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CHAPTER 5

PHYTOCHEMICAL ANALYSIS AND

CHARACTERIZATION OF CALLUS

CULTURES

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Introduction

Medicinal plants are becoming an important research area for novel and

bioactive molecules for drug discovery. Novel therapeutic strategies and agents are

urgently needed to treat different incurable diseases. Many plant derived active

compounds are in human clinical trials. Ursolic acid and oleanolic acid (OA) are

triperpene acids having a similar chemical structure and is distributed widely in plants

all over the world (Takeoka et al., 2000; Zhu et al., 2002). They are of interest to

scientists because of their biological activities. UA and OA also possess liver-

protection (Yim et al., 2001) and anti-inflammatory effects (Ismaili et al., 2001). In

recent years, it was found that they had marked anti-tumor effects and exhibited

cytotoxic activity toward many cancer cell line in culture (Hollosy et al., 2001).

The pharmaceutical industry has synthesized more than 3 million chemicals

for designing new drugs. Despite of their success in developing new drugs, effective

treatment for most diseases has not been a complete success due to complexity of

diseases. These drugs are known to affect the immune system, in addition possess

adverse sideffects. Now a days drug discovery from phyto-medicine or herbal therapy

is mainly focused, rather than synthetic chemicals for the treatment of most disease,

due to no side effects and also it enhances immune system (Pandey et al., 2011). Plant

based drugs provide outstanding contribution to modern therapeutics. The potential

for screening new compounds from plants is enormous, till date only 1% of tropical

plants has been studied for their pharmaceutical potential (Jachak & Saklani, 2007).

During 1950-1970, about 100 plant based drugs were introduced in USA drug market.

During 2000- 2006, 26 percent novel molecule based and plant derived drugs were

approved and launched by USA drug market (Table 5.1) list of chemical mentioned

below (Saklani & Kutty, 2008).

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Table 5.1: Drugs approved/launched based on plant natural products during the

period of 2000–2006 (Saklani & Kutty, 2008).

Year Generic Name Lead Compound Disease area

2000 Exelon (Rivastigmine tartrate) Physostigmine Dementia–Alzheimer’s

2000 Arteether (Artemotil) Artemisinin Antimalarial

2000 Galanthamine HBr (Reminyl) Galanthamine Alzheimer’s disease

2000 Bexarotene Retenoic acid derivatives

Cutaneous T cell lymphoma

2000 L -dopa-methyester (Levomet)

L -Dopa Parkinson’s diseases

2000

Malarone (Atovaquone; proguanil hydrochloride)

Quinine Antimalaria

2000

Rapacuronium bromide (Raplon)

Tubocurarine Neuromuscular blocking agent/anaesthesia

2001

Galanthamine HBr (Reminyl) Galanthamine Dementia–Alzheimer’s

2002 Nitisinone (Orfadin) Leptospermone Antityrosinaemia

2002

Tiotropium bromide Tiotropium Chronic obstructive pulmonary disease

2002 Avinza (Morphine sulfate) Morphine Pain

2003 Miglustat (Zavesca) 1-Deoxynojirimycin Type1 Gaucher disease

2004

Spiriva HandiHaler (Tiotropium bromide)

Tiotropium

Chronic obstructive pulmonary disease

2004 Apokyn (apomorphine HCl) Apomorphine Parkinson’s diseases

2004 Palladone (hydromorphone) Moderate-to-severe pain

2004

DepoDur (morphine sulfate) extended release

Morphine Post-surgical pain relief

2004 Belotecan Campthotecin Ovarian & small lung cancer

2005 Tamibarotene (Amnolake) Retenoic acid derivatives

Acute myelogenous leukaemia

2005

Abraxane (paclitaxel protien-bound particles)

Paclitaxel Breast cancer

2005 THC:CBD (Sativex) THC, CBD MS pain

2006

Taxotere (docetaxel) injection Docetaxel

Head and neck cancer and stomach cancer

2006 Duodote (atropine and pralidoxine chloride) injection

Atropine

Exposure to organophosphorous nerve agents (Antidote)

2006 Exelon (rivastigmine tartrate Phytostigmine Dementia–Parkinson’s

2006 Hycamtim (topotecan HCl) Camptothecin Cervical cancer

2006

Cesamet (nabilone) Delta-9-THC Chemotherapy nausea and vomiting

2006 Polyphenon E (Veregen) Ointment

Green tea polyphenol (catechin) extract

Genital and perianal warts

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Isolation of the analgesic morphine from Papaver somniferum in 1816

(Benyhe, 1994), serpentine from Rauwnolfia serpentina in 1953 provides as sources

of new drug and new chemical entities (NCE). During 1981-2002, 61% of 877 small

molecules (NCE) were introduced as drugs. Viz, natural products (6%); derivied

(27%); Natural product derived pharmacophore (5%) and synthetic compounds

mimics natural products (Newman et al., 2003; Jachak & Saklani, 2007).

