 6.33 6.87 13.02 16.64 1731.1112a 1.63 2.35 5.06 64.19 317.5912b 4.15 5.06 8.13 11.21 4221.3314a 25.67 34.9 45.26 84.61 164.1516b 14.64 19.71 36.34 47.55 327.2118a 45.56 56.05 79.56 80.83 69.14Negative control 0 0 0 0 0Ascorbic acid 83.79 88.99 85.41 91.25 6.50

aResults are the mean of three independent experiments.

Figure 1

0

20

40

60

80

100(a)

0 50 100 150 200 250 300 350

%

µg/mL

Scavenging activity (%)

2a

6b

8a

9a

12a

VC

0

20

40

60

80

100

0 50 100 150 200 250 300 350

%

µg/mL

Scavenging activity (%)

12b

14a

16b

18a

VC

(b)

Scavenging activity % on DPPH radicals of the most active synthesizedcompounds.


12 Mandour AH, El-Sawy ER, Ebaid MS, Hassan SM. Synthesis and potentialbiological activity of some novel 3-[(N-substituted indol-3-yl)methyleneami-no]-6-amino-4-aryl-pyrano(2,3-c)pyrazole-5-carbonitriles and 3,6-diamino-4-(N-substituted indol-3-yl)pyrano(2,3-c)pyrazole-5-carbonitriles. Acta Pharm2012; 62:15–30.

13 El-Sawy E, Mandour A, Mahmoud K, Islam I, Abo-Salem H. Synthesis,antimicrobial and anti-cancer activities of some new N-ethyl, N-benzyl andN-benzoyl-3-indolyl heterocycles. Acta Pharm 2012; 62:157–179.

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15 Mandour A, El-Sawy E, Shaker K, Mustafa M. Synthesis, anti-inflammatory,analgesic and anticonvulsant activities of 1,8-dihydro-1-ary1-8-alkylpyrazolo(3,4-b)indoles. Acta Pharm 2010; 60:73–88.

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17 James PN, Snyder HR. Indole-3-aldehyde. Organic Syntheses 1959; 39:30–31.

18 Mndzhoyan AL, Papayan GL, Zhuruli LD, Karagezyan SG, Galstyan LS,Sarafyan VG. Synthesis and biological study of hydrazinohydrazones of indolealdehydes and ketones series. Arm Khim Zh (USSR) 1969; 22:707–713.

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Synthesis and antihypertensive activity of certain substituted

dihydropyridines and pyrimidinonesWageeh S. El-Hamoulya, Kamelia M. Amineb, Hanaa A. Tawfika

and Dina H. Dawooda

aDepartment of Chemistry of Natural and MicrobialProducts, National Research Centre and bDepartmentof Pharmaceutical Chemistry, Faculty of Pharmacy,Cairo University, Giza, Egypt

Correspondence to Hanaa A. Tawfik, PhD, Departmentof Chemistry of Natural and Microbial Products,National Research Centre, Dokki, Giza 12311, EgyptTel: + 20 201 224 2709 16; fax: + 20 233 370 931;e-mail: [email protected]

Received 17 July 2012Accepted 10 October 2012


2013,12:20–27

Background and objective

Some bulky substituted aromatic aldehydes reacted with urea and ethyl acetoacetate

in the presence of acetic acid as a catalyst to yield solely substituted dihydropyridines

(Hantzsch-type molecule). In the presence of p-toluene sulfonic acid as a catalyst, the

products were only dihydropyrimidines (Biginelli compounds). The same aldehydes

yielded dihydropyrimidinones on using acetyl acetone instead of ethyl acetoacetate

whatever the catalyst used. These two classes of molecules represent a heterocyclic

system of a remarkable antihypertensive effect. The aim of this study was to synthesize

certain dihydropyridine and pyrimidinone derivatives with aromatic moiety with bulky

substituents to be evaluated for their antihypertensive effect.

Methods

The aldehydes 3-(substituted-phenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde 3–5, 4-

oxo-4H-chromene-3-carbaldehyde (6), and substituted phenylazo-benzaldehyde 7–9

reacted with ethyl acetoacetate and urea in ethanol in the presence of acetic acid to

yield dihydropyridines 10–15. Aldehydes 3–9 reacted with ethyl acetoacetate and urea

in the presence of p-toluene sulfonic acid to yield dihydropyrimidinones 16–22.

Furthermore, the reaction of the aldehydes 3–9 with ethyl acetoacetate and urea in the

presence of either acetic acid or p-toluene sulfonic acid yielded the corresponding

dihydropyrimidinones 23–29.


The hypotensive activity of compounds 10–14 and 16–20 indicated that the 4-aryl-

dihydropyridine derivatives 10–14 showed higher activity than the pyrimidinones

16–20. The most active compound was 4-(1,3-diphenyl-1H-pyrazol-4-yl)-2,6-dimethyl-

1,4-dihydropyridine-3,5-dicarboxylic acid diethyl ester (10) at dose levels of 0.6, 1.2,

and 2.4 mg/kg. It showed more or less similar hypotensive activity as the reference

drug nifedipene at doses of 1.2 and 2.4 mg/kg. Its LD50 = 298 mg/kg body weight.

Keywords:

antihypertensive activity, bulky substituted aldehydes, dihydropyridines,

dihydropyrimidinones

Egypt Pharm J 12:20–27& 2013 Division of Pharmaceutical and Drug Industries Research, National Research Centre1687-4315

IntroductionThe one-pot acid-catalyzed Biginelli [1,2] condensation

is the most commonly used reaction to produce

dihydropyrimidines (DHPMs, 1). This very simple

reaction involves three component cyclocondensation of

urea, an aldehyde and a b-oxoester or 1,3-dicarbonyl

compound using ethanol as a solvent and catalytic

amounts of HCl, AcOH, or H2SO4 among other

acids [3–7]. In contrast, in the Hantzsch reaction

discussed, more than a century ago [8], the main way

to obtain dihydropyridines (DHPs, 2) and is commonly

carried out as a one-pot condensation of a b-dicarbonyl

compound with an aldehyde but with ammonia instead of

urea using ethanol as a solvent.

These two classes of molecules (1 and 2) represent a

heterocyclic system with remarkable pharmacological

properties that include antiviral [9,10], antitumor [11,12],

antibacterial [13,14], and anti-inflammatory [15–18] ac-

tivities. In addition, a number of these heterocyclic

NH

H

MeMe

Hantzsch dihydropyridines

COOEtEtOOC

R

HN

Biginelli dihydropyrimidines

NH

H

Me

COOEt

O

R

1 2

20 Original article


DOI: 10.7123/01.EPJ.0000426587.41764.d4

systems have emerged as exerting orally active antihy-

pertensive effects or to act as a-1A-adrenoceptor-selective

antagonists [19,20], for example nifedipene and amlude-

pine. It is worth mentioning that several examples of

highly substituted DHPMs and DHPs are reported to

show high antihypertensive activity, for example doxazo-

sin [20] and nicardipine [21,22].

The aim of this work was to synthesize some DHPs and

pyrimidinones with the aromatic moiety bearing bulky

substituents to be evaluated for their antihypertensive

activity.

ExperimentalChemistry

All melting points were determined in open capillary tubes

using silicon oil on a Gallen Kamp Apparatus (Finsbury,

London, England) and were uncorrected. 1H-NMR spectra

were determined using a JEOL EX-270 NMR spectrometer

(Musashino 3-chome, Akishima, Tokyo, Japan) with tetra-

methylsilane as an internal standard. Mass spectra were

performed using a GC-MS-QP 1000EX Schimadzu Gas

Chromatography MS Spectrometer (Columbia, Maryland,

USA). The infrared spectra were recorded on an FT/

IR330E infrared spectrophotometer using KBr discs.

Elemental analyses were carried out at the Micro analytical

Laboratory of the National Research Center, Dokki, Cairo,

Egypt. The reactions were followed up by thin layer

chromatography (TLC) using chloroform/methanol (9 : 1)

as an eluent and detected using a UV lamp.

General procedure for the preparation of substituted

dihydropyridine compounds (10–15)

A mixture of the appropriate aldehydes 3–9 (6 mmol), urea

(0.9 g, 15 mmol), ethyl acetoacetate (1.17 ml, 9 mmol), and

glacial acetic acid (2 ml) in absolute ethanol (50 ml) was

heated under reflux for several hours (12–18 h) (monitored

by TLC). After the completion of the reaction, the solvent

was removed under vacuum and the precipitated product

was treated with water, filtered off, washed with water,

dried, and crystallized from methanol.

4-(1,3-Diphenyl-1H-pyrazol-4-yl)-2,6-dimethyl-1,4-

dihydropyridine-3,5-dicarboxylic acid diethyl ester (10)

Yield 72%, m.p. 154–1561C, IR (KBr, cm – 1): 3343 (NH),

1682 (CO); 1H-NMR (d6-DMSO, d, ppm): 0.91 (t, 6H,

2CH3), 2.22 (s, 6H, 2CH3), 3.84 (q, 4H, 2CH2), 5.16 (s,

1H, C4-H), 7.26–7.88 (m, 10H, Ar-Hs), 8.00 (s, 1H,

pyrazole), 8.78 (s, 1H, NH, D2O exchangeable); Ms: m/z(%): 469 [(M + -2, (62)], 441 (100%), 397 (83), 326 (71),

251 (93), 220 (90), 206 (22), 179 (32), 77 (99). Analysis:

for C28H29N3O4 (471.55), calcd: C, 71.32; H, 6.20; N,

8.91%. Found: C, 71.45; H, 6.30; N, 8.71%.

2,6-Dimethyl-4-[3-(4-nitrophenyl)-1-phenyl-1-H-

pyrazole-4-yl]-1,4-dihydropyridine-3,5-dicarboxylic acid

diethyl ester (11)


1683 (CO); 1H-NMR (d6-DMSO, d, ppm) 0.87 (t, 6H,

2CH3), 2.24 (s, 6H, 2CH3), 3.87 (q, 4H, 2CH2), 5.18 (s,

1H, C4-H), 7.31–8.36 (m, 10H, 9Ar-Hs and 1H pyrazole),

8.81 (s, 1H, NH); Ms: m/z (%): 514 [M + -2, (22)], 486

(70), 442 (100), 251 (52). Analysis: for C28H28N4O6

(516.55), calcd: C, 65.11; H, 5.46; N, 10.85%. Found: C,

65.33; H: 5.19; N, 10.67%.

4-[3-(2-Hydroxy-phenyl)-1-phenyl-1H-pyrazol-4-yl]-2,

6-dimethyl-1,4-dihydro-pyridine-3,5-dicarboxylic acid

diethyl ester (12)

Yield 68%, m.p. 98–1001C, IR (KBr, cm – 1): 3357 (OH),

3249 (NH) and 1693 (CO); 1H-NMR (d6-DMSO, d,

ppm), 0.98 (t, 6H, 2CH3), 2.13 (s, 6H, 2CH3), 3.87 (q,

4H, 2CH2), 5.10 (s, 1H, C4-H), 6.91–7.77 (m, 9H, Ar-

Hs), 8.12 (s, 1H, pyrazole-H), 8.54 (s, 1H, NH) and 9.59

(s, 1H, OH); Ms: m/z (%): 485 [(M + -2, (94%)], 457 (24),

438 (100), 413 (43), 394 (20), 252 (16), 236 (27).

Analysis: for C28H29N3O5 (487.55), calcd: C, 68.98; H,

6.00; N, 8.62%. Found: C, 68.86; H, 5.79; N, 8.52%.

2,6-Dimethyl-4-(4-oxo-4H-chromen-3-yl)-1,4-

dihydropyridine-3,5-dicarboxylic acid diethyl ester (13)

Yield 65%, m.p. 213–2151C; 1H-NMR (d6-DMSO, d,

ppm) 1.10 (t, 6H, 2CH3), 1.12 (t, 6H, 2CH3), 2.22 (s,

6H, 2CH3), 2.25 (s, 6H, 2CH3), 3.96 (q, 4H, 2CH2), 4.02

(q, 4H, 2CH2), 4.82 (s, 1H, C4-H), 5.24 (s, 1H, C4-H),

7.43 (t, 1H, H-6), 7.50 (t, 1H, H-6), 7.55 (d, 1H, H-8),

7.57 (d, 1H, H-8), 7.64 (t, 1H, H-7), 7.73 (t, 1H, H-7),

7.93 (s, 1H, H-2), 8.14 (s, 1H, H-2), 8.00 (d, 1H, H-5),

8.02 (d, 1H, H-5), 8.82 (s, 1H, NH), 9.18 (s, 1H, NH);

Ms m/z (%) 397 (M + , 12%), 352 (7), 324 (100), 294 (10),

252 (32), 223 (17). Analysis: for C22H23NO6 (397.42),

calcd: C, 66.49; H, 5.83; N, 3.52%. Found: C, 66.80; H,

5.70; N, 3.41%.

Doxazosin

O

O

ONN

N

N

NH2

MeO

MeO

O

NO

O2N

NH

Me Me

Me

EtOOC

Nicardipine

Synthesis and antihypertensive activity El-Hamouly et al. 21

2,6-Dimethyl-4-(2-hydroxy-3-methoxy-5-phenylazo-

phenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid diethyl

ester (14)

Yield 75%, m.p. 124–1261C; IR (KBr, cm – 1): 3448 (OH),

3344 (NH), 1693 (CO); 1H-NMR (d6-DMSO, d, ppm):

1.10 (t, 3H, CH3), 2.28 (s, 3H, CH3), 3.84 (s, 3H,

OCH3), 4.00 (q, 2H, CH2), 5.17 (s, 1H, C4-H), 7.03 (s,

1H, Ar-H), 7.22 (s, 1H, Ar-H), 7.56 (t, 3H, Ar-Hs), 7.80

(s, 1H, N3H, D2O exchangeable), 7.98 (d, 2H, Ar-Hs),

9.26 (s, 1H, N1H, D2O exchangeable), 10.99 (s, 1H,

OH); Ms: m/z (%): 477 [M + -2, (34)], 431 (12), 372 (38),

354 (32), 252 (81), 238 (41), 105 (55), 93 (86) and 77

(100). Analysis: for C26H29N3O6 (479.52), calcd: C,

65.12; H, 6.10; N, 8.76%. Found: C, 65.29; H, 6.12; N,

8.95%.

2,4-Dimethyl-5-oxo-9-phenylazo-5H-chromeno[3,4-

c]pyridine-1-carboxylic acid ethyl ester (15)

Yield 66%, m.p. 203–2061C; IR (KBr, cm – 1): 1730 (CO),

1684 (CO); 1H-NMR (d6-DMSO, d, ppm): 1.33 (t, 3H,

CH3), 2.69 (s, 3H, CH3), 2.93 (s, 3H, CH3), 4.58 (q, 2H,

CH2), 7.61 (t, 3H, Ar-Hs), 7.64 (d, 1H, Ar-H), 7.88 (d,

1H, Ar-H), 8.22 (d, 2H, Ar-Hs), 8.31 (s, 1H, Ar-H); Ms:

m/z (%), 400 [M + -1, (27)], 356 (10), 329 (17), 268 (37),

250 (60), 224 (24), 169 (91), 105 (55), 77 (100). Analysis:

for C23H19N3O4 (401.41), calcd: C, 68.82; H, 4.78; N,

10.47%. Found: C, 68.63; H, 4.91; N, 10.60%.

4-(Aryl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-

carboxylicacid ethyl ester (16–22)

General procedure

A mixture of the appropriate aldehydes 3–9 (10 mmol),

urea (1.5 g, 25 mmol), ethyl acetoacetate (1.95 ml, 15 mmol),

and p-toluene sulfonic acid (1.72 g, 10 mmol) in absolute

ethanol (35 ml) was heated under reflux for 6–8 h

(monitored by TLC). After completion of the reaction,

the solvent was removed under vacuum and the pre-

cipitated product was treated with water, filtered, washed

with water, and dried. Crystallization from the appropriate

solvent yielded the desired compounds 16–22.

4-[1,3-Diphenyl-1H-pyrazole-4-yl]-6-methyl-2-oxo-

1,2,3,4-tetrahydropyrimidine-5-carboxylic acid ethyl

ester (16)

Yield 74%, m.p. 178–1801C (methanol); IR (KBr, cm – 1):

3349 (NH), 3222 (NH), 1693 (CO), 1642 (CO); 1H-

NMR (d6-DMSO, d, ppm): 0.82 (t, 3H, CH3), 2.23 (s,

3H, CH3), 3.80 (q, 2H, CH2), 5.38 (s, 1H, C4-H),

7.27–7.90 (m, 11H, 10Ar-Hs and 1H pyrazole), 8.35 (s,

1H, N3H) and 9.16 (s, 1H, N1H). Analysis: for

C23H22N4O3 (402.45), calcd: C, 68.64; H, 5.51; N,

13.92%. Found: C, 68.80; H, 5.34; N, 13.71%.

6-Methyl-4-[3-(4-nitro-phenyl)-1-phenyl-1H-pyrazol-4-

yl]-2-oxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylic acid

ethyl ester (17)

Yield 83%, m.p. 190–1931C; IR (KBr, cm – 1): 3439 (OH),

3210 (NH), 3122 (NH), 1713 (CO), 1657 (CO); 1H-


3H, CH3), 3.82 (q, 2H, CH2), 5.44 (s, 1H, C4-H),

6.87–7.89 (m, 10H, 9Ar-Hs and 1H pyrazole), 8.34 (s, 1H,

N3H), 9.20 (s, 1H, N1H). Analysis: for C23H21N5O5

(447.44), calcd: C, 61.74; H, 4.73; N, 15.65%. Found: C,

61.96; H, 4.53; N, 15.85%.

4-[3-(2-Hydroxy-phenyl)-1-phenyl-1H-pyrazol-4-yl]-6-

methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic

acid ethyl ester (18)

Yield 79%, m.p. 201–2041C; IR (KBr, cm – 1): 3223 (NH),

3109 (NH), 1698 (CO), 1649 (CO); 1H-NMR (d6-

DMSO, d, ppm): 0.83 (t, 3H, CH3), 2.26 (s, 3H, CH3),

3.82 (q, 2H, CH2), 5.44 (s, 1H, C4-H), 7.13–8.50 (m,

10H, 9Ar-Hs and 1H pyrazole), 7.85 (s, 1H, N3H, D2O

exchangeable), 9.23 (s, 1H, N1H, D2O exchangeable).


5.30; N, 13.39%. Found: C, 66.37; H, 5.49; N, 13.21%.

6-Methyl-2-oxo-4-(4-oxo-4H-chromen-3-yl)-1,2,3,4-

tetrahydropyrimidine-5-carboxylic acid ethyl ester (19)


3281 (NH), 1710 (CO), 1669 (CO), 1638 (CO); 1H-


3H, CH3), 3.98 (q, 2H, CH2), 5.23 (s, 1H, C4-H), 7.24

(s, 1H, H-2), 7.45 (t, 1H, H-6), 7.63 (d, 1H, H-8), 7.78

(t, 1H, H-7), 8.12 (d, 1H, H-5), 8.23 (s, 1H, N3H), 9.31

(s, 1H, N1H); Ms: m/z (%): 328 (M + , 12), 269 (17%),

255 (100%), 169 (18%); Analysis: for C17H16N2O5

(328.32), calcd: C, 62.19; H, 4.91; N, 8.53%. Found: C,

62.37; H, 4.79; N, 8.37%.

4-(2-Hydroxy-3-methoxy-5-phenylazo-phenyl)-6-methyl-

2-oxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylic acid

ethyl ester (20)


3214 (NH), 3198 (NH), 1689 (CO), 1640 (CO); 1H-

NMR (d6-DMSO d, ppm): 1.10 (t, 3H, CH3,), 2.28 (s,

3H, CH3), 3.84 (s, 3H, OCH3), 4.00 (q, 2H, CH2), 5.17

(s, 1H, C4-H), 7.03 (s, 1H, Ar-H), 7.22 (s, 1H, Ar-H),

7.56 (t, 3H, Ar-Hs), 7.80 (s, 1H, N3H, D2O exchange-

able), 7.98 (d, 2H, Ar-Hs), 9.26 (s, 1H, N1H, D2O

exchangeable), 10.99 (s, 1H, OH, D2O exchangeable);

Ms: m/z (%), 410 [M + (12)], 302 (44), 210 (32), 105

(42), 93 (52), 77 (100). Analysis: for C21H22N4O5

(410.43), calcd: C, 61.46; H, 5.40; N, 13.65%. Found: C,

61.35; H, 5.35; N, 13.68%.

4-(2-Hydroxy-5-phenylazo-phenyl)-6-methyl-2-oxo-

1,2,3,4-tetrahydropyrimidine-5-carboxylic acid ethyl

ester (21)

m.p. 167–1701C, IR (KBr, cm – 1): 3455 (OH), 3220

(NH), 3210 (NH), 1690 (CO), 1662 (CO); 1H-NMR (d6-

DMSO, d, ppm) 1.05 (t, 3H, CH3), 2.24 (s, 3H, CH3),

3.98 (q, 2H, CH2), 5.52 (s, 1H, C4-H), 6.93 (d, 1H, Ar-

H), 7.37 (s, 1H, N3H, D2O exchangeable), 7.51 (t, 3H,

Ar-Hs), 7.63 (s, 1H, Ar-H), 7.75 (d, 1H, Ar-H), 7.84 (d,

2H, Ar-Hs), 9.23 (s, 1H, N1H, D2O exchangeable), 10.61

(s, 1H, OH, D2O exchangeable); Ms: m/z (%), 380 (M + ,

20), 183 (22), 105 (21), 93 (28), 77 (100). Analysis: for

C20H20N4O4 (380.40), calcd: C, 63.15; H, 5.30; N,

14.73%. Found: C, 63.38; H, 5.40; N, 14.87%.


4-[2-Hydroxy-5-(4-nitrophenylazo)phenyl]-6-methyl-2-

oxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylic acid

ethyl ester (22)

Yield 74%, m.p. 158–1611C, IR (KBr, cm– 1): 3356 (OH),

3234 (NH), 3114 (NH), 1687 (CO), 1651 (CO); 1H-NMR

(d6-DMSO, d, ppm): 1.07 (t, 3H, CH3), 2.30 (s, 3H, CH3),

3.97 (q, 2H, CH2), 5.50 (s, 1H,C4-H), 7.00 (d, 1H, Ar-H),

7.38 (s, 1H, N3H), 7.69 (s, 1H, Ar-H), 7.72–8.07 (m, 5H,

Ar-Hs), 9.21 (s, 1H, N1H), 10.90 (s, 1H, OH). Analysis: for

C20H19N5O6 (425.39), calcd: C, 56.47; H, 4.50; N, 16.46%.

Found: C, 56.66; H, 4.23; N, 16.64%.

Preparation of 5-acetyl-4-(3-aryl-1-phenyl-1H-pyrazole-

4-yl)-6-methyl-3,4-dihydro-1H-pyrimidin-2-one (23–29)

General procedure

A mixture of the selected aldehyde, 3–9 (10 mmol), urea

(1.5 g, 25 mmol) and acetylacetone (1.5 ml, 15 mmol) in

ethanol (50 ml) acidified with glacial acetic acid (2 ml) or

p-toluene sulfonic acid (1.72 g, 10 mmol) was heated

under reflux for 5–6 h. The solvent was then evaporated

under reduced pressure and the residue formed was

treated with water, filtered off, washed with water, dried,

and crystallized from methanol.

5-Acetyl-4-(1,3-diphenyl-1H-pyrazol-4-yl)-6-methyl-3,4-

dihydro-1H-pyrimidin-2-one (23)


3222 (NH), 1696 (CO), 1671 (CO); 1H-NMR (d6-

DMSO, d, ppm): 2.16 (s, 3H, CH3), 2.25 (s, 3H,

COCH3), 5.43 (s, 1H, C4-H), 7.30–7.87 (m, 11H, 10Ar-

Hs and 1H pyrazole), 8.28 (s, 1H, N3H), 9.12 (s, 1H,

N1H); MS: m/z (%): 372 (M + , 93), 357 (38), 329 (36),

254 (8), 221 (100), and 153 (43). Analysis: for

C22H20N4O2 (372.42), calcd: C, 70.95; H, 5.41; N,

15.04%. Found: C, 70.79; H, 5.51; N, 15.19%.

5-Acetyl-6-methyl-4-[3-(4-nitrophenyl)-1-phenyl-1H-

pyrazol-4-yl]-3,4-dihydro-1H-pyrimidin-2-one (24)


3235 (NH), 3165 (NH), 1655 (CO), 1620 (CO); 1H-

NMR (d6-DMSO, d, ppm); MS: m/z (%): 386 (M + -2,

10), 345 (8), 235 (11), 221 (21), 154 (17) and 66 (100).


4.59; N, 16.78%. Found: C, 63.47; H, 4.68; N, 16.92%.

5-Acetyl-4-[3-(2-hydroxy-phenyl)-1-phenyl-1H-pyrazol-4-

yl]-6-methyl-3,4-dihydro-1H-pyrimidin-2-one (25)


3114 (NH), 1656 (CO), 1619 (CO); 1H-NMR (d6-

DMSO, d, ppm): 2.07 (s, 3H, CH3), 2.33 (s, 3H,

COCH3), 5.50 (s, 1H, C4-H), 7.12–8.36 (m, 10H, 9Ar-

Hs and 1H pyrazole), 7.83 (s, 1H, N3H, D2O exchange-

able), 9.20 (s, 1H, N1H, D2O exchangeable); MS, m/z(%): 416 (M + -1, 41), 373 (40), 326 (17), 266 (72), 235

(15), 153 (100) and 124 (50). Analysis: for C22H19N5O4

(417.42), calcd: C, 68.03; H, 5.19; N, 14.42%. Found: C,

68.23; H, 5.32; N, 14.61%.

5-Acetyl-6-methyl-4-(4-oxo-4H-chromen-3-yl)-3,4-

dihydro-1H-pyrimidin-2-one (26)

Yield 75%, m.p. 218–2201C; IR (KBr, cm – 1): 3340 (NH),

3273 (NH), 1703 (CO), 1671 (CO), 1645 (CO); 1H-

NMR (d6-DMSO, d, ppm): 2.15 (s, 3H, CH3), 2.31 (s,

3H, COCH3), 5.34 (s, 1H, C4-H), 7.25 (s, 1H, H-2), 7.45

(t, 1H, H-6), 7.63 (d, 1H, H-8), 7.78 (t, 1H, H-7), 8.12

(d, 1H, H-5), 8.25 (s, 1H, N3H), 9.32 (s, 1H, N1H); Ms:

m/z (%), 255 (100), 239 (8), 153 (18), 146 (26), 121 (31),

105 (35). Analysis: for C16H14N2O4 (298.29), calcd: C,

64.42; H, 4.73; N, 9.39%. Found: C, 64.56; H, 4.42; N,

9.61%.

5-Acetyl-4-(2-hydroxy-3-methoxy-5-phenylazo-phenyl)-

6-methyl-3,4-dihydro-1H-pyrimidin-2-one (27)


3255 (NH), 3112 (NH), 1707 (CO), 1663 (CO). 1H-

NMR (d6-DMSO, d, ppm); MS, m/z (%): 379 (M + _1, 7),

350 (52), 335 (21), 322 (27), 258 (39), 244 (9), 153 (17),

93 (100), 124 (43). Analysis: for C20H20N4O4 (380.40),

calcd: C, 63.15; H, 5.30; N, 14.73%. Found: C, 63.40; H,

5.31; N, 14.55%.

5-Acetyl-4-(2-hydroxy-5-phenylazo-phenyl)-6-methyl-

3,4-dihydro-1H-pyrimidin-2-one (28)


3235 (NH), 3150 (NH), 1681 (CO), 1621 (CO); 1H-

NMR (d6-DMSO, d, ppm): 2.11 (s, 3H, CH3), 2.33 (s,

3H, COCH3), 5.63 (s, 1H, C4-H), 7.00 (d, 1H, Ar-H),

7.04 (s, 1H, N3H, D2O exchangeable), 7.53 (t, 3H, Ar-

Hs), 7.62 (s, 1H, Ar-H), 7.72 (d, 1H, Ar-H), 7.82 (d, 2H,

Ar-Hs), 9.27 (s, 1H, N1H, D2O exchangeable), 10.59 (s,

1H, OH, D2O exchangeable); MS m/z (%): 350 (M + , 13),

307 (10), 198 (23), 153 (16), 93 (100). Analysis: for

C19H18N4O3 (350.37), calcd: C, 65.13; H, 5.18; N,

15.99%. Found: C, 65.33; H, 5.28; N, 16.25%.

5-Acetyl-4-[2-hydroxy-5-(2-nitro-phenylazo)-phenyl]-6-

methyl-3,4-dihydro-1H-pyrimidin-2-one (29)


3281 (NH), 3230 (NH), 1697 (CO), 1650 (CO); MS m/z(%): 396 (M + + 1, 10), 350 (12), 337 (17), 257 (30), 243

(13), 337 (17), 257 (30), 243 (13), 226 (15), 153 (20%),

93 (100). Analysis: for C19H17N5O5 (395.37), calcd: C:

NHNH2

CH3

O

HN

N

CH3

NN

CHO

R RR

+

POCl3DMF

3) R = H; 4) R = 4-NO2;5) R = 2-OH


57.72; H, 4.33; N, 17.71%. Found: C, 57.58; H, 4.41; N,

17.63%.

Chemistry

The aldehydes 3-(substituted-phenyl)-1-phenyl-1H-pyr-

azole-4-carbaldehyde 3–5 [23] 4-oxo-4H-chromene-3-

carbaldehyde (6) [24] and substituted phenylazo-ben-

zaldehyde 7–9 [25] reacted with ethyl acetaoaetate and

urea in ethanol in the presence of acetic acid to yield

DHPs 10–15.

Compound 8 reacted similarly but underwent intra-

molecular condensation and aromatization to yield 2,4-

dimethyl-5-oxo-9-phenylazo-5H-chromeno[3,4–c]pyridine-

1-carboxylic acid ethyl ester (15). Similar behavior has

been reported previously [26].

Also, compound 9 yielded a mixture of products that were

hardly separable; perhaps, decomposition occurred be-

cause of the long reaction time.

Moreover, aldehydes 3–9 reacted with urea and ethyl

acetoacetate in the presence of p-toluene sulfonic acid to

yield dihydropyrimidinones 16–22.

Furthermore, reaction of the aldehydes 3–9 with urea and

acetyl acetone in alcohol as a solvent in the presence of

either acetic acid or p-toluene sulfonic acid yielded the

corresponding dihydropyrimidinones 23–29.

Antihypertensive activity

Ten of the newly synthesized substituted DHPs 10–14

and tetrahydropyrimidines 16–20 were screened for their

hypotensive activity using normotensive cat models [27].

Materials and methodsMale cats of local strains weighing from 2.5 to 4.0 kg were

housed (one per cage) in the animal facility (Faculty of

OH

COCH3

O

O

CHO

POCl3/DMF

6

R

N2HCl. NN

R

R1

OH

NN CHO

ROH

R1

OH

R1

+

7, R = H, R1 = OMe8, R = R1 = H,9, R = NO2 R1 = H

R NN

NH

CH3H3C

CO2EtEtO2C

H

NN

NH

CH3CH3

CO2E tE tO2CH

MeO

OH

NH

CH3CH3

CO2E tE tO2C

O

OH

10) R = H11) R = 4 -NO2

12) R = 2-OH

13 14


Medicine, El-Azhar University) for 7 days before the

experiment. Animals were always kept at 22 ± 2 h and a

12 h light/12 h dark cycle. Stressful conditions or manip-

ulation were avoided. Cats were divided into groups; each

group included four cats and one group was used as a

control. All cats were anesthetized with phenobarbital

sodium (35 mg/kg, intraperitoneally) and their blood

pressures (BP) were recorded from the carotid artery.

BP of each cat was measured before and 30 min after the

intravenous injection of the tested compounds. The

tested compounds were dissolved in DMSO and admi-

nistered at different doses (0.6, 1.2, 2.4 mg/kg) in 0.5 ml

volume in the same way as the reference drug nifedipine.

The same volume of DMSO was administered to animals

in the control group. The reduction of BP between two

measurements was recorded as mmHg. These results

were expressed as mean ± SEM; analysis variance (two-

way) was used for statistical analysis. LD50 was preformed

according to the procedure described in the study

conducted by Kerber [28].

Results and discussionThe hypotensive effect of the tested DHP derivatives

10–14 and DHPMs 16–20 is shown in Table 1 in

comparison with nifedipine as a reference drug. In the

DHP series, the test compounds showed significant

hypotensive activity at all dose levels (0.6, 1.2, and

2.4 mg/kg). The 4-(1,3-diphenyl-1H-pyrazolyl) derivative

10 was the most active at all dose levels. Also, it had more

N

N

NH

NH

Me

CO2E t

H

O

OH

R1

R

O

O

NH

NH

MeO

CO2E tH

R NN

NH

NH

MeO

CO2E tH

16, R = H, 17, R = 4- NO2

18, R = 2-OH

19 20, R = H, R1 = OMe21, R = R1 = H22, R = NO2, R1 = H

N N

HN

NH

MeO

R1R

HN

NH

O

R NN

HN

NH

MeO

H

23, R = H,24, R = 4-NO225, R = 2-OH

26 27, R = NO2, R1 = 2-OH, R2 = H28, R = H, R1 = 2-OH, R2 = H29, R = H, R1 = 4-OH, R2 =3-OCH3

Me

O

Me

O

OR2

Me

O

Table 1 Effect of tested compounds (10–14 and 16–20) on the

mean blood pressure of anesthetized normotensive cats

compared with the reference drug nifedipine

Dose (mg/kg) Compounds Mean reduction in BP

0.6 mg/kg Control (DMSO) 100.17 ± 1.82Nifedipine 44.17 ± 1.4510 55.40 ± 1.4011 79.00 ± 1.2412 61.67 ± 2.2313 65.00 ± 1.7114 75.00 ± 1.5916 76.23 ± 2.6017 74.17 ± 1.6018 76.50 ± 1.2419 61.00 ± 1.1020 89.50 ± 1.28

1.2 mg/kg Nifedipine 22.17 ± 1.4210 27.17 ± 1.9111 77.83 ± 0.8712 51.50 ± 1.6113 64.33 ± 1.7114 67.00 ± 0.4516 68.33 ± 1.8917 70.00 ± 1.1918 62.33 ± 0.8719 58.00 ± 1.4620 88.83 ± 1.71

2.4 mg/kg Nifedipine 15.17 ± 1.0110 13.00 ± 0.8211 62.00 ± 1.5312 15.50 ± 0.9213 60.33 ± 0.8814 60.50 ± 1.1816 64.50 ± 1.3417 68.00 ± 1.3918 57.00 ± 1.5119 47.67 ± 1.0920 85.67 ± 0.92

BP, blood pressure; DMSO, dimethyl sulfoxide.


or less similar potency as nifidipine (refrerence standerd)

at doses of 1.2 and 2.4 mg/kg. The other tested DHPs 11

and 12 bearing 3-aryl-1-phenyl-1H-pyrazolyl as well as

the chromonyl derivative 13 and 4-hydroxy-3-methoxy-5-

(phenylazo)-phenyl substituent at the 4-position 14

showed weak activities compared with the reference

drug. For tetrahydropyrimidine series 16–20, the eval-

uated data showed that the 4-chromonyl derivative 19

had significant hypertensive activity (61.00 ± 1.10),

which was higher than the 4-pyrazolyl analogous 16–18

at a dose of 0.6 mg/kg. A nonsignificant change was

observed in the presence of 4-[4-hydroxy-3-methoxy-5-

(phenylazo)-phenyl] derivative 20 when administered at

the same dose level. The hypotensive values of this series

were negligible compared with those of nifedipine at

doses of 0.6, 1.2, and 2.4 mg/kg.

Moreover, Table 2 shows that LD50 of the most active

compound 10 was equal to 298 mg/kg body weight.

Conclusively, the 4-aryl-DHP derivatives 10–14 showed

higher hypotensive activity than the tetrahydropyrimi-

dines 16–20 carrying the same aryl substituents at the

same position. The most active compound was 4-(1,3-

diphenyl-1H-pyrazol-4-yl)-2,6-dimethyl-1,4-dihydropyri-

dine-3,5-dicarboxylic acid diethyl ester 10 at dose levels

of 0.6, 1.2, and 2.4 mg/kg. It showed more or less similar

hypotensive activity as the reference drug nifedipine at

doses of 1.2 and 2.4 mg/kg.