The search for anti-cancer agents from plant sources started in earnest in the

1950s with the discovery and development of the vinca alkaloids, vinblastine and

vincristine, and the isolation of the cytotoxic podophyllotoxins. As a result, the United

States National Cancer Institute initiated an extensive plant collection program in

1960, focused mainly in tempenate regions. This led to the discovery of many novel

chemotypes showing a range of cytotoxic activities (Cassady & Douros, 1980).

OA and UA are well known for their hepatoprotective effect for both acute

chemically induced liver injury and chronic liver fibrosis and cirrhosis (Liu, 1995).

UA, a pentacyclic triterpene acid, concentrated in sage leaves (Liu, 1995), inhibit

inflammation related changes in human gingival cells (Zdarilova et al., 2009) and in

other model (Liu, 1995).This compound also has anti-mutagenic activity (Young et

al., 1994).

Moulisha et al., (2010) isolated a pentacyclic triterpenoid, UA from the

methanolic extract of the leaves of Terminalia arjuna – a medicinal plant. They

demonstrated that the compound possess in vitro anti-leishmanial and anti-cancer

activities. Currently UA is in human clinical trial for treating cancer, tumor and skin

wrinkles (Sultana, 2011). Wu et al., (2012a) suggested that UA, derived from a variety

of medicinal plants, exhibits potent anticancer activity against many types of cancer

cells. Kim et al., (2011a) indicated that UA could be used as a potential anticancer

drug for breast cancer. They induced apoptoxic cell death by UA through

mitochondrial death pathway and extrinsic death receptor pathway in MDA-MB-231

cells. Messner et al., (2011) had shown that UA inhibits endothelial proliferation and

is a potent inducer of endothelial cell death. It also causes DNA-damage and oral

application in mice potently stimulated atherosclerotic plaque formation in vivo.

Plants have played an important role as a source of effective anticancer agents,

and it is significant that over 60% of currently used anti cancer agents are derived in

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one way or another from natural sources, including plants, marine organisms and

micro-organisms (Newman et al., 2003; Cragg & Newman, 2005a). Manikrao et al.,

(2011) focused on plant originated natural products UA that could offer better relief

from inflammation than currently used commercial drugs. The Docking analysis

reveals that UA inhibit COX-2 enzyme by hydrophobic and hydrogen bonding

interactions. UA also plays the role of chemical marker in some medicinal plants,

nutraceutical (Cui et al., 2006) and phytopharmaceuticals (Dufour et al., 2007).

Es-Saady et al., (1994) working on the effects of UA and its analogues on

soybean is lipoxygenase activity suggested that UA probably acts on physical and

functional properties of cell membranes and may be an additional class of membrane

active agents with potential anti-cancer activity. It may be a pleiotropic membrane

active agent that seeps into the lipidic layers and affects multiple signal transduction

pathways in mammalian cells.

During the past decade research activities in many disciplines, such as

phytochemistry, food sciences, biotechnology, medicine, etc., broadened the hitherto

narrow view on betalains. The challenge to bringing together knowledge from all

these different areas is considered to be most fruitful (Stintzing & Carle, 2008).

Review of Literature

Scientific documentation of phytochemical constituents of several plant

species have been reviewed in the light of recent literature as follows.

Several workers investigated chemical constituents of leaves, roots, flowers

and seeds of Asteraceae members. Chopra et al., 1956 observed the presence of

triterpenes, saponins, amino acids, ascorbic acid, citric acid and helianthic acid in

Helianthus annuus. Murakani 1973 isolated kirenol from Siegesbeckia pubescens

(Dev et al., 1982).

Similarly, thiophene derivatives from Eclipta species (Singh, 1988),

polyphenols from Psiadia trinervia (Wang & Hostettmann, 1990), sesquiterpene

lactone from Tanacetum densum (Goren et al., 1993), alkaloids, flavanoids, Steroids,

tannins and phenols screened from Desmodium gangeticum and Premna tomentosea.

These phytochemical screening provides biochemical basis for ethnopharmacological

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claims for treatment and prevention of various diseases and disorders (Shanthanayaki

& Suriyavathana, 2010). Essential oil from Siegesbeckia jorulensis and S. orientalis

showed inhibitory activity against protein tyrosine phosphatase (Lee et al., 2002b);

Anti-inflammatory properties of essential oils from Artimisia glabella

(Seidakhmetova et al., 2002); mycotoxigenic inhibitory activity from Achillea

millefolium and Achillea fragrantissima oils (Soliman & Badeaa, 2002) and

antioxidant activity of compounds from the medicinal herb Aster tataricus (Lin et al.,

2003) are some of the screened phytochemical constitutents used in

ethnopharmacological treatments for the prevention or curing of some diseases /

disorders.

Luo et al., (2010) conducted preliminary phytochemical investigation and

antimycobacterial evaluation of the medicinal plants Maerua edulis, Securidaca

longepedunculata, Zanthoxylum capense, and Tabernaemontana elegans. They have

reported that n-Hexane extracts of Maerua edulis and Securidaca longepedunculata,

ethyl acetate extract of Tabernaemontana elegans and dichloromethane extract of

Zanthoxylum capense possess antimycobacterial activity against Mycobacterium bovis

BCG and Mycobacterium tuberculosis H37Ra. They also reported Tabernaemontana

elegans showed strong activity when compared other 3 plant extracts. Linear chain

unsaturated fatty acids (Maerua edulis and Securidaca longepedunculata) and indole

alkaloids (Tabernaemontana elegans) were prominent identified compounds.

There has been an increasing trend towards replacement of synthetic colorants

by natural pigments in the last 25 years because of natural pigment’s safety and health

benefits. Natural pigments are generally less stable and have higher cost than

synthetic colorants and more and more attention has been paid towards their

utilization and development. Betalains are of great taxonomic significance in higher

plants. The presence of betalains in members of the order Caryophyllales has been an

important criterian for their classification. Betalains are water-soluble nitrogenous

pigments. They occur only in the plants from 10 families of the order Caryophyllales.

So far it has been found that betalains in nature comprise approximately 50 red

betacyanins and 20 yellow betaxanthins (Francis, 1999). The presence of betalains

and anthocyanins is mutually exclusive in the Angiosperms (Strack et al., 1993;

Stafford, 1994).

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Betalains attractiveness for use as colorant of food has increased recently due

to their reportedly high anti oxidative free radical scavenging activities and concerns

about the use of various synthetic activities. Recent reports state that betalains and

betalain-containing plant extract have high anti oxidant capacities and have

significantly increased scientific interest in them (Cai et al., 2003; Stintzing et al.,

2005). However, it should be noted that some authors have attributed the high

antioxidant activity of crude betalain-containing extract to their high concentration of

flavonoids (Lee et al., 2002a). Betalains reportedly have diverse, desirable activities

(Lila, 2004), including anti-inflammatory (Lee et al., 2006), hepatoprotective (Galati

et al., 2005), cancer chemo-preventative activities (Kapadia et al., 1996) and the

ability to reduce oxidative stress (Tesoriere et al., 2004a) and protect low density

lipoproteins from oxidation (Tesoriere et al., 2004b).

Recently, Sreekanth et al., (2007) have reported that betanin induces apoptosis

in human chronic mylloid leucemia cells. Studies of the renal extraction of betalains

are of great importance and have shown that renal clearance is a minor pathway in

their overall elimination (Netzel et al., 2005). Hence, betalains are likely to be highly

suitable in natural colorants for preparing healthy foods and their consumption is

likely to increase. Plant cell and tissue cultures are attractive alternative sources of

bioactive plant substances, including betalain pigments (Ramachandra Rao &

Ravishankar, 2002). The biotechnological production of food colorants using plant in

vitro cultures offers several advantages over the conventional cultivation of whole

plants, notably the ability to maintain aseptic, controlled conditions (Vanisree et al.,

2004).