ConclusionThe synthesis of substituted DHPs 10–15 and pyrimi-

dinones 16–29 was achieved. The comparison of the

tested compounds 10–14 and 16–20 for their hypoten-

sive activity using the nonselective cat models led to the

conclusion that the 4-aryl-DHP derivatives 10–14

showed higher hypotensive activity than the pyrimidi-

nones derivatives carrying the same aryl substituent at

the same position. The most active compound was 4-(1,

3-diphenyl-1H-pyrazol-4-yl)-2,6-dimethyl-1,4-dihydropy-

ridine-3,5-dicarboxylic acid diethyl ester 10 at dose levels

of 0.6, 1.2, and 2.4 mg/kg. It showed more or less similar

hypotensive activity as the reference drug nifedipine at

doses of 1.2 and 2.4 mg/kg. Its LD50 is 298 mg/kg body

weight, which would present a fruitful matrix for the

development of a potent antihypertensive agent.


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Table 2 LD50 in male mice after an intraperitoneal

administration of compound 10

Groupnumber

Oral doses(mg/kg body

weight)Number of

dead animalsDose

difference Meana Productb

1 240 – – – –2 260 1 20 0.5 103 280 3 20 2 405 300 5 20 4 806 320 7 20 6 1207 340 10 20 8.5 170Total 420

Number of animals/group = 10 mice.LD50: 340 – (420/10) = 298 mg/kg body weight.LD50, lethal dose, 50%.aInterval mean of the number of dead animals (mice).bProduct of the interval mean and the dose difference.


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Immobilization of Mucor racemosus NRRL 3631 lipase and

characterization of silica-coated magnetite (Fe3O4) nanoparticlesAbeer A. El-Hadia,c, Hesham I. Salehb,d, Samia A. Moustafab

and Hanan M. Ahmeda

Departments of aChemistry of Natural and MicrobialProducts, bInorganic Chemistry, National ResearchCentre, Giza, Egypt, cDepartment of Biology, Faculty ofScience, Taif University, Taif and dDepartment ofChemistry, Northern Border University, Arar, SaudiArabia

Correspondence to Abeer A. El-Hadi, Department ofChemistry of Natural and Microbial Products, NationalResearch Centre, El-Behoos St.33, Dokki, Giza 12311,EgyptTel: + 20 233 54974; fax: + 20 233 70931;e-mail: [email protected]



2013,12:28–35

Introduction and purpose

The uncoated magnetite (M) and silica-coated magnetite (MS) nanoparticles have

been suggested as carriers for the immobilization of enzymes to improve their activity

and stability. The objective of this study was to demonstrate the potential use of

magnetic nanoparticles in bioengineering applications, using Mucor racemosus NRRL

3631 lipase as the model enzyme.


The magnetite (Fe3O4) particles were synthesized by the chemical coprecipitation

technique, that is, Massart’s process with minor modifications, using stable ferrous and

ferric salts with ammonium hydroxide as the precipitating agent. The uncoated and

coated magnetite nanoparticles for immobilizing the lipase were characterized

according to the particle sizes, as measured from the transmission electron

microscope images. The infrared and X-ray powder diffraction spectra can well explain

the bonding interaction and crystal structures of various samples, respectively.


Different concentrations of silica-coated magnetite (MS) nanoparticles were used as

cross-linking agents. A silica concentration of 1% was proven to be more suitable, with

an immobilization efficiency of 96%. The transmission electron microscope images

revealed the diameters of the uncoated magnetite particles to be 10–16 nm and those

of the coated particles to be about 11 nm. The optimal pH and temperature of the

immobilized lipase were 5–6 and 401C, respectively. There was a slight decrease in

the residual activity of the immobilized lipase at 601C for 1 h. The kinetic constants

Vmax and Km were determined to be 250 U/mg protein and 20 mmol/l, respectively, for

the immobilized lipase. The residual activity of the immobilized lipase remained over

51% despite being used repeatedly seven times. It can be concluded that Fe3O4

magnetic nanoparticles and silica-coated magnetite (MS) nanoparticles have been

successfully prepared with excellent properties using the chemical coprecipitation

technique with some modifications. The silica coating appeared to be effective in

protecting the magnetite from being converted to other oxide species. The results of

the X-ray powder diffraction indicate that the composites were in the nanoscopic

phase. The resulting immobilized lipase had better resistance to pH and temperature

inactivation compared with free lipase and exhibited good reusability.

Keywords:

Fe3O4/SiO2, immobilization efficiency, magnetic nanoparticles, Mucor racemosus

NRRL 3631 lipase, stability


Introduction

In recent years, the use of nanophase materials offers

many advantages because of their unique size and

physical properties. Magnetic nanoparticles have become

very popular when used in conjunction with biological

materials such as proteins, peptides, enzymes, antibodies,

and nucleic acids because of their unique properties [1];

this application is mainly based on the magnetic feature

of the solid phase that helps in achieving a rapid and

easy separation from the reaction medium in a magnetic

field. Previous studies have reported that magnetic

nanoparticles tend to lose their magnetizability when

biopolymer-coated nanoparticles are circulated in the

body [2]. Consequently, inorganic carrier materials

including magnetite and silica gels were being focused

on because of their thermal and mechanical stability,

nontoxicity, and high resistance against microbial attacks

and solutions of organic solvents [3]. Silica and its

derivatives when coated onto the surface of magnetic

nanoparticles may help to change their surface properties.

With the appropriate coating, the magnetic dipolar

attraction between the magnetic nanoparticles may be

screened, thus minimizing or even preventing aggrega-

tion. The coating film could also provide a chemically

inert layer against the nanoparticles, which is particularly

28 Original article


DOI: 10.7123/01.EPJ.0000427102.64865.32

useful in biological systems [1,4]. The larger specific

surface area and surface reactive groups that are

introduced by further modification of silica materials are

favorable during the preparation of silica carriers for

immobilized enzymes, and these carriers are very suitable

for adsorption and immobilization of the adsorbed protein

abundantly and steadily [5]. Lipases from different

sources are currently used in enzymatic organic synth-

esis [6,7]. The expanding interest in lipases mainly lies

on their wide industrial applications, including detergent

formulation, oil/fat degradation, pharmaceutical synth-

esis, cosmetics, paper manufacture, and oleochemis-

try [8]. To use lipases more economically and efficiently

in aqueous as well as in nonaqueous solvents, their

activity and operational stability needs to be improved by

immobilization. In addition, the enzyme immobilization

onto magnetic supports such as nanosized magnetite

particles allows an additional merit, namely, the selective

and easy enzyme recovery from the medium under a

magnetic force, compared with other conventional sup-

port materials. Hence, there is no need for expensive

liquid chromatography systems, centrifuges, filters, or

other equipment. In contrast, lipases obtain the highest

activity when their molecules are immobilized onto

nanoparticles because of their relatively high specific

area; this promises results on immobilizing lipases onto

surface-modified nano-sized magnetite particles [9].

The objective of this study was to demonstrate the

potential use of magnetic nanoparticles in bioengineering

applications. Mucor racemosus NRRL 3631 lipase was used

as the model enzyme in this study. The uncoated and

silica-coated magnetite nanoparticles were characterized

by X-ray powder diffraction (XRD), transmission electron

microscopy, and Fourier transform-infrared (IR) spectro-

scopy. The properties of the immobilized lipase such as

activity, recovery, protein analysis, and thermal stability

were investigated.

Materials and methodsCommercial lipase enzyme was prepared from M.racemosus NRRL 3631. All other materials were of

analytical grade and used without further purification;

these materials included tetraethyl orthosilicate

(TEOSZ98%), ammonia solution (NH3, 28 wt%), ferrous

dichloride tetrahydrate (FeCl2 � 4H2O), ferric trichloride

hexahydrate (FeCl3 � 6H2O), glucose (C6H6O6), potas-

sium chloride (KCl), potassium dihydrogen phosphate

(KH2PO4), magnesium sulfate (MgSO4 � 7H2O), disodium

hydrogen phosphate (Na2HPO4), sodium dihydrogen

phosphate (NaH2PO4), peptone from animal protein, olive

oil, gum arabic, and acetone.

Microorganisms, medium, and growth conditions

M. racemosus NRRL 3631 was maintained on potato

dextrose agar (PDA) slants. The microorganism was

grown in 250 ml Erlenmeyer flasks containing 100 ml of

the medium. The medium was inoculated with 4 ml of

spore suspension, and the flasks were incubated for 72 h

in an orbital shaker operating at 200 rpm at 301C. For

lipase production, the composition of the basal medium

(9% w/v) was: glucose, 1; olive oil, 1; peptone, 30;

KH2PO4, 0.2; KCl, 0.05; and MgSO4 � 7H2O, 0.05%, with

an initial pH of 6.5 [10]. The medium was heat sterilized

at 1211C for 15 min.

Standard method for enzyme activity assay

The lipase assay was performed using an olive oil

emulsion according to the procedure described by

Starr [11]. The olive oil emulsion was prepared as

follows: 10 ml of olive oil and 90 ml of 10% arabic gum

were emulsified using a homogenizer for 6 min at

20 000 rev/min. The reaction mixture composed of 3 ml

of olive oil emulsion, 1 ml of 0.2 mol/l Tris-buffer (pH

7.5), 2.5 ml of distilled water, and 1 ml of enzyme solution

was incubated at 371C for 2 h with shaking. The emulsion

was destroyed by the addition of 10 ml of acetone (95% v/v)

immediately after incubation, and the liberated free fatty

acids were titrated with 0.05 N.

Analytical procedure of protein determination

Protein measurements were carried out according to the

method of Lowry et al. [12], using BSA as the standard.

The amount of bound protein was determined indirectly

from the difference between the amount of protein

present in the filtrate and that in washing solutions after

immobilization.

Partial purification of M. racemosus lipase using

ammonium sulfate

Ammonium sulfate (60% saturation) was added to 900 ml

of the culture supernatant at 41C. The precipitate was

collected by centrifugation at 12 000g at 41C for 20 min

and dissolved in a constant amount of distilled water. The

lipase activity and protein concentrations were deter-

mined [13].

Synthesis of magnetite nanoparticles

The nanoparticles were prepared according to the

method described by Massart [14] but without the use

of hydrochloric acid. A total of 4.05 g of FeCl3 � 6H2O and

1.98 g of FeCl2 � 4H2O was dissolved in 100 ml of distilled

water; the solution was purged with nitrogen to agitate

the mixture and prevent the oxidation of Fe2 + ions. After

30 min of purging, 143 ml of 0.7 mol/l NH4OH was added

dropwise into the solution and the now basified solution

was purged for an additional 10 min. During the addition

of NH4OH, it was noticed that the solution changed color

from the original brown to dark brown and then to black.

The precipitate was magnetically separated using a

permanent magnet and then washed with distilled water

several times and allowed to dry in air. The resulting

product was defined as M.

Synthesis of silica-coated magnetite nanoparticles

The above-mentioned experiment was repeated until the

step in which the solution was purged with nitrogen to

agitate the mixture. After this step, the precursor

TEOS (3 ml) was carefully dropped into the reaction

mixture of iron using a syringe, with mechanical stirring.

Lipase immobilization on Fe3O4 nanoparticles El-Hadi et al. 29

The homogenization was performed for 15 min. After

sonication for 15 min, 143 ml of 0.7 mol/l NH4OH was

added dropwise into the mixture with continuous

mechanical stirring for 30 min. The coated particles were

finally separated from the liquid using a permanent

magnet, washed with distilled water several times, and

allowed to dry in air. Finally, we also determined the

effect of silica coating by varying the amount of TEOS

added to the reaction mixture. In this regard, we studied

the effect of five different amounts of TEOS, 1.04, 2.08,

4.22, 8.33, and 12.5 ml, which are equivalent to 0.5, 1, 2,

4, and 6% molar ratios, respectively. The determine

parameter of silica-coated magnetite nanoparticles is

labeled as MS1, MS2, MS3, MS4 and MS5.

Characterization

XRD was used to investigate the crystal structure of the

magnetic nanoparticles. The size and shape of the

nanoparticles were examined using a transmission elec-

tron microscope (TEM) (Model JEOL-1230, Japan). The

IR spectra were recorded using a Fourier transform-

infrared spectrophotometer (FT-IR). The sample and

KBr were pressed to form a tablet.

Immobilization of lipase

Because of the epoxy groups of the magnetite silica

nanoparticles, lipase immobilization was carried out by

treatment of the lipase solution with the nanoparticles

directly. The particles (200 mg of Fe3O4 coated with 1%

silica nanoparticles) were added to 40 ml of phosphate

buffer (0.1 mol/l, pH 6.5) containing lipase (1 ml). The

mixture was placed in a shaking incubator at 301C with

continuous shaking at 150 rpm for 6 h to finish the

immobilization of lipase. The immobilized lipase was

recovered by magnetic separation and washed with

phosphate buffer (0.1 mol/l, pH 6.5) three times to

remove excess enzyme. The resulting immobilized lipase

was held at 41C before use. The enzymatic activities of

the free and immobilized lipases were measured by

titrating the fatty acids that were obtained from the

hydrolysis of olive oil. One unit of lipase activity (U) is

defined as the amount of enzyme that hydrolyzes olive

oil, liberating 1.0 mmol of fatty acid per minute under the

assay conditions. The relative recovery (%) was the ratio

between the activity of the immobilized lipase and that of

free lipase [15].

Biochemical characterization of the free and

immobilized lipases and their reusability

Thermal stability of the free and immobilized lipase was

studied by incubating the biocatalyst at 30–801C for 15,

30, and 60 min in a water bath. Similarly, to determine the

stability at varying pH values, the immobilized enzyme

was reinsulated separately in 0.2 mol/l of citrate buffer at

pH 3–7 and in tris-HCl buffer at pH 7.6–9 for 1 h, and the

residual activities were determined under standard assay

conditions. The residual activity in the samples without

incubation was considered to be 100%. The inactivation

rate constant (K) and the half-life time (t1/2) were

calculated using the following formula: Half-life = 0.693/K,

in which K is the deactivation rate constant = slope of the

straight line [16].

The kinetic parameters Vmax and Km were determined for

the immobilized lipase. In addition, the reusability of the

immobilized lipase was determined by hydrolysis of olive

oil by the immobilized lipase recovered using magnetic

separation and compared with the first run (activity

defined as 100%).

Results and discussionStructure and shape of the support for nanoparticles

The XRD pattern (Fig. 1) of the Fe3O4 (M) nanoparticles

prepared under standard conditions revealed diffraction

peaks at 111, 220, 311, 400, 422, 511, 440, etc., which

were the characteristic peaks of Fe3O4 crystals with a

cubic spinel structure [17]. It was clear that only the

phases of Fe3O4 were detectable and there were no other

undesired diffraction maximas of the impurities that

could be observed in the spectra. From the relatively wide

half-peak breadth, it could be estimated that the particle

size is quite small. From the XRD patterns, the average

diameter that was calculated to be 13.8 nm using the

Scherrer equation (D = Kl/b cosy, in which K is constant,

l is X-ray wavelength, and b is the peak width of half-

maximum) [18,19]. Interestingly, it was observed that

the diffraction patterns for the samples MS1 and MS2

Figure 1

X-ray powder diffraction patterns of (a) pure Fe3O4 nanoparticles and(b) MS1 and MS2.


consisted of an amorphous structure, which was attrib-

uted to the amorphous silica matrix, as clearly indicated

in Fig. 1 [20]. The XRD patterns of the remaining

samples MS3, MS4, and MS5 (not presented here) also

showed an amorphous structure. The relatively low

intensity reflections and absence of significant sharp

diffraction peaks for the MS1 and MS2 patterns are

probably due to the presence of SiO2 on the surface of

the magnetic nanoparticles. Xu et al. [21] also suggested

that the low intensity of the reflection peaks could be

attributed to the ultrafine crystalline structure of the

magnetite particles used for the generation of silica-

coated nanoparticles. The particle size and morphology of

Fe3O4, Fe3O4/SiO2, and Fe3O4/SiO2/enzyme were eval-

uated from the TEM micrographs. It is noteworthy that

the size distribution is 10–16 nm, which matched the

value calculated using the Scherrer equation, and that the

nanoparticles are spherical in shape (Fig. 2a) and their

aggregation can be discerned clearly. In Fig. 2b and c, the

coated silica layer can be observed as a typical core–shell

structure of the Fe3O4/SiO2 nanoparticles. The dispersity

of the Fe3O4/SiO2 nanoparticles was also improved, and

the average size increased to about 32 nm. After lipase

adsorption, the degree of particle aggregation increased;

however, a change in the particle size was not observed

(Fig. 2d).

FT-IR spectra of the magnetite nanoparticles

The FT-IR spectra of magnetite are shown in Fig. 3. A

factor group analysis, reported in a classic IR study on

spinels, suggested that there were four IR-active bands;

however, in most cases, including magnetite, only two of

them are observed between 400 and 800 cm – 1 [22]. In

this study, Fe3O4 showed a broad band that consisted of

two slightly split peaks identified at 573 and 621 cm – 1;

these peaks were attributed to the stretching vibration of

Figure 2

Transmission electron microscope images of (a) Fe3O4 nanoparticles, (b) MS1 nanoparticles, (c) MS1 nanoparticles with immobilized lipase, and(d) MS1 nanoparticles without lipase.


the Fe–O bond and confirmed the occupancy of Fe3 +

ions at tetrahedral sites in a manner consistent with that

reported in the literature [23–25]. On the low-frequency

side of the broad band, we observed that the weak peaks

appearing at 432 and 453 cm – 1 corresponded to the

presence of the Fe3 + –O2 – bond at octahedral sites [26].

In contrast, we found a broad peak near 3380 cm – 1 and a

sharp peak near 1635 cm – 1, which were attributed to the

stretching and binding vibrations of the hydroxyl groups.

These peaks confirm the presence of adsorbed water on

the surface of magnetite [27]. However, the peaks at

1383 and 1453 cm – 1 resulted from the stretching

vibration of the C–O bonds in CO2, which might come

from air.

FT-IR spectra of the silica-coated magnetite

nanoparticles

Figure 3 shows the IR spectrum of the silica-coated

magnetite nanoparticles. It was clear that the character-

istic adsorption bands of the Fe–O bond (Fe3 + –O2 – ) of

the silica-coated magnetite nanoparticles shift to higher

wave numbers of 585, 637, 441, and 483 cm-1, respec-

tively, compared with that of uncoated nanoparticles (in

573, 621, 432, and 453 cm – 1). The absorption bands at

around 1030, 800, and 470 cm – 1 reflect the Si–O–Si

asymmetry, Si–O–Si symmetric stretching vibrations, and

deformation mode of Si–O–Si, respectively [28]. The

bands at 569 and 965 cm – 1 are possibly because of

the Fe–O–Si and Si–O–Si stretching vibrations caused by

the perturbation of the metallic ion in the SiO4 tetra-

hedra [29], respectively. The FT-IR spectra of the lipase

on the silica-coated magnetite nanoparticles (Fig. 3)

showed a spectra similar to the IR spectra of the silica-

coated magnetite nanoparticles with immobilized lipase.

It was observed that the characteristic bands of lipase at

1655 and 1535 cm – 1 [30] revealed that it was immobi-

lized on the silica-coated magnetite nanoparticles.

The amount of enzyme added and the corresponding

immobilization efficiency

The relationship between the amounts of enzyme

(0.5–3 ml) and immobilization efficiency has been shown

in Fig. 4. When the enzyme amount added was 1 and

1.5 ml, with 300 mg of the magnetite coated with 1%

silica, the maximal immobilization efficiency was 87 and

96%, respectively. The curve in Fig. 4 illustrates that the

immobilization efficiency gradually decreases when the

amount of enzyme added is more than 1.5 ml. This could

be explained by an overall amount of the added enzyme

formed an intermolecular space hindrance of the immobil-

ized enzyme, which will not only the active site of the

enzymes but also restrain the dispersion of the substrate

and product [3].

Effect of different concentrations of SiO2 coating the

magnetite nanoparticles on the immobilization

efficiency of lipase

To solve the leaching problems of the adsorbed lipase and

improve the conventional way for lipase immobilization,

different concentrations of MS nanoparticles ranging

from 0.5–4% were used as cross-linking agents for

immobilization in 300 mg of magnetite nanoparticles.

The experimental results have been given in Fig. 5. It was

shown that the immobilization efficiency decreased

slightly from 96 to 84% with an increase in the SiO2

concentration from 1 to 4% and then decreased sharply

with a further increase in the concentration of SiO2 to

6% (76%). Although a higher amount of lipase binding

occurred when a low concentration of SiO2 was used for

silica coating the magnetic nanoparticles, there was a

substantial loss of enzyme activity.

Biochemical properties of the free and immobilized

lipase

The effect of pH on the specific enzyme activity of lipase

immobilized by silica was studied by varying the pH of

the reaction medium from 3–9 using a 0.1 mol/l citrate

phosphate buffer (3–7) and a 0.1 mol/l Tris (hydroxy

Figure 3

(a) Fourier transform-infrared spectrophotometer spectra of Fe3O4,(b) MSI without immobilized lipase, and (c) MSI with immobilized lipase.

Figure 4

Effect of different amounts of Mucor racemosus NRRL 3631 lipase onthe immobilization efficiency.


methyl) amino methane buffer (7.5–9), and the pH

profile has been shown in Fig. 6a. Generally, the binding

of enzymes to polycationic supports would result in an

acidic shift in the optimum pH [31,30]; similarly, after

silica immobilization, the optimum pH of lipase exhibited

an acidic shift (5–6). The variation in the residual activity

of the free and immobilized lipase with pH is shown

in Fig. 6b. The immobilized lipase was stable in the pH

range of 3–5 as compared with the free enzyme; this

indicated that immobilization appreciably improved the

stability of lipase in the acidic region.

The thermal stabilities of the free and immobilized lipase

in terms of the residual activities have been compared

in Fig. 7. Lipase immobilized on MS nanoparticles

remained fully active up to 401C. These results are

similar to those obtained by Huang et al. [30], who found

that binary immobilized lipase from Candida rugosa was

fully active at 401C; however, inactivation of the enzyme

occurred on treatment at higher temperatures. About

40% of the residual activity of free lipase was preserved at

601C for 1 h; however, about 72.9% residual activity was

preserved in case of the immobilized enzyme. At 801C,

the free enzyme was fully inactivated, whereas the

immobilized form preserved about 37.8% of its residual

activity for 15 min. Hiol et al. [32] studied the thermo-

stability of the free enzyme of Rhizopus oryzae and found

that it was highly inactivated at 451C and almost all

activity was lost at 501C after a 40 min incubation. This

thermal stabilization could be explained by the location

of the lipase inside the micropores of the support,

wherein the enzyme is protected against alterations of

the microenvironment. The Michaelis–Menten kinetics

of the hydrolytic activity of the free and immobilized

lipases have been represented in Table 1, using varying

initial concentrations of olive oil as the substrate. The

Michaelis constant (Km) and the maximum reaction

velocity (Vmax) were evaluated from the double reciprocal

plot. The Vmax value of 250 U/mg protein exhibited by

the immobilized lipase was found to be higher than that

of free lipase (50 U/mg protein). The Km value (20 mmol/l)

determined for the immobilized lipase was about three-

folds higher than that of free lipase (6.66 mmol/l), which

indicated a lower affinity toward the substrate. This

increase in Km might be either due to the structural

changes induced in the enzymes by the immobilization or

the lower accessibility of the substrate to the active

sites [33,30]. The inactivation temperature of the soluble

and immobilized lipase was observed to be between 50 and

701C. In general, the immobilization processes protected

the enzymes against heat inactivation, for example, the

calculated half-life values at 50, 60, and 701C for the

immobilized enzyme were 630, 533, and 391.5 min,

respectively, which are higher than those (231, 198, 187,

and 3 min, respectively) of the free enzyme as shown

in Table 2, that is, the free enzyme showed a half-life of

10.5 h at 501C, 8.88 h at 601, and 6.5 h at 701C. Our results

are nearly similar to those obtained by Kumar et al. [34],

who reported the half-life of Bacillus coagulans BTS3 lipase at

55 and 601C to be 2 h and 30 min, respectively; moreover,

they reported the half-life of lipase from another meso-

philic bacteria (Bacillus spp.) to be 2 h at 601C. They

reported that the deactivation rate constants of 1.1� 10–3,

1.3� 10–3, and 1.7� 10 – 3 for the experimental immobi-

lized enzyme at temperatures of 50,60, and 701C,

respectively were lower than those (3� 10–3, 3.77� 10–3,

Figure 5

Effect of different concentration of SiO2-coated magnetic nanoparticleson the immobilization efficiency of Mucor racemosus NRRL 3631lipase.

Figure 6

Effect of pH values on the activity (a) and stability (b) of free and immobilized Mucor racemosus lipases.


and 4.8� 10–3, respectively) of the free enzyme at the

same temperatures. These results could be related to a

hydrophilic or hydrophobic environment. A hydrophilic

microenvironment allowed the immobilized derivatives to

follow a double experimental decay in their activities,

wherein the hydrophobic microenvironment makes the

enzymatic activity suffer a single experimental decay

during storage conditions [35].

Variations in the enzyme activity with repeated batch

enzyme reactions

Operational stability was the most important parameter

in the immobilization of enzymes because inactivation is

inevitable when the free enzyme is exposed to inade-

quate ambient conditions. The recycling efficiency of the

immobilized lipase has been presented in Fig. 8. It was

observed that the immobilized lipase retained 51% of its

original activity even after the seventh reuse; this

indicated that the resultant bound lipase had a better

reusability, which was desirable for applications in

biotechnology. The loss of activity may be ascribed to

conformational changes in the enzyme, blocking of the

lipase active sites, or the gradual loss of the bound lipase

during the reaction procedures.

ConclusionFrom these results it can be concluded that Fe3O4

magnetic nanoparticles and silica-coated magnetite (MS)

nanoparticles with excellent properties have been suc-

cessfully prepared using the chemical coprecipitation

technique with some modifications. The XRD results

indicate that the composites were in the nanoscopic

phase. Based on the TEM images, the diameters of the

uncoated magnetite particles were determined to be

around 10–16 nm and those of the coated particles to be

about 11 nm. The silica coating appeared to be effective

in protecting the magnetite from being converted to

other oxide species. The thermal and pH stabilities of the

immobilized lipase increased on immobilization. The

optimal pH and temperature of the immobilized lipase

were 5–6 and 401C, respectively. There was a slight

decrease in the residual activity of the immobilized

lipase. The operational stability of the immobilized lipase

Figure 7

Thermal stability of free and immobilized Mucor racemosus NRRL 3631 lipases.

Table 1 Kinetic parameters (Vmax and Km) for the free and

immobilized enzymes

Types Vmax (U/mg protein) Km (mmol/l)

Immobilized lipase 250 20Free lipase 50 6.66

Table 2 Kinetic parameters (half-life and the deactivation rate

constant) for the free and immobilized enzymes

Half-life (min) Deactivation rate constant

Types 501C 601C 701C 501C 601C 701C

Immobilizedlipase

630 533 391.5 1.8�10–3 1.3�10–3 1.7�10–3

Free lipase 231 198 187.3 3�10–3 3.77�10–3 4.8�10–3

Figure 8

Operational stability of the immobilized Mucor racemosus NRRL lipaseon the hydrolysis process.


over repeated cycles could substantially save on the cost

of the enzyme. The residual activity of the enzyme even

after seven repeated uses was over 51%. Conclusively,

magnetic nanoparticles provide an economically efficient

and selective system for enzyme immobilization.


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Extracellular polysaccharides produced by the newly discovered

source Scopularis spp.Siham A. Ismail

Department of Chemistry of Natural and MicrobialProducts, Division of Pharmaceutical and DrugIndustries, National Research Centre, Cairo, Egypt

Correspondence to Siham A. Ismail, Department ofChemistry of Natural and Microbial Products, Divisionof Pharmaceutical and Drug Industries, NationalResearch Centre, El-Behowth St., PO Box 12311,Dokki, 12622 Cairo, EgyptTel: + 20 122 357 1676; fax: + 20 233 370 931;e-mail: [email protected]

Received 26 August 2012Accepted 15 November 2012


2013,12:36–39

Background

Microorganisms are better and cheaper sources for the production of polysaccharides.

Therefore, there has been an increasing interest in isolating and identifying new

microbial polysaccharides.

Objective

The aim of this study was to produce new extracellular polysaccharides, with better

rheological properties and varied applications, from the newly discovered fungal strain

Scopularis spp., using different carbon sources.

Methods

Fourier transform infrared spectroscopy, carbohydrate analysis, and thin layer

chromatography were the methods used for the preliminary characterizing of the

produced polysaccharides.

Results

Among the 10 examined carbon sources, fructose, raffinose, sucrose, and maltose

were found to produce an appreciable amount of extracellular polysaccharides (0.90,

0.87, 0.86, and 0.74 g/l, respectively), whereas arabinose, lactose, and mannitol

produced a minimal amount of extracellular polysaccharides (0.22, 0.17, 0.12 g/l,

respectively). However, all the tested sugars enhanced the growth of the fungal strain.

The analytical method proved that the polymer was a heteropolysaccharide with six

sugar moieties, all different in their relative ratios from one carbon source to another.

Glucose was found to be the most abundant monosugar in all the polymer samples.

Galactose, rhamnose, and glucuronic acid also appeared on the thin layer

chromatography plate.

Conclusion

A new extracellular heteropolysaccharide was produced from the new source,

Scopularis spp. The produced polysaccharide contained glucose, galactose,

glucuronic acid, rhamnose, and two other unidentified sugars as indicated from the

thin layer chromatography plate.

Keywords:

acid hydrolysis, carbon source, extracellular polysaccharides, Scopularis spp.


IntroductionPolysaccharides are highly valued, biologically active

polymers with many industrial applications in food, feed,

textile, cosmetic, and pharmaceutical industries and are

also used as depolluting agents [1]. Owing to their

bioactive nature, they have many medicinal applications

as anticancer, antiviral, antioxidant, antibacterial, anti-

inflammatory, and prebiotic agents [2–8]. However, most

of the commercial polysaccharides are produced from

plants and algae and a small proportion is produced from

microbial sources [9]. Fungi are currently an interesting

source of biologically active compounds. Most of the

mould-produced polysaccharides are obtained from

mushrooms [8,10,11].

Microorganisms are better and cheaper sources for the

production of polysaccharides compared with plants or

algae because of their high growth rate, ability to grow in

cheaper nutrient media within a few days, lower space

requirement, and ease of manipulation [12]. Therefore,

there has been an increasing interest in isolating and

identifying new microbial polysaccharides that may

compete with traditional polysaccharides.

Therefore, the aim of this study was to examine the

ability of the new fungal strain Scopularis spp. to produce

high yields of extracellular polysaccharides (EPS), with

better rheological properties and varied applications,

using different carbon sources.

Materials and methodsMicroorganisms and media

The Scopularis spp. used in this study was obtained from

the culture collection of the National Research Centre

(Egypt). The strain was maintained by subculturing on

36 Original article


DOI: 10.7123/01.EPJ.0000427067.33748.ea

potato dextrose agar slants monthly (PDA; Merck,

Darmstadt, Germany). The slants were incubated at

28–301C for 7 days before storage at 41C.

The inoculum cultures were grown in 250 ml Erlenmeyer

flasks containing 50 ml of sterilized medium comprising

(g/l): lactose, 7.5; NaN03, 1.0; yeast extract, 1.5;

MgSO4.7H2O, 0.5; KH2PO4, 1.0; KCl, 0.5; and FeSO4.7-

H2O, 0.01 at pH 5 [13] in a rotatory incubator shaker at

150 rpm and 28–301C for 3 days before using.

Fermentation

Fermentation was carried out in 250 ml Erlenmeyer flasks

containing 50 ml of the above mentioned medium with

different sugars as the carbon source. The tested sugar

solutions were sterilized separately and mixed aseptically

with the other components before inoculation with 5%

(v/v) of the inoculums. The flasks were incubated

at 28–301C in a rotary shaker at 150 rpm for 7 days.

Mycelial dry weight

The mycelial pellets were separated from the viscous

liquid culture by centrifugation (6000 rpm, 20 min). After

the removal of the supernatant, the mycelia was washed

thoroughly with distilled water and dried to a constant

weight to attain the mycelial dry weight.

Isolation of the extracellular polysaccharides

The viscous supernatant obtained from the above men-

tioned step were collected and dialyzed against tap water

for 2 days using a 10 000–12 000 MWCO membrane (VWR

Scientific, Spectrum Companies, Goshen Parkway, West

Chester, USA), changing it three times daily; it was then

dialyzed against distilled water in the same way, after which

the solution was centrifuged again as indicated above. The

dialyzed cultures were mixed with three volumes of chilled

absolute ethanol (v/v) with stirring. The precipitated

polysaccharide was collected together as viscous filaments

and could easily be separated from the liquid and the other

compact particles that settled quickly to the bottom. The

collected EPS were washed with a water : ethanol mixture

(1 : 1, v/v) to remove the residue of the liquid culture; it

was then dried and weighted as crude EPS.

All the experiments were conducted in triplicate and the

results are the averages of these three independent trials.

Monosaccharide composition analysis

Acid hydrolysis of the crude polysaccharides was carried

out according to the procedure described by Fischer and

Dorfel [14]. In brief, 0.05 g of the crude EPS was mixed

with 0.5 ml of 80% sulfuric acid and left overnight at room

temperature; it was then diluted with 6.5 ml of distilled

water and boiled in a water bath for almost 6 h. The

mixtures were cold neutralized with excess BaCO3 and

subjected to thin layer chromatography for primary

investigation.

Thin layer chromatography

Silica gel plates (Merck) were used to identify the

composition of the hydrolyzed polysaccharides. The sam-

ples were spotted onto the plates along with different

standard monosugars. The plates were developed at room

temperature in a saturated chamber containing n-propanol :

water (85 : 15, v/v). The sugars were detected by spraying

the dried plates with 3% phenol reagent, followed by

incubation at 1001C in an oven for 10 min [15].

Fourier transform infrared analysis

The crude polysaccharide was mixed with KBr powder,

ground, and pressed into 1 mm pellets for Fourier transform

infrared (FTIR 6100; Jasco, HoChi Minh City, Japan)

spectroscopy in the frequency range of 4000–400 cm – 1.

Results and discussionCarbohydrates are very important nutritional require-

ments for the growth and development of all fungi.

However, different fungal species vary in their ability to

utilize different carbon sources. The results shown

in Fig. 1 indicate that the tested fungus had the ability

to grow in all the used carbon sources, but the production

of the EPS was quite distinct for each sugar used. Among

the 10 sources examined, fructose, raffinose, sucrose, and

maltose enhanced the production of EPS (0.90, 0.87,

0.86, and 0.74 g/l, respectively), whereas arabinose,

lactose, and mannitol produced minimal amounts of

EPS (0.22, 0.17, 0.12 g/l, respectively). The effect of the

different carbon sources on the amount of polysacchar-

ides produced was recorded by all the researchers and was

found to be related to the microorganism used. The

mycelial growth did not parallel with the production of

EPS; this has also been reported by other research-

ers [16–18]. It has been observed that the production of

EPS increased with an increase in the concentration of

the sugars used and when the morphology of the fungal

growth was in the form of pellets rather than fibers

(unpublished data). The amount of EPS produced during

Figure 1

The effect of different sugars on the production of extracellularpolysaccharides (EPS) and cell growth [expressed as constant dryweight (CDW)].

Polysaccharide new microbial source Ismail 37

our study is within the range that has been published by

other authors [17–19].

Fourier transform infrared spectroscopic analysis

The configuration of the crude EPS produced from four

different sugars has been shown in Fig. 2a–d.The spectra

clearly indicate that all the samples had a broad band

around 3400 cm – 1 representing a large number of

hydroxyl groups and a sharp band at 2922 cm – 1 for the

C–H bending vibration of the CH2 groups, and the two

bands are characteristic of carbohydrate polymers. The

bands near 1736 cm – 1 and those around 1250 cm – 1 may

be attributed to the stretching vibration of the C = O and

C–O–C of the acyl groups. The bands at 1420 cm – 1 and

those around 1606–1621 cm – 1 have been suggested to

represent the carboxyl groups of acids, whereas the bands

in the range of 820–955 cm – 1 represent the linkages

between the mono sugars. All the data were within the

range that has been reported by other authors [5,6,20–23].

Effect of different carbon sources on the composition of

the extracellular polysaccharides

The monosugar composition of the crude EPS produced

from the different carbon sources was identified using

thin layer chromatography as shown in Fig. 3. The plates

indicate the presence of more than six distinguishable

spots in most of the samples. However, the relative ratio

of the monosugars was entirely different. All the samples

mainly contain glucose, galactose, glucuronic acid, and

rhamnose. Although glucose is the main monosugar

component of the produced EPS, neither glucose nor

Figure 2

50

60

70

80

90

100(a)

40080012001600200024002800320036004000

Tra

nsm

ittan

ce (

%)

Wavenumber (cm-1)

FT-IR of crude EPS from fructose

50

60

70

80

90

100

40080012001600200024002800320036004000

Tra

nsm

ittan

ce (

%)

Wavenumber (cm-1)

FT-IR of crude EPS from glucose

50

60

70

80

90

100

40080012001600200024002800320036004000

Tra

nsm

ittan

ce (

%)

Wavenumber (cm-1)

FT-IR of crude EPS from raffinose

50

60

70

80

90

100

40080012001600200024002800320036004000

Tra

nsm

ittan

ce (

%)

Wavenumber (cm-1)

FT-IR of crude EPS from Surose

(b)

(c) (d)

(a) Fourier transform infrared (FTIR) of the crude extracellular polysaccharides (EPS) produced from fructose as the carbon source in the culturemedium. (b) FTIR of the crude EPS produced from glucose as the carbon source in the culture medium. (c) FTIR of the crude EPS produced fromraffinose as the carbon source in the culture medium. (d) FTIR of the crude EPS produced from sucrose as the carbon source in the culture medium.

Figure 3

Thin layer chromatography plate for the different samples: 1, 7, 8, 9, 12,and 13 for glucuronic acid, fucose, glucose, N-acetyl glucosamine,galactose, and rhamnose standards, respectively, and 2, 3, 4, 5, 6, 10,and 11 for fructose, raffinose, maltose, sucrose, glucose, lactose, andarabinose samples, respectively.


its isomer galactose gave the highest yield of the

produced EPS, when used in the culture medium as

the carbon source. However, both glucose and galactose

gave an appreciable amount of EPS (0.6 and 0.56 g/l,

respectively).The influence of the carbon source on the

production and composition of the EPS has been

reported by other authors as well [5,17,24]. The presence

of different sugar moieties suggests that the produced

EPS was a heteropolysaccharide.

ConclusionA new extracellular heteropolysaccharide was produced from

a newly discovered source, Scopularis spp. The strain has the

ability to grow and produce EPS in the presence of all the

tested sugars. The produced polysaccharide contains

glucose, galactose, glucuronic acid, rhamnose, and two other

unidentified sugars. This study will open doors for further

studies on attaining a greater production of EPS from this

newly discovered source and also for clarifying their exact

composition, structures, and biological activities. Moreover,

the oligosaccharides and low-molecular-weight polysacchar-

ides that come out of the dialysis bag have to be identified.

AcknowledgementsThis work was supported by National Research Centre, Cairo, Egypt;as part of the project of the Department of Chemistry of Natural andMicrobial Products. Thank’s for the responsible professors of thefinancial department.

Conflicts of interestThere are no conflicts of interest.

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Polysaccharide new microbial source Ismail 39

Biotransformation of soybean saponin to soyasapogenol B by

Aspergillus parasiticusHala A. Amina, Yousseria M. Hassanb and Soad M. Yehiaa

aDepartment of Chemistry of Natural and MicrobialProducts, National Research Center, Dokki andbDepartment of Microbiology, Faculty of Science,Ain shams University, Cairo, Egypt

Correspondence to Hala A. Amin, PhD, Department ofChemistry of Natural and Microbial Products, NationalResearch Center, Dokki, 12311 Cairo, EgyptTel: + 20 233 464 472; fax: + 20 237 622 603;e-mail: [email protected]

Received 7 November 2012Accepted 15 January 2013


2013,12:40–45

Objectives

The aim of this study was to select of the most potent fungus that is able to hydrolyze

soybean saponin (SS) to soyasapogenol B (SB). The selected fungus was cultivated

under different physiological conditions to evaluate its ability to transform SS to

achieve the maximal conversion output.


Within 72 h, the biotransformation of SS by Aspergillus parasiticus, followed by

isolation and purification of SB as a main product were carried out. The identity of SB

was established by determination of its RF value and IR, mass spectra, and NMR

spectra. Furthermore, different sets of experiments were carried out to enhance the

activity of the tested organism and consequently, SB production.


Screening of different fungal isolates for transformation of SS to SB revealed that

A. parasiticus produced the highest yield of SB. The maximum SB yield was obtained

using a production medium composed of (%, w/v): malt extract, 4; yeast extract, 2;

KH2PO4, 0.2; (NH4)2SO4, 0.2; MgSO4 �7H2O, 0.03; CaCl2 �2H2O, 0.03; galactose,

0.5; and SS, 3 (pH 8). The medium was inoculated with 6% (v/v) inoculum of a 72 h

old culture and incubated on a rotary shaker (150 rpm) at 301C for 72 h. Under these

optimal conditions, the cell biotransformation efficiency was increased from 13.44 to

65%.

Keywords:

Aspergillus parasiticus, biotransformation, soyasapogenol B, soybean saponin


IntroductionSaponins are structurally diverse molecules that are

chemically referred to as triterpenes and steroid glycosides.

They consist of nonpolar aglycones coupled with one or

more monosaccharide moieties [1]. This combination of

polar and nonpolar structural elements in their molecules

explains their soap-like behavior in aqueous solutions.

Soyasaponins are a group of oleanane triterpenoids found

in soy and other legumes. They are divided into three

groups, based on the structure of the aglycone moiety, the

A, B, and E saponins [2]. Soyasapogenols A, B, and E are

conjugated as glycosides in soy [3,4]. The current

consensus is that soyasapogenols A, B, and E are true

aglycons, whereas soyasapogenols C, D, and E are artifacts

of hydrolysis that occur during the isolation process of

A, B, and E soyasapogenols .

Soyasaponins have various physiological effects including

hepatoprotective [5], anticarcinogenic [6], antiviral [7],

and anti-inflammatory [8] activities. Soyasapogenol B (SB),

obtained from soybean saponin (SS), is known to have

hepatoprotective [9], antiviral [10], antimutagenic [11],

anti-inflammatory [8], and growth suppressing effects on

cells derived from human colon and ovarian cancer [11,12].

Results from in-vitro fermentation suggest that colonic

microflora readily hydrolyzed SS to aglycones [2]. These

observations suggested that the dietary chemopreventive

effects of SS against colon cancer may involve alteration

by the microflora [12]. There is some evidence, as with

many other saponins, that bioactivity of SS is increased as

sugar moieties are eliminated from the saponin structure,

thereby reducing the polarity.

Aglycones, soyasapogenols, are produced by acid hydrolysis

of saponins, but there have been reports of aglycone

production by microorganisms. Kudou et al. [13] cultured

158 strains of the genus Aspergillus in a medium containing

SS and reported that 26 of them had a marked SS hydrolase

activity. Watanabe et al. [14] isolated a SS hydrolase from

Neocosmospora vasinfecta var. vasinfecta PF1225, a filamentous

fungus that can degrade SS and generate SB. Recently,

Amin and Mohamed [15] reported the production of SB

(86.3%) from SS using immobilized Aspergillus terreus on a

loofah sponge. The aim of this study was to select the most

potent fungus that is able to hydrolyze SS to SB. The

selected isolate was cultivated under different physiological

conditions to evaluate its ability to transform SS to achieve

the maximal conversion output.

Materials and methodsCultivation of fungal isolates

The different fungal isolates used in this work (Table 1)

were donated by the Center of Cultures of Chemistry of

40 Original article


DOI: 10.7123/01.EPJ.0000427332.31862.10

Natural and Microbial Products Department, National

Research Center (Cairo, Egypt). They were maintained

on potato dextrose agar slants at 41C and subcultured

at intervals of 1–2 months. Unless otherwise stated, the

fermentations were carried out in 250 ml Erlenmeyer

flasks containing 100 ml of the fermentation medium

composed of (%, w/v): malt extract, 4; yeast extract, 2;

KH2PO4, 0.2; (NH4)2SO4, 0.2; MgSO4 � 7H2O, 0.03;

CaCl2 � 2H2O, 0.030; and SS, 1 (pH 5.7) [16]. The flasks

were inoculated with 6% inoculum and agitated on a

rotary shaker at 150 rpm at 30 ± 21C for 72 h.

General assessment of the chemicals and instruments

used

SS (50%) was purchased from Organic Technologies Co.

(Coshocton, Ohio, USA). Potato dextrose agar and yeast

extract were products of Biolife Italiana (Milano, Italy).

Bacto malt extract and bacto peptone were purchased

from Difco Laboratories (New Jersey, USA). 1H NMR

and 13C NMR spectra were measured using a Bruker

AMX 500 instrument (Weizmann Institute of Science

Chemical, Rehovot, Israel) operating at 500 MHz for 1H

NMR and at 125 MHz for 13C NMR. Samples were dis-

solved in fully deuterated dimethyl sulfoxide (DMSO-d5).

The chemical shifts (d) are reported in ppm and the

coupling constants (J) in Hz. Mass spectra were measured

using a Finnigan mat. SSQ 7000 instrument at an

ionization voltage of 70 eV and EI mode.

Quantitative analysis of soyasapogenol B

At the end of the biotransformation period, the reaction

mixture was extracted twice with double the volume of

ethyl acetate. Thereafter, the organic layer was dried over

anhydrous sodium sulfate and concentrated under

reduced pressure. The residue was dissolved in a

chloroform–methanol mixture (1 : 1) and mounted on

thin-layer chromatography (TLC) plates. The plate was

first chromatographed for soyasapogenols using the

above-mentioned solvent system and then for SS using

a solvent system comprising chloroform–methanol–acetic

acid (10 : 20 : 1, v/v). SS and SB were detected on TLC

plates by spraying with 10% H2SO4 and then heating for

10 min at 1101C; they were then quantitatively analyzed

using a TLC-scanner (Shimadzu CS-9000 dual wave-

length flying spot, thin layer chromato-scanner, Tokyo,

Japan) at l equal to 530 nm [16]. The obtained weight of

SB was calculated by calibration of the line obtained from

the standard sample using the area under the curve for

the biotransformation products in each chromatogram.

SB molar yield %ð Þ¼

Weight of soyasapogenol B/MW of soyasapogenol B

Weight of soyasaponin I/MW of soyasaponin I�100;

where MW is the molecular weight and soyasaponin I

represents SS.

Separation and identification of the biotransformation

products

After cultivation of Aspergillus parasiticus on the biotrans-

formation culture medium containing 1% SS, the result-

ing filtrate (500 ml) was extracted twice with ethyl

acetate, and the organic layer was concentrated under

reduced pressure to obtain an oily sample (415 mg). A

preparative silica gel plate (silica gel 60 F-254 aluminum

plates; Merck, Darmstadt, Germany) was spotted and

developed using the same solvent system (benzene :

ethyl acetate : acetic acid; 24 : 8 : 1, v/v). The areas

containing soyasapogenols were detected by a slight

discoloration on the plates, and these sections were

scraped, extracted with chloroform : methanol (1 : 1), and

evaporated to dryness. This led to isolation of compound

I (56 mg) as the main product.

Compound I

Compound I was identified as SB, with a melting point of

2301C. The H1 NMR (DMSO-d5) results were as follows:

d at 5.18 (t, 1H, J12,11a= J12,11b= 4 Hz, H-12), 4.85 (d, 1H,

J24a-24b = 4.6 Hz, H-24a), 4.14 (dd, 2H, H-3, and H-21), 4.05

(d, 1H, J24b-24a = 4.6 Hz, H-24b), 3.82 (d, 1H, J22a-21b= 8.4

Hz, J22a,21a= 2.4 Hz, H-22a), 1.2 (s, 3H, H-23), 1.18 (s, 3H,

H-27), 0.95 (s, 3H, H-28), 0.90 (s, 3H, H-26), 0.84 (s, 6H,

H-25), 0.82 (s, 3H, H-29), and 0.80 (s, 3H, 30) and the 13C

NMR (CD3Cl) results were shown in Table 2.

Optimization of soybean saponin biotransformation

using Aspergillus parasiticus

Optimization of the environmental conditions for micro-

bial biotransformation processes on a laboratory scale

is important to obtain information for the scaled-up

production of the target product in a large-scale

fermentor. The parameters assessed were pH (4, 5, 5.7,

6, 7, 8, and 9) of the medium, inoculum size (1, 2, 3, 4, 5,

6, 8, and 10%, v/v) and age (24, 48, 72, and 96 h), duration

of the biotransformation process (24, 48, 72, 96, 120, and

144 h), SS concentration (0.5, 1, 2, 3, and 4%, w/v),

incubation temperature (20, 25, 30, 35, and 401C), and

shaking incubator speed (static,100, 150, 200, and

250 rpm). For examining the effect of the cultivation

medium composition on the biotransformation process,

different levels of either malt extract (2, 3, 4, 5, and 6%,

Table 1 Bioconversion of soybean saponin to soyasapogenol B

by different fungal strains

Soyasapogenol B

Fungal isolates mg/100 ml Molar yield (%)

Aspergillus fumigatus 6.8 2.85Aspergillus flavus 22.34 9.38Aspergillus niger 18.58 7.8Aspergillus parasiticus 32 13.44Aspergillus ruber 4.92 2.06Rhizopus riori 14.6 6.13Penicillium aurantiacum – –Penicillium waksmanii 1.86 0.78Penicillium frequentans 9.6 4.03Penicillium cyclopium 8.12 3.41Trichoderma harzianum 3.9 1.63Trichoderma viride 3.82 1.6

Strains were cultivated on a transformation culture medium consistingof (%, w/v): malt extract, 4; yeast extract, 2; KH2PO4, 0.2; (NH4)2SO4,0.2; MgSO4 �7H2O, 0.03; CaCl2 �2H2O, 0.03; and SS, 1 (pH 5.7) at150 rpm and 30 ± 21C for 72 h.SS, soybean saponin

SS biotransformation to SB Amin et al. 41

w/v) or yeast extract (0.5, 1, 2, 2.5, and 3%, w/v), different

carbon sources (glucose, galactose, mannose, sucrose,

arabinose, and starch), and different concentrations of

galactose (0.5, 1, 2, 3, 4, and 5% w/v) were individually

used.

Results and discussionScreening experiments

Twelve fungal isolates were screened for their saponin-

hydrolyzing abilities to produce SB from the SS that was

added to the culture medium. Results in Table 1 indicate

different capacities of the tested cultures to produce SB.

P. aurantiacum failed to perform the desired reaction,

whereas the other fungal isolates (Aspergillus flavus,Aspergillus fumigates, Aspergillus niger, Aspergillus parasiticus,Aspergillus ruber, Penicillium cyclopium, Penicillium frequentans,Penicillium waksmannii, Rhizopus riori, Trichoderma harzianum,

and Trichoderma viride) could. Among the 12 examined

fungal cultures, A. parasiticus produced the highest yield

of SB; it could transform about 13.44% of the added SS,

with the formation of 32 mg/100 ml SB. In this connec-

tion, Kudou et al. [13] reported that 26 of 158 strains of

the genus Aspergillus had a marked SS hydrolase activity

when cultured in a medium containing SS. Moreover,

Watanabe et al. [17] purified a SS hydrolase from

Aspergillus oryzae PF1224.

Identification of the biotransformation products

As A. parasiticus was cultivated for 72 h on a medium

containing 1% SS; compound I was isolated as a major

product (about 80%) in addition to some other minor

by-products. Physicochemical characteristics and various

spectral data of the obtained compound I were identical

to those of standard SB. Compound I produced red color

with sulfuric acid alone or with Liebermann–Burchard

reagent for the triterpenes. The molecular formula was

assigned to be C30H50O3 from the EI-mass spectra

(458 m/z). The presence of seven tertiary methyl singlets

(d 0.8–1.2) and a triplet olefinic proton at d 5.18 (t, 1H,

J12,11a = J12,11b = 5 Hz, H-12) in the NMR spectra

suggested a olean-12-en structure with three hydroxyl

groups. The hydroxyl groups were identified as being

attached at C-3, C-22, and C-24 from the H1 and 13C

NMR spectral data. The downfield shift of both C-3 and

C-22 (d 78.57 and 73.98, respectively) in the 13C NMR

spectrum suggested that two hydroxyl groups were

attached at these positions. The third hydroxyl group

was supported at C-24 by the presence of two signals at

d 4.85 (d, 1H, J24a-24b = 4.6 Hz, H-24a) and d (d, 1H,

J24b-24a = 4.6 Hz, H-24b), in addition to a methylene carbon

signal at 62.94 ppm in the 13C NMR spectrum. The signal

at d 3.82 (d, 1H, J22a-21b= 8.4 Hz, J22a,21a= 2.4 Hz, H-22a)

was assigned as the H-22a proton, which suggested a

b-orientation of the oxygen atom. Therefore, compound I

was identified as 3 b, 22 b, 24-trihydroxyolean-12 (13)-ene

(SB). All spectral data were in agreement with those

published by Kitagawa and colleagues [18,19].

Optimization of soybean saponin biotransformation by

Aspergillu sparasiticus

Effect of pH

Results presented in Table 3 show that the highest SS

conversion activities were maintained within the pH

range of 7–9; however, the biotransformation process was

markedly impedd at pH values below 5.7. In addition, the

initial pH values of the medium (4–9) were found to be

shifted toward more acidic values (3.39–6.83) at the end

of the bioconversion process. A maximum concentration

of SB (89.39 mg/100 ml) corresponding to a molar yield of

37.59% was obtained at pH 8. These findings supported

the data reported by Amin et al. [19] for the bioconversion

of SS to SB by A. terreus. Kudou et al. [20] found that

saponin hydrolase enzyme from A. oryzae KO-2 was stable

at pH values ranging from 5.0 to 8.0.

Effect of inoculum size

Results illustrated in Fig. 1 indicate that the yield of SB

was positively correlated to the increase in the inoculum

size up to 6% inoculum (v/v), corresponding to 0.0568 mg

cell dry weight, which led to the highest yield of SB

(37.59%). In contrast, an increase or decrease in the

inoculum size led to a gradual decrease in the SB yield.

Effect of the incubation period

The capacity of A. parasiticus to transform SS proved to be

markedly affected by the duration of the transformation

process. As shown in Fig. 2, biotransformation of SS to SB

increased gradually with increase of the incubation period

until the maximum value of 37.59% after 72 h was

reached, giving an SB yield of 89.5 mg/100 ml. However,

this yield sharply decreased upon increasing the time

more than 72 h, probably due to a further metabolism of

Table 213

C NMR assignments of soyasapogenol B

Carbon number Soyasapogenol B

1 38.132 27.143 78.574 42.055 55.346 18.547 32.778 41.149 47.1110 36.2411 23.1312 121.4613 144.0314 41.5615 25.4516 27.8317 36.8718 44.5119 46.0120 30.1121 41.5622 73.9823 22.8024 62.9425 15.5726 16.5027 24.9928 28.2329 32.5930 20.23


the product. Watanabe et al. [14] isolated a SS hydrolase

from Neocosmospora vasinfecta var. vasinfecta PF1225 after a

72 h incubation period. Moreover, the cell biomass yields

were determined at different time intervals (24, 48, 72,

96, 120, and 144 h) and were found to be 2.118, 3.04,

4.558, 4.566, and 5.386 g/100, respectively. Therefore, the

trends of SB production and cell growth were roughly

equivalent.

Effect of the culture medium composition

Results given in Figs 3 and 4 indicate that A. Parasiticusacts optimally at malt extract concentrations of 40 g/l and

yeast extract concentrations of 20 g/l, producing an SB

yield of 37.59%. Lower or higher levels of malt or yeast

extract gave lower yields of SB. Watanabe et al. [14] used

the same concentrations of malt and yeast extracts to

isolate a SS hydrolase from Neocosmospora vasinfecta var.vasinfecta PF1225.

As regards the additional carbon sources, results illu-

strated in Fig. 5 clearly indicate that the maximum yield

of SB (41.6%) was achieved when galactose was added to

the transformation medium; this is may be due to the

enhanced growth of the fungus by using lactose as the

carbon source. In contrast, the other tested carbon

sources supported comparatively lower conversion esti-

mates and were thus excluded.

Moreover, the effect of different levels of galactose on SB

production was studied. Data given in Fig. 6 reveal that a

low level of galactose (0.5%) supported maximum SB

production (49%), whereas increasing galactose levels

over 1% resulted in a dramatic decrease in SB production,

possibly because the cells preferred the easily oxidizable

galactose as an exclusive carbon source and repressed the

induction of saponin-hydrolyzing activity [19].

Effect of soybean saponin levels

Kudou et al. [20] reported that saponin hydrolase was an

enzyme induced by the existence of SS as it has high

substrate specificity for the glucuronide bonds of glyco-

sides. Thus, to enhance the productivity, different

substrate (SS) concentrations ranging from 0.5 to 4%

(w/v) were supplemented to the transformation culture

medium at the inoculation time. Results given in Fig. 7

indicate that molar yields of SB increased on increasing

the amounts of SS supplemented to the culture medium

up to the 3% level. Above the latter concentration, the

yield of SB decreased gradually; this is may be due to

inhibition of the SS hydrolase on increasing the substrate

concentration to more than 3%. Kudou et al. [20]

indicated that SS hydrolase from A. oryzae KO-2 is

inhibited by increasing the substrate level above the

optimum concentration (2.5 mmol/l).

Effect of incubation temperature

Results in Fig. 8 show that relatively high SB yields were

maintained at temperatures ranging from 25 to 351C.

Maximum SS conversion (65%) was achieved at 301C,

leading to a production of 464.24 mg/100 ml SB. Watanabe

et al. [14] cultivated Neocosmospora vasinfecta var. vasinfectaPF1225 on an MY medium at 251C to isolate a SS

hydrolase; this means that the optimal incubation

temperature depends on the type of organism used.

Table 3 Effect of different initial pH values on production of

soyasapogenol B from soybean saponin by Aspergillusparasiticus

Soyasapogenol B

Initial pH Final pH mg/100 ml Molar yield (%)

5 4.10 11.38 4.785.7 4.96 32 13.446 5.03 64.33 27.017 5.71 83.16 34.928 7.22 89.5 37.599 6.83 83.39 35.02

Initial medium pH was adjusted using 1N HCl and 1N KOH at differentpH values. Aspergillus parasiticus was cultivated on a transformationculture medium at 150 rpm and 30 ± 21C for 72 h.

Figure 1

0 1 2 3 4 5 6 7 8 9 10 11

20

40

60

80

100

"SB" Molar Yield

Inoculum size (ml)

SB (

mg/

100m

l)

0

10

20

30

40

Molar Y

eild (%)

Effect of inoculum size on production of soyasapogenol B (SB) fromsoybean saponin by Aspergillus parasiticus. Biotransformation wasperformed on a transformation culture medium (pH 8) inoculatedseparately with different inoculum sizes. Flasks were incubated at150 rpm and 30 ± 21C for 72 h.

Figure 2

20 40 60 80 100 120

0

20

40

60

80

100 "SB" MolarYeild Cell Biomass

Time (hour)

SB (

mg/

100m

l)

0

10

20

30

40

50

60

(Molar Y

eild)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

cell

biom

ass

(gm

/50m

l)

Duration of soyasapogenol B (SB) accumulation during hydrolysis ofsoybean saponin by Aspergillus parasiticus. A. parasiticus wascultivated on a transformation culture medium at pH 8, 150 rpm, and30 ± 21C. Molar yield of soyasapogenol B and cell dry weight weredetermined at different time intervals.


Figure 3

2 3 4 5 670

75

80

85

90 SB Molar yield

Malt extract (%)

SB (

mg/

100m

l)

32

34

36

38

40

42

44

Molar yield (%

)

Effect of malt extract concentration on production of soyasapogenol B(SB) from soybean saponin by Aspergillus parasiticus. A. parasiticuswas cultivated on a transformation culture medium supplemented withvarying amounts of malt extract (2–6%, w/v) at pH 8, 150 rpm, and30 ± 21C for 72 h. Control treatment: using 4% malt extract.

Figure 4

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.50

20

40

60

80

100

"SB" Molar Yield

Yeast extract (%)

SB (

mg/

100m

l)

10

20

30

40

Molar Y

eild (%)

Effect of yeast extract concentration on production of soyasapogenol B(SB) from soybean saponin by Aspergillus parasiticus. A. parasiticuswas cultivated on a transformation culture medium supplemented withvarying amounts of yeast extract (0.5–3%,w/v) at pH 8, 150 rpm, and30 ± 21C for 72 h. Control treatment: using 2% yeast extract.

Figure 5

Glucose Galactose Mannose Arabinose Sucrose Starch control60

70

80

90

100

110 "SB" Molar Yeild

Carbon sources

SB (

mg/

100m

l)

28

30

32

34

36

38

40

42

Molar yield (%

)

Effect of adding different carbon sources to the fermentation mediumon soyasapogenol B (SB) production. Aspergillus parasiticus wascultivated on a transformation culture medium supplemented with 1%(w/v) of one of these carbon sources at pH 8, 150 rpm, and 301C for72 h. Control treatment: without addition of the carbon source.

Figure 6

0 1 2 3 4 560

80

100

120 "SB" Molar Yield

Galactose (%)

SB (

mg/

100m

l)

30

40

50

60

70

Molar yield (%

)

Effect of galactose concentration on soyasapogenol B (SB) production.Aspergillus parasiticus was cultivated on a transformation culturemedium supplemented with different concentrations of galactose(0.5–5%, w/v) at pH 8, 150 rpm, and 301C for 72 h. Control treatment:using 1% galactose.

Figure 7

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.50

100

200

300

400

500

" SB" Molar Yield

Soybean saponin (SS, %)

SB (

mg/

100m

l)

45

50

55

60

65

70

75

Molar yield (%

)

Effect of substrate concentration on production of soyasapogenol B(SB) from soybean saponin (SS) by Aspergillus parasiticus. A.parasiticus was cultivated on a transformation culture mediumsupplemented with different levels of SS (0.5–4%, w/v) at pH 8,150 rpm, and 30 ± 21C for 72 h. Control treatment: using 1% SS.

Figure 8

20 25 30 35 400

100

200

300

400

500

Temperature (°C)

"SB" Molar Yeild

SB (

mg/

100m

l)

0

20

40

60

80

Molar yeild(%

)

Effect of different temperature values on production of soyasapogenol B(SB) from soybean saponin (SS) by Aspergillus parasiticus. A.parasiticus was cultivated on a transformation culture medium composedof (%, w/v): malt extract 4; yeast extract, 2; KH2PO4, 0.2; (NH4)2SO4,0.2; MgSO4 �7H2O, 0.03; CaCl2 �2H2O, 0.03; and SS, 3 (pH 8). Flaskswere incubated at different temperatures and 150 rpm for 72 h.


ConclusionA. parasiticus was screened and selected on the basis of

its ability to hydrolyze SS, producing a high yield of SB.

A maximum conversion value of 65% was obtained using

a production medium composed of (%, w/v): malt extract,

4; yeast extract, 2; galactose, 0.5; and SS, 3 (pH 8). The

medium was inoculated with 6% (v/v) inoculum and

incubated at 301C for 72 h. Under these optimal condi-

tions, the SB molar yield increased from 13.44 to 65%.


References1 Oleszek WA. Chromatographic determination of plant saponins. J Chroma-

togr 2002; A967:147–162.

2 Hu J, Reddy MB, Hendrich S, Murphy PA. Soyasaponin I and sapongenol Bhave limited absorption by Caco-2 intestinal cells and limited bioavailabilityin women. J Nutr 2004; 134:1867–1873.

3 Kudou S, Tonomura M, Tsukamoto C, Shimoyamada M, Uchida T, Okubo K.Isolation and structural elucidation of the major genuine soybean saponin.Biosci Biotechnol Biochem 1992; 56:142–143.

4 Shiraiwa M, Harada K, Okubo K. Composition and structure of ‘group Bsaponin’ in soybean seed. Agric Biol Chem 1991; 55:911–917.

5 Kinjo J, Hirakawa T, Tsuchihashi R, Nagao T, Okawa M, Nohara T, Okabe H.Hepatoprotective constituents in plants 14. Effects of soyasapogenol B,sophoradiol, and their glucuronides on the cytotoxicity of tert-butyl hydro-peroxide to HepG2 cells. Biol Pharm Bull 2003; 26:1357–1360.

6 Zhang W, Popovich DG. Effect of soyasapogenol A and soyasapogenol Bconcentrated extracts on Hep-G2 cell proliferation and apoptosis. J AgricFood Chem 2008; 56:2603–2608.

7 Hayashi K, Hayashi H, Hiraoka N, Ikeshiro Y. Inhibitory activity of soyasaponinII on virus replication in vitro. Planta Med 1997; 63:102–105.

8 Ahn K-S, Kim J-H, Oh S-R, Min B-S, Kinjo J, Lee H-K. Effects of oleanane-type triterpenoids from fabaceous plants on the expression of ICAM-1.Biol Pharm Bull 2002; 25:1105–1107.

9 Sasaki K, Minowa N, Kuzuhara H, Nishiyama S, Omoto S. Synthesis andhepatoprotective effects of soyasapogenol B derivatives. Bioorg Med ChemLett 1997; 7:85–88.

10 Kinjo J, Yokomizo K, Hirakawa T, Shii Y, Nohara T, Uyeda M. Anti-herpes virusactivity of fabaceous triterpenoidal saponins. Biol Pharm Bull 2000; 23:887–889.

11 Berhow MA, Wagner ED, Vaughn SF, Plewa MJ. Characterizationand antimutagenic activity of soybean saponins. Mutat Res 2000; 448:11–22.

12 Gurfinkel DM, Rao AV. Soyasaponins: the relationship between chemi-cal structure and colon anticarcinogenic activity. Nutr Cancer 2003; 47:24–33.

13 Kudou S, Tsuizaki I, Shimoyamada M, Uchida T, Okubo K. Screening formicroorganisms producing soybean saponin hydrolase. Agric Biol Chem1990; 54:3035–3037.

14 Watanabe M, Sumida N, Yanai K, Murakami T. A Novel saponin hydrolasefrom Neocosmospora vasinfecta var. vasinfecta. Appl Environ Microbiol2004; 70:865–872.

15 Amin HA, Mohamed SS. Immobilization of Aspergillus terreus on loofasponge for soyasapogenol B production from soybean saponin. J Mol CatalB: Enzymatic 2012; 78:85–90.

16 Sullivan C, Sherma J. Development and validation of an HPTLC-densitometry method for assay of glucosamine of different forms in dietarysupplement tablets and capsules. Acta Chromatographica 2005; 15:119–130.

17 Watanabe M, Sumida N, Yanai K, Murakami T. Cloning and characterizationof saponin hydrolases from Aspergillus oryzae and Eupenicillium brefeldia-num. Biosci Biotechnol Biochem 2005; 69:2178–2185.

18 Kitagawa I, Yoshikawa M, Wang HK, Saito M, Tosirisuk U, Fujiwara T, Tomita K.Revisions of the structure of the sapogenols. Chem Pharm Bull 1982;30:2294.

19 Amin HAS, Hanna AG, Mohamed SS. Comparative studies of acidic andenzymatic hydrolysis for production of soyasapogenols from soybean sa-ponin. Biocatalysis Biotransformation 2011; 29:311–319.

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Characterization of ternary solid dispersions of nimesulide

with Inutec SP1 and b-cyclodextrin and evaluation

of anti-inflammatory efficiency in ratsRawia M. Khalila, Mamdouh M. Ghorabb, Noha Abd El Rahmana

and Silvia Kocova El-Arinia

aDepartment of Pharmaceutical Technology, NationalResearch Centre, Cairo and bDepartment ofPharmaceutics, Faculty of Pharmacy, Suez CanalUniversity, Ismailiya, Egypt

Correspondence to Rawia M. Khalil, PhD, Departmentof Pharmaceutical Technology, National ResearchCentre, El-Bohowth St., PO Box 12622, Dokki,11371 Cairo, EgyptTel: + 20 233 335 456; fax: + 20 233 370 931;e-mail: [email protected]

Received 5 November 2012Accepted 26 January 2013


2013, 12:46–56

Objective

The objective of this investigation is to enhance the physicochemical properties

of nimesulide (NS) and the stability of NS solid dispersions in order to improve

the anti-inflammatory activity of the drug.

Background

NS – a NSAID – is sparingly soluble in water and this low aqueous solubility in addition

to its poor wettability leads to variability in the bioavailability of the drug.


In the present study, ternary dispersions of NS were investigated using a new

polymeric carrier, Inutec SP1 (Inutec), in combination with b cyclodextrin (b-CD).

The ternary dispersions were prepared using different ratios of NS and b-CD (2 : 1;

1 : 1; 1 : 2), to which a fixed amount of Inutec (20% w/w of total formula) was added

using different methods of incorporation of the drug. Physical mixtures of equivalent

compositions were prepared by physically mixing the ingredients. The optimal

formulation obtained with a full factorial experimental design was used for the

evaluation of anti-inflammatory activity.

Results

In the ternary dispersions, the dissolution behavior improved in comparison with the

physical mixtures and was found to be dependent on the technique of incorporation

of the drug, the method of preparation, and the molar ratio of drug to b-CD. Physical

characterization of the ternary dispersions by infrared spectroscopy (FTIR), differential

scanning calorimetry, and X-ray powder diffraction indicated a decrease in crystallinity

because of partial inclusion in b-CD and the effect of Inutec, which promoted the

formation of microcrystals or partial amorphization of the drug during the processing

of the dispersions by kneading. Differential scanning calorimetry and X-ray powder

diffraction curves of the dispersions prepared by the solvent method indicated the

presence of a polymorphic form of NS with a lower melting point. The optimized ternary

dispersion predicted by the full factorial design showed good physical stability

following an accelerated stability test. The ternary dispersion of NS, Inutec, and

b-CD was found to show better anti-inflammatory efficiency in rats compared with

a commercial tablet of NS.

Conclusion

It can be concluded that the dissolution properties and the anti-inflammatory efficacy of

the ternary dispersions of NS with b-CD and Inutec were enhanced because of a

secondary solubilization of the inclusion by the polymeric surfactant.

Keywords:

accelerated stability, b-cyclodextrin, Inutec SP1, in-vivo evaluation in rats, nimesulide,

ternary solid dispersion


Introduction

Nimesulide (NS) is an important anti-inflammatory drug

and shows selective COX-2 inhibition, which contributes

toward its good gastrointestinal tolerability. Moreover,

despite concerns over its potential hepatotoxicity, it

remains approved for the market because of the

beneficial action overweighing the risks associated with

the drug [1]. However, the very poor aqueous solubility of

NS is a huge hurdle in formulation development.

Therefore, enhancement of water solubility has been an

ongoing challenge for pharmaceutical researchers as it can

lead to more efficient and safer formulations of this

important medicament.

46 Original article


DOI: 10.7123/01.EPJ.0000427333.85411.b9

Numerous studies have dealt with the use of different

carriers for the preparation of solid dispersions (SD) of

NS [2–8]. Considerable amount of research has been

published on complexation of NS with b-cyclodextrin

(b-CD). Nalluri and colleagues [9,10] studied binary

systems in 1 : 1 and 1 : 2 molar ratios of drug and carrier.

They reported that the increase in dissolution properties

was because of the formation of a 1 : 1 complex in

solution. Further increase in b-CD led to the formation of

a 1 : 2 complex in the solid state. However, despite the

true inclusion formed, the dissolution rate and efficiency

values obtained were not as anticipated. The reason for

this is the formation of crystalline inclusion complexes

(IC) [9,10].

To overcome the drawback of the limited aqueous

solubility of b-CD, Dutet et al. [6] examined the effect

of double hydrophilization in ternary systems of NS and

b-CD and PEG 6000, but found no improvement in the

bioavailability of NS in rats.

Generally, these and other attempts have failed to

produce a stable marketable NS product, and thus there

is a need for novel, more efficient carriers for NS. Recent

advances in excipient technology have resulted in new

surfactants such as Inutec SP1 (Inutec) of Orafti Non-

Food. It is derived frominulin by grafting alkyl groups on

a polyfructose backbone (Fig. 1).

In this way, a structure is obtained, with polyfructose loops

providing steric stability. The polymeric nature of this

surfactant has made Inutec very useful as an emulsifier in

cosmetic and food industries [11]. Its application in

pharmaceutical formulations has been reported by Van den

Mooter et al. [12] as a carrier for SD of itraconazole, a drug

with very low aqueous solubility. A 20/80 w/w SD of

itraconazole and Inutec led to an improved dissolution rate.

The dissolution efficiency (DE) depended more on the

method of preparation than on the degree of amorphization.