Flavonoids have been reported to possess antibacterial, antioxidant, anti-

inflammatory, antiallergic, antimutagenic and vasodilatory activity (Alan & Miller,

1996). Saponins showed hypocholesterolemic and antidiabetic properties, while

steroids and triterpenoids displayed analgesic properties (Sayyah et al., 2004). The

presence of terpenoids in T. decandra has also been reported by other researchers, and

this plant is widely used in herbal medicine (Hayashi et al., 1993).

UA occurs abundantly in Danshen (Saivia miltiorrhiza L.) (Kong, 1989) and

some other plants, such as S. officinalis (Baricevic et al., 2001), Lepechinia

caulescens (Aguirre-Crespo et al., 2006) and in Silphium trifoliatum (Kowalski,

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2007). A recent functional study demonstrated that much higher concentrations of UA

(and a methanolic extract of Lepechinia caulescens) relaxed rat aorta in NOS-

dependent manner (Aguirre-Crespo et al., 2006). Using radiochrmatographic

procedures and blood cells, Najid et al., (1992) have investigated the direct effects of

UA an anachidonic acid metabolism in comparison with classical lipoxygenase and

cyclooxygenase inhibitors.

Kim et al., (2011) have demonstrated that ursolic acid inhibits tumorogenesis,

tumor promotion and suppress angiogenesis and in their study they have found that

ursolic acid decreased cell proliferation rate and induce apoptosis in human breast

cancer cell line, MDA-MB-231 clearly indicating that UA could be used as a potential

anticancer drug for breast cancer. Wang et al., (2011b) in their study assessed the

protective effect of UA against the lipopolysaccharide induced cognitive deficits in

mice.

Species of Trianthema are known for anti-inflammatory, anti-hyperglycemic,

hepatoprotective and antioxidant application in traditional system viz Ayurveda and

Unani (Geethalakshmi et al., 2010c); anticarcinogenic potential (Bhattacharya &

Chatterjee, 1998b; Bhattacharya & Chatterjee, 1998a).

Many herbal remedies individually or in combination have been recommended

in various medical treatises for the cure of different diseases. T. decandra has been

recognized in different system of traditional medicines for the treatment of diseases

and ailments of human beings. It has been known since ancient times for curative

properties and has been utilized for treatment of various ailments such as burns and

wounds, known for antimicrobial properties, many infectious condition and bacterial

infections, fever, tooth ache, hepatoprotective, analgesic, anti-inflammatory,

antidiabetic and other skin disorders. In the traditional systems of medicine such as

Ayurveda and Unani, T. decandra and its species are used for anti-inflammatory, anti-

hyperglycemic, hepatoprotective and antioxidant. A wide range of phytochemical

compounds including terpenoid, alkaloid and flavonoids have been isolated from this

genus (Geethalakshmi et al., 2010c).

The protective role of T. portulacastrum against diethylnitrosoamine –

induced experimental hepatocarcinogenesis was evaluated (Bhattacharya &

Chatterjee, 1999). Morphometric evaluation of focal lesions showed a reduction of

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altered liver cell foci/cm2 and a reduction of the average focal area. A decrease in the

percentage of liver parenchyma occupied by foci seems to suggest the

anticarcinogenic potential of the plant extract in DENA-induced hepatocarcinogenesis

(Bhattacharya & Chatterjee, 1998b; Bhattacharya & Chatterjee, 1998a).

The result of other preliminary phytochemical studies concluded that the

Euphorbia neriifolia possesses the significant antioxidant activity compared to other

well characterized, standard antioxidant systems in vitro and could serve as free

radical inhibitors or scavengers, acting possibly as primary antioxidants, which might

be due to the presence of alkaloids, tannins, flavonoids, proanthocynidin and

sapogenin (Pracheta et al., 2011a).

An in vitro study on phytochemical composition and antioxidant potential of

Ruta graveolens L. a medicinal plant has been done and the result showed that content

of furanocoumarin-bergapten in the extracts showed good correlation with free radical

scavenging, transition metal reduction and reducing power, while total phenolic

content showed good correlation with nitric oxide reduction potential. Antioxidant

activity of in vitro cultures was significantly higher compared to in vivo plant material

(Diwan et al., 2012).