In a recent study, Janssens et al. [13] investigated further the

effect of Inutec on itraconazole in ternary dispersions with

polyvidone-vinylacetate 64 (PVPVA 64) and found that the

improvement vis-a-vis the binary systems depended on the

incorporation of a sufficient amount of PVPVA 64 required

for the molecular dispersion of itraconazole. Ibrahim

et al. [14] used Inutec and hydroxypropyl-b-CD for the

preparation of chewable tablets of etodolac. The authors

reported that the dissolution rate of etodolac at pH 1.2 and

6.8 was improved compared with a pure drug and physical

mixture (PM) as a result of loss of crystallinity.

We reported in a previous publication [15] on the effect

of Inutec on the dissolution behavior of NS in binary

dispersions of NS with increasing amounts of Inutec. The

dissolution rate was enhanced proportionally with the

increase in the Inutec concentration and a ratio of drug to

Inutec of 1 : 3 led to a maximum of 87% DE after 180 min.

The aim of the present study is to evaluate the effect of

Inutec at a low concentration (20% w/w) to act as a second

hydrophilization factor in ternary dispersions based on

NS –b-CD complexes. Another aim is to use an experi-

mental design for optimization of the formula to conduct

accelerated stability tests and consequently for its use in

the evaluation of anti-inflammatory efficiency in rats.

Materials and methodsMaterials

NS was obtained as a gift sample from Sigma (Monofia,

Egypt). Inutec SP1 was generously provided by Orafti

Non-Food (Tienen, Belgium). b-CD (MW 1135) was

purchased from Sigma Chemical Company (St Louis,

Missouri, USA). All other materials were of analytical

grade.

Preparation of ternary systems from inclusion complex

and Inutec (ICSD)

First, the IC of NS and b-CD were prepared in 2 : 1, 1 : 1,

and 1 : 2 molar ratios using two methods: solvent and

kneading methods.

Solvent method (IC/S)

The alcoholic solution of NS was added to an aqueous

solution of b-CD. The resulting mixture was stirred for

30 min and evaporated under reduced pressure at a

temperature of 601C until dry. The dried mass was

ground in a mortar and passed through a sieve (250 mm).

Kneading method (IC/K)

A mixture of NS and b-CD was wetted with water and

kneaded thoroughly for 30 min in a glass mortar.

The resulting paste was dried under vacuum for 24 h.

Figure 1

Chemical structure of Inutec SP1.

Ternary solid dispersions of nimesulide Khalil et al. 47

The dried mass was ground in a mortar and passed

through a sieve (250 mm).

Second, the IC of NS–b-CD was mixed with a fixed

amount of Inutec (20% w/w of the total formula), and

then wetted together with water and kneaded as

discussed in the kneading method.

These systems are distinguished by the preparation

method of the binary IC.

It should be noted that by maintaining the amount of

Inutec added to the ternary systems constant, the ratio of

Inutec to NS increases with an increase in the molar ratio

of b-CD to drug.

Preparation of ternary solid dispersions

NS, b-CD, and Inutec were dispersed together using

either the solvent or the kneading method.

Solvent method (SD/S)

The aqueous solution of b-CD was added to an alcoholic

solution of NS and Inutec. The solvents were evaporated

using the rotavapor as discussed previously.

Kneading method (SD/K)

A mixture of NS, b-CD, and Inutec was wetted with

water and kneaded as discussed previously.

Preparation of physical mixtures

The corresponding PM were obtained by mixing the

various components together in a mortar by trituration for

5 min, followed by sieving (250 mm).

Determination of NS content in the prepared

formulations

An accurately weighed amount of NS formulation was

dissolved in phosphate buffer (pH = 7.4) and sonicated

for 30 min to ensure complete extraction of the drug from

the dispersion. The content of NS was determined

spectrophotometrically at 392 nm using a UV spectro-

photometer. Each preparation was tested in triplicate.

Wettability study

A powder sample (3 g) was placed in a sintered glass

funnel (33 mm internal diameter). The funnel was

plunged into a beaker containing water such that the

surface of water in the beaker remained at the same level

as the powder in the funnel. Methylene blue powder

(100 mg) was layered uniformly on the surface of the

powder in the funnel. The time required for wetting of

the methylene blue powder was measured. The average

of three observations was used for drawing the conclu-

sions [3].

Phase solubility study

Solubility studies were carried out as described by

Higuchi and Connors [16]. An excess amount of NS

was added to screw-capped vials containing different

concentrations of the carrier solution. The vials were

shaken mechanically at 37 ± 0.51C for 72 h until reaching

equilibrium. Filtration of the suspension was carried out

using 0.45 mm millipore filters. An aliquot portion of the

filtrate was diluted with phosphate buffer (pH = 7.4) and

analyzed for drug content by measuring its absorbance

spectrophotometrically at 392 nm against a blank solution

containing the same concentrations of the carrier. Each

experiment was conducted in triplicate.

Solid-state characterization

Fourier transform infrared spectroscopy

Fourier transform infrared (FTIR) spectra were recorded

using an FTIR-6100 type A spectrophotometer (Jasco,

Tokyo, Japan) equipped with a deuterated triglycine

sulfate detector. Samples were prepared in KBr disks

using a hydrostatic press. The scanning range was

between 4000 and 400 cm–1 at 4 cm–1 resolution.

Differential scanning calorimetry

Differential scanning calorimetry (DSC) was performed

using a Pyris5 instrument (Perkin Elmer, Waltham,

Massachusetts, USA) equipped with an intercooler. A

dry purge of nitrogen gas was used at 20 ml/min. The

instrument was calibrated with pure indium. Samples

(2–3 mg) were analyzed in closed Al pans from 50 to

2201C at a heating rate of 101C/min.

X-ray powder diffraction

X-ray powder diffraction (XRPD) patterns of the pure

ingredients and all of the SD containing varying propor-

tions of NS in the matrix were recorded using an X-ray

diffractometer (Scintag Inc., Cupertino, California, USA)

equipped with CuKa as the source of radiation.

Measurements were carried out using 45 kV voltage and

9 mA current. The 2y values and the intensities of the

peaks were compared for pure ingredients, the PM, and

the SD systems.

Solubility study

The solubility was determined in distilled water at 371C.

A sample equivalent to 25 mg of NS (excess amount of

NS) was added to 10 ml of distilled water in a vial with a

teflon-lined screw cap. The vials were shaken mechani-

cally at 37 ± 0.51C for 72 h until reaching equilibrium.

Filtration of the suspension was carried out using 0.45 mm

millipore filters. An aliquot portion of the filtrate was

diluted with phosphate buffer (pH = 7.4) and analyzed

for drug content by measuring its absorbance spectro-

photometrically at 392 nm. Each experiment was con-

ducted in triplicate.

In-vitro dissolution study

The dissolution rate was determined in the USP

Dissolution Tester, Apparatus I, at 37 ± 0.51C. The

dissolution medium was 900 ml of phosphate buffer

(pH = 7.4) at a rotation speed of 50 rpm. Powder samples

containing 25 mg of NS or its equivalent of PM or SD

were filled in transparent zero-sized hard gelatin

capsules. Aliquots, each of 5 ml, from the dissolution

medium were withdrawn at time intervals 15, 30, 45, 60,

90, 120, 150, and 180 min and replaced by an equal

volume of fresh dissolution medium. The samples were

filtered through a 0.45 mm millipore filter and assayed


spectrophotometrically for NS at 392 nm using fresh

dissolution medium as a blank.

The DE was calculated according to Khan [17] and is

defined as the area under the dissolution curve up to the

time, t, expressed as a percentage of the area of the

rectangle described by 100% dissolution at the same

time. The DE can have a range of values depending on

the time interval chosen. The DEs at 30, 60, and 180 min.

were calculated from the dissolution profiles. The

experiments were conducted in triplicate.

Experimental design

An experimental design was generated to estimate the

effects on the dissolution properties of the following

experimental variables: method of preparation at two

levels (solvent, kneading) and the NS : b-CD : Inutec

ratio at three levels (2 : 1 : 20%, 1 : 1 : 20%, and 1 : 2 : 20%).

Accelerated stability study

An accelerated stability study was carried out by

subjecting the SD to stressed conditions at 401C and

75% relative humidity (maintained using a saturated

solution of NaCl) for a period of 3 months. The effect of

the stressed conditions was determined by measuring

in-vitro dissolution and by DSC and XRPD studies.

Evaluation of anti-inflammatory activity of selected

nimesulide solid dispersions

Twenty-four adult female albino rats 150 ± 20 g were used.

The rats were randomly allocated into four groups, each

including six animals. Carrageenan was used to induce rat’s

paw edema. This effect was determined according to the

method described previously in the literature [18,19]. The

animals were kept on a standard laboratory diet. The rats

were kept fasted for 16 h before the experiment, but were

allowed free access to water. The samples were adminis-

tered orally as a suspension to the respective animal groups

at a dose of 50 mg/kg [20,21].

One hour after administration, edema was induced by an

injection of 0.1 ml of 1% (w/v) carrageenan solution in

distilled water into the planter aponeurosis of rats’ right

hind paws. The volume of the injected paw was measured

immediately after carrageenan injection and after 1, 2, 3,

4, and 5 h using a plethsymometer. The percentage

increase in paw volume was calculated according to the

equation given by Delporte et al. [22].

% increase in paw volume¼ Vf�Við Þ/Vi�100;

where Vf and Vi are the final and the initial paw volume

of an animal, respectively.

In addition, the percentage inhibition of edema volume for

each time was calculated from the mean effect in control and

in treated animals according to the following equation [23].

% inhibition of edema volume¼ 1�Vt/Vcð Þ�100;

where Vt and Vc are the mean increase in the volume of the

carrageenan-injected paw of the treated group and the

control group, respectively. The one-way analysis

of variance test was carried out on the area under percentage

increase in edema volume versus time curve.

Statistical analysis of data

All data were analyzed statistically using the analysis of

variance test for a P value of 0.05 using the social package

for statistical study Software (SPSS Company, IBM

Corporation, New York, USA). Differences were consid-

ered statistically significant at a value of P less than 0.05.

Results and discussionThe composition and method of preparation of all the

systems studied are listed in Table 1.

Nimesulide content in the prepared formulations

The drug content and the percentage recovery were

determined in all prepared formulations in order to

confirm that there was no drug loss during preparation

and that the SD showed good content uniformity.

Wettability study

The mean wetting times of representative PM and

dispersions are shown in Fig. 2. It can be seen that the

wetting time for pure NS (8 h) was significantly reduced

in the PM. The wettability was further improved in the

dispersions and the best results were obtained for the

dispersions prepared using the SD technique [SD/S

(1 : 2 : 20%) and SD/K (1 : 2 : 20%) in Fig. 2]. It is also

evident from Fig. 2 that the wetting times decreased

significantly (Po0.05) on adding Inutec to the binary

complexes (compare ternary ICSD/S; ICSD/K to binary

IC/S; IC/K in Fig. 2). This confirms the secondary

hydrophilization effect of Inutec in the ternary systems.

Phase solubility study

The phase solubility diagrams of NS and b-CD with and

without 0.5% Inutec can be classified as AL-type according

to Higuchi and Connors [16] as shown in Fig. 3. The

aqueous solubility of NS increased linearly (R2 = 0.9952

and 0.9971 in the absence and in the presence of 0.5% of

Inutec, respectively) as a function of the b-CD concentra-

tion. The phase solubility of NS in aqueous solutions of

b-CD increased in the presence of 0.5% of Inutec as

reflected by the small increase in the stability constant

from Kc = 354 to Kc = 430 mol/l. This is in agreement with

the increased wettability of the PM (Fig. 2).

Solid-state characterization

Studies were carried out to determine the nature of the

products obtained.

Fourier transform infrared spectroscopy

FTIR data were obtained to determine whether chemical

interactions occurred during the preparation of the SD.

Figure 4 shows the FTIR spectra of the individual

components, their PM, and the different dispersions. The

FTIR spectra of the PM showed the patterns of each

component. In the FTIR spectra of the ICSDs and the SDs,

the peak of the N–H function at 3290 cm–1 was slightly

pronounced or invisible. Otherwise, no other new bonds


were observed, which indicates that there was no interaction

between NS and the carriers at the molecular level.


DSC thermograms were generated to test for the

possibility of the inclusion of NS in b-CD.

Figure 5 shows the DSC thermograms of the individual

components and their ternary systems at a 1 : 2 molar ratio

prepared by the solvent (S) and kneading (K) methods.

The thermogram of NS showed a single endothermic

peak with onset at 148.81C and a peak at 1501C

corresponding to its melting point. These results were

also reported by Chowdary and Nalluri [24] and by

Abdelkader et al. [5]. The DSC thermogram of b-CD

showed a broad endothermic effect with a peak at 971C.

Inutec showed a small endothermic peak at 103.51C at

the tail of the water evaporation endotherm at 601C and a

glass transition signal at 1431C as also reported by Van

den Mooter et al. [12].

The thermogram of the PM is a combination of the DSC

curves of the individual components without changes in

the melting peaks. The ternary ICSDs prepared by both

methods (S, K) and the ternary SD prepared by the

kneading method (K) showed a marked reduction in the

intensity of the NS endotherm when compared with that

of the PM, indicating progressive partial inclusion of NS

within the b-CD cavity. The thermogram of the ternary

SD prepared by the solvent method was characterized by

a split endotherm indicating that the NS showed

polymorphism because of the use of an organic solvent

(methanol) in the preparation of the dispersions. As we

reported earlier, the use of a solvent induced a different

polymorphic form of NS that melts at a slightly lower

temperature [15]. Bergese et al. [25] also reported such a

polymorphic form of the drug and Di Martino et al. [26]

obtained a split endotherm because of the use of an

organic solvent in their study.

The ICSD (solvent) system, in contrast, did not show the

double-peaked endotherm. This might be attributed to

the use of a smaller amount of alcoholic solvent used in

its preparation (Table 1).

X-ray powder diffraction

XRPD patterns were obtained to determine the crystal-

linity of the products obtained and to confirm the results

of the DCS study.

Figure 2

480.00

49.22 50.1325.49 24.19 22.33 23.52 20.22 20.59

050

100150200250300350400450500

NS

PM (

1:2)

PM (

1:2:

20%

)

IC/S

(1:

2)

IC/K

(1:

2)

ICSD

/S (

1:2:

20%

)

ICSD

/K (

1:2:

20%

)

SD/S

(1:

2:20

%)

SD/K

(1:

2:20

%)

Tim

e (m

inut

es)

NS Formulation

Wettability of selected NS–b-CD and NS–b-CD–Inutec SP1 formula-tions: comparison of different methods of preparation.

Figure 3

0

0.0005

0.001

0.0015

0.002

0.0025

0.003

0 0.05 0.1 0.15 0.2

β-CD Concentration (M)

NS

Con

cent

ratio

n (M

)

Phase solubility diagrams of NS in different concentrations of b-CDwith and without 0.5% Inutec SP1 at 371C: K, NS–b-CD:y= 0.0112x+ 2E – 05, R 2 = 0.9952; ’, NS–b-CD–Inutec: y= 0.0136x +8E – 05, R 2 = 0.9971. b-CD, b cyclodextrin; NS, nimesulide.

Table 1 Composition of physical mixtures and solid dispersions

prepared using different methods

NS formulationNS : b-CD(mol/mol)

Inutec %(w/w) Method of preparationa

PM (2 : 1) 2 : 1 0 Physical mixingPM (1 : 1) 1 : 1 0 Physical mixingPM (1 : 2) 1 : 2 0 Physical mixingPM (2 : 1 : 20%) 2 : 1 20 Physical mixingPM (1 : 1 : 20%) 1 : 1 20 Physical mixingPM (1 : 2 : 20%) 1 : 2 20 Physical mixingIC/S (2 : 1) 2 : 1 0 IC/solvent (50 ml)IC/S (1 : 1) 1 : 1 0 IC/solvent (50 ml)IC/S (1 : 2) 1 : 2 0 IC/solvent (50 ml)IC/K (2 : 1) 2 : 1 0 IC/kneadingIC/K (1 : 1) 1 : 1 0 IC/kneadingIC/K (1 : 2) 1 : 2 0 IC/kneadingICSD/S (2 : 1 : 20%) 2 : 1 20 ICSD/solvent (50 ml)ICSD/S (1 : 1 : 20%) 1 : 1 20 ICSD/solvent (50 ml)ICSD/S (1 : 2 : 20%) 1 : 2 20 ICSD/solvent (50 ml)ICSD/K (2 : 1 : 20%) 2 : 1 20 ICSD/kneadingICSD/K (1 : 1 : 20%) 1 : 1 20 ICSD/kneadingICSD/K (1 : 2 : 20%) 1 : 2 20 ICSD/kneadingSD/S (2 : 1 : 20%) 2 : 1 20 SD/solvent (70 ml)SD/S (1 : 1 : 20%) 1 : 1 20 SD/solvent (100 ml)SD/S (1 : 2 : 20%) 1 : 2 20 SD/solvent (150 ml)SD/K (2 : 1 : 20%) 2 : 1 20 SD/kneadingSD/K (1 : 1 : 20%) 1 : 1 20 SD/kneadingSD/K (1 : 2 : 20%) 1 : 2 20 SD/kneading

b-CD, b cyclodextrin; IC/K, inclusion complex prepared using thekneading method; IC/S, inclusion complex prepared using the solventmethod; ICSD/K, inclusion complex in solid dispersion prepared usingthe kneading method; ICSD/S, inclusion complex in solid dispersionprepared using the solvent method; NS, nimesulide; PM, physicalmixture; SD/K, solid dispersion prepared using the kneading method;SD/S, solid dispersion prepared using the solvent method.aValues in parentheses represent the amount of methanol used in thepreparation (for 1 mol NS).


Figure 6 shows the diffractograms of NS, b-CD, Inutec,

their ternary PM, and the different SD (ICSD and SD) at

1 : 2 molar ratios with b-CD, prepared using the solvent

and the kneading methods.

NS showed the characteristic diffraction pattern with

numerous distinctive peaks, indicating the highly crystal-

line nature of the drug. The most abundant peaks were

observed at 2y values of 19.3 and 23.11. The diffraction

pattern of b-CD showed numerous peaks, with a major

peak at 2y= 12.481, whereas the diffraction pattern of

Inutec was characterized by very small peaks protruding

from the halo around 2y= 16–201.

The XRPD pattern of the PM represents a combination

of the individual patterns of the drug and the carriers and

the intensities of the peaks reflect the fraction of the

drug in the mixture. The diffractograms of ICSD

prepared using the solvent and kneading methods and

SD prepared using the kneading method showed a

notable reduction in the intensity of the characteristic

peaks of the drug in comparison with the PM. This

reduction in peak intensity is a result of loss of

crystallinity of the drug in the preparation, indicating

partial inclusion of NS within the b-CD cavity. The

XRPD patterns of the ternary SD prepared using the

solvent method showed a diffraction peak at 2y= 18.91,

which was not observed in the XRPD pattern of the pure

NS. This indicates the presence of polymorphism

because of the use of an organic solvent (methanol).

The diffractograms of the binary and ternary systems

were compared quantitatively with the diffractogram of

the PM. For this purpose, the values of the relative

intensity (I/Io) were used, which were calculated from

the intensity (I) of a selected peak (2y= 23.11) and the

intensity (Io) of the major peak (2y= 19.31). The relative

intensity values decreased to I/Io = 85 and 88% in the ICs

and further to 80 and 82% in the ternary ICSDs

depending on the method of preparation (solvent vs.

kneading). The highest decrease in the relative intensity

was observed in the ternary systems prepared using the

SD technique, using the kneading method (I/Io =

69.8%), indicating the highest degree of amorphization

compared with the PM and the other binary and ternary

formulations. However, the I/Io value of ternary disper-

sion prepared using the SD technique with methanol was

not calculated because of the appearance of a new peak

indicative of a different polymorphic form of NS. Janssens

et al. [13] investigated the diffractograms of ternary

systems of itraconazole with PVPVA 64 and Inutec SP1 in

different ratios of polymer to Inutec. They reported that

all the systems showed XRPD amorphous behavior,

except for the one with the lowest ratio. They therefore

concluded that itraconazole was molecularly dispersed

in the PVPVA, whereas Inutec did not interact with any

Figure 4

Inutec

NS

5001000150020002500300035004000

%T

Wavenumber [cm-1]

β-CD

PM (1:2:20%)

ICSD/S (1:2:20%)

ICSD/K (1:2:20%)

SD/S (1:2:20%)

SD/K (1:2:20%)

FTIR spectra of NS, Inutec SP1, b-CD, their PM and their ternary ICSD,and SD at 1 : 2 molar ratio prepared by solvent (S) and kneading (K)methods. b-CD, b cyclodextrin; FTIR, Fourier transform infraredspectroscopy; ICSD, inclusion complex in solid dispersion;NS, nimesulide; PM, physical mixture; SD, solid dispersions.

Figure 5

Temperature °C

NS

β-CD

PM (1:2:20%)

ICSD/S (1:2:20%)

ICSD/K (1:2:20%)

SD/S (1:2:20%)

SD/K (1:2:20%)

Inutec

Hea

t Fl

ow (

mW

)

DSC thermograms of NS, Inutec SP1, b-CD, their PM, and their ternaryICSD and SD at a 1 : 2 molar ratio prepared by solvent (S) and kneading(K) methods at a heating rate of 101C/min. b-CD, b cyclodextrin; DSC,differential scanning calorimetry; ICSD, inclusion complex in soliddispersion; NS, nimesulide; PM, physical mixture; SD, solid dispersions.


of the components on a molecular level. This is in

agreement with the present study that is NS interacts

with the b-CD by means of partial inclusion. Further

decrease in crystallinity in ternary systems compared with

binary IC may be attributed to Inutec, which promoted

the formation of microcrystals in the ternary systems.

Consequently, the addition of this polymeric surfactant

increased the saturation solubility and this could lead to

better dissolution rates of the ternary dispersions vis-a-vis

the binary systems. The XRPD findings are in full

agreement with the DSC results (Fig. 5).

Solubility study

The aqueous solubility in distilled water of pure NS and

the ternary ICSDs and SDs is shown in Fig. 7. The

solubilities obtained for the PMs and the binary ICs are

also shown for comparison. The solubility obtained for

pure drug was 14.3 mg/ml. In the PM, the solubility

increased as a result of the IC formed in the solution. The

addition of Inutec led to a slight increase in solubility

(compare ternary PMs with binary PMs in Fig. 7). This is

in agreement with the results from the phase solubility

study.

In the dispersions, the solubility increased compared with

the PMs, indicating further interaction between NS and

the carriers in the solid state at all drug to b-CD molar

ratios. It can be noted that the addition of Inutec to the

preformed IC of NS and b-CD did not induce a change in

saturation solubility in any significant way (compare ICs

with ICSDs). However, direct dispersion of the compo-

nents enhanced the solubility significantly (Po0.05). Of

the two methods of dispersion, the use of solvent yielded

better results (compare ternary SD/S with ternary SD/K

in Fig. 7).

In-vitro dissolution study

The dissolution rate (%D) and the DEs at 30, 60, and

180 min of all the systems studied are summarized

in Table 2. Maximum values were obtained after

180 min of dissolution testing. It should be noted that

although the maximum values obtained did not reach

100% after 3 h of dissolution testing, conducting the tests

for a longer period was not considered practical.

The dissolution enhancement in the PM was 2.2-fold

compared with pure drug, which is in agreement with the

improved wettability and complexation of the drug with

b-CD in solution. The difference between the dissolu-

tion rates of the ternary PM and the binary PM was not

statistically significant (P40.05).

The dissolution profiles of all the dispersions showed a

significant improvement (Po0.05) compared with the

PM because of the progressive inclusion in the b-CD and/

or the secondary hydrophilization action of Inutec. It can

be seen from Table 2 that the increase in dissolution

could be related to both the method of preparation and

the presence of Inutec in the systems.

The dissolution profiles of the IC also increased

compared with the PM but to a lesser degree than when

compared with the dispersions. Similar to the saturation

solubility, the dissolution rate is also the highest for

solvent SD.

The improvement in the dissolution rate and the DE of

the PM in comparison with the pure drug can be

attributed to the formation of a soluble 1 : 1 complex of

NS with b-CD as confirmed by the phase solubility

studies and the results of the solubility tests. The

addition of Inutec did not affect the dissolution of the

ternary PM significantly (P40.05), although this would

have been anticipated from the improved wettability

because of the solubilizing action of this polymeric

surfactant. In the dispersion systems, however, the

improved wettability resulting from the addition of

Inutec significantly increased the dissolution of the

ternary dispersions in comparison with the binary

dispersions (Po0.05). This is because by physical

mixing, the polymer is only deposited on the drug,

whereas during the kneading process, there is deeper

entrapment of the drug in the polymer network. Because

of the unique action of Inutec to adsorb onto hydrophobic

substrates with its alkyl chains, more hydrophobic

particles become occupied by Inutec with its hydrophilic

fructose loops in the solution. This leads to an increase in

Figure 6

0 5 10 15 20 25 30 35 40 45 50

2�

Inte

nsity

NS

β-CD

PM (1:2:20%)

ICSD/S (1:2:20%)

ICSD/K (1:2:20%)

SD/S (1:2:20%)

SD/K (1:2:20%)

Inutec

X-ray powder diffractograms of NS, Inutec SP1, b-CD, their PM, andtheir ternary ICSD and SD at a 1 : 2 molar ratio prepared by solvent (S)and kneading (K) methods. b-CD, b cyclodextrin; ICSD, inclusioncomplex in solid dispersion; NS, nimesulide; PM, physical mixture;SD, solid dispersions.


wettability and consequently to better dissolution proper-

ties of the ternary systems.

Generally, all dispersions showed better solubility and

dissolution rate when compared with the PM. This can be

attributed to the changes in the solid states as shown by

the results of the physical characterization of the SD. In

the kneaded dispersions, the predominant factor is the

reduced crystallinity of the drug because of partial inclusion

in the b-CD or the formation of microcrystals dispersed in

the polymer network or the formation of some amorphous

drug during the processing of the formulations. This was

evidenced by the decrease in the melting endotherm on

the DSC curves and by the reduced relative intensity of

the characteristic peaks on the X-ray diffractograms. In

addition to this, the dispersions prepared using the solvent

method showed polymorphism as indicated by the split

endotherm in the DSC curves and by the presence of a

new peak on the X-ray diffractograms. Polymorphism of NS

has been reported in the literature [25, 26] and the

enhanced dissolution of the solvent dispersions found in

the present study could be because of the polymorphic

form with a lower melting point.

On the basis of the data obtained, it can be concluded

that several factors contributed toward the enhanced

dissolution rate and DE of the ternary dispersions of NS,

b-CD, and Inutec, that is increased wettability because of

a second solubilization of the inclusion by the polymeric

surfactant Inutec, decreased crystallinity because of

partial inclusion of NS in b-CD, and increased solubility

because of polymorphism.

The use of Inutec may offer advantages not found with the

more commercial carriers used for the preparation of SD.

Evaluation of the results of an experimental design

Three characteristic points on the dissolution curves

(responses) were used for the evaluation of the effects of

the experimental variables (factors). They are D30, D60,

and DE180, representing the percentage drug dissolved at

30, 60 min and the DE at 180 min, respectively (Table 2).

A 2� 2� 3 experimental design was generated for the

three factors (technique, method, and ratio) at the

selected levels.

The least square model was used in order to predict the

optimal values of the responses within the ranges of the

factors used in the experimental design. The main effects

represent the values of the estimates of the parameters

calculated from the model that was used to fit the data.

The largest effect on the DE180 (7.4) was because of the

ratio. The second highest effect (3.2) was because of the

method of preparation of the ternary system, whereas

the effect of the technique (i.e. SD or ICSD) exerted the

smallest effect, with a value of the estimate of 2.8.

The optimal formulation was predicted to be at the

following levels of the experimental variables: techni-

que = SD; method = solvent; ratio = 1 : 2 : 20%.

This formula was used for further studies (accelerated

stability and in-vivo evaluation of anti-inflammatory

activity).

Accelerated stability study

The physical stability was investigated by comparing the

dissolution profiles as well as the solid-state character-

istics of the freshly prepared samples and of samples aged

1, 2, and 3 months. For this study, the formula optimized

by the factorial design was used, that is the ternary SD

Figure 7

0

10

20

30

40

50

60

70

80

90

100N

S

PM (

2:1)

PM (

1:1)

PM (

1:2)

PM (

2:1:

20%

)

PM (

1:1:

20%

)

PM (

1:2:

20%

)

IC/S

(2:

1)

IC/S

(1:

1)

IC/S

(1:

2)

IC/K

(2:

1)

IC/K

(1:

1)

IC/K

(1:

2)

ICSD

/S (

2:1:

20%

)

ICSD

/S (

1:1:

20%

)

ICSD

/S (

1:2:

20%

)

ICSD

/K (

2:1:

20%

)

ICSD

/K (

1:1:

20%

)

ICSD

/K (

1:2:

20%

)

SD/S

(2:

1:20

%)

SD/S

(1:

1:20

%)

SD/S

(1:

2:20

%)

SD/K

(2:

1:20

%)

SD/K

(1:

1:20

%)

SD/K

(1:

2:20

%)

Nimesulide Formulations

Solu

bilit

y of

NS

(µg/

ml)

PM

ICICSD

SD

Solubility (mg/ml) of physical mixtures and dispersions of NS with b-CD and Inutec SP1.


obtained using the solvent method, SD/S (1 : 2 : 20%). In

addition, the ternary SD obtained using the kneading

method, SD/K (1 : 2 : 20%), was also investigated.

The dissolution rates of fresh and aged samples using the

solvent method and those using the kneading method are

shown in Fig. 8a and b, respectively. Statistical analysis

showed that under the conditions of the accelerated test,

no significant changes occurred in the dissolution

behavior of the ternary dispersions of NS, b-CD, and

Inutec (P40.05).

The thermal (by DSC) and crystalline (by XRPD)

characteristics of the aged samples after 3 months of

accelerated stability testing did not show any significant

changes compared with those of the fresh samples,

indicating good stability in the solid state.

Evaluation of the anti-inflammatory activity in rats

The presence of edema is one of the prime signs of

inflammation [27]. It has been documented that carragee-

nan-induced rat paw edema is a suitable in-vivo model to

predict the efficacy of anti-inflammatory agents, which act

by inhibiting the mediators of acute inflammation [28]. The

efficiency of NS in the inhibition of the edema volume was

determined using the method described in the experi-

mental part.

The samples used in the study included the optimized

ternary SD obtained using the solvent method [SD/S

(1 : 2 : 20%)] and the commercially available NS tablet

(designated as market tablet) as well as the control. The

anti-inflammatory effect was monitored during 5 h

following carrageenan injection. The results are pre-

sented in Fig. 9.

The results shown in Fig. 9 indicate that there is a

marked increase (Po0.05) in the mean percentage

inhibition of edema volume with the SD when compared

with the commercially available tablet.

With respect to the pharmacodynamic parameters, it can be

seen that the maximum percentage inhibition of edema

volume for all samples occurred 1 h after dosing. Also, it can

be seen in Fig. 9 that the dispersion inhibited the increase

in paw volume during the early phase of inflammation

(1–3 h after carrageenan injection) and also showed a good

inhibitory effect at a later phase (up to 5 h). This is in

agreement with the studies of Garcia-Pastor et al. [29], who

suggested a biphasic model in carrageenan-induced edema.

The first phase begins immediately after injection and

decreases within 1–1.5 h. The second phase remains

through 3 h. The delayed phase is considered to result

from the effect of prostaglandins on mediator release.

ConclusionThe effects of the carriers investigated in this study that

resulted in the enhancement of the dissolution properties

and the anti-inflammatory activity of the water-insoluble

drug NS represent potential incentive toward the

development of a stable formulation that can lead to a

reduction in the dose without the need to modify the

basic molecule of the drug. The addition of a hydrophilic

polymeric surfactant (Inutec) in a small concentration

markedly enhanced the dissolution rate of NS compared

with the binary IC with b-CD.

Table 2 Dissolution rate (%D) and dissolution efficiency of different formulations

%D (min) DE (%) (min)

NS formulation 30 60 180 30 60 180

PM (2 : 1) 22.35 ± 2.1 30.6 ± 1.5 40.6 ± 3.9 12.7 ± 0.1 19.7 ± 0.2 29.9 ± 1.4PM (1 : 1) 27.25 ± 1.6 35.0 ± 3.4 47.3 ± 2.2 16.3 ± 1.1 23.9 ± 1.6 36.3 ± 0.3PM (1 : 2) 33.78 ± 2.5 42.1 ± 2.6 54.6 ± 1.9 19.0 ± 1.9 27.0 ± 2.4 40.1 ± 3.3PM (2 : 1 : 20%) 23.74 ± 2.2 31.7 ± 1.5 41.5 ± 4.0 13.7 ± 0.2 20.3 ± 0.2 30.9 ± 1.5PM (1 : 1 : 20%) 28.62 ± 1.7 35.6 ± 3.4 49.6 ± 2.3 17.1 ± 1.1 24.9 ± 1.7 37.5 ± 0.3PM (1 : 2 : 20%) 36.51 ± 2.7 43.9 ± 2.8 55.2 ± 1.9 21.7 ± 0.5 31.3 ± 1.5 44.6 ± 1.8IC/S (2 : 1) 43.62 ± 2.2 62.4 ± 2.3 77.2 ± 1.0 25.4 ± 1.0 40.2 ± 1.2 61.3 ± 0.9IC/S (1 : 1) 51.66 ± 2.8 68.9 ± 2.6 80.8 ± 1.8 28.0 ± 2.4 45.3 ± 2.2 65.5 ± 2.2IC/S (1 : 2) 64.71 ± 3.8 78.3 ± 2.5 84.9 ± 2.5 36.4 ± 2.1 54.5 ± 2.5 73.0 ± 2.8IC/K (2 : 1) 39.90 ± 3.6 54.9 ± 3.5 75.1 ± 4.2 20.5 ± 2.3 34.5 ± 2.3 56.5 ± 2.9IC/K (1 : 1) 44.77 ± 3.4 61.1 ± 2.9 78.3 ± 4.2 24.7 ± 2.4 39.8 ± 3.1 60.9 ± 3.5IC/K (1 : 2) 50.71 ± 2.5 72.4 ± 3.0 83.6 ± 2.9 26.2 ± 2.4 45.6 ± 2.7 66.8 ± 2.4ICSD/S (2 : 1 : 20%) 47.81 ± 4.5 66.0 ± 3.4 80.0 ± 3.7 26.0 ± 1.7 41.7 ± 1.0 62.5 ± 0.6ICSD/S (1 : 1 : 20%) 48.94 ± 2.6 65.6 ± 4.3 81.9 ± 3.1 23.0 ± 2.2 41.3 ± 2.1 64.9 ± 2.5ICSD/S (1 : 2 : 20%) 59.18 ± 2.1 79.4 ± 3.2 88.9 ± 2.1 32.5 ± 1.6 51.8 ± 2.1 73.8 ± 2.1ICSD/K (2 : 1 : 20%) 47.05 ± 3.3 58.6 ± 4.8 70.5 ± 3.2 26.0 ± 2.4 39.5 ± 2.3 57.4 ± 1.6ICSD/K (1 : 1 : 20%) 47.68 ± 3.3 65.8 ± 3.3 81.7 ± 3.8 25.0 ± 2.6 41.2 ± 2.9 63.1 ± 0.7ICSD/K (1 : 2 : 20%) 53.88 ± 3.3 75.2 ± 3.5 85.2 ± 3.3 31.7 ± 2.3 48.3 ± 2.6 69.6 ± 2.5SD/S (2 : 1 : 20%) 56.08 ± 2.9 69.4 ± 2.9 82.3 ± 3.6 35.9 ± 2.0 50.4 ± 0.8 67.5 ± 1.7SD/S (1 : 1 : 20%) 65.51 ± 3.8 77.6 ± 2.3 86.5 ± 3.3 40.7 ± 2.5 56.7 ± 2.5 73.6 ± 2.7SD/S (1 : 2 : 20%) 75.68 ± 3.2 86.7 ± 4.3 98.6 ± 2.8 48.5 ± 1.9 65.1 ± 2.8 84.5 ± 3.1SD/K (2 : 1 : 20%) 46.34 ± 2.7 59.9 ± 2.3 73.8 ± 2.3 25.3 ± 2.2 39.3 ± 2.7 59.0 ± 2.7SD/K (1 : 1 : 20%) 49.39 ± 4.4 69.3 ± 2.9 83.7 ± 1.2 28.7 ± 2.4 44.6 ± 2.4 66.5 ± 1.9SD/K (1 : 2 : 20%) 61.68 ± 2.3 77.5 ± 3.7 89.1 ± 3.9 33.2 ± 2.4 52.1 ± 1.6 74. 0 ± 2.5

DE, dissolution efficiency; IC/K, inclusion complex prepared using the kneading method; IC/S, inclusion complex prepared using the solvent method;ICSD/K, inclusion complex in solid dispersion prepared using the kneading method; ICSD/S, inclusion complex in solid dispersion prepared usingthe solvent method; NS, nimesulide; PM, physical mixture; SD/K, solid dispersion prepared using the kneading method; SD/S, solid dispersionprepared using the solvent method.


The polymeric surface-active agent Inutec, which was

shown to improve the anti-inflammatory activity of NS

but that has not as yet been fully investigated or reported

in the literature, might have huge potential as a carrier for

other water-insoluble drugs.


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7 Dhanaraju MD, Thirumurugan G. Dissolution profiling of nimesulide soliddispersions with polyethylene glycol, Talc and their combinations as disper-sion carriers. Int J Pharm Tech Res 2010; 2:480–484.

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10 Nalluri BN, Chowdary KP, Murthy KV, Becket G, Crooks PA. Tablet for-mulation studies on nimesulide and meloxicam-cyclodextrin binary systems.AAPS PharmSciTech 2007; 8:E1–E7.

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13 Janssens S, Humbeeck JV, Mooter GVd. Evaluation of the formulation of soliddispersions by co-spray drying itraconazole with Inutec SP1, a polymericsurfactant, in combination with PVPVA 64. Eur J Pharmaceut Biopharmaceut2008; 70:500–505.

14 Ibrahim MM, El-Nabarawi M, El-Setouhy DA, Fadlalla MA. Polymericsurfactant based etodolac chewable tablets: formulation and in vivo eva-luation. AAPS PharmSciTech 2010; 11:1730–1737.

15 Khalil RM, Ghorab MM, Abd El-Rahman N, Kocova El-Arini S. Enhancementof dissolution of nimesulide using a novel surface-active polymeric carrier,Inutec SP1. Egypt Pharm J (NRC) 2010; 9:181–201.

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23 Abdel-Allah DH. Formulation and quality improvement of tablets ofantirheumatic activity [PhD thesis]. Egypt: Cairo University; 2008.

Figure 9

0

10

20

30

40

50

60

70

80

20 1 3 4 5 6

MTSD/S (1:2:20%)

Time (h)

Inhi

bitio

n of

oed

ema

(%)

Mean percentage inhibition of edema volume after administration of theselected NS formulations in Carrageenan-induced paw edema in rats:K, market tablet; ’, solid dispersion prepared using the solventmethod [SD/S (1 : 2 : 20%)]. NS, nimesulide.

Figure 8

0

10

20

30

40

50

60

70

80

90

100(a)

(b)

0 50 100 150 200

0

10

20

30

40

50

60

70

80

90

100

0 50 100 150 200

Time (minutes)

% D

rug

diss

olve

d%

Dru

g di

ssol

ved

Time (minutes)

Dissolution profiles of fresh and aged solid dispersions prepared usingthe solvent method [(a) SD/S (1 : 2 : 20%)] and the kneading method[(b) SD/K (1 : 2 : 20%)]:m, fresh sample; ’, after 1 month; ~, after 2months; K, after 3 months. Data represent mean ± SD (n = 3).


24 Chowdary KPR, Nalluri BN. Nimesulide and b-cyclodextrin inclusion com-plexes: physicochemical characterization and dissolution rate studies. DrugDev Ind Pharm 2000; 26:1217–1220.

25 Bergese P, Bontempi E, Colombo I, Gervasoni D, Depero LE. Microstructuralinvestigation of nimesulide-crospovidone composites by X-ray diffraction andthermal analysis. Composites Sci Tech 2003; 63:1197–1201.

26 Di Martino P, Censi R, Barthelemy C, Gobetto R, Joiris E, Masic A, et al.Characterization and compaction behaviour of nimesulide crystal forms. Int JPharm 2007; 342 (1–2):137–144.

27 Sur TK, Pandit S, Battacharyya D, Kumar CKA, Lakshmi SM, Chatttopadhyay D,Mandal SC. Studies on the antiinflammatory activity of Betula alnoides bark.Phytother Res 2002; 16:669–671.

28 Morebise O, Fafunso MA, Makinde JM, Olajide OA, Awe EO. Anti-inflammatory and analgesic property of leaves of Gongronema latifolium.Phytother Res 2002; 16:S75–S77.

29 Garcia-Pastor P, Randazzo A, Gomez-Paloma L, Alcaraz MJ, Paya M. Effectsof petrosaspongiolide M, a novel phospholipase A2 inhibitor, on acute andchronic inflammation. J Pharmacol Exp Ther 1999; 289:166–172.


DNA fingerprinting and profile of phenolics in root and root calli

of Arctium lappa L. grown in EgyptElsayed A. Aboutabla, Mona El-Tantawyb, Nadia Sokkara and Manal M. Shamsb

aDepartment of Pharmacognosy, Faculty of Pharmacy,Cairo University and bDepartment of Pharmacognosy,National Organization for Drug Control and Research(NODCAR), Cairo, Egypt

Correspondence to Elsayed A. Aboutabl, Departmentof Pharmacognosy, Faculty of Pharmacy, CairoUniversity, Kasr-el-Aini Str., 11562 Cairo, EgyptTel: + 20 100 242 8817; fax: + 20 223 628 426;e-mail: eaboutabl@ yahoo.com

Received 17 December 2012Accepted 19 February 2013


2013,12:57–62

Aim

The aim of this study was the establishment of an efficient and promising protocol for

callus production from Arctium lappa L. roots (family Asteraceae) and comparison of

the metabolic profile of their phenolic and flavonoid content. DNA fingerprinting of

A. lappa L. was carried out using the molecular generic marker technique (random

amplification of polymorphic DNA-PCR), which was newly introduced in Egypt, for

identification and authentication of the plant.

Methods

The effect of different concentrations of benzyladenine and naphthalene acetic acid

added to MS media on initiation of root callus production and mass of callus produced

was investigated. The presence or absence of various secondary metabolites of the

root and calli was also determined using colorimetric methods and high performance

liquid chromatography.


The growth parameters of the callus were determined. Each callus differs from the root

in the profile of phenolic and flavonoid content. The calli have a higher phenolic content

than the root and differ in the flavonoid profile.

Keywords:

Arctium lappa L, DNA fingerprinting, flavonoids, phenolics, root callus


IntroductionArctium lappa L. or burdock (Asteraceae) is native to

Europe and north Asia. Traditionally, it has been used as a

safe and edible food product [1,2] and for the treatment

different ailments [3–5]. Phytochemical investigation of

different organs of the plant revealed the presence of

fixed oil, phenolic acids, flavonoids, lignans [2,6], resin,

mucilage, essential oil [7], polyacetylenes [7], and

caffeoylquinic acid derivatives [8]. In a previous study [9],

bioactive lignans and phenolics and the biological

activities of extracts from different organs of A. lappa L.

cultivated in Egypt were studied. PCR sequencing was

carried out for six A. lappa L. breeds from southern

Taiwan using two primers, ITS1-5.8S and rRNA-ITS2,

which revealed that they all had an amplified fragment

that was 358 bp in length [10]. Automatic sequence

analysis showed that the DNA sequences for different

breeds of Arctium can differ [10]. Hypocotyls and cotyls of

the plant were induced to produce callus for high

frequency plant regeneration [11]. In the current

literature, few studies on tissue culture and DNA

fingerprinting of the plant were found, but no reports

dealing with the phenolic profile of the callus were found.

Accordingly, the aim of the present work was to carry out

PCR sequencing for the identification and authentication

of A. lappa L., a plant grown in Egypt, and to study the

root callus metabolites, as the accumulation of secondary

products in plant cell cultures depends on the composi-

tion of the culture medium.

Subjects and methodsPlant material

Authentic seeds of A. lappa L. were kindly provided to

Prof. Dr E.A. Aboutabl by the Botanic Garden, Bonn,

Germany and were cultivated in the Experimental

Station of Medicinal and Aromatic Plants, Faculty of

Pharmacy, Cairo University. For tissue culture, seeds were

collected from the cultivated plant during the fruiting

stage.

Plant material for DNA fingerprinting

Freeze-dried leaves of A. lappa (10 g) were powdered in

liquid nitrogen, and genomic DNA was extracted by a

modification of the cetyltrimethylammonium bromide

method [12].

Reference standards

Rutin, daidzein, genistein, isorhamnetin, luteolin, bio-

chanin A, hyperoside, gallic acid, chlorogenic acid, caffeic

acid, ferulic acid, and coumarin were obtained from the

Department of National Organization of Drug Control

and Research Standards.

Primers were obtained from Operon Technologies Inc.

(Almeda, California, USA).

Methods

DNA amplification was carried out using the random

amplification of polymorphic DNA (RAPD) technique

Original article 57


DOI: 10.7123/01.EPJ.0000428269.66909.9a

with 15 primers (the sequences are shown in Table 1).

The GeneAmp PCR system 9700 (Perkin Elmer,

Cambridge, UK) and a gel documentation system

(Bio-Rad Gel Doc-2000, Bio-Rad Laboratories, GmbH,

Munich, Germany) were used for photographing of PCR

products.

PCR reactions[13,14] were carried out in a total volume

of 25 ml with 10 ng/ml of genomic DNA as a template, 3 ml

of random primer, 2.5 ml of 2 mmol/l dNTP mix (Abgene,

Surrey, UK), 2.5 ml of 10� PCR buffer, 2 ml of 25 mmol/l

MgCl2, and 0.3–5 U/ml of Taq DNA polymerase. An

aliquot of 22 ml of master mix solution was dispensed in

each PCR tube (0.2 ml Eppendorf tube) containing 3 ml

of the appropriate template DNA. The reaction involved

initial denaturation by heating for 4 min at 941C.

Complete denaturation of DNA indicated efficient

utilization of the template in the first amplification cycle

and a good yield of the PCR product. The reaction

mixture was then subjected to 40 cycles of the following

program: a denaturation step at 941C for 45 s, an

annealing step at 361C for 1 min, and an elongation or

extension step at 721C for 2 min. After the last cycle, the

mixture was subjected to a final extension step for 7 min

at 721C, followed by soaking at 41C until removal of the

reaction mixture from the PCR machine. The amplifica-

tion products were resolved by electrophoresis on a 1.4%

agarose gel containing ethidium bromide (0.5 mg/ml) in

1� tris-borate-EDTA buffer. A total of 15 ml of each PCR

product was mixed with 3 ml of loading buffer (tracking

dye) and loaded into the wells of the gel. The gel

was run at 85 V for about 3 h or until the tracking dye

reached the gel. An ultraviolet (UV) Polaroid camera

was used for visualization of RAPD. Polaroid camera was

used for 6 visualization of RAPD; markers being scored

as DNA fragments present in some lanes and absent

in others.

Tissue culture

The seeds were washed thoroughly with running tap

water for about 15 min and surface-sterilized by immer-

sion in 10% commercial Savlon solution (10th of

Ramadan, Sharkiah, Egypt) (an antiseptic solution

containing 0.3% w/v chlorhexidine gluconate and 3% w/v

cetrimide) for about 5 min with shaking. The seeds were

then washed three times with sterile distilled water and

immersed in 30% commercial Clorox solution (10th of

Ramadan) (a disinfectant containing 1.5% sodium hypo-

chlorite) with 1–2 drops of wetting agent (Tween 80)

while shaking on a shaker for 10 min. Thereafter, the

seeds were washed three times with sterile distilled

water. They were then cultured in a jar containing sterile

solid MS control media without a plant growth regulator

and incubated at 22–281C with a photoperiod of 16 h/day

(200–2500 lx). After 6–8 weeks, the plantlets grown were

used to obtain the explants used for callus cultures. The

6–8-week-old seedlings grown in vitro on sterile MS

medium (Fig. 1) were used as a sources of explants [15].

Dissection of uniformly-sized explants (about 0.5 cm in

length) from different organs – that is, shoot tips, leaves,

roots, and stems – was performed under aseptic condi-

tions using a sterile scalpel and forceps [16]. The

different explants were cultured in jars containing sterile

medium supplemented with different concentrations of

various plant growth regulators such as benzyladenine

(BA), kinetin, naphthalene acetic acid (NAA), indolebu-

tyric acid, 2,4-dichlorophenoxy acetic acid, and indole

acetic acid. For each condition, 30 jars were prepared,

each jar containing five explants. The cultures were

incubated at 211C ( ± 21C) with a photoperiod of

16 h/day (1500–2000 lx) for a period of 6 weeks.

Determination of total phenolics

Air-dried plant root calli 1 and 2 (1 g each) were defatted

with petroleum ether and extracted with 70% methanol

by sonication at room temperature. Stock solution

(concentration: 1 mg/ml) was prepared from the concen-

trated residue by dissolving in distilled water. The

phenolic compound in the root was found to be gallic

acid on Folin–Ciocalteau colorimetry [17] using a

Shimadzu 1601 spectrophotometer at 730 nm, and the

total phenolic content of the root was compared with that

Table 1 Sequence of 15 primers assayed using the RAPD-PCR

technique

Primer Sequences (50–30)

A-01 50-CAGGCCCTTC-30

A-11 50-CAATCGCCGT-30

B-06 50-TGCTCTGCCC-30

B-08 50-GTCCACACGG-30

B-15 50-GGAGGGTGTT-30

B-18 50-CCACAGCAGT-30

P-01 50-GTAGCACTCC-30

O-02 50-ACGTAGCGTC-30

O-09 50-TCCCACGCAA-30

E-08 50-TCACCACGGT-30

E-05 50-TCACCACGGT-30

E-11 50-GAGTCTCAGG-30

G-06 50-GTGCCTAACC-30

Z-13 50-GACTAAGCCC-30

G-17 5’- ACGACCGACA-3’

RAPD, random amplification of polymorphic DNA.

Figure 1

The random amplification of polymorphic DNA electrophoretic profileof Arctium lappa L., cultivated in Egypt, generated by 15 primers(M: 100 bp plus fermentas).


of the calli. Determination of total the phenolic content

of the cultivated roots, leaves, and seeds was carried out

in our previous work [9].

Colorimetric determination of total flavonoid content

Powdered, air-dried (2 g) plant root calli 1 and 2 were

defatted with petroleum ether, extracted with 70%

methanol till exhaustion, and evaporated to dryness.

The combined methanolic extract was adjusted to 50 ml.

A 5 ml aliquot of each extract was treated with a 5 ml

aliquot of 0.1 mol/l AlCl3 reagent [18]. The absorbance of

the color developed was measured at lmax 422 nm against

a blank, and the corresponding amount of rutin was

recorded.

HPLC determination of isoflavones

Dried root (1 g) and root calli (1 and 2, 0.25 g) were

separately defatted, filtered, and extracted with 50%

ethanol. The ethanol was evaporated under vacuum at

351C, and the phenolics in the remaining aqueous

solution were extracted with ethyl acetate (1 : 1). The

phenolic fractions were stored in the dark at 41C until

analysis by high performance liquid chromatography

(HPLC). An Aglient 1100 system (Agilent Technologies

Deutschland GmbH, Germany) equipped with a column

compartment, quaternary pump, degasser, auto sampler,

and UV detector was used for HPLC analysis. Elution was

performed at a flow rate of 1 ml/min with a mobile phase

of water/acetic acid (98 : 2 v/v, solvent A) and methanol/

acetonitrile (50 : 50 v/v, solvent B), starting with 5% B and

increasing the level of B to 30% at 25 min, 40% at 35 min,

52% at 40 min, 70% at 50 min, and 100% at 55 min; the

UV detector was set at 254 nm [19]. Retention times

were compared with those of certain standard isoflavones.

Before injection into the HPLC system, each sample was

filtered through a 0.4 mm membrane filter into the sample

vial for injection.

HPLC determination of phenolics

Extraction and HPLC analysis of phenolics were carried

out under the same conditions as those for isoflavones,

but measurements were made with a detector set at

330 nm. Retention times were compared with those of

available phenolic standards.

ResultsTotal genomic DNA profiling of A. lappa L., grown

in Egypt, was performed using 15 random primers.

The number of banding patterns generated by each

primer was recorded to obtain the DNA profile of A. lappaunder investigation, in order to compare it with

previously reported phenotypic characters as well as for

chemical investigations. Molecular size, in base pairs, of

amplified DNA fragments produced by 15 decamer

primers in A. lappa L. is listed in Table 1, and their

reproducible RAPD profiles generated are shown

in Fig. 1. The total number of bands generated by the

15 primers was 93, the smallest size of amplified product

being 245 bp, whereas the largest size of the amplified

product being 3030 bp. Primer P1 produced nine bands,

with 245 bp being the smallest size and 3030 bp being the

largest size; primer A1 was the least reproducible and

generated three bands with molecular sizes 1739, 724,

and 276 bp.

Callus production

Figure 2 shows 6–8-week-old seedlings of A. lappa grown

in vitro on sterile MS media; these were used as a source

of explants for callus production. Trials using different

explants (shoot tips, leaves, stems, and roots) and

different growth regulators were carried out for initiation

of callus. Calli were obtained successfully on MS media

supplemented with plant growth regulators for roots:

MS + 0.5 mg/l BA + 1 mg/l NAA (callus 1) and MS + 0.5

mg/l BA + 0.1 mg/l NAA (callus 2). The different callus

Figure 2

Six- to eight-week-old seedling of Arctium lappa L. grown in vitro in MSmedium.

Table 2 The effect of plant growth regulators on callus growth parameters of Arctium lappa L. root and total phenolic and flavonoid

content of Arctium lappa L. root and two calli

Root calli (greenish brown compact undifferentiated callus)

Characteristics Root Callus 1 Callus 2

Callus fresh weight (g) – 5.01 + 0.3 4.22 + 0.2Callus dry weight (g) – 0.48 + 0.05 0.36 + 0.03Total phenolic content (%; calculated as gallic acid content in dried material) 5.33 6.53 7.98Total flavonoid content (%; calculated as rutin content in dried material) 0.05 0.003 0.002

Phenolics of Arctium lappa root and calli Aboutabl et al. 59

growth parameters are listed in Table 2, and callus types

are presented in Fig. 3a and b.

Determination of total phenolic content of the root calli

compared with that of the root

Colorimetric determination showed that there was a

variation in the phenolic content of the root compared

with that of the calli (Table 2). Callus 2 showed a higher

phenolic content than callus 1 and the root because of the

effect of plant growth regulators (BA and NAA) on the

biosynthesis of polyphenols [15].

Determination of the flavonoid compounds of the root

calli compared with those of the A. lappa L. root

The flavonoid content in each of the two calli was less

than that in the root (Table 2).

HPLC determination of isoflavones in the root callus

compared with those in the main plant parts

The concentration of isoflavones (in mg/g; Table 3)

indicates that the root contains only genistein and differs

in metabolic profile compared with root callus 1 (MS +

0.5 mg/l BA + 1 mg/l NAA), which contains isorhamnetin

and biochanin A, and root callus 2 (MS + 0.5 mg/l

BA + 0.1 mg/l NAA), which contains daidzein and genis-

tein. The flavonoid content in callus culture differs

qualitatively and quantitatively from that in the parent

plant [20].

HPLC determination of the phenolic content of the root

callus compared with that of the root

The root differs in its phenolic metabolic profile

compared with the two calli. The phenolic compounds

present in the root were identified as gallic acid, ferulic

acid, chlorogenic acid, hyperoside, rutin, coumarin, and

luteolin. Callus 1 was found to contain gallic acid,

chlorogenic acid, caffeic acid, ferulic acid, and coumarin,

whereas callus 2 was found to contain gallic acid,

chlorogenic acid, caffeic acid, coumarin, and luteolin.

The corresponding concentrations are listed in Table 3

(in mg/g; Table 4).

Figure 3

(a) Callus 1 (�0.76; MS + 0.5 mg/l BA + 1 mg/l NAA). (b) Callus 2 (�1; MS + 0.5 mg/l BA + 0.1 mg/l NAA).

Table 3 Phenolics identified by high performance liquid

chromatography in Arctium lappa L. root and calli

Concentration (mg/g)

Compounds Rt (min) Root Callus 1 Callus 2

Gallic acid 2.51 0.49 0.36 0.78Daidzein 3.03 – – 0.054Genistein 3.57 0.005 – 0.014Isorhamnetin 4.29 – 0.080 –Chlorogenic acid 5.54 0.62 0.06 0.58Caffeic acid 6.15 – 0.06 0.70Biochanin A 7.17 – 0.018 –Hyperoside 7.95 0.31 – –Rutin 8.11 0.22 – –Ferulic acid 9.17 0.01 0.06 –Coumarin 9.70 0.02 0.22 0.66Luteolin 11.78 0.01 – 0.62


ConclusionFrom the present study, it was deduced that using BA and

NAA for the induction of root callus production caused an

increase in the phenolic content compared with that

of the main root. Decreasing the amount of NAA in callus

2 (MS + 0.5 mg/l BA + 0.1 mg/l NAA) resulted in a higher

phenolic content than that in callus 1 (MS + 0.5 mg/l

BA + 1 mg/l NAA). In addition, HPLC results for callus 2

show a marked increase in caffeic acid, coumarin, and

luteolin content; however, the flavonoid content in the two

calli decreased, and the metabolic profile of isoflavones

showed great variation. DNA fingerprinting helps in the

authentication and identification of A. lappa L., which is

grown in Egypt. This is the first report on tissue culture

and molecular biological study of this plant. The current

literature, our previous work [9], and also the results of the

present work prove the importance of the plant; hence,

the authors recommended that the study on the effects of

plant growth regulators, precursors, and other factors that

increase the main active constituents of the plant, which

can be used as a source of natural raw material for

phytopharmaceuticals, be continued.

Table 4 Molecular size, in base pairs, of amplified DNA fragments produced by 15 decamer primers in Arctium lappa L.

Molecular sizeof DNA marker (bp) A1 A11 O2 O9 E5 E8 E11 B6 B8 B15 B18 G6 G17 Z13 p1

245 + +268 +276 +310 +359 +370 +440 + +453 +467 +525 + +556 +573 +590 +644 + + +683 + + +703 + + + +724 +745 + + +813 +838 +862 +888 + + +914 +941 +969 + +998 + + + +1028 +1058 + + +1090 + +1122 + + +1155 +1189 + +1225 +1298 + +1337 +1377 +1417 + + + +15031547 + +1593 + + +1640 + + +1989 +1739 +1791 +1844 +1899 + + +2013 +2073 +2134 + +2197 + +2330 +2399 +2776 +2858 + + +3030 +Sum 3 7 5 7 4 9 8 7 5 4 7 6 5 6 9

Phenolics of Arctium lappa root and calli Aboutabl et al. 61


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Influence of formulation parameters on the physicochemical

properties of meloxicam-loaded solid lipid nanoparticlesRawia M. Khalila, Ahmed Abd El-Baryb, Mahfoz A. Kassema,Mamdouh M. Ghorabc and Mona Bashaa

aDepartment of Pharmaceutical Technology, NationalResearch Centre, bDepartment of Pharmaceutics,Faculty of Pharmacy, Cairo University, Cairo andcDepartment of Pharmaceutics, Faculty of Pharmacy,Suez Canal University, Ismailia, Egypt

Correspondence to Rawia M. Khalil, Departmentof Pharmaceutical Technology, National ResearchCentre, El-Bohowth St., PO Box 12622, Dokki,12311 Cairo, EgyptTel: + 20 1006935895/ + 1006550825;fax: + 20 233370931;e-mail: [email protected]

Received 26 November 2012Accepted 4 February 2013


2013, 12:63–72

Objective

The aim of this research was to investigate novel particulate carrier systems such as

solid lipid nanoparticles (SLNs) for topical delivery of a lipophilic drug, meloxicam

(MLX).

Methods

MLX-loaded SLNs were prepared using a modified high-shear homogenization and

ultrasonication technique using different types of lipids and surfactants. Lipid

nanoparticles were characterized in terms of entrapment efficiency, particle size,

Zeta potential, differential scanning calorimetry, transmission electron microscopy,

and in-vitro release studies.

Results

The lipid nanoparticles showed mean diameters of 210–730 nm, whereas the

entrapment efficiency ranged from 50 to 84% depending on emulsifier and lipid

concentration or type. MLX-loaded SLNs showed spherical particles with Zeta

potentials varying from – 15.7 to – 30.5 mV. A differential scanning calorimetry study

revealed that MLX encapsulated in SLNs was in the amorphous form. All nanoparticle

formulations exhibited sustained release characteristics, and the release pattern

followed the Higuchi’s equation. The analysis of results revealed that the type and

concentration of the emulsifier or lipid used had a significant effect on the

physicochemical properties on the investigated SLNs formulations.

Conclusion

The present study indicates that SLNs could potentially be exploited as carrier systems

for MLX, with improved drug loading capacity and controlled drug release.

Keywords:

differential scanning calorimetry, in-vitro release study, meloxicam, solid lipid

nanoparticles, topical delivery


IntroductionIn recent years, significant effort has been devoted to

develop nanotechnology for drug delivery. Solid lipid

nanoparticles (SLNs) are aqueous colloidal dispersions,

the matrix of which comprises solid biodegradable lipids.

SLNs combine the advantages and avoid the drawbacks

of several colloidal carriers of their class such as physical

stability, protection of incorporated labile drugs from

degradation, controlled release, and excellent tolerabil-

ity [1]. SLNs offer a suitable means of delivering drugs

for various application routes; they attract great attention

as novel colloidal drug carriers for topical use [2]. The

advantages of these carriers include negligible skin

irritation, controlled release, and protection of active

substances [3]. Because they are composed of nonirrita-

tive and nontoxic lipids, SLNs seem to be well suited for

use on inflamed and damaged skin. Moreover, SLNs have

distinct occlusive properties because of the formation of

an intact film on the skin surface upon drying, which

decreases transepidermal water loss and favors drug

penetration through the stratum corneum [4]. Besides

having a highly specific surface area, nanometer-sized

SLNs also facilitate the contact of the encapsulated drug

with the stratum corneum [4]. The nanometer-sized

particles can make close contact with superficial junctions

of corneocyte clusters and furrows between corneocyte

islands, which may favor accumulation for several hours,

allowing for sustained drug release [5]. Other advantages

of SLNs include a high drug payload and incorporation of

lipophilic and hydrophilic drugs [2]. SLNs have been

used to improve skin/dermal uptake of several drugs [6,7],

which supports the idea that SLNs can be used as carriers

for topical delivery of meloxicam (MLX).

MLX is a potent, nonsteroidal anti-inflammatory

water-insoluble drug [8,9]. It inhibits cyclooxygenase

(COX). MLX is more selective for the COX-2 isoform

of prostaglandin synthetase compared with the COX-1

form. Therefore, MLX has been labeled a ‘preferential’

inhibitor instead of a ‘selective’ inhibitor of COX-2

[10].

Original article 63


DOI: 10.7123/01.EPJ.0000428643.74323.d9

The intention of this study was to prepare and evaluate

MLX-loaded SLNs and to optimize the formulation

parameters in order to fabricate SLN dispersions of

desired characteristics for topical delivery of MLX, aiming

to improve skin uptake and reduce systemic absorption

and dermal irritation.

Materials and methodsMaterials

MLX was supplied by Medical Union Pharmaceuticals

(Ismailia, Egypt). Geleol (glyceryl monostearate 40–55;

40–55% monoglycerides, 30–45% diglycerides, melting point

(m.p.) 54.5–58.51C), Compritol 888 ATO (glyceryl behe-

nate; 15–23% monoglycerides, 40–60% diglycerides, 21–35%

triglycerides, m.p. 69.0–74.01C), and Precirol ATO5 (glycer-

yl palmitostearate; 8–22% monoglycerides, 40–60% digly-

cerides, 25–35% triglycerides, m.p. 50–601C) were kindly

donated by Gattefosse (Saint-Priest, France). Tween 80

(polysorbate 80), methanol Chromasolv, and dialysis tubing

cellulose membrane (molecular weight cutoff 12 000 g/mole)

were purchased from Sigma Chemical Company (St. Louis,

Missouri, USA). Cremophor RH40 (polyoxyl 40 hydrogenated

castor oil) was kindly donated by BASF (Ludwigshafen,

Germany). All other chemicals and reagents used were of

analytical grade.

Methods

Preparation of solid lipid nanoparticles

SLNs were prepared by a slight modification of the

previously reported high-shear homogenization and ultra-

sonication technique [11,12]. Briefly, the lipid phase

consisted of Geleol, Compritol, or Precirol as the solid

lipid was melted 51C above the melting point of the lipid

used. MLX (0.5%w/w) was dissolved therein to obtain a

drug–lipid mixture. An aqueous phase was prepared by

dissolving the surfactant in distilled water and heated up

to the same temperature of the molten lipid phase.

The hot lipid phase was poured onto the hot aqueous

phase and homogenization was carried out at 25 000 rpm

for 5 min using a Heidolph homogenizer (Heidolph

Instruments, Schwabach, Germany). The resultant hot

oil-in-water emulsion was sonicated for 30 min (Digital

Sonicator; MTI, Michigan, USA). MLX-loaded SLNs

were finally obtained by allowing the hot nanoemulsion to

cool to room temperature. Blank SLNs were prepared

using the same procedure variables.

Meloxicam entrapment efficiency

The entrapment efficiency percentage (EE%), which

corresponds to the percentage of MLX encapsulated

within the nanoparticles, was determined by measuring

the concentration of free MLX in the dispersion medium.

The unentrapped MLX percentage was determined by

adding 500ml of MLX-loaded nanoparticles to 9.5 ml of

methanol and centrifuging this dispersion at 9000 rpm

(Union 32R; Hanil Science Industrial, Gangwondo, Korea)

for 30 min. The supernatant was filtered through a

Millipore (Sigma-Aldrich, St. Louis, USA) membrane filter

(0.2mm) and analyzed for unencapsulated MLX at 360 nm

using a validated UV-spectrophotometric method (model

2401/PC; Shimadzu, Kyoto, Japan) after suitable dilution.

The EE% was calculated using the following equation [13]:

EE %¼Winitial drug�Wfree durg

Winitial drug

�100;

where Winitial drug is the initial mass of the drug used and

Wfree drug is the mass of the free drug detected in the

supernatant after centrifugation of the aqueous dispersion.

Particle size analysis

Particle size analysis of MLX-loaded nanoparticles was

performed using a laser diffraction (LD) particle size anal-

yzer (Master Sizer X; Malvern Instruments, Worcestershire,

UK) at 251C. The LD data obtained were evaluated using

volume distribution as diameter values of 10, 50, and 90%

and span values. The diameter values indicate the

percentage of particles possessing a diameter equal to

or lower than the given value. The span value is a

statistical parameter used to evaluate the particle size

distribution: lower the span value, narrower is the particle

size distribution. It is calculated using the following

equation [14]:

Span¼LD90 %�LD10 %

LD50 %

:

Zeta Potential and pH measurement

The z potential was measured in folded capillary cells

using a Laser Zetameter (Malvern Instruments). Measure-

ments were performed in distilled water adjusted with a

solution of 0.1 mmol/l NaCl at 251C. The z potential

values were calculated using the Smoluchowski equation.

The pH values of MLX lipid nanoparticles were

measured at 251C using a digital pH meter (Jenway,

Staffordshire, UK).

Transmission electron microscopy

Morphological examination of MLX-loaded SLNs was per-

formed using transmission electron microscopy (TEM)

(model JEM-1230; Jeol, Tokyo, Japan). One drop of the

diluted sample was deposited onto the surface of a carbon-

coated copper grid and negatively stained with a drop of

2% (w/w) aqueous solution of phosphotungstic acid for

30 s. Excess staining solution was wiped off with filter

paper, leaving a thin aqueous film on the surface. After

staining, the samples were allowed to dry at room

temperature for 10 min for analysis [15].


Differential scanning calorimetry (DSC) analysis was

carried out using a Shimadzu Differential Scanning

Calorimeter (DSC-50; Shimadzu). About 10 mg of sample

was added into a 40 ml aluminum pan, which was sealed

and heated in the range of 30–3001C at a heating rate of

101C/min. An empty aluminum pan was used as a

reference standard. The analysis was carried out under

nitrogen purge.


Rheological study

The rheological properties of the prepared lipid nano-

particles were determined using Brookfield’s Viscometer

(Brookfield LV-DV II + ; Brookfield, Massachusetts,

USA). The sample (20 g) was placed in a beaker and

allowed to equilibrate for 5 min. The measurements were

carried out at ambient temperature using the suitable

spindle. The spindle speed rate was increased in

ascending order from 1 to 100 rpm and then decreased

in descending order from 100 to 1 rpm, with each kept

constant for 10 s before a measurement was made.

In-vitro release study

The in-vitro release of MLX was evaluated using the

dialysis bag diffusion technique described by Yang

et al. [16]. The release studies of MLX from SLNs were

performed in phosphate buffer (pH 5.5) and methanol

(75 : 25). Aqueous nanoparticulate dispersion equivalent

to 2 mg of MLX was placed in a cellulose acetate dialysis

bag and sealed at both ends. The dialysis bag was

immersed in the receptor compartment containing 50 ml

of dissolution medium, which was stirred in a water bath

shaker at 100 rpm (Memmert GmbH, Schwabach,

Germany) and maintained at 32 ± 21C. The receptor

compartment was covered to prevent evaporation of the

dissolution medium. A 2 ml sample of the receiver

medium was withdrawn at predetermined time intervals

(0.5, 1, 2, 3, 4, 5, 6, 8, 24, and 48 h) and replaced by an

equivalent volume of fresh medium to maintain constant

volume. The samples were analyzed for drug content

spectrophotometrically at 360.5 nm. The data were

analyzed using linear regression equations, and the order

of drug release from the different formulations was

determined.

Statistical analysis

All experiments were repeated three times, and data were

expressed as mean value ± SD. The statistical analysis

was carried out using one-way analysis of variance.

A P value of less than 0.05 was considered statistically

significant.

Results and discussionPreparation of solid lipid nanoparticles

In the present study, MLX-loaded SLNs dispersions were

composed of Geleol, Compritol 888 ATO, or Precirol

ATO5 as core matrices used in different concentrations of

5, 7.5, and 10% (w/w). These lipid-based carrier systems

were stabilized using 0.5, 1, 2.5, and 5% (w/w) Tween 80

or Cremophor RH40. MLX was incorporated at a constant

concentration of 0.5% (w/w). The w/w percentage

composition of the investigated MLX SLNs is shown

in Tables 1 and 2.

Meloxicam entrapment efficiency

The entrapment efficiencies of all SLN formulations are

presented in Tables 1 and 2. The entrapment efficiencies

varied from 50.42 ± 2.07 to 84.38 ± 0.65%. It can be

observed that increasing the amount of surfactant from

0.5 to 1 to 2.5 to 5% (w/w) at a constant amount of lipid

(5% w/w) resulted in a gradual significant decrease

(Po0.05) in the entrapment efficiencies. However, no

change in EE% was observed (Table 1) for Compritol

(SLN7 and SLN8) and Precirol SLNs (SLN13 and

SLN14) on increasing the Tween 80 concentration from

0.5 to 1%. Moreover, for Geleol SLNs (SLN3 and SLN4),

no significant decrease in EE% was observed on increas-

ing the Tween 80 concentration above 2.5% (w/w)

(P40.05). Table 2 shows that using Cremophor RH40

resulted in the same gradual decrease in EE% (Po0.05);

however, in case of Geleol SLNs (SLN21 and SLN22)

and Precirol SLNs (SLN33 and SLN34), a further

increase in the Cremophor RH40 concentration from

2.5 to 5% did not result in significant changes in EE%

(P40.05). This observed decrease in EE% could be

Table 1 Composition and entrapment efficiency of meloxicam solid lipid nanoparticles (%w/w) of different lipids using Tween 80

Lipid

Formulas Type Concentration Tween 80 (%) Entrapment efficiency %a

SLN1 Geleol 5 0.5 59.78 ± 1.04SLN2 1 56.63 ± 0.88SLN3 2.5 51.03 ± 0.96SLN4 5 50.42 ± 2.07SLN5 7.5 0.5 62.30 ± 0.23SLN6 10 67.49 ± 1.27SLN7 Compritol 5 0.5 62.47 ± 0.25SLN8 1 62.22 ± 1.03SLN9 2.5 57.31 ± 1.92SLN10 5 54.79 ± 0.21SLN11 7.5 0.5 65.76 ± 1.77SLN12 10 72.63 ± 1.66SLN13 Precirol 5 0.5 65.68 ± 0.09SLN14 1 65.53 ± 0.40SLN15 2.5 62.00 ± 0.39SLN16 5 58.51 ± 0.71SLN17 7.5 0.5 70.02 ± 0.89SLN18 10 75.99 ± 3.36

SLN, solid lipid nanoparticle.aValues represent mean ± SD.