Phytochemical investigation of the stem bark of Terminalia mollis afforded

friedelin, catechin with epicatechin, gallocatechin with epigallocatechin and 3-O-

methylellagic acid 4'-O- α-rhamnopyranoside. Arjunolic acid with 2α, 3β, 23-

trihydroxy-urs-12-en-28-oic acid, 2α-hydroxyursolic acid, gallic acid, chebulanin and

2''-O-galloylvitexin were isolated from the leaf. Chebulanin, betulinic acid, ursolic

acid, catechin, isoorientin, orientin, isovitexin and punicalagin were isolated from

Terminalia brachystemma leaf (Liu et al., 2009 ).

Another plant derived anticancer compound is podophyllotoxin, it is found in

Podophyllum sp. that is used to produce semi-synthetic anticancer pharmaceuticals

such as etoposide, teniposide, and etoposide phosphate (Lata et al., 2009).

Most of the work published are of in vivo hither to no attempt has been made

to evaluate or compare in vitro. Analysis and characterization of bioactive compound

is responsible for these activities. Hence present chapter aims to evaluate this

objective from ethnopharmaceutical importance.

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Materials and Methods

Chemicals

All the chemicals used were of analytical grade purchased from Hi media

(Mumbai, India). Solvents were purchased from Merck, Mumbai, India.

Material

Callus of Trianthema decandra was harvested, dried at room temperature and were

used for solvent extraction (Fig 5.1).

Fig 5.1: Harvested calli for extraction

Preparation of plant extracts:

Soxhlet extraction: Shade dried callus was powdered with the help of Waring

blender. 25 grams of shade dried callus powder was filled in the thimble and extracted

with 150 ml of methanol upto 48 hours. Final solvent extract was concentrated

separately under reduced pressure (Harborne, 1998).

Fractionation of extracts:

The dried methanol extract was redissolved in 100ml of 70 % aqueous

methanol. The 70% aqueous extract was taken in a separating funnel, 100 ml of

hexane was added to the separating funnel, the funnel was shaken for few minutes and

allowed for phase separation, the organic hexane phase was separated, the lower

methanolic phase was collected. methanolic aqueous phase was taken again in

separating funnel 100 ml of chloroform was added and the funnel was shaken for few

minutes and allowed for phase separation. The lower chloroform phase was collected.

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10-20 ml of water was added to the aqueous phase and taken in a separating

funnel, 100 ml of ethyl acetate was added in the separating funnel and shaken for few

minutes. Ethyl acetate phase was collected and the methanolic fraction was retained.

All the fractions were collected and dried using rotary evaporator and subjected for

preliminary phytochemical analysis.

Preliminary phytochemical analysis

Qualitative Chemical Analysis (Harborne, 1998; Edeoga et al., 2005b)

All the extracts were subjected to qualitative chemical tests to detect the presence of

various phytoconstituents.

Tests for Sterols and Triterpenoids

Libermann-Burchard test:

Extracts treated with few drops of acetic anhydride, boil and cool,

concentrated sulphuric acid is added from the side of the test tube, a brown ring at the

junction of two layers and the upper layer turns green indicates the presence of sterols

and formation of deep red color indicates the presence of triterpenoids.

Salkowski’s test:

Treat the extract in chloroform with few drops of concentrated sulphuric acid,

shake well and allow to stand for some time, red color appears in the lower layer

indicates the presence of sterols and formation of yellow colored lower layer indicates

the presence of triterpenoids.

Tests for Glycosides

Keller kiliani’s test:

� Test I: Extracts 200 mg of the drug by warming in a test tube with 5 ml of dilute

(10%) sulphuric acid on water both at 100 0C for two minutes, centrifuge or filter,

pipette out supernatant or filtrate. Neutralize the acid extract with 5% solution of

sodium hydroxide (noting the volume of NaOH). Add 0.1 ml of Fehling’s solution

A and B until alkaline (test with pH paper) and heat on a water bath for 2 minutes.

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Note the quantity of red precipitate formed and compare with that formed in

Test II.

� Test ll: Extract 200 mg of the drug using 5 ml of methanol and boil on water bath.

After boiling add equal volume of water to the volume of NaOH used in the above

test. Add 0.1 ml of Fehling’s A and B until alkaline (red litmus changes to blue)

and heat on water bath for 2 minutes. Note the quantity of the red precipitate

formed.

Compare the precipitate of Test II with Test I. If the precipitate in test ll is greater

than in test l, then Glycoside may be present. Since test l represents the amount of free

reducing sugar already present in the drug, whereas Test ll represents the glycosides

after acid hydrolysis.