Meloxicam loaded solid lipid nanoparticles Khalil et al. 65

explained by the partition phenomenon. High surfactant

levels in the external phase might increase the partition

of the drug from the internal to the external phase of the

medium. This increased partition is due to the increased

solubilization of the drug in the external aqueous phase

such that more volumes of the drug can disperse and

dissolve in it [17]. However, some cases in which further

increase of surfactant concentration did not lead to a

significant change in EE% could suggest that an optimum

concentration of the surfactant was reached, sufficient to

cover the surface of the nanoparticles effectively. The

data also clearly showed that the formulations prepared

using Cremophor RH40 as a surfactant had higher EE%

compared with those prepared using Tween 80. Similar

results were reported by Lv et al. [18] for penciclovir-

loaded SLNs.

The structure of the lipid used has a great influence on

the capacity for drug incorporation. Therefore, the effect

of lipid type and concentration on the entrapment

efficiency of MLX SLNs was also investigated (Tables 1

and 2). Geleol SLNs exhibited the lowest entrapment of

MLX when compared with Compritol and Precirol. This

can be attributed to the difference in composition and

chain length of the three lipids used. The higher drug

entrapment efficiency observed with Precirol and Com-

pritol was attributed to the high hydrophobicity due to

the long chain fatty acids attached to the triglycerides,

resulting in increased accommodation of lipophilic

drugs [19].

The results also showed that increasing the lipid

concentration from 5 to 7.5 to 10% (w/w) led to a gradual

increase in the entrapment efficiency, which was

observed for lipids used at constant concentrations of

Tween 80 and Cremophor RH40 (Po0.05). However,

this increase in the entrapment efficiency is not

proportional to the increase in lipid content, which can

be observed for the three lipids. An exception was

observed for SLN31 and SLN35 wherein a significant

increase in EE% occurred only on increasing Precirol

concentrations from 7.5 to 10% (w/w). A possible

explanation for these observations is that the increase

in lipid content can afford more space to encapsulate

more drug, thus reducing drug partition in the outer

phase [18,20]. This may also be due to an increase in the

viscosity of the medium, resulting in faster solidification

of nanoparticles, which would further prevent drug

diffusion to the external phase of the medium [21].

Particle size analysis

The LD 90% of the formulated SLNs is presented

in Table 3. In case of Tween 80 and Cremophor RH40,

the nanoparticulate dispersions showed sizes ranging

from 210 ± 35.36 to 740 ± 14.14 nm and from 235 ± 21.21

to 730 ± 14.14 nm, respectively. The low span values of

different formulations indicate a narrow particle size

distribution. The results clearly showed that there was a

gradual decrease in particle size with an increase in

surfactant concentration from 0.5 to 1 to 2.5 to 5% (w/w)

(Po0.05). This was observed for all formulations except

for SLN1 and SLN2 and for SLN19 and SLN20, in which

an initial increase in surfactant concentration from 0.5 to

1% did not lead to a significant decrease in particle size

(P40.05). However, a further increase in surfactant

concentration above 2.5% for SLN33 and SLN34 did not

result in a significant change in particle size (P40.05).

The decrease in size of nanoparticles at high surfactant

concentrations might be due to an effective reduction in

the interfacial tension between the aqueous and lipid

phases, leading to the formation of emulsion droplets of

smaller sizes [22]. Higher surfactant concentrations

effectively stabilize the particles by forming a steric

barrier on the particle surface and thereby protect smaller

particles and prevent their coalescence into bigger

ones [17]. For the formulations in which further increase

of surfactant concentration above 2.5% did not reduce the

particle size significantly, the data clearly suggest that an

optimum concentration of the surfactant was reached,

Table 2 Composition and entrapment efficiency of meloxicam solid lipid nanoparticles (%w/w) of different lipids using

Cremophor RH40

Lipid

Formulas Type Concentration (%) Cremophor RH40 (%) Entrapment efficiency %a

SLN19 Geleol 5 0.5 63.31 ± 1.11SLN20 1 58.33 ± 1.42SLN21 2.5 52.28 ± 1.89SLN22 5 53.34 ± 1.20SLN23 7.5 0.5 65.89 ± 0.83SLN24 10 69.10 ± 0.42SLN25 Compritol 5 0.5 68.63 ± 0.34SLN26 1 62.16 ± 1.64SLN27 2.5 59.34 ± 0.32SLN28 5 56.64 ± 0.91SLN29 7.5 0.5 70.41 ± 0.58SLN30 10 77.47 ± 0.93SLN31 Precirol 5 0.5 78.77 ± 0.85SLN32 1 73.33 ± 1.31SLN33 2.5 67.71 ± 2.76SLN34 5 66.79 ± 0.92SLN35 7.5 0.5 79.51 ± 0.24SLN36 10 84.38 ± 0.65

SLN, solid lipid nanoparticle.aValues represent mean ± SD.


sufficient to cover the surface of nanoparticles effectively

and prevent agglomeration during the homogenization

process [23].

The results also showed that increasing the lipid content

from 5 to 7.5 to 10% (w/w) led to a subsequent increase

in particle size (Table 3). Statistical analysis of the data

showed no significant increase in particle size in case of

SLN19 and SLN23 on increasing the lipid concentration

from 5 to 7.5%. A similar result was obtained on increasing

the lipid concentration from 7.5 to 10% in case of SLN11

and SLN12 and in SLN29 and SLN30. This increase in

particle size may partially be related to the viscosity of

the samples, as viscosity is a key factor affecting the

ability to create a fine dispersion. At higher lipid

contents, the efficiency of homogenization decreases

because of a higher viscosity of the sample, resulting in

larger particles. Moreover, a high particle concentration at

high lipid contents increases the probability of particle

contact and subsequent aggregation [24]. The LD 90%

values of MLX SLNs of different lipids at a constant

surfactant concentration (0.5% w/w) are shown in Fig. 1.

For both surfactants used, Compritol showed the largest

particle sizes, followed by Precirol and then Geleol.

These differences in sizes may be due to differences in

the chain lengths and viscosities of the lipids used [25].

Compritol 888 ATO (m.p. 69.0–74.01C) is a solid lipid

based on glycerol esters of behenic acid (C22), in which

the main fatty acid is behenic acid (485%) but other fatty

acids (C16–C20) are also present. Precirol ATO5 (m.p.

50.0–60.01C) and Geleol (m.p. 54.5–58.41C) are com-

posed mainly of palmitic (C16) and stearic acids (C18)

(490%). A high melting temperature resulting in higher

viscosity and the long hydrocarbon chain length of

Compritol might result in larger particle sizes in

comparison with Precirol and Geleol.

f Potential analysis and pH measurements

As shown in Table 3, all formulations were negatively

charged; the z potential varied from – 15.7 mV (SLN14)

to – 30.5 mV (SLN18), indicating relatively good stability

and dispersion quality. It was noticeable that as the

amount of surfactant increased in the formulation the

z potential became more negative. However, the influ-

ence of surfactant type is less pronounced.

Tween 80 and Cremophor RH40 being nonionic surfac-

tants could successfully be used in the production of

relatively stable dispersions. This behavior could be a

result of the strong effect of surfactants in an emulsion

system on the adsorbed layer thickness [26]. Although

nonionic surfactants could not ionize into charged groups

like ionic ones, they still demonstrated an effect on the

z potential. This might be due to molecular polarization

and adsorption of emulsifier molecules onto the charge in

water: they were absorbed onto the emulsifier layer of the

Table 3 Particle size, f potential, and pH values of meloxicam solid lipid nanoparticles

Formulas LD 90% Span z potential (mV) pH Formulas LD 90% Span z potential (mV) pH

SLN1 420 ± 14.14 0.51 – 15.9 6.15 ± 0.03 SLN19 425 ± 17.68 1.39 – 15.8 5.97 ± 0.01SLN2 385 ± 7.07 0.63 – 16.0 5.68 ± 0.04 SLN20 370 ± 14.14 1.23 – 17.5 5.82 ± 0.02SLN3 250 ± 28.28 0.18 – 17.9 5.61 ± 0.06 SLN21 265 ± 7.07 1.10 – 19.8 5.88 ± 0.05SLN4 210 ± 35.36 0.34 – 20.9 5.53 ± 0.05 SLN22 235 ± 21.21 1.21 – 19.1 5.93 ± 0.08SLN5 480 ± 14.14 0.67 – 25.5 5.72 ± 0.16 SLN23 490 ± 28.28 1.61 – 20.5 5.85 ± 0.01SLN6 555 ± 7.07 1.15 – 25.5 5.80 ± 0.04 SLN24 565 ± 7.07 1.64 – 25.2 5.84 ± 0.07SLN7 580 ± 14.14 0.80 – 18.8 5.77 ± 0.02 SLN25 565 ± 3.54 1.64 – 15.9 5.87 ± 0.01SLN8 545 ± 7.07 1.27 – 21.1 6.26 ± 0.10 SLN26 505 ± 7.07 1.84 – 17.8 5.71 ± 0.03SLN9 440 ± 28.28 1.06 – 21.0 5.96 ± 0.08 SLN27 465 ± 21.21 1.72 – 21.6 6.08 ± 0.02SLN10 385 ± 7.07 0.80 – 22.3 6.09 ± 0.01 SLN28 390 ± 14.14 1.30 – 21.7 5.95 ± 0.07SLN11 680 ± 28.28 1.28 – 23.0 5.56 ± 0.01 SLN29 685 ± 10.61 1.90 – 19.8 5.92 ± 0.01SLN12 740 ± 14.14 1.27 – 27.1 5.67 ± 0.08 SLN30 730 ± 14.14 1.95 – 22.8 5.53 ± 0.02SLN13 470 ± 14.14 0.88 – 16.9 5.91 ± 0.01 SLN31 490 ± 14.14 1.36 – 20.2 6.24 ± 0.08SLN14 415 ± 21.21 0.83 – 15.7 6.42 ± 0.04 SLN32 435 ± 3.54 1.55 – 20.0 5.26 ± 0.03SLN15 310 ± 14.14 0.81 – 18.6 5.70 ± 0.05 SLN33 315 ± 7.07 1.29 – 21.4 5.63 ± 0.02SLN16 265 ± 7.07 0.48 – 22.4 5.70 ± 0.03 SLN34 285 ± 21.21 1.27 – 22.6 5.94 ± 0.03SLN17 570 ± 28.28 1.03 – 29.8 5.76 ± 0.06 SLN35 580 ± 28.28 1.30 – 20.4 5.56 ± 0.13SLN18 685 ± 7.07 1.34 – 30.5 5.49 ± 0.08 SLN36 685 ± 7.07 1.71 – 24.3 5.48 ± 0.28

LD, laser diffraction; SLN, solid lipid nanoparticle.

Figure 1

0100200300400500600700800

0.5 7.5 10.0Lipid conc.(%)

GeleolCompritolPrecirol

Part

icle

siz

e (n

m)

0

100

200

300

400

500

600

700

800(b)

(a)

0.5 7.5 10.0Lipid conc.(%)

GeleolCompritolPrecirol

z

Part

icle

siz

e (n

m)

Effect of lipid concentration and type on particle size measured by laserdiffraction 90% of meloxicam solid lipid nanoparticles using (a) Tween80 and (b) Cremophor RH40.


particle/water interface, and an electric double layer

similar to an ionic layer was formed. Considering the

effect of lipid type and concentration on the z potential

of the produced SLN formulations, the results showed no

direct relationship between the type of lipid used and the

measured z values. In contrast, as the lipid concentration

increased, the z potential was found to become more

negative. Rahman et al. [17] reported the same observa-

tion when studying the effect of increasing Compritol

concentrations in the final formulation.

The bulk pH values of the stratum corneum and upper

viable epidermis have been measured to be 4.0–4.5 and

5.0–7.0, respectively [27]. For a topical preparation to be

applied safely onto the skin, its pH should lie within this

range. The pH values of different MLX SLN formula-

tions ranged from 5.26 ± 0.03 to 6.42 ± 0.04 (Table 3)

and hence were in the required range.

Transmission electron microscopy

TEM was used to investigate the morphology of MLX-

loaded SLNs. It was evident from the TEM images that

the nanoparticles were almost spherical with smooth

morphology, appeared as black dots, and were well

dispersed and separated on the surface (Fig. 2). This

description is in agreement with a previous observation

that the use of chemically heterogeneous lipids in

combination with heterogeneous surfactants favors the

formation of ideally spherical lipid nanoparticles [11].

The figure illustrates the presence of a very thin layer

surrounding the particles, which suggests a drug-enriched

core model. This model can be achieved if during the

lipid solidification process, the drug precipitates first,

which results in a drug-enriched core covered with a lipid

shell that has a lower drug concentration. This drug

distribution within the nanoparticles will have its impact

on the in-vitro drug release profile discussed.

Differential scanning calorimetry analysis

Figure 3 shows the DSC thermograms of pure MLX, bulk

lipids (Geleol, Compritol 888 ATO, and Precirol ATO5),

and MLX-loaded SLNs. Pure MLX showed a sharp

endothermic peak at 259.541C, corresponding to its

melting point, indicating its characteristic crystalline

nature. Bulk Geleol showed a distinctive melting peak at

66.011C, whereas Compritol 888 ATO showed a sharp

peak at 74.221C. The bulk Precirol ATO5 exhibits a sharp

endothermic event, ascribing to melting, around 63.351C,

with a small but well-defined shoulder at 57.371C, which

might be due to melting of the a-polymorphic form [28].

These sharp melting endothermic peaks of bulk lipids

indicate that the starting materials were crystalline. As

observed in Fig. 3, the thermograms of all investigated

SLN systems did not show the melting peak of MLX,

indicating the conversion of crystalline MLX to the

amorphous form, which could be attributed to complete

dissolution of the drug in the molten lipid matrix. The

melting points of Geleol, Compritol 888 ATO, and

Precirol ATO5 in the SLN form were depressed, showing

a slight shift toward the lower temperatures when

compared with the corresponding bulk lipids. This

Figure 2

SLN4 SLN10 SLN16

SLN22 SLN28 SLN34

Transmission electron micrographs of meloxicam solid lipid nanoparticles.


melting point depression could be due to the small

particle size (nanometer range), the high specific surface

area, and the presence of a surfactant. In other words, the

depression can be attributed to the Kelvin effect [4].

Kelvin realized that small, isolated particles would melt at

a temperature lower than the melting temperature of

bulk materials. In the same way, the melting enthalpy

values of different lipids in SLN formulations showed

drastic depression compared with those of their bulk

lipids. These lower melting enthalpy values should

suggest a less-ordered lattice arrangement of the lipid

within the nanoparticles compared with those of the bulk

materials [13]. For the less-ordered crystalline or

amorphous state, the melting of the substance requires

less energy compared with the perfectly crystalline

substance, which needs to overcome the lattice force.

Rheological study

The rheological properties of MLX SLNs were presented

by plotting the shear stress (SS) versus the shear rate

(SR) (flow curves) and the viscosity versus the shear rate

(viscosity curves) curves [29,30]. The rheograms of

selected different SLN formulations are shown

in Fig. 4. As shown from the continuous shear rheometry,

SLN dispersions revealed a non-Newtonian flow. The

viscosity of non-Newtonian fluids changes according

to the shear rate, that is, has no constant viscosity [31].

This flow was characterized by the shear-thinning

behavior, in which the viscosity of the SLN dispersions

decreased with an increase in the shear rate. At present,

shear-thinning materials are also considered thixotropic,

because it always takes time, even though limited, to

regroup the microstructural elements [32]. In addition,

the type of lipid affected the viscosity of the final

product. For both surfactants used, Geleol SLNs showed

lower viscosities compared with Precirol and Compritol

SLNs.

In-vitro release studies

To compare the drug release profile from the prepared

SLN formulations, the release efficiency (RE%) after

48 h was used. The data clearly showed that the release of

the drug from the investigated SLN formulations can

be influenced by the type and concentration of the

surfactant, in addition to the nature and concentration of

lipid matrix used. Some formulations of Tween 80 and

Cremophor RH40 SLNs were selected, representing

those of highest and lowest surfactant and lipid

concentrations. The selected formulations of Tween 80

SLNs were SLN1, SLN4, SLN6, SLN7, SLN10, SLN12,

SLN13, SLN16, and SLN18, whereas those of Cremo-

phor RH40 SLNs were SLN19, SLN22, SLN24, SLN25,

SLN28, SLN30, SLN31, SLN34, and SLN36. The

percentage of MLX released during B48 h ranged from

Figure 3

Hea

t flo

w (

W/g

)

Hea

t flo

w (

W/g

)

Temperature (oC) Temperature (oC)

(a) (b)

Differential scanning calorimetry thermograms of pure meloxicam (MLX), bulk lipids (Geleol, Compritol, and Precirol), and MLX solid lipidnanoparticles (SLNs) using. (a) Tween 80 and (b) Cremophor RH40.


Figure 4

0

20

40

60

80

100

0

SS (dyne/cm2)

SR (

sec-1

)

upward curvedownward curve

0

10

20

30

40

50

0

SR (sec-1)

Vis

cosi

ty (

cP)

upward curve

downward curve

0

20

40

60

80

100

0

SS (dyne/cm2)

SR (

sec-1

)


0

10

20

30

40

50

60

SR (sec-1)

Vis

cosi

ty (

cP)

upward curve

downward curve

0

20

40

60

80

100

0

SS (dyne/cm2)

SR (

sec-1

)


010203040506070

SR (sec-1)

Vis

cosi

ty (

cP)

upward curve

downward curve

0

20

40

60

80

100

120

0

SS (dyne/cm2)

SR (

sec-1

)


0

10

20

30

40

50

SR (sec-1)

Vis

cosi

ty (

cP)


0

20

40

60

80

100

120

0

SS (dyne/cm2)

SR (

sec-1

)


0

10

20

30

40

50

60

SR (sec-1)

Vis

cosi

ty (

cP)

Vis

cosi

ty (

cP)


0

20

40

60

80

100

120

0

SS (dyne/cm2)

SR (

sec-1

)


0

10

20

30

40

50

SR (sec-1)


SLN4

SLN10

SLN16

SLN22

SLN28

SLN34

20015010050

400300200100

25020015010050

600400200

300200100

20015010050

15010050

0 15010050

0 15010050

0 15010050

0 15010050

0 15010050

Rheograms of meloxicam solid lipid nanoparticles (SLNs). SR, shear rate; SS, shear stress.


29.42 (SLN18) to 76.61% (SLN4) in case of Tween 80

SLNs and from 29.33 (SLN31) to 72.72% (SLN28) in

case of Cremophor RH40 SLNs (Fig. 5). Interestingly,

the amount of surfactant used had a great influence on

the release pattern of SLNs. Increasing the surfactant

concentration from 0.5 to 5% (w/w) led to an increase in

the percentage of MLX released and the RE% (Po0.05)

(Fig. 5 and Table 4).

The fast or rapid release and higher release efficiency

observed at higher surfactant concentrations could be

explained by the partitioning of the drug between the

melted lipid phase and aqueous surfactant phase during

particle production. During particle production by the

hot homogenization technique, the drug partitions from

the liquid oil phase to the aqueous water phase. The

amount of drug partitioning to the water phase will

increase with the increase of drug solubility in the water

phase as a result of increasing the temperature of

the aqueous phase and surfactant concentration. Higher

the temperature and surfactant concentrations, greater

is the solubility of the drug in the water phase. During

cooling of the produced O/W nanoemulsion, the solubility

of the drug in the water phase decreases continuously

with decrease in the temperature of the water phase,

which implies a repartitioning of the drug into the lipid

phase. When reaching the recrystallization temperature

of the lipid, a solid lipid core starts forming, including the

drug that is present at this temperature in this lipid

phase. Reducing the temperature of the dispersion

further increases the pressure on the drug because of

its reduced solubility in water to further repartition into

the lipid phase. The already crystallized core is not

accessible anymore for the drug; consequently, the drug

concentrates in the still liquid outer shell of the SLN

and/or on the surface of the particles. The amount of drug

in the outer shell is released relatively rapidly, whereas

the drug incorporated into the particle core is released

gradually [33].

As regards the type of lipid matrix, the results clearly

showed that among the glycerides used, the highest

release was achieved with Geleol when compared with

Compritol and Precirol. Being the lipid of highest

monoglyceride content, Geleol showed the highest

release efficiency and consequently lower t50%. In case

of Compritol and Precirol, the relatively slow release and

higher t50% can be attributed to the hydrophobic long

chain fatty acids of the triglycerides that retain the

lipophilic drug, resulting in a more sustained re-

lease [23,34]. This effect was evident in Tween 80

SLN formulations, whereas in case of Cremophor RH40

SLNs the difference between the three lipids was less

pronounced (Fig. 5 and Table 4).

The results also indicate the effect of lipid concentration

on SLNs’ release profile: increasing the lipid concentra-

tion from 5 to 10% (w/w) resulted in a corresponding

decrease in the percentage of MLX released and a

consequent increase in t50% for Tween 80 and Cremophor

RH40 SLNs (Fig. 5 and Table 4). However, in case of

Geleol SLNs (SLN1, SLN6, SLN19, and SLN24), a

slight increase in RE% was observed (Table 4). This

observed decrease in the release profile can be attributed

to the higher lipid content encapsulating the drug, thus

reducing drug partition in the outer phase and conse-

quently its release in the receiver media. The release

profiles of these SLNs resemble the drug-enriched core

model [35]. In such a model, the drug-enriched core is

surrounded by a practically drug-free lipid shell. Because

of the increased diffusional distance and hindering effects

by the surrounding solid lipid shell, the drug has a

sustained release profile.

The release pattern of the drug from all SLN formula-

tions followed the Higuchi’s equation. The R2 values

Figure 5

0102030405060708090

100(a)

(b)Times (h)

% M

LX r

elea

sed

SLN1SLN4SLN6SLN7SLN10SLN12SLN13SLN16SLN18

0102030405060708090

100

0 10 20 30 40 50 60

0 10 20 30 40 50 60Times (h)

% M

LX r

elea

sed

SLN19SLN22SLN24SLN25SLN28SLN30SLN31SLN34SLN36

The release profile of meloxicam (MLX) from solid lipid nanoparticles(SLNs) using (a) Tween 80 and (b) Cremophor RH40 as surfactants.

Table 4 Release efficiency and t50% (h) of the selected

meloxicam solid lipid nanoparticles formulations

Surfactants

Tween 80 Cremophor RH40

Formulasa RE 48 (%) t50% (h) Formulaa RE 48 (%) t50% (h)

SLN1 32.42 ± 1.28 33.72 SLN19 33.74 ± 2.50 33.10SLN4 55.55 ± 1.70 18.73 SLN22 50.18 ± 1.70 23.28SLN6 38.93 ± 4.37 43.46 SLN24 37.08 ± 4.31 44.17SLN7 26.62 ± 0.72 56.19 SLN25 34.97 ± 0.89 39.39SLN10 39.98 ± 1.82 25.12 SLN28 48.00 ± 0.88 20.65SLN12 20.58 ± 1.98 92.44 SLN30 24.67 ± 1.52 68.74SLN13 29.05 ± 1.77 47.14 SLN31 20.78 ± 1.51 117.54SLN16 51.08 ± 0.77 20.14 SLN34 48.95 ± 3.49 24.03SLN18 21.58 ± 2.57 132.75 SLN36 23.35 ± 3.23 45.501

RE, release efficiency; SLN, solid lipid nanoparticle; t50% (h), timerequired to release 50% of the drug.aSee Tables 1 and 2 for the description of the formulations.


ranged from 0.9151 to 0.9977 in case of Tween 80 and

from 0.9115 to 0.9984 in case of Cremophor RH40. This

result is generally in agreement with many studies that

reported that drug-loaded SLNs provide a controlled release

pattern following Higuchi’s square root model [36,37].

ConclusionIn this study, the MLX-loaded SLNs were successfully

prepared using modified high-shear homogenization and

ultrasound techniques. Physicochemical characterization

revealed that the prepared drug-loaded SLNs were of

spherical shape and homogenously distributed. The DSC

analysis showed the amorphous state of MLX in SLNs.

SLNs achieved high drug incorporation with small-sized

particles (nanosize) and showed shear-thinning rheologi-

cal behavior. The in-vitro release behavior was greatly

affected and can be controlled by optimizing the

compositional variables. The sustained release behavior

of MLX-loaded SLNs together with the favorable

physicochemical characteristics supports that SLNs are

promising delivery systems for poorly water-soluble drugs

such as MLX and can form a foundation for further

clinical studies for the topical delivery of MLX.


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Effect of pollution on the chemical content and secondary

metabolites of Zygophyllum coccineum and Tamarix niloticaHanan E. Osmana and Reham K. Badawyb

aDepartment of Plant and Microbiology, Facultyof Science (Girls Branch), Al-Azhar University andbEnvironmental Pollution Unit, Department of PlantEcology and Range Management, Desert ResearchCenter, Cairo, Egypt

Correspondence to Hanan E. Osman, PhD,Department of Plant and Microbiology, Facultyof Science (Girls Branch), Al-Azhar University,Nasr City 11651, Cairo, EgyptTel: + 20 22633998; fax: + 20 22633101;e-mail: [email protected]

Received 4 November 2012Accepted 14 February 2013


2013,12:73–82

Objectives

This study investigated the uptake and translocation pattern of trace metals from two

medicinal plant species namely: Zygophyllum coccineum and Tamarix nilotica from two

contaminated sites and a noncontaminated (NC) site. The effects of heavy metals on

the amino acids and secondary metabolites of the tested plant species were assessed.


Medicinal plant samples and soil samples were collected from three different sites: two

contaminated and one NC site. The concentration levels (mg/kg) of the selected trace

metals (Al, B, Cr, Cu, Fe, Mn, Mo, Pb, V, and Zn) were estimated in the tested plant

species and associated soil.

Results

Heavy metal contents in the investigated plant species reflected the metal

concentration in the soil samples. The highest content of the determined heavy metals

were detected in both tested plants from contaminated sites in comparison with those

from the NC site.

The concentrations of free amino acids in T. nilotica and Z. coccineum plants from the

contaminated sites were higher compared with those in plants from the NC site. Moreover,

the concentration of free amino acids in plants from the wastewater-contaminated sites

was higher compared with that in plants from the Suez industrial emission site.

The content of secondary metabolites (tannins, saponins, and alkaloids) was

decreased in plants from polluted sites compared with those from the NC site. The

concentration of tannins ranged from 0.07 to 0.33 g, saponins from 9.99 to 8.22%,

and alkaloids from 7.95 to 1.00%. Moreover, the maximum tannins and alkaloid content

was detected in Z. coccineum from the noncontaminated site.

Conclusion

The plants collected from the investigated sites pose a serious danger. However, a

periodical assessment of plants used for traditional medicine should be encouraged as

this will assist in ensuring their quality and safety in herbal use, especially for people

living in urban areas where the level of pollution may be very high.

Keywords:

free amino acid, heavy metals, medicinal plant, secondary metabolites, Tamarix nilotica,

Zygophyllum coccineum


IntroductionMedicinal plants are widely used as home remedies and

raw materials for pharmaceutical industries. The past

decade has seen a significant increase in the use of herbal

medicine. The environmental conditions in developing

countries; pollution in irrigation water, atmosphere, and

soil; sterilization methods; and storage conditions all play

an important role in the contamination of medicinal

plants by pesticides and heavy metals. The sources of

environmental pollution with toxic metals are quite

varied, ranging from industrial and traffic emissions to

the use of purification mud and agricultural expedients,

such as cadmium-containing dung, organic mercury

fungicides, and the insecticide lead arsenate [1].

Heavy metal contamination in agricultural environments

can result from an atmospheric fallout, pesticide for-

mulations, contamination by chemical fertilizers, and

irrigation with water of poor quality [2]. Heavy metals

rank high among the chief contaminants of leafy

vegetables and medicinal plants [3].

Uptake of trace elements by plants varies and depends

largely on several factors such as soil pH and organic matter

content. Plant uptake is one of the major routes of exposure

of the food chain to trace elements in the soil [4].

Trace elements play an important role in the chemical,

biological, metabolic, and enzymatic reactions in the

living cells of plants, animals, and human beings [5].

However, the release of trace metals through human

activities into the environment has increased over the

years, and the excess of these metals in the environment

has been reported to be extremely dangerous to human

health [6]. The accumulation of trace metals by plants is

Original article 73


DOI: 10.7123/01.EPJ.0000428268.89779.59

one of the most serious environmental concerns. This is

as a result of the harmful effects of toxic metals on animal

and human health [7].

Evidence of severe poisoning caused by some metal

compounds and the proven carcinogenicity of some metal

ions has fostered intensive research into the different

uptake and translocation patterns in food crops [8]. The

broad use of traditional medicines by rural communities

because of the accessibility and affordability of herbal

medicine has also necessitated a further research into the

uptake and translocation pattern of trace metals by some

medicinal plants from urban areas [3].

Zygophyllum coccineum belongs to the Zygophyllaceae

family. The leaves, stems, and fruits of this plant are

used in folk medicine as a drug active against rheuma-

tism, gout, asthma, and hypertension. It is also used as a

diuretic, local anesthetic, antihistaminic, and antidiabetic

agent [9].

Several species of plants belonging to the genus Tamarix(Family: Tamaricaceae) have been used in traditional

medicine. Antioxidant and antimicrobial activities of T.hispida [10] and T. aphyla [11] have also been described.

Tamaricaceous plants produce a unique class of hydro-

lysable tannins with diverse structures [12].

The environmental conditions, atmosphere, pollution,

soil, and harvesting and handling are some of the factors

that may play important roles in the contamination of

medicinal plants by metals and microbial growth [3]. It is

therefore of major interest to evaluate the composition

of some metallic elements in herbal plants, because at

elevated levels, these metals can be dangerous and

toxic [13,14].

Although some trace metals may have both curative and

preventive roles in combating diseases, it has been

established that an overdose or prolonged ingestion of

medicinal plants may lead to chronic accumulation of

different elements that may cause various health

problems [15].

The overall objectives of this research were to determine

the concentrations of the 10 tested heavy metals in

Tamarix nilotica and Z. coccineum plant biomass from

contaminated and noncontaminated (NC) sites and to

determine the effect of heavy metal contamination from

industrial emissions or by wastewater irrigation on the

content of secondary metabolites and amino acids of both

tested plant species.

Materials and methodsSite description

This study was carried out at three sites: two contami-

nated and one NC. The NC site was located at Sokhna

Road, 35 km from Cairo governorate.

The first contaminated site is a wastewater-contaminated

(WWC) site near the domestic wastewater channel. This

site is located at El-Saff, Cairo governorate, which is

south of the industrial complex of Helwan (including the

Iron and Steel Factory and Weaving, Coke, and fertilizer

industries). These industrial activities produce large

amount of wastes that are usually dumped into an

artificial canal extending over a large area behind the

factories. The source of irrigation in this site is the

sewage effluent, which comes from the sewage treatment

station at Helwan since the past 23 years (according to

the report of the committee preparing the Egyptian code

for reuse of wastewater, 2004). The second contaminated

site, the Suez industrial emission (SIE) contaminated

site (SEC), is located near the fertilizer and ceramic

factories in Ain Sokhna, Suez governorate. The fertilizer

plant of the Egyptian Fertilizers Company (EFC)

manufactures granulated urea.

Soil and plant sampling

During June 2009, Z. coccineum and T. nilotica plant

samples, based on their coverage at the site, together

with the associated soil samples were collected. The

tested medicinal plants were collected from their natural

habitats. The plants were not exposed to any agricultural

treatments. Five random samples were collected from

each site to obtain a comprehensive profile of the site for

statistical analysis.

The soil samples were collected from a depth of 0–60 cm.

The collection of plant samples was based on plant

coverage at the site and plant health.

Soil and plant analysis

Soil samples were air dried at room temperature and then

sieved using a 2-mm stainless steel sieve. The soil : water

extracts (1 : 2.5) were prepared and used in the

determination of pH, electrical conductivity, and cationic

and anionic compositions according to the methods

described by Richards [16] and by Jackson [17]. The

total carbonates were determined according to the

methods described by Piper [18]. The organic matter

was determined according to the method described by

Nelson and Sommers [19]. The available nitrogen in

the soil was extracted using a solution of 2 mol/l KCl

according to the method described by Keeney and

Nelson [20]. The available phosphorus was extracted

using a solution of 0.5 mol/l NaHCO3, pH 8.5, according

to the method described by Watanabe and Olsen [21].

The soil samples were analyzed for the total content of

the studied elements in the filtered soil extracts obtained

from samples digested by HNO3, H2SO4, and 60%

HClO4, as outlined by Hesse [22]. Total tested heavy

metals were determined by inductively coupled plasma

optical emission spectrometry (ICP).

The plant samples were washed with distilled water to

remove any adhering soil. After washing, the plant

samples were oven dried at 651C and then ground to a

powder. The plant samples were digested with H2O2 and

H2SO4 [23] and then subjected to analysis of nitrogen

and phosphorus. The nitrogen content was determined

using a modified Micro-Kjeldahl method, as described by

Peach and Tracey [24]. The phosphorous content was


determined according to the method described by

Rowell [25]; this method depends on the formation of a

blue complex between phosphate and ammonium molyb-

date in the presence of ascorbic acid (reducing agent). The

samples were measured with a spectrophotometer at an

absorbance of 880 nm. The plant samples were analyzed

for the total content of the studied elements using the

digested extracts, which were obtained with 0.5 g of

concentrated HNO3 and H2O2 [26]. The heavy metal

content in all the samples was determined by aspirating

directly to ICP. The alkaloid content was determined

according to the method described by Jenkins et al. [27].

The saponin content was determined according to the

method described by Wall et al. [28]. The tannin content

was determined according to the method described by

Claus [29]. The free amino acid content was determined

according to the method described by Block et al. [30].

Metal translocation factor

The root-to-shoot translocation factor (TF) was de-

scribed as the ratio of heavy metals in the plant shoot

to that in the plant root [31]. The TF is determined

according to the equation: BF = C [HM in shoot]/C [HM

in root].

Statistical analysis

The experiment was laid out in a randomized complete

block design with three replications. There were two

factors in the study: three sites (NC, WWC, and SEC)

and two types of plant species (Z. coccineum and T. nilotica).

Data were subjected to analyses using M-STATC., as

described by Russell [32]. The mean values were

compared using the Duncan New Multiple range test

as described by Waller and Duncan [33]. Mean values

indicated by the same alphabetical letters in the same

column are not significantly different at P = 0.05.

The data on the TF, alkaloid content, tannin content,

and saponin content of the samples were presented as

mean ± SD of the three replicates and were analyzed

using Excel 2007 for Windows.

Results and discussionSoil properties and heavy metal concentrations

Chemical properties of the soil from the three tested sites

are presented in Table 1. The data shows that salinity of

the saturated extract from the soil, as evidenced by the EC

values, was very high in soil from the WWC site

(11.28 mMho). The values of soil pH ranged from 8.83 in

the soil from the WWC site to 8.71 in that from the

industrial emission site, indicating that the soils are alkaline

in these locations. The soil from the NC site was slightly

alkaline with a pH of 7.97. Schipper et al. [34] reported that

after long-term wastewater irrigation, the soil pH increased

and that this may be due to the high content of cations

such as Na, Ca, and Mg in the wastewater.

The organic matter content was high in the soil from the

contaminated sites; it was 1.24% at the WWC site and

0.69% at the SIE site compared with 0.43% at the NC

site. The cationic composition of the total salts is mostly

dominated by Na + , followed by Ca2 + and Mg2 + , and

then by K + . The most dominant anion was SO42 – ,

followed by Cl – , and then by HCO3. The highest OM,

Ca2 + , Mg2 + , Na2 + , K + , Cl – , and SO4– concentrations

were detected in the WWC sample, whereas the highest

HCO3 content was detected in the SIE sample.

Accumulation of K in the soil with wastewater application

was attributed to the original content of this nutrient in

the wastewater applied [35]. Irrigation with wastewater

increased the total cation concentration of Ca and

Mg [36].

As shown in Table 2, the available N and P content in the

soil samples from the contaminated sites is significantly

higher compared with those from the NC site as a result

of contamination with wastewater at the WWC site and

Table 1 Electrical conductivity (EC), pH, concentration organic matter content (OM) and some anions and cations (mEq/l) in the

studied soil samples from the noncontaminated (NC) site, El-Saff wastewater-contaminated (WWC) site, and Suez industrial

emission (SIE) site

Cations (mEq/l) Anions (mEq/l)

Sites EC (mMho) pH OM (%) Ca2 + Mg2 + Na + K + CO32 – HCO3

– Cl– SO42 –

NC 1.206 7.97 0.43 5.5 3 3.65 1.01 0 0.8 3.125 9.235WWC 11.28 8.83 1.24 32 22 140.50 2.05 0 0.8 86.25 109.5SIE 0.468 8.71 0.69 3 2.5 9.90 0.75 0 1.2 1.25 13.7

Table 2 Interaction effects of the site and plant species on nitrogen, phosphorus, and heavy metal contents (mg/kg) of the studied

soil samples from the noncontaminated (NC) site, El-Saff wastewater-contaminated (WWC) site, and Suez industrial emission (SIE)

site

Site Plant N P Al B Cr Cu Fe Mn Mo Pb V Zn

NC T. nilotica 24.4 d 4.6 b 449.4 d 51.1 d 23.0 d 32.4 d 163.4 c 22.8 c 1.4 d 21.7 b 3.3 b 25.5 bZ. coccineum 21.0 d 2.7 b 368.0 d 42.8 d 17.3 d 35.8 d 119.5 c 30.4 c 1.6 d 21.7 b 3.9 b 25.3 b

WWC T. nilotica 65.1 c 5.5 b 3221.0 b 111.7 b 54.8 a 117.9 a 1533.5 a 131.0 ab 4.1 c 123.3 a 16.3 a 122.5 aZ. coccineum 194.5 b 4.9 b 3493.1 a 128.7 a 49.2 ab 112.3 a 1215.1 b 127.6 ab 5.0 b 121.9 a 15.1 a 124.6 a

SIE T. nilotica 228.9 a 18.8 a 2306.6 c 94.4 c 36.8 c 96.2 b 1264.3 ab 116.6 b 6.1 a 113.9 a 12.6 a 135.2 aZ. coccineum 34.9 d 5.0 b 2303.3 c 103.3 bc 41.6 bc 83.9 c 1510.7 a 141.3 a 5.3 b 115.6 a 14.3 a 125.8 a

Mean values for each column having common letters are not significantly different at the 0.05 level.

Pollution effect on two wild plants Osman and Badawy 75

with fertilizer factory effluent at the SIE site. These

elements are essential nutrients for plant growth.

Heavy metal contents of the three sites are represented

in Table 2. The total heavy metal contents were increased

significantly many folds in the samples from the

contaminated sites compared with those from the NC

site. Heavy metal concentrations of the contaminated

sites were increased by 8.21, 2.56, 2.58, 3.38, 9.72, 4.86,

3.14, 5.65, 4.36, and 4.86 times at the WWC site, whereas

they were increased by 5.64, 2.11, 1.95, 2.64, 9.81, 4.84,

3.94, 5.29, 3.74, and 5.14 times at the SIE site for Al, B,

Cr, Cu, Fe, Mn, Mo, Pb, V, and Zn, respectively compared

with the NC site.

The results show a great variability in the heavy metal

content according to site of plant collection. The

maximum concentrations of Al, B, Cr, Cu, Mn, Pb, and

V were found at the WWC site: significantly for Al, B, Cr,

and Cu and nonsignificantly for Mn, Pb, and V. Mean-

while, the maximum but not significant concentrations of

Fe, Mo, and Zn were detected in plants from the SIE site.

Soils, especially those found in or near the metalliferous sites

and metal smelters, are highly contaminated with heavy

metals, including Cd, Cr, Cu, Pb, Ni, and Zn. For example,

soils sampled from a former Zn/Cd smelter site contained up

to 99 500 mg/kg Zn in addition to 1005–7220 mg/kg Pb,

2500–4500 mg/kg Cu, and 28–578 mg/kg Cd [37].

Heavy metal concentrations in plants

Metal concentrations in plants vary with plant spe-

cies [38]. Plant uptake of heavy metals from soil occurs

either passively with the mass flow of water into the roots

or through active transport across the plasma membrane of

root epidermal cells. Under normal growing conditions,

plants can potentially accumulate certain metal ions an order

of magnitude greater than the surrounding medium [39].

The plant species has a considering effect on the heavy

metal content in both roots and shoots of T. nilotica and Z.coccineum plants. The contents of Al, B, and Fe in T. niloticaroots and those of Al, B, Cr, Cu, Fe, Pb, and Zn in T.nilotica shoots were significantly higher compared with

those in Z. coccineum roots and shoots, respectively.

Meanwhile, the contents of Cu, Mn, and Zn in Z.coccineum roots were higher compared with those in

T. nilotica roots (Figs 1 and 2). The contents of B, Cr,

Mo, and V and Mn, Mo, and Zn in roots and shoots,

respectively for both plants were the same.

The effect of the site on the heavy metal concentrations

in both T. nilotica and Z. coccineum plants are depicted

in Figs 3 and 4. The results showed that, in most cases,

the concentrations of the tested heavy metals in plants

from the WWC site were significantly higher compared

with those in plants from the SIE site. The increase in Al,

B, Cr, Cu, Fe, Mn, Mo, Pb, V, and Zn concentrations was

7.74, 3.10, 4.36, 3.81, 4.17, 7.42, 4.22, 9.30, 6.10, and 5.30-

fold, respectively in plant shoots from the WWC site and

was 6.57, 1.96, 3.39, 2.73, 3.91, 5.35, 6.31, 7.35, 5.55, and

4.39-fold, respectively in plants from the SIE site

compared with that in plants from the NC site.

Figure 2

Effect of plant species on shoot heavy metal content (mg/kg) of Tamarixnilotica and Zygophyllum coccineum. Values followed by differentletters within columns are significantly different at the 0.05 probabilitylevel.

Figure 3

Effect of the site on shoot heavy metal content (mg/kg) of Tamarixnilotica and Zygophyllum coccineum. Values followed by differentletters within columns are significantly different at the 0.05 probabilitylevel.

Figure 1

Effect of plant species on root heavy metal content (mg/kg) of Tamarixnilotica and Zygophyllum coccineum. Values followed by differentletters within columns are significantly different at the 0.05 probabilitylevel.


On comparing the two contaminated sites, mostly there

was a significant increase in the determined heavy metal

content in plants from the WWC site compared with

plants from the SIE site (Figs 3 and 4).

The data in Table 3 shows the interaction effect of the

plant species and site on the tested heavy metal contents

for T. nilotica and Z. coccineum. The high heavy metal

contents for both roots and shoots, mostly, were detected

in plants from the WWC site.

The content of heavy metals in industrialized regions were

determined by Januz et al. [40], who reported that the plants

growing in an industrialized region have higher contents of

heavy metals compared with plants growing in a second less

industrialized region. Some metals such as Cu, Mn, and

Zn are the natural essential components of enzymes and

coenzymes and are important for growth, photosynthesis,

and respiration, although other metals such as Pb and Cd

have no biochemical or physiological importance, therefore

they are considered as very toxic pollutants.

Although the concentrations of the tested heavy metals

in soils at contaminated sites were above the critical

concentrations in soil for these elements [41], no visual

phytotoxicity symptoms on both tested plants were

observed.

The Al, Cr, Cu, Fe, Mn, Mo, and Pb concentrations were

all above the normal range for roots and shoots of both

tested plants from the contaminated sites, whereas the

concentrations of B and Zn were within the permissible

level (Table 3).

The variation in the elemental content from plant to

plant is mainly attributed to the differences in the

botanical structure and mineral composition of the soil in

which the plants are cultivated. Other factors responsible

for a variation in the elemental content are preferential

absorbability of the plant, use of fertilizers, irrigation

water, and climatic conditions [38].

Translocation factor of heavy metals

A plant’s ability to translocate metals from the roots to

shoots is measured using the TF, which is defined as the

ratio of metal concentration in the shoots to that in the

roots. The TF index showed that the both tested plant

species most efficiently translocated the tested heavy

metals to the shoot system. The mean TF (average TF

values for each metal in different sites for both tested

plants) values revealed that T. nilotica showed great

efficiency for translocating metals from the roots to

shoots. The TF values for T. nilotica for all tested metals

under study were higher than 1, except for B and V

(Figs 5 and 6). The trends of the TF values for heavy

metals in T. nilotica were in the order of Cr4Cu4Mo4Fe4Pb4Zn4Mn4Al4V4B. Meanwhile, Z. coccineumhad a TF higher than 1 for Cr, Cu, Pb, and V. The results

in Figs 5 and 6 show that TF of Z. coccineum for these

considered metals were in the order of

Cr4Cu4Pb4V4Zn4Fe = Mo4Al = B4Mn. A TF

higher than 1 indicated a very efficient ability to transport

metals from the roots to shoots, most likely due to

efficient metal transport systems [43].

Figure 4

Effect of the site on root heavy metal contents (mg/kg) of Tamarixnilotica and Zygophyllum coccineum. Values followed by differentletters within columns are significantly different at the 0.05 probabilitylevel.

Table 3 Interaction effect of the site and different plant species on heavy metal contents (mg/kg) in roots and shoots of T. niloticaand Z. coccineum plants from the noncontaminated (NC) site, El-Saff wastewater-contaminated (WWC) site, and Suez industrial

emission (SIE) site

Site Plant Al B Cr Cu Fe Mn Mo Pb V Zn

RootNC T. nilotica 84.83 d 24.37 b 1.29 c 19.68 c 113.6 d 8.32 d 0.33 d 6.92 c 0.75 d 21.64 d

Z. coccineum 104.00 d 24.12 b 1.66 c 23.84 c 161.1 c 11.7 d 0.37 d 6.88 c 1.47 d 32.25 cWWC T. nilotica 644.20 a 67.26 a 6.02 a 58.88 b 564.8 a 82.97 a 1.84 b 82.24 a 8.49 a 58.23 b

Z. coccineum 545.10 b 44.28 ab 5.19 b 74.65 a 494.4 b 85.28 a 1.39 c 86.38 a 7.59 b 62.21 abSIE T. nilotica 438.80 c 43.75 ab 5.65 ab 55.55 b 556.2 a 41.08 c 1.7 b 81.25 a 6.62 c 56.55 b

Z. coccineum 402.20 c 33.78 b 5.01 b 68.31 b 502.9 b 52.32 b 2.45 a 60.54 b 6.55 c 69.80 aShootNC T. nilotica 77.80 e 15.49 d 1.47 c 20.38 e 131.6 d 10.71 d 0.45 e 8.55 d 0.74 e 11.39 d

Z. coccineum 58.92 e 19.11 d 1.77 c 16.00 e 139.30 d 8.17 d 0.26 f 9.04 d 1.62 d 11.79 dWWC T. nilotica 570.00 a 66.18 a 8.70 a 79.72 a 655.8 a 69.28 a 1.37 d 88.89 a 7.60 a 67.84 a

Z. coccineum 488.20 c 41.04 b 5.42 b 58.79 c 474.50 c 67.54 a 1.67 c 74.84 b 6.71 bc 55.05 bSIE T. nilotica 530.5 b 31.79 c 5.73 b 63.88 b 559.5 b 43.27 c 2.39 a 71.84 b 6.92 ab 53.88 b

Z. coccineum 368.30 d 35.97 bc 5.24 b 35.52 d 498.90 c 55.30 b 2.15 b 57.43 c 6.55 c 47.98 cPL 135a 14–78a 5b 1.1–33.1a 450b 44.25b Up to 1a 0.3–18.8a – 6–126a

PL, permissible limits according to Kabata Pendias & Pendias [41]a and FAO/WHO [42]b standards for metal concentrations in consumablevegetables and edible parts.Mean values for each column having common letters are not significantly different at the 0.05 level.


The mean TF for the tested heavy metals ranged from 0.62

to 1.21 and 0.83 to 1.21 for T. nilotica and Z. coccineum,

respectively. According to Baker [44], there are three basic

types of tolerance strategies to heavy metals (accumulation,

exclusion, and indication), which describe the relationship

between the total soil and plant metal concentration and

that excluder and accumulator plants could grow together

in the same environment. The relationships between the

soil and plant metal concentrations should be thoroughly

tested for each plant species separately to understand the

physiological mechanisms.

Accumulation and exclusion are two basic strategies by

which plants respond to elevated concentrations of heavy

metals [45]. In metal accumulator species, TFs greater

than 1 were common, whereas in metal excluder species

the TFs were typically lower than 1 [44].

Nitrogen and phosphorus content in plants

Nitrogen (N) is the essential mineral element required

in the greatest amount by plants, comprising 1.5–2% of

plant dry matter [46]. Phosphorus (P) is the second

nutritional element after nitrogen that limits plant growth,

having a concentration of about 0.2% of the total plant dry

weight [47]. P is a macronutrient that is a key component

in many molecules (i.e. nucleic acids, phospholipids, and

ATP) that participates in basic plant processes [48].

The concentration of nitrogen and phosphorus were

significantly higher in tested plants from the contami-

nated sites compared with those from the NC site. The

highest content of N was detected in plants from the

WWC site, whereas the highest P content was detected

in plants from the SIE site (Fig. 7).

Amino acid contents

Under heavy metals stress, plants exhibit a number of

physiological changes in their cells [49,50]. Several

mechanisms allow plants to tolerate the presence of

Figure 7

Interaction effect of the site [noncontaminated (NC) site, El-Saff wastewater-contaminated (WWC) site, and Suez industrial emission (SIE) site] andplant species (Tamarix nilotica and Zygophyllum coccineum) on nitrogen and phosphorus contents (ppm) in plants. Values followed by differentletters within columns are significantly different at the 0.05 probability level.

Figure 6

Translocation factors with SDs of Al, B, Cr, Cu, Fe, Mn, Mo, Pb, V, andZn in Zygophyllum coccineum plants from the noncontaminated (NC)site, El-Saff wastewater-contaminated (WWC) site, and Suez industrialemission (SIE) site. Error bars represent ± SE of the mean values forthree separate plant extractions.

Figure 5

Translocation factors with SDs of Al, B, Cr, Cu, Fe, Mn, Mo, Pb, V, andZn in Tamarix nilotica from the noncontaminated (NC) site, El-Saffwastewater-contaminated (WWC) site, and Suez industrial emission(SIE) site. Error bars represent ± SE of the mean values for threeseparate plant extractions.


heavy metals inside the cells, and synthesis of phyto-

chelatins has been particularly concerned, as phytoche-

latins may chelate heavy metals, leading to detoxification

of these metals in cells [51]. The interaction of heavy

metals with sulfhydryl-containing amino acids and pep-

tides/proteins plays a major role in their environmental

and biochemical behavior [52].

Sixteen types of amino acids were detected in the shoots

of the tested plant species from the three sites (NC,

Table 4 Mean free amino acid (FAA) contents of Tamarix nilotica and Zygophyllum coccineum from the noncontaminated (NC) site,

El-Saff wastewater-contaminated (WWC) site, and Suez industrial emission (SIE) site

NC WWC SIE

FAA (%) T. nilotica Z. coccineum T. nilotica Z. coccineum T. nilotica Z. coccineum

Aspartic Acidic 0.3109 0.3637 0.4752 0.6929 0.3893 0.6369Glutamic 0.3549 0.4244 0.6011 0.9076 0.4433 0.6742Histidine Alkali 0.1642 0.2343 0.2725 0.3422 0.2005 0.2515Arginine 0.1828 0.2866 0.3877 0.3321 0.4278 0.2527Lysine 0.1658 0.2139 0.2912 0.4333 0.2768 0.2422Threonine Neutral 0.1224 0.1481 0.2433 0.3333 0.1931 0.1568Serine 0.1446 0.1756 0.2816 0.3608 0.1926 0.2127Proline 0.4923 0.4405 0.6181 0.8159 0.5669 0.6279Glycine 0.1314 0.1756 0.2669 0.2898 0.2017 0.2086Alanine 0.1796 0.1914 0.2899 0.4014 0.2510 0.2735Valine 0.1193 0.1455 0.2327 0.3260 0.2070 0.1451Methionine 0.0005 0.0026 0.0005 0.0210 0.0147 0.0014Isoleucine 0.0947 0.1145 0.1336 0.2393 0.1453 0.1125Leucine 0.1860 0.2191 0.3622 0.4993 0.3056 0.2357Tyrosine 0.0543 0.0601 0.1538 0.1623 0.0868 0.0794Phenylalanine 0.1192 0.1400 0.2240 0.3264 0.1488 0.1596

Table 5 Correlation coefficients between the contents of free amino acids and heavy metals in shoots of Tamarix nilotica

Amino acid Al B Cr Cu Fe Mn Mo Pb V Zn

Aspartic 0.888 0.984 0.992* 0.958 0.930 0.996* 0.449 0.940 0.896 0.952Threonine 0.938 0.955 1.00** 0.986 0.968 0.999* 0.556 0.975 0.944 0.983Serine 0.813 0.999* 0.963 0.907 0.667 0.972 0.315 0.881 0.823 0.898Glutamic 0.818 0.999* 0.965 0.911 0.872 0.975 0.325 0.886 0.828 0.903Proline – 0.958 – 0.586 – 0.803 – 0.887 – 0.924 – 0.779 – 0.944 – 0.913 – 0.953 – 0.897Glycine 0.909 0.975 0.997* 0.971 0.947 0.999* 0.491 0.956 0.916 0.966Alanine 0.960 0.932 0.998* 0.996* 0.984 0.995* 0.613 0.988* 0.965 0.993*Valine 0.989* 0.873 0.980 0.999* 0.999* 0.971 0.716 1.00** 0.992 1.00**Methionine 0.436 – 0.202 0.103 0.260 0.343 0.064 0.881 0.315 0.420 0.280Isoleucine 0.957 0.582 0.800 0.885 0.923 0.776 0.945 0.911 0.951 0.895Leucine 0.969 0.918 0.995* 0.998* 0.989* 0.990* 0.641 0.993* 0.974 0.997*Tyrosine 0.797 1.00** 0.955 0.896 0.854 0.966 0.290 0.869 0.807 0.886Histidine 0.767 0.999* 0.940 0.873 0.827 0.952 0.243 0.844 0.778 0.863Lysine 0.802 1.00** 0.958 0.900 0.959 0.968 0.299 0.873 0.813 0.891Arginine 0.955 0.577 0.797 0.883 0.920 0.773 0.947 0.908 0.950 0.892

*Correlation is significant at the level 0.05.**Correlation is significant at the level 0.01.

Table 6 Correlation coefficients between the contents of free amino acids and heavy metals in shoots of Zygophyllum coccineum

Amino acid Al B Cr Cu Fe Mn Mo Pb V Zn

Aspartic 0.993 0.998* 0.993 0.915 0.976 0.999* 0.919 0.995 0.998* 1.00**Threonine 0.744 0.709 0.572 0.908 0.483 0.693 0.313 0.732 0.615 0.658Serine 0.835 0.806 0.687 0.960 0.608 0.792 0.450 0.825 0.725 0.762Glutamic 0.974 0.961 0.896 0.998* 0.845 0.954 0.731 0.969 0.918 0.939Proline 0.969 0.955 0.887 0.999* 0.834 0.948 0.717 0.964 0.910 0.932Glycine 0.883 0.858 0.753 0.982 0.680 0.846 0.532 0.874 0.786 0.820Alanine 0.931 0.911 0.822 0.997* 0.759 0.901 0.625 0.924 0.851 0.880Valine 0.714 0.677 0.536 0.889 0.445 0.661 0.271 0.701 0.580 0.625Methionine 0.677 0.638 0.491 0.864 0.397 0.620 0.220 0.663 0.536 0.583Isoleucine 0.706 0.669 0.526 0.884 0.434 0.652 0.260 0.693 0.570 0.615Leucine 0.752 0.717 0.581 0.913 0.493 0.701 0.324 0.739 0.623 0.666Tyrosine 0.828 0.799 0.679 0.957 0.598 0.785 0.440 0.818 0.717 0.755Histidine 0.779 0.746 0.616 0.930 0.530 0.731 0.364 0.768 0.656 0.698Lysine 0.811 0.780 0.657 0.948 0.574 0.766 0.413 0.801 0.696 0.735Arginine 0.993 0.985 0.940 0.984 0.899 0.981 0.802 0.991 0.956 0.971

*Correlation is significant at the level 0.05.**Correlation is significant at the level 0.01.


WWC, and SIE) (Table 4). Amino acids are divided into

three types (i.e. acidic, alkali, and neutral) on the basis of

their characters [53].

The concentrations of amino acids in T. nilotica and Z.coccineum plants from the contaminated sites were higher

compared with those in plants from the NC site. The

most abundant amino acid in all the plant tissues was

glutamic acid. Moreover, the concentration of amino acids

in plants from the domestic wastewater site was higher

compared with that in plants from the SIE site for both

tested plants. These results are in agreement with those

of Wu et al. [54] and of Kovacik et al. [55].

On computing correlation coefficients it was revealed that

levels of aspartic acid and threonine in shoots of T. niloticawere significantly positively correlated with their respective

Cr and Mn concentrations (Table 5). As regards the levels

of serine, glutamic acid, tyrosine, histidine, and lysine,

only boron (B) showed a positive relationship. In case of

levels of proline, methionine, isoleucine, and arginine, no

correlations were detected. Levels of valine, alanine, and

leucine were positively and significantly correlated with

more than one metal. Concentrations of Al, Cu, Fe, Pb, and

Zn; Cr, Cu, Fe, Mn, Pb, and Zn; and Cr, Cu, Mn, Pb, and

Zn, respectively were correlated with levels of valine,

leucine, and alanine, respectively.

In Z. coccineum, a significant positive correlation was

detected between levels of aspartic acid and concentra-

tion of B, Mn, V, and Zn in the shoot, whereas levels of

glutamic, proline, and alanine correlated with shoot

concentrations of Cu (Table 6).

In most agricultural soils, nitrate (NO3– ) is the most

important source of N for plants [56]. For nitrogen

metabolism, the nitrate must be taken up across the

plasma membrane. Once inside the symplast of a plant,

Figure 8

Content of secondary metabolites (alkaloids, saponins, and tannins) and fat (%) of Tamarix nilotica and Zygophyllum coccineum plants from thenoncontaminated (NC) site, El-Saff wastewater-contaminated (WWC) site, and Suez industrial emission (SIE) site. Mean values for each columnhaving common letters are not significantly different at the 0.05 level.


NO3– is reduced to NO2

– by nitrate reductase (NR), and

NO2– is converted to NH4-N by nitrite reductase. The

resulting NH4-N is then assimilated into amino acids,

nucleic acids, proteins, chlorophylls, and other metabo-

lites [57]. Factors influencing the enzymatic regulation

responsible for N assimilation include: contents of

Mo [58] and Cu [59].

The content of amino acids in shoots of T. nilotica and Z.coccineum plants from the three tested sites were in the

order of WWC4SIE4NC, in line with the nitrogen and

phosphorus concentrations in plants. The amino acid

content (acidic, alkali, and neutral amino acids) showed

an increase in plants from the WWC site compared with

those from the other sites, which may be due to an

elevation of nitrogen, phosphorus, Mo, and Cu concen-

trations in shoots of the plants (Table 3).

Cruz et al. [60] reported that activities of nitrogen

metabolism-related enzymes such as nitrate reductase are

considerably lower in a low nitrate supply compared with

a high supply of nitrates.

Mo, one of the essential microelements for plant growth

and the metal component of the Mo cofactor, is

responsible for the catalytic activity of NR, aldehyde

oxidase, xanthine dehydrogenase, and sulfite oxidase. Mo

promotes N accumulation and utilization in wheat plants,

which is directly related to nitrate reductase. A higher Mo

status also results in higher accumulation and utilization

of plant N [58]. Cu exposure results in increase in the

concentration of free amino acids [59]. It can be observed

that there is superiority of Z. coccineum plants in terms of

amino acid content compared with T. nilotica; this may be

due to the higher content of shoot Mo in Z. coccineumcompared with T. nilotica and a genetic variation between

the two plants.

Effect of heavy metals on secondary metabolites

Phytochemicals are divided into two main groups

according to their function in the plant body: primary

and secondary constituents. The primary constituents are

sugars, amino acids, proteins, and chlorophyll and the

secondary constituents consist of alkaloids, terpenoids,

saponins, flavonoids, tannins, and phenolic com-

pounds [61].

The content of secondary metabolites (tannins, saponins,

and alkaloids) and fat were lower in plants from the

polluted sites compared with those from the NC site.

The tannin content ranged from 0.07 to 0.33 g, saponin

from 9.99 to 8.22%, and alkaloids from 7.95 to 1.00%.

Moreover, the maximum tannin and alkaloid contents

were detected in Z. coccineum from the NC site (Fig. 8).

Heavy metal-induced changes in the phenolic com-

pounds may further affect their functions in plant cells.

Phenolic compounds, including tannins, are often in-

volved in responses to different kinds of abiotic and biotic

stresses [62].

Cobbett and Goldsbrough [63] hypothesized that sec-

ondary metabolism may be an integral part of the plant’s

capacity to modify metabolic processes to survive and

grow in adverse conditions, including in the presence of

phytotoxic metals.

Individual plant species differ in their capacity to modify

their metabolism to tolerate or accumulate heavy metals.

The modifications may involve sequestration of the

metals in vacuoles, biosynthesis of organic compounds

that detoxify these metals, or synthesis of modified

tissues to exclude the contaminant [64]. These processes

often alter the uptake and distribution of other metal

ions, as was seen in the present study with altered heavy

metal concentrations in both tested plant tissues. A

consequence of this modified metabolism may include

the loss of specific enzymes or nonessential biomolecular

synthetic processes such as secondary metabolite bio-

synthesis.

ConclusionThese results prove that industrial pollutants and their

metal contamination can change the chemical composi-

tion of the soil and its properties, which reflects on some

medicinal plants, thereby, seriously impacting the quality,

safety, and efficacy of natural plant products produced by

medicinal plant species. The plants from polluted areas

cannot be used as herbal medicine. It is also important to

implement good quality control practices for screening

of herbal medicines to protect consumers from toxicity.

The data presented in this study provide the evidence

of the detrimental effects of naturally occurring or

industrially generated metal contamination in T. niloticaand Z. coccineum.

The plants collected from the investigated sites pose a

serious danger; however, a periodical assessment of plants

used for traditional medicine should be encouraged as

this will assist in ensuring their quality and safety in

herbal use, especially for people living in urban areas

where the level of pollution may be very high.

Amino acids are well-known biostimulants that have

positive effects on plant growth and yield. The higher

content of amino acids in the studied plant species from

the contaminated sites led us suggest extraction of amino

acid and their usage as foliar sprays for different plant

species (agricultural uses), especially plants of Z. coccineumthat have a short life cycle. Further studies are warranted

to extract these amino acids and to ensure the safety and

heavy metal-free status of these amino acids for their use.


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Optimization of growth conditions and continuous production

of inulinase using immobilized Aspergillus niger cellsNagwa A. Atwaa and Enas N. Daniala,b

aDepartment of Chemistry of Natural and MicrobialProducts, Division of Pharmaceutical Industries,National Research Centre, Cairo, Egypt andbDepartment of Biochemistry, Faculty of Girls Science,King Abdulaziz University Jeddah, Saudi Arabia

Correspondence to Nagwa A. Atwa, PhD, Departmentof Chemistry of Natural and Microbial Products,Division of Pharmaceutical Industries, NationalResearch Centre, El-Behoos St. 33, Dokki, Cairo12622, EgyptTel: + 20 100 522 7200; fax: + 20 234 25490;e-mail: [email protected]

Received 7 January 2013Accepted 17 March 2013


2013,12:83–89

Aim

The aim of the study was the optimization of growth conditions for the production

of inulinase as well as the continuous production of the enzyme in an airlift bioreactor

using Aspergillus niger cells.

Methods

First, inulinase production by A. niger cells, using different carbon and nitrogen

sources, was studied on a shake flask level. Second, the cells were adsorbed onto

different carriers, and their production over several successive batches was tested.

Finally, the economically-favorable continuous production of inulinase by A. niger cells

immobilized onto linen fibers was carried out in an airlift bioreactor using crude inulin

juice as the fermentation medium.

Results

Although all tested substances resulted in the biosynthesis of certain amounts of

inulinase enzyme, the highest titer of 163.5 U/ml was obtained when the producing

cells were incubated for 96 h at 271C and 180 rpm in a fermentation medium

containing both inulin and peptone as sole carbon and nitrogen sources, respectively.

Moreover, when the cells of the tested microorganism were adsorbed onto different

carriers, especially linen fibers, their productivity was also successfully maintained, to

different extents, for five successive batches. However, as commercially pure inulin is

very expensive and available in only small quantities, the fermentation medium was later

substituted by a crude inulin extract obtained by mechanical crushing and filtration of

Jerusalem artichoke tubers. The crude inulin juice was able to sustain inulinase

production during the second batch cultivation of A. niger cells that were immobilized

by their adsorption onto linen fibers to a satisfactory level of about 122 U/ml.

Furthermore, the use of the previously mentioned crude inulin preparation was also

compared with the use of either complete or minimal media, composed solely of 1%

pure inulin, for the continuous production of inulinase enzyme by A. niger cells that

were immobilized in their maximum production phase and packed inside an external

loop airlift bioreactor. The results of this experiment were very encouraging as, using

this technique, an inulinase production of about 838 U/ml over an incubation period of

48 h was obtained compared with a production of about 996 and 1013 U/ml, which

resulted from the use of either minimized or complete media, respectively, for the same

incubation period.

Conclusion

The method adopted in this study for inulinase production is simple, economic, time

saving, and nontoxic to the microorganism. Moreover, the loaded linen fiber pads are

reusable.

Keywords:

airlift bioreactor, Aspergillus niger, inulin, inulinase


IntroductionInulin is a widespread polyfructan, naturally occurring in

more than 30 000 edible plant species [1]. It consists of

linear chains of b(2,1)-linked fructose residues attached

to a terminal sucrose molecule [2]. Apart from its role as a

food component, inulin has also received great impor-

tance as a raw material for the production of fructose

syrup [3,4] and inulooligosaccharides [5]. Fructose is a

safe alternative to sucrose, which is known to be the

cause of many health problems including corpulence,

carcinogenicity, atherosclerosis, and diabetes [2]. In

addition, fructose also increases the absorption of iron,

as it forms an iron–fructose complex whose absorption

was found to be much better than that of inorganic

Original article 83


DOI: 10.7123/01.EPJ.0000428964.32893.44

iron [6]. Fructose can be produced from inulin either

enzymatically or chemically through acid hydrolysis.

The latter method is not recommended because of

the undesirable coloring of inulin hydrolysate and the

formation of difructose anhydride, which has practically

no sweetening properties [2]. Moreover, the enzymatic

production of organic products, especially those used in

food and pharmaceutical industries, has many advantages

over chemical processes: The productivity is generally

higher, because of the high specificity of the enzymes for

their substrates; the production cost is relatively lower;

and most importantly it creates less pollution. Therefore,

many efforts have been made to replace chemical

processes with enzymatic ones [7]. Unfortunately, the

conventional enzymatic production of fructose from

inulin involves many steps and results in only a 45%

fructose yield. In contrast, the almost complete hydrolysis

of inulin (90–95%) into fructose can be performed in a

single step using inulinase enzyme [b(2,1)-fructan

fructanohydrolase] [6,8]. Inulinase is produced by many

microorganisms, including filamentous fungi, yeasts, and

bacteria. The fermentation of inulinase by these micro-

organisms can be greatly improved by modifying some

parameters, including the physiochemical and nutritional

conditions of growth required by the producing cells. In

this study, Jerusalem artichoke (Helianthus tuberosus) was

used as a cheap source of inulin, as about 80% w/w (dry

weight basis) of the tuber acts as a store for carbohy-

drates [7]. Moreover, in comparison with conventional

fermentations, immobilization of living cells provides

several important advantages such as a faster production

rate, easier purification of products, and a higher

productivity over a certain period of time [9]. One of

the most reliable, safe, and easy methods of immobiliza-

tion is the adsorption of the producing cells onto an inert,

suitable support [10–12]. Therefore, the present study

was carried out to examine the inulinase productivity of

Aspergillus niger cells under different cultivation conditions

and to study the effect of adsorption immobilization of

these cells onto different carriers on the productivity.

Finally, the continuous production of inulinase by the

producing cells that were immobilized onto linen fibers

and packed inside an external loop airlift bioreactor was

also investigated over a number of successive batches,

using either complete or minimal media as well as crude

inulin juice.

Materials and methodsMicroorganism

The production of inulinase was carried out using a locally

isolated strain of A. niger. The microorganism was

maintained on Czapek’s Dox (CD) agar medium [13]

at 301C for 7 days and then stored in the refrigerator

until use.

Authentic enzyme and chemicals

Inulinase (EC.3.2.1.7) was supplied by NOVO Industry

(A/S, Seoul, Korea). Pure inulin and the remaining

chemicals used were obtained from Sigma (St. Louis,

Missouri, USA). All solvents (analytical grade) were

obtained from Merck (Darmstadt, Germany).

Supports tested for cell immobilization

The immobilization of Aspergillus cells, and eventually

their inulinase productivity, was tested by the adsorption

method using three different carriers: glass wool (Pyrex

fiber glass, Sliver 8 mm; Corning Glass Work, Corning,

New York, USA), linen, and synthetic fibers (the latter

two were locally provided).

Preparation of crude inulin solution

Twenty grams of Jerusalem artichoke tubers (H. tuberosus),

collected locally, were washed, sliced, and grinded using a

blender along with 100 ml of distilled water, then filtered

through a fine gauze. The pH of the solution was

adjusted to 6.2 by addition of concentrated sodium

hydroxide. The resulting juice was sterilized at 1211C and

1.5 atmospheric pressure for 15 min [14]. The raw inulin

extract was analyzed, and its inulin content concentration

was estimated, according to the method described by

Ashwell [15], to be B1.5% (w/v).

Recovery and activity assay of inulinase

Inulinase activity was assayed by measuring the amount

of reducing sugars released from inulin [16]. The

fermentation broth was centrifuged at 3000g and 41C

for 5 min. The obtained supernatant was used as the

crude enzyme. A reaction mixture of 0.1 ml of the enzyme

sample and 0.9 ml of acetate buffer (0.1 mol/l, pH 5.0)

containing 2% inulin was incubated at 501C in a water

bath for 15 min. The mixture was then kept at 1001C for

10 min to inactivate the enzyme. The same mixture to

which the same amount of inactivated crude enzyme

(heated at 1001C for 10 min) was added before the

reaction was used as a control. The reaction mixture was

assayed for reducing sugars according to the method of

Nelson–Somogyi cited by Spiro [17]. The calibrating

curve was drawn with fructose (10–100 mg). One unit of

inulinase was defined as the amount of enzyme that

released one micromole of fructose from inulin per min

under assay conditions.

Media and cultivation conditions

Shake flask fermentation of free cells

Unless otherwise mentioned, inulinase production was

carried out in 250 ml Erlenmeyer flasks, each containing

50 ml of basal CD medium [13] comprising (g/l): inulin,

10; NaNO3, 3; K2HPO4, 1; MgSO40.7H2O, 0.5; KCl, 0.5;

and Fe2SO40.7H2O, 0.01 (pH 6.5). The flasks were then

sterilized, inoculated with about 2� 109 spores/ml of the

producing microorganism, and incubated for 96 h at

120 rpm and 301C. The effect of various carbon sources,

such as fructose, glucose, maltose, starch, and lactose, was

investigated. Each carbon source was added to the basal

medium (without inulin) at a concentration of 10 g/l

either individually or in combination with inulin, which

was then supplemented at a concentration of either 1 or

5 g/l. Similarly, various organic and inorganic nitrogen

sources were individually added to the basal medium as a

substitute for NaNO3 in order to study their effect on


inulinase production. The tested organic nitrogen sources

(peptone, urea, and yeast, beef, and meat extracts) were

added at a concentration of 50 g/l. The inorganic nitrogen

sources under study (NH4SO4 and NH4Cl) were added

according to their nitrogen content such that it was

equivalent to that of NaNO3, which was omitted from the

medium.

Shake flask fermentation of immobilized cells

The immobilization of A. niger was studied using the

adsorption method [11,12]. A total of 1.4 g of each tested

support (glass wool and synthetic and linen fibers) was

added to a 250 ml flask containing 50 ml of the optimized

medium composed of (g/l): inulin, 10; peptone, 50; K2HPO4,

1; MgSO40.7H2O, 0.5; KCl, 0.5; and Fe2SO40.7H2O, 0.01

(pH 6.5). The flasks were then sterilized, inoculated with

about 2� 109 spores/ml of the producing microorganism,

and incubated for 96 h at 120 rpm and 301C. To assess the

productivity of the immobilized cells for another batch, the

loaded pads were washed thoroughly with normal saline,

carefully squeezed under sterile conditions, and used to

inoculate 50 ml of a fresh sterile medium, which was then

reincubated under the former conditions but for a shorter

incubation period of 72 h.

An experiment was carried out as an attempt to reduce

the quantity of the constituents of the fermentation

medium used during repeated batch cultivation of the

cells previously adsorbed onto linen fibers in their

maximum inulinase production phase. This was achieved

to decrease the growth of the escaped cells and to

produce inulinase using the cheapest possible medium.

Therefore, as described previously, A. niger cells were

inoculated in 50 ml of sterile medium along with 1.4 g of

linen fibers in each flask. After 96 h of incubation at 301C

and 120 rpm, the linen fiber pads saturated with the cells

in their maximum production phase were washed

thoroughly with normal saline solution and carefully

squeezed using sterilized forceps. These pads were then

transferred to new flasks containing different ratios of the

constituents of the main medium as shown in Table 1.

Crude inulin, obtained by mechanical crushing and

filtration of Jerusalem artichoke tubers, as previously

explained, was also tested. These flasks were then

reincubated at 301C and 120 rpm for another 72 h. At

the end of this incubation period, inulinase production

and the cell dry weight of unadsorbed cells in each flask

were estimated.

Airlift bioreactor fermentation of immobilized cells

The production of inulinase enzyme by A. niger cells

immobilized onto linen fibers was investigated in an

external loop airlift bioreactor [12], using either complete

or minimized fermentation media as well as raw inulin

juice. A schematic diagram of the designed apparatus is

illustrated in Fig. 1. The bioreactor consists mainly of a

riser column with a height of 40 cm and a downcomer

tube with a diameter of 1 cm. The riser column is

composed of an inner perforated column with an internal

diameter of 3 cm jacketed by an outer column that has an

internal diameter of 4 cm. The inner column was

designed to hold up the linen fiber pads, on which the

producing cells were previously immobilized, and to

prevent their fluidization. The perforation of the column

allowed the fermentation broth to come in contact with

the immobilized cells in many parts and also helped

achieve a good oxygen transfer to the packed fibers.

Table 1 Optimization of the fermentation medium used in the

second batch production of inulinase by Aspergillus niger cells

immobilized onto linen fibers

Inulin (g/l) Peptone (g/l) Salt content (%)

Control medium number1 10 50 1002 10 50 100

Second batch medium number1 10 50 1002 10 25 1003 10 5 1004 10 1 1005 10 0 1006 10 50 507 10 50 258 10 50 09 8 50 10010 5 50 10011 2 50 10012 7.5 37.5 7513 5 25 5014 2.5 12.5 2515 10 0 016 10 0 0

Controls 1 and 2, inulinase production by free cells and by the firstbatch of cultivated immobilized cells, respectively.

Figure 1

Schematic diagram of the airlift bioreactor and its accessories used forthe production of inulinase by immobilized Aspergillus niger cells. 1,riser column; 2, inner perforated column; 3, downcomer tube; 4, one-way valve; 5, air sparger; 6, air inlet; 7, air outlet; 8, medium inlet; 9,medium outlet; C1–C5, clamps; F1, F2, F3, air filters; P1, P2, peristalticpump; P3, air pump; R1, medium feeding reservoir; R2, productcollection reservoir.

Optimization of inulinase production Atwa and Danial 85

The whole system was mounted inside an incubator

adjusted at 301C.

The inoculum was in the form of eight firmly squeezed

linen pads supporting Aspergillus cells that were previously

immobilized by their cultivation in the optimized

medium for 96 h at 120 rpm and 301C. The linen pads

loaded with the immobilized cells were packed, under

aseptic conditions, inside the inner column of the

bioreactor. A working volume of 360 ml of each tested

medium was fed one at a time. The aeration rate of the

bioreactor was adjusted at 0.5 v/v/m. The fermentation

medium once introduced by a peristaltic pump P1 into

the riser column was left without circulation for 30 h in

contact with the immobilized cells. This phase was

performed to reinitiate the inulinase production of the

immobilized cells. Thereafter, peristaltic pump P2 was

adjusted such that the medium could circulate at a rate of

30 ml/h. During the experiment, 20 ml aliquots of the

culture were systematically withdrawn with a syringe

through an inline air filter. These samples were assayed to

monitor inulinase production and cell escapement.

Results and discussionOptimization of growth and inulinase production

parameters of free cells on shake flask level

Effect of different incubation periods

The production of inulinase by A. niger cells on inulin

basal CD fermentation medium was monitored over a

period of 120 h under the previously mentioned shaking

cultivation conditions of 120 rpm and 301C. The results

illustrated graphically in Fig. 2 show that the activity of

inulinase was 0.88 U/ml in the fermentation medium after

6 h of incubation. This recorded enzyme activity was

found to increase linearly with time by a production rate

(Qp) of 1.6 U/ml/h and reached a maximum volumetric

production of 139.349 U/ml after about 96 h of incuba-

tion. After this incubation period, a gradual decrease in

inulinase activity was observed. The reported production

decrease rate (– Qp) was about 0.69 U/ml/h. Cell growth

was also studied during the course of fermentation and

was found to increase gradually with time by a specific

growth rate (m) of about 0.35 g/l/h. A maximum cell dry

weight (Xmax) of about 9.98 g% was recorded after 96 h of

incubation. Thereafter, a slight cell lysis was observed

with a specific degradation rate (– m) of about 0.02 g/l/h,

resulting in a cell dry weight of 9.51 g/l after 120 h of

incubation. This result showed that inulinase production

was growth-dependent and that the maximum inulinase

productivity of the producing organism was just before

the onset of its stationary phase of growth. Moreover, a

maximum yield coefficient (units of inulinase per gram of

cell mass formed) of 1396.3 U/g cells was recorded after

96 h of incubation.

Effect of different carbon sources

Different carbon sources were tested for their ability to

sustain substantial amounts of inulinase enzyme produc-

tion (Fig. 3). Among them, inulin resulted in a maximum

enzyme production of about 140 U/ml, followed by

sucrose, which resulted in B114 U/ml of the enzyme.

Lower enzyme titers ranging between 96 and 80 U/ml

were recorded upon using other carbon sources including

(in descending order of enzyme activity recorded):

fructose, glucose, maltose, starch, and finally lactose.

However, because the use of inulin as a sole carbon source

in the fermentation medium was inconvenient owing to

its high cost, it was therefore added to the medium

containing each individual carbon source, in small

percentages of 0.1 and 0.5%, as an attempt to initiate

higher inulinase production. This goal was achieved as

the addition of inulin in these percentages resulted in

significant increases in enzyme production (results

ranging from 5.6 to 12%). However, none of these

enzyme titers could exceed the level obtained when

inulin was added as a sole carbon source in the

fermentation medium.

Figure 2

Effect of different incubation periods on the growth of and inulinaseproduction by free Aspergillus niger cells cultivated in basal Czapek’sDox medium.

Figure 3

Effect of different carbon sources, added either individually or inaddition to 0.1 or 0.5% inulin, on inulinase production by freeAspergillus niger cells.


Effect of different nitrogen sources

Different nitrogen sources, either organic or inorganic,

were also tested for inulinase productivity. The results

in Fig. 4 show that a maximum production of about

163.5 U/ml could be achieved when peptone was used as

a sole nitrogen source in the fermentation medium. Much

lower yields ranging between 124 and 93 U/ml were

recorded upon using other organic nitrogen sources

including (in descending order of enzyme productivity):

yeast and beef extracts, urea, and then finally meat

extract. In contrast, the use of inorganic nitrogen sources

such as NaNO3, NH4SO4, and NH4Cl resulted in en-

zyme titers of approximately 131, 125, and 99.5 U/ml,

respectively.

Optimization of growth and inulinase production

parameters of immobilized cells on shake flask level

Effect of immobilizing Aspergillus niger cells on different

carriers

A. niger cells were tested for their ability to produce

inulinase while immobilized by adsorption onto different

carriers including glass wool and synthetic and linen

fibers. The enzyme production results were compared

with those obtained when free cells of the fungus were

cultivated on the same optimized culture medium under

similar cultivation conditions. The results in Fig. 5

indicate that the cells immobilized onto linen fibers

were only slightly affected by the immobilization process,

as they were able to produce a satisfactory enzyme

concentration of about 131 U/ml compared with 164 U/ml

produced by cultivation of free cells for the same

incubation period of 96 h. Successive batch cultivation

of the immobilized cells was performed to test their

inulinase productivity. This result was very promising

because this previously mentioned inulinase titer was,

more or less, attained after a much shorter incubation

time of 72 h, as the cells were inoculated in their

maximum production phase. The obtained yield of

inulinase could also be more or less sustained within

appropriate ranges for five consecutive batches, resulting

in a total enzyme yield of 630 U/ml within a combined

serial incubation period of 384 h. In contrast, the first

batch cultivation of the A. niger cells adsorbed onto either

synthetic fibers or glass wool resulted in lower inulinase

yields of 112 and 103 U/ml, respectively. It was observed

that these titers were maintained, with only a slight

decrease, during the experiment.

Optimization of the fermentation medium used for the second

batch production of inulinase by Aspergillus niger cells

immobilized onto linen fibers

The results illustrated in Fig. 6 show that a maximum

inulinase production of about 162 U/ml was obtained

when free cells were cultivated for 96 h (control 1).

Moreover, the first batch cultivation of immobilized cells

(control 2) resulted in a satisfactory inulinase production

of 134.5 U/ml for the same incubation period. However,

the results showed no significant differences between the

inulinase titers estimated in the fermentation broths of

media no. 1 to 8 (used in the second batch cultivation of

72 h), which ranged between 130.28 and 120.44 U/ml.

Figure 4

Effect of different organic and inorganic nitrogen sources on inulinaseproduction by free Aspergillus niger cells.

Figure 5

Inulinase production during repeated batch cultivation of Aspergillusniger cells immobilized onto different support materials. Control,inulinase production by free cells.

Figure 6

Optimization of the fermentation medium used in the second batchproduction of inulinase by Aspergillus niger cells immobilized onto linenfibers. Controls 1 and 2, inulinase production by free cells and by thefirst batch of cultivated immobilized cells, respectively.


This means that the inulinase productivity of the cells

was more or less maintained in the second batch even

when the peptone or salt content of the medium was

reduced or even eliminated. However, it was found that

the inulin content of the medium was critical for both the

growth of the producing organism and its productivity, as

its reduction, keeping the percentage of the other

constituents constant, affected the inulinase titer and

cell growth greatly (media no. 9 to 11). The critical effect

of inulin on inulinase production was also revealed when

different percentages ranging between 75 and 25% were

used in the media (media no. 12 to 14), as the production

of inulinase decreased to 87.16 and 47.21 U/ml, respec-

tively. However, medium 15, composed of only pure

inulin (10 g/l), and medium 16, composed of crude inulin

solution (15 g/l), resulted in satisfactory inulinase levels

of 126 and 122 U/ml, respectively. Relying on these

results, the complete medium could be substituted by

either medium 15 (minimal medium) or medium 16 (raw

inulin extract) for the production of inulinase during the

repeated batch cultivation of A. niger cells immobilized

onto linen fibers.

Optimization of fermentation medium used for the

continuous production of inulinase by immobilized cells

in an airlift bioreactor

Inulinase production using complete medium

The results illustrated in Fig. 7 show that inulinase

production increased gradually at the rate of 1.56 U/ml/hr

and reached a volumetric production of 102.6 U/ml after

only 48 h of incubation. This maximum inulinase

production level was maintained until 78 h of incubation.

Inulinase production using minimal medium

The experiment was repeated using minimal medium as

previously described. Although this medium was only

composed of 10 g/l pure inulin, lacking any other media

component, the inulinase productivity of the cells, of

about 100 U/ml, was satisfactorily restored after only 24 h

of incubation wherein the recorded productivity rate was

2.54 U/ml/h. This titer was more or less maintained until

the end of the fermentation time (Fig. 7). The obtained

results could be attributed to the fact that immobilized

cells need nutrients that will only maintain their

inulinase productivity on the expense of their growth.

It was also observed that the use of a minimal medium

resulted in a reduction in unwanted growth of escaping

cells, which favors recovery of the produced enzyme.

Inulinase production using crude inulin solution

The experiment was finally performed using the crude

inulin solution, prepared as previously mentioned in the

Material and methods section. The latter resulted in a

slightly reduced inulinase yield compared with that

obtained using either complete or minimal media

(Fig. 7). The recorded productivity rate under these

conditions was 2.03 g/l/h for the first 36 h of the

incubation period. However, a satisfactory production

level ranging between 85.9 and 79.9 U/ml was then

reached and approximately sustained for another 42 h.

These results were very encouraging as, using this

technique, a combined production of about 838 U/ml of

inulinase was obtained from a very economic crude

extract of inulin in only 48 h, which is comparable with

yields of 996 and 1013 U/ml that were obtained when

immobilized cells were cultivated using pure inulin in

either minimized or complete media, respectively, for the

same incubation period.

ConclusionFrom these experiments, we can conclude that the

production of inulinase by A. niger cells immobilized by

their adsorption onto the surface of linen fibers, using

crude inulin extraction, is a very promising method that

could be performed on large scales for economic,

industrial production of the enzyme. The main advantage

of this method is the higher productivity of the

immobilized cells compared with that of the free cells,

considering the possibility of their repeated batch

cultivation. It was also observed that the production time

during the repeated batch cultivations reduced by more

than half. Moreover, with the use of crude inulin juice, a

low percentage of cell growth, and eventually cell

escapement, was attained. The latter made the recovery

and purification of the enzyme much easier. As a final

conclusion, this method is simple, economic, time saving,

and nontoxic to the microorganism. In addition, the

loaded linen pads are reusable.

AcknowledgementsThe authors express their deepest gratitude to Prof. Dr. A.I. El-Diwanyand Prof. Dr. M.A. Farid for their generous participation in designing andfunding the airlift bioreactor and for their continuous invaluable support.

Conflicts of interestThere are no conflicts of interest.

Figure 7

Continuous production of inulinase by Aspergillus niger cells immobi-lized onto linen fibers in an airlift bioreactor, using either complete orminimized media as well as crude inulin juice.


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11 Farid MA, El-Batal AI, El-Diwany AI, El-Anshasy HA. Optimization of glutamicacid production with immobilized cells of Corynebacterium glutamicum.Adv Food Sci 1996; 18:34–38.

12 Atwa NA. Microbial and biochemical studies on the production of anti-biotics for veterinary uses [PhD thesis]. Microbiology Department, Faculty ofSciences, Cairo University; 2003..

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Chemical constituents from the aerial parts of Salsola inermisFatma S. Elsharabasya,c and Ahlam M. Hosneyb

Departments of aChemistry of Natural and MicrobialProducts, bTheraputic Chemistry, National ResearchCenter, Dokki, Egypt and cCollage of Science andHumanities, Salman bin Abdul Aziz University, AlkharjCity, Kingdom of Saudi Arabia

Correspondence to Fatma S. Elsharabasy, PhD,Department of Chemistry of Natural and MicrobialProducts, National Research Center, El-Behooth St,Dokki 12311, EgyptTel: + 20 101 468 5611; fax: + 20 233 370 931;e-mail: [email protected]



2013,12:90–94

Background and objective

The hydroalcoholic extract from the aerial parts of Salsola inermis exhibited antioxidant,

anti-inflammatory, and antinociceptive effects. The present study deals with the

isolation and identification of the chemical constituents of this hydroalcoholic extract.


The aerial parts of S. inermis (Forsskal) were collected from wild plants growing near

the El-Alamein area in October 2005. Air-dried and powdered aerial parts of S. inermis

were extracted with 70% alcohol in H2O. The extract was partitioned successively with

CHCl3, EtOAc, and n-BuOH. The structures of the isolated compounds were

determined by chemical and spectroscopic analyses.


Phytochemical investigation of the alcoholic extract from the aerial parts of

S. inermis revealed 12 compounds, identified as long chain hydroxyl fatty acid

9,12,13-trihydroxydecosan–10,15,19-trienoic acid; trans-N-feruloyl tyramine-40 0 0-O-b-

D-glucopyranoside; umbelliferone; scopoletin; 3-methyl kaempferol; olean-12-en-3,28-

diol; olean-12-en-28-oic acid; stigmasterol-3-b-O-D-glucopyranoside; 3-O-[b-D-

glucopyranosyl]oleanolic acid; kaempferol 3-O-b-glucopyranoside; and isorhamnetin

3-O-b-glucopyranoside, in addition to b-sitosterol, stigmasterol, and stigmastanol.

Some of these compounds have hydroxyl groups, which help in scavenging free

radicals and inhibit COX and various mediators involved in the pathogenesis of

pain relief.

Keywords:

aerial parts, coumarins, flavonoids, NMR, Salsola inermis, terpenes


IntroductionThe genus Salsola, family Chenopodiaceae (Goosefoot

family), includes over 100 species found in the dry

regions of Asia, Europe, and Africa [1]. The Salsolaspecies represents 16 species in Egypt, most of which

grow in the Egyptian deserts [2]. Previous phytochemical

investigation of the genus resulted in the isolation of

alkaloids, saponins, sterols and their glucosides, comarino-

lignan, isoflavonoids, and flavonoids [3–10]. Some Salsolaplants are widely used as folk medicine for the treatment

of hepatitis [11] or infections caused by tapeworm and

parasites [12]; they also have pronounced vasoconstric-

tive, hypertensive, and cardiac stimulant action [13]

and can act as an allergenic substance [14,15]. Reactive

oxygen species (ROS) are always present in cells as

metabolic products of normal cellular respiration. How-

ever, oxidative stress, an imbalance caused by excessive

ROS originating from endogenous and exogenous sources,

might cause inflammation and therefore play a pivotal

role in many diseases [16]. Cytopreventive antioxidants

prevent the formation of free radicals and scavenge them

or promote their decomposition [17]. In chemical terms,

polyhydroxy flavonoids efficiently modulate the redox

status and thus may play a critical role in regulating the

inducible gene expression of inflammatory mediators in

the lipopolysaccharide-stimulated mouse leukemic mono-

cyte macrophage cell line (RAW 2647macrophages) [18].

As a continuation of our previous studies that showed that

the ethanol extract of Salsola inermis has antioxidant and

anti-inflammatory properties [19], the present study

deals with the isolation and identification of chemical

constituents of the hydroalcoholic extract from the aerial

parts of S. inermis.

Materials and methodsElectron impact mass spectra (EIMS) were obtained using

Varian MAT 711 (Germany), Finnigan SSQ 7000 (San Jose,

California, USA), and OMM 7070 E spectrometers

(Maryland, USA). 1H-NMR and 13C-NMR spectra were

recorded at 500 MHz on a JEOL 500 A spectrometer

(JEOL Inc., USA). The 1H-NMR and 13C-NMR chemical

shifts are expressed in ppm relative to tetramethylsilane.

Infrared (IR) spectra were measured on a Perkin Elmer FT-

IR1700 spectrometer (Perkin Elmer, USA) at the National

Research Centre, Cairo, Egypt. Ultraviolet (UV) spectra

were recorded on a Shimadzu UV-Vis spectrophotometer

(Shimadzu, USA). Thin layer chromatography (TLC)

plates (aluminum sheets) precoated with silica gel G 60

(F 254; Merck) were used for chromatography. Special

reagents used were iodine–potassium iodide for detection

of coumarins and chlorosulfonic acid spray reagent for the

detection of sterols and triterpens. The two-dimensional

paper chromatographic technique using the solvent system

90 Short communication


DOI: 10.7123/01.EPJ.0000428060.24957.95

BuOH : HOAc : H2O (4 : 1 : 5) and HOAc (15%) was also

used [20].

Plant material

The aerial parts of S. inermis (Forsskal) were collected

from wild plants growing near the El-Alamein area in

October 2005. The plant specimen was authenticated by

Dr N. El-Hadidi, Faculty of Science, Cairo University, and

was compared with reference herbarium specimens.

General procedure for extraction and isolation

Air-dried and powdered aerial parts of S. inermis were

extracted with 70% alcohol in H2O after evaporation of

the solvent under reduced pressure. It was essential that

the extract (200 g) be partitioned successively with

CHCl3, EtOAc, and n-BuOH.

The CHCl3 fraction (8 g) was applied onto a silica gel

column and eluted with a gradient of n-hexane, CHCl3, and

MeOH (100–0, 90–10, 80–20, 70–30, 60–40, 50–50, 40–60,

20–80, 0–100) to give five fractions A1–A5. Further

purification of A1 (0.8 g) by preparative TLC with

n-hexane/CHCl3 as an eluent afforded compounds I

(0.05 g) and II (0.02 g). Moreover, column chromatography

of A2 with CHCl3 afforded compounds III (0.28 g) and IV

(0.10 g). Column chromatography of A4 with gradient

elution using EtOAc/MeOH yielded compound V

(0.18 g). The EtOAc fraction (7 g) was chromatographed

over silica gel with successive petroleum ether/EtOAc

(80–20, 20–80, 0–100) and EtOAc/MeOH (90–10, 0–100)

elution to give eight fractions, B1–B8. Column chromato-

graphy of B6 (1.24 g) with CHCl3/MeOH elution (9–1 and

9–2) afforded compounds VI (5 mg) and VII (3 mg).

Further, column chromatography of B8 with CHCl3/MeOH

elution (9–1 and 8–2) afforded compound VIII (6 mg).

BuOH (12 g) applied onto a flash column chromatography

column with H2O/MeOH gradient elution afforded three

fractions, C1–C3. Purification of C2 and C3 carried out on a

Sephadex LH-20 column with CHCl3/MeOH elution (1–9

and 0–10) afforded compounds IX, X, XI, and XII.

The physical and spectral data of the isolated compounds

are as follows.

Compound I

Gummy white solid, EIMS, m/z 386: [M] + calculated for

C22H42O5; IR (KBr) nmax cm – 1 3437, 2925, 2854, 1740,

929; 1H-NMR (500 MHz, CDCl3) dH 0.9 (3H, t, J = 7.3,

H-22), 1.35 (11 H, bs, H-4, H-5, H-6, H-7, H-8a), 1.45

(1H, m, H-8b), 1.61 (2H, m, H-3), 2.05 (2H, t, J = 6,

8 Hz, H-21), 2.17 (1H, m, H-14a), 2.33 (1H, m, H-14b),

3.46 (1H, m, H-13), 3.98 (1H, t, J = 5.3 Hz, H-12), 4.05

(1H, m, H-9), 5.42, O, (1H, J = 11.2, 5.2 Hz, H-16), 5.47,

O, (1H, J = 11.2, 5.2 Hz, H-15), 5.68 (1H, dd, J = 15.7,

5.2 Hz, H-11), 5.73 (1H, dd, J = 15.7, 5.2 Hz, H-10).

Compound II

Amorphous powder, IR (KBr) nmax cm – 1 3416, 2925,

1725, 1646, 1515, 1269, 1074. EIMS, m/z: 476 [M] + calcd

for C24H30NO9. UV lmax (MeOH) nm (loge): 225 (3.12),

278 (2.99), 311 (3.1). 1H-NMR and 13C-NMR spectral

data are presented in Table 1.

Compound III

White crystals, m.p. 225–2281C, Rf 0.42 (TLC, S1); UV

lmax nm (MeOH) 217, 245, 260sh, 279sh, and 322 nm;

EIMS m/z 162 [M] + , C9H6O3. 1H-NMR (500 MHz,

CDCl3) dH 6.15 (1H, d, J = 9.6 Hz, H-3), 6.58 (1H, d,

J = 2.6 Hz, H-8), 6.85 (1H, dd, J = 8.6 Hz, H-6), 7.35

(1H, d, J = 8.6 Hz, H-5), 7.81 (1H, d, J = 9.3 Hz, H-4).

Compound IV

Colorless needle crystals (CHCl3), m.p. 221–2231C, Rf

0.5 (TLC, S1); UV lmax nm (MeOH) 229, 250sh, 260sh,

295sh and 342 nm. 1H-NMR (500 MHz, CDCl3) dH 6.26

and 7.58 (2H, d, J = 9.6 Hz, H-3 and H-4), 6.85 and 6.92

(2H, s, H-8 and H-5) and 3.92 (3H, s, Me-6).

Compound V

Colorless needles, m.p. 131–1321C, showed [M+] peak at

m/z 412 (25.0%), 414 (17%), and 416 (1.40%) and

characteristic fragmentation peaks at m/z 275, 255, 231, 213.

Compound VI

White needles (0.22 g), m.p. 2541C. M + peak at m/z 441

(9.30%), corresponding to C30H50O, and an intensive

peak at m/z 411 (18.92%), corresponding to M + –

CH2OH. IR showed characteristic absorption bands at

3395 (OH), 2925 cyclic (CH2), 1730, and 1446 (C = C).

D12 double bond proved to be readily recognizable by

mass spectra and 1H-NMR shows seven tertiary methyl

proton singlets at 0.81, 0.82, 0.84, 0.86, 1.18, 1.22, and

1.84, an olefin proton at d 5.4 (br.s.), and a hydroxyl

methylene proton at 5.14.

Compound VII

Isolated as white crystals (0.01 g), m.p. 259–601C, IR

spectrum showed strong bands near 3415 cm – 1 (OH),

1735 cm – 1 (CO), two bands 1390–1375 and 1369–1354

cm – 1 in the ‘A-region’, and three bands at 1328–1318,

1303–1296, and 1267–1248 cm – 1 in the ‘b-region’; its

Table 11H-NMR (300 MHz) and

13C-NMR (300 MHz) for

compound II (CHCl3-d6)

Position dH dC

1 1752 6.47 121.73 7.43 140.9810 129.1820 7.17 br d (1.6) 114.030 14840 148.650 6.74 d (8.5) 115.6460 6.95 dd (8.2, 1.7) 121.410 0 3.34 t (7.3) 40.5520 0 2.76 t (7.3) 40.3910 0 0 132.020 0 0, 60 0 0 7.17 d (8.5) 13030 0 0, 50 0 0 7.1 2 d (8.5) 115.040 0 0 156Glc-1 4.2 d (7.4) 101.32 3.33 (overlap) 74.013 3.39 (overlap) 77.444 3.25 (overlap) 70.625 3.25 (overlap) 67.936 3.99 dd (12.0, 1.9) 63.61

3.73 dd (12.0, 5.5)OCH3 3.81s 56.42

Chemical constituents of Salsola inermis Elsharabasy and Hosney 91

mass gave an M + + 1 peak at m/z 457 (2.02%), corres-

ponding to C30H48O3, fragmentation characteristic with

respect to oleanane triterpenoids having D12 : 13 unsaturation.

The ion at m/z 189 stands for rings A and B in the

dehydrated form [21].

Compound VIII

Yellow powder, m.p. 275–2781C, Rf 0.47 (TLC, S2); UV

lmax nm (MeOH) 268 and 363, (MeOH/NaOMe) 279

and 423, (MeOH/AlCl3) 269 and 423; 1H-NMR

(500 MHz, DMSO) dH 6.16 and 6.42 (2H, d,

J = 2.2 Hz, H-6 and H-8), 6.90 and 8.0 (each 2H, d,

J = 8.7 Hz, H-30, -50 and H-20,-60).

Compound IX

White powder, m.p. 265–2681C, Rf 0.47 (TLC, S2);

EIMS, M + peak at m/z 576, 9.45%, corresponding to the

molecular formula C35H60O6, m/z 163 (13.54) of one

hexose sugar; IR spectrum (KBr) Vmax cm – 1, 3421 (OH),

1730–1446 (C = C), 1129, 1076, 1055, and 1015 (ether

linkage of glycoside); 1H-NMR (500 MHz, DMSO-d6) dH

0.64 and 1.02 (each 3H, s, H-18 and H-19), 0.78–0.85

(9H, m, H-26, 27 and H-29), 0.89 (3H, d, J = 6.6 Hz,

H-21), 0.94 (3H, m, H-29), 4.39 (1H, m, H-3), 5.38 (1H,

broad s, H-6), and 4.30 (1H, d, J = 7.7 Hz, H-10).

Compound X

White powder, m.p. 260–2631C, Rf 0.57 (TLC, S2); EIMS,

M+ at m/z 618 compatible with C36H58O8, m/z 456 ascribe

to the mass of triterpene (aglycone), corresponding to

C30H47O3, m/z 438 (aglycone-H2O), 426 (aglycone-2Me),

410 (aglycone-COOH + Me), 248 and 189, 133, the ion at

m/z 161 stands for a hexose sugar.

Compound XI

Yellow powder, UV lmax nm: (MeOH) 256, 267.1, 292sh,

357; MeOH + NaOMe, 272, 291, 325sh, 415; MeOH +

NaOAc, 273, 315, 390; MeOH + AlCl3, 274, 292, 340sh,

425 MeOH + AlCl3 + HCl; 272, 303sh, 360sh, 403.1H-NMR (500 MHz, DMSO) dH 7.82 (2H, d,

J = 8.2 Hz, H-20, 60), 6.84 (2H, d, J = 8.2 Hz, H-30,50),

6.28 (1H, d, J = 1.9 Hz, H-6), 5.30 (1H, d, J = 7.6 Hz,

H-100 of glucose), 3.27–3.57 (m, rest of glucose protons).

Acid hydrolysis gave kaempferol and glucose.

Compound XII

Yellow powder, m.p. 224–226oC; brown fluorescence in

UV, Rf 0.34, UV lmax nm: (MeOH) 254, 265sh, 353;

(NaOMe) 270, 331sh, 415; (NaOAc) 271, 311sh, 394;

(AlCl3) 264, 296, 366sh, 400; (AlCl3 + HCl) 262, 300, 366,

400. Acid hydrolysis gave isorhamnetin and glucose.


Results and discussionsThe aqueous ethanolic extract was successively parti-

tioned in H2O/CHCl3, H2O/EtOAc, and H2O/n-BuOH.

The three fractions were then subjected to a sequence of

column chromatography procedures to yield compounds

I–V, VI–VIII, and IX–XII, respectively.

9,12,13-Trihydroxydocosan–10,15,19-trienoic acid (I) was

isolated as a white solid with the molecular formula

C22H42O5, calculated from the [M + ] peak at m/z 386. Its

IR spectrum showed OH and CO absorptions at 3437 and

1740 cm – 1, respectively. 13C-NMR was characteristic of

an unsaturated long chain fatty acid with a methyl group

at dC 14.3, several methylene carbons from 23.2 to 39.91,

two sp2carbons at 139.3 and 157.4, and a substituted

carboxyl carbon at dC 166.98, in addition to three low-

field oxygenated carbons at dC 72.99 and 77.0, bearing

methane protons at dH 4.2, 3.89, and 3.58, respectively,

which confirmed the presence of three hydroxyl groups;

an olefinic proton signal appeared at d 5.27. Analysis of

the spectra provided evidence for the fragment and

established the structure of compound I [22].

Trans-N-feruloyl tyramine-4000-O-b-D-glucopyranoside (II)

showed EIMS, M + at m/z 476 calculated for the

molecular formula C24H30NO9. Its IR spectrum exhibited

characteristic absorption bands for a hydroxyl group

(3416 cm – 1), conjugated carbonyl group (1646 cm – 1),

and conjugated double bond (1515 cm – 1). Acid hydrolysis

of II afforded D-glucose as determined by comparing the

Rf of the hydrolysis product with that of an authentic

sample using the paper chromatographic technique. The1H-NMR spectrum (Table 1) indicated the presence of

one 1,4-disubstituted aromatic ring at dH 7.19 (2H, d,

J = 8.4 Hz, H-2000, 6000) and dH 7.19 (2H, d, J = 8.5 Hz, H-

3000, 5000); one 1,3,4-trisubstituted aromatic ring at dH 6.95

(1H, dd, J = 8.2, 1.7 Hz, H-60) and dH 6.75 (1H, d,

J = 8.2 Hz, H-50); one trans olefin at dH 6.68 (1H, d,

J = 15.2 Hz, H-3) and dH 4.22 (1H, d, J = 12.1 Hz, H-2);

and one methoxy proton at dH 3.99 (3H). From the

coupling constant of the anomeric proton at dH 4.24

(1H, d, J = 7.4 Hz, Glc-1), C-1 of the D-glucopyranose

was determined to be in the b-configuration. Analysis of

the 13C-NMR (Table 1; dC-1" 175, dC-1 40.5) and the

molecular formula of II revealed that C-100 and C-1were

linked by a nitrogen atom. The current analysis and

comparison with the data in the literature suggested the

structure of compound II [23].

The two coumarins III and IV were isolated from the

CHCl3 extract. Umbelliferone (III) showed shine blue

fluorescence under UV light (366) and when sprayed

with I2/KI reagent turned into a colorless spot. From

the results of 1H-NMR analysis and by cochromato-

graphy with the reference substance, compound III was

identified.

Scopoletin (IV) showed strong blue fluorescence under

UV light (366) and when sprayed with I2/KI reagent

turned into brown spot. The UV spectrum of IV in

MeOH showed absorption bands at 229, 250sh, 260sh,

295sh, and 342 nm, which suggested a 6,7-dioxgenated

coumarin skeleton. From the results of 1H-NMR analysis

and by cochromatography with the reference substance,

compound IV was identified [24].

Three known sterols (V) isolated from the CHCl3 extract

gave positive results for the Liebermann test for sterols

and showed an [M + ] peak at m/z 412 (25.0%), 414

(17%), and 416 (1.40%) corresponding to C29H48O,

C29H50O, and C29H52O, respectively. Because of its

occurrence with the identified sterols [25], the sterol

with M + at m/z 414 (17.0%) was identified as b-

sitosterol, the sterol with M + at m/z 412 (25.0%) was

identified as stigmasterol, and the sterol with M + at m/z416 was identified as sitostanol.

Three compounds VI, VII, and VIII were isolated from

the EtOAc extract.

Olean-12-en-3,28 diol (VI) gave a positive Liebermann

test for triterpenes. The compound with M + at m/z 441

(8.02%) was identified as C30H50O2, with a peak at 411

(45.0%). Spectral analysis suggested the structure of the

compound [21].

Olean-12-en-28-oic acid (VII): the IR spectrum showed

strong bands near 3415 (OH) and 1735 cm – 1 (CO): two

bands, 1390–1375 and 1369–1354 cm – 1, in the so called

‘A-region’ and three bands at 1328–1318, 1303–1296, and

1267–1248 cm – 1 in the ‘b-region’; its mass gave an

M + + 1 peak at m/z 457 (2.02%), corresponding to

C30H48O3, fragmentation characteristic with respect to

oleanane triterpenoids having D12 : 13 unsaturation. The

ion at m/z 189 represents rings A and B in the dehydrated

form. Previous spectral data and chemical analysis

elucidate the structure of this compound [21].

3-Methyl kaempferol (VIII) was identified from the

analysis of its UV spectra in MeOH before and after the

addition of different shift reagents and from the analysis

of its 1H-NMR spectral data [20]; this was further

confirmed by cochromatography with a reference sub-

stance.

Stigmasterol-3-b-O-D-glucopyranoside (IX) showed an

EIMS M + peak at m/z 576 (9.45%), corresponding to

the molecular formula C35H60O6, m/z 163 (13.54) of one

hexose sugar. IR spectroscopy revealed bands Vmax cm – 1,

3421 OH, 1730–1446 (C = C), 1129, 1076, 1055, and

1015 (ether linkage of glycoside). 1H-NMR revealed one

anomeric proton at 4.43 (d, J = 6.78), indicating the sugar

to be in the b-configuration. Thus, from the large JH1,H2

Chemical constituents of Salsola inermis Elsharabasy and Hosney 93

coupling constant, the structure of this compound was

elucidated as stigmasterol-3-b-O-D-glucopyranoside [25].

3-O-[b-D-glucopyranosyl]oleanolic acid (X) showed an

M + peak at m/z 618, corresponding to the molecular

formula C36H58O8, and a fragment ion at m/z 456,

corresponding to H30H48O3. This is ascribed to the mass

of triterpene acid having a D12 aglycone. IR revealed

bands Vmax cm – 1, 3421 OH, 1730–1446 (C = C), 1129,

1076, 1055, and 1015 (ether linkage of glycoside).

Kaempferol 3-O-b-glucopyranoside (XI) and isorhamne-

tin 3-O-b-glucopyranoside (XII) gave typical brown

fluorescence under UV for the C-3-substituted flavonoid

glycosides. Acid hydrolysis yielded glucose and kaemp-

ferol or isorhamnetin, respectively. The structures of

compounds XI and XII were confirmed by 1H-NMR and

cochromatography with authentic reference samples [26].

Isolation of these compounds from S. inermis has not been

reported previously.

ConclusionTwelve compounds were isolated and identified for the

first time from the 70% ethanolic extract of S. inermis.Some of these compounds contain different hydroxyl

groups and the others were terpenoids, which help

scavenge free radicals and inhibit COX and various

mediators involved in the pathogenesis of pain relief.

The chloroform fraction showed more potent inhibitory

activity than the ethanol extract, whereas the 70%

ethanolic extract was more potent than the chloroform

fraction in antinociceptive activity.


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