Test for Alkaloids

Mayers test: (Potassium mercuric iodide solution).

To the extract/sample solution, add few drops of mayers reagent, creamy

white precipitate is produced.

Dragendorff’s test: (Potassium bismuth iodide solution).

To the extract/sample solution, add few drops of Dragendorff’s reagent,

reddish brown precipitate is produced.

Wagner’s test: (Solution of iodine in potassium iodide).

To the extract/sample solution, add few drops of Wagners reagent, reddish

brown precipitate is produced.

Tests for phenolic compounds

Ferric chloride test: Extract solution gives blue-green color with few drops of FeCl3.

Shinoda test: (Magnesium hydrochloride reduction test)

To the extract solution, add few fragments of magnesium ribbon and concentrated

hydrochloric acid drop wise, yellowish; yellow –orange occasionally orange colour

appears after few minutes.

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Zinc – Hydrochloride reduction test:

To the extract solution, add a mixture of zinc dust and concentrated

hydrochloric acid. It gives yellowish, yellow – orange occasionally orange colour

appears after few minutes.

Tests for Flavonoids

Shinoda test: (Magnesium hydrochloride reduction test)

To the extract solution add few fragments of magnesium ribbon and

concentrated hydrochloric acid drop wise, pink, scarlet, crimson red or occasionally

green to blue colour appears after few minutes.

Zinc – Hydrochloride reduction test:

To the extract solution, add a mixture of zinc dust and concentrated

hydrochloric acid. It gives red color after few minutes.

Alkaline reagent test:

To the extract solution, add few drops of sodium hydroxide solution,

formation of an intense yellow color that turns to colorless on addition of few drops of

dilute acetic acid which indicates the presence of flavonoids.

Tests for Tannins

Gelatin test:

Extract solution with 1% gelatin solution containing 10% sodium chloride

gives white precipitate.

Ferric chloride test:

Extract solution gives blue-green color precipitate with FeCl3.

Vanillin hydrochloride test:

Extract solution when treated with few drops of vanillin hydrochloride reagent

gives purple red color.

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Test for betalains

For realizing the pigments the following experiment has been done. The

weight of violet red coloured callus obtained has been measured. 862 mg of fresh

weight (FW) of callus having violet red colour was aseptically removed from the

culture flask, macerated and extracted the pigment with cold water (temperature <10).

The extract (16 ml final volume) was centrifuged at 10000 g at 4°C for 15 min.

Optical density of the supernatant was scanned over a range (200 to 600 nm) of

wavelength using a spectrophotometer. The λmax was observed at 537 nm with an

optical density (OD) of 0.164. When 0.1 N HCl was added the colour was red

whereas on adding 0.1 N NaOH the colour was yellow which was not reversible to

red on further addition of acid. From the previous observations,the pigment was

confirmed as betalains. Total pigment content (mg/g FW) = OD × DF × final volume/

1120 × 0.862, where 1120 is a constant factor based on betanin molar absorptivity.

The total pigment content was calculated using the previous equation as 2.72 mg/g

FW.

Separation of compounds by Thin Layer Chromatography

TLC studies were carried out to find the number of compounds present in the

partition fractions of ethyl acetate, hexane, chloroform and also in the methanolic

extract.

Sample Preparation: Small quantities of each fraction were dissolved

separately in methanol. Chromatography of the active extracts was used on silica gel

60 F254, Merck. The plates were activated in an hot air oven at 110 oC for 20 min.

Samples were applied to the adsorbent surface at about 2 cm from the bottom using a

capillary tube and developed in glass chambers (6X25 cm) previously saturated with

the vapours of the respective solvent systems. Butanol: Acetic acid: Water (4:1:5);

Chloroform: Acetone (7:1); Chloroform: Methanol (9:1); Chloroform: Methanol:

Water (6:3:1); Chloroform:Methanol: Acetic acid (7:2:1): Methanol: Water: Acetic

acid (9:0.5:0.5), Chloroform: Methanol: Acetic acid (9:0.5:0.5), v/ v/ v were the

solvent systems used. The plate was removed when the solvent travelled four fifth of

the length of the adsorbent on the plate. Visualization was done initially with UV light

(366–254 nm). Three replicates of each sample were examined and mean Rf Values

were taken. Finally visualized under iodine vapours.

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The retention factor, or Rf, is defined as the distance travelled by the compound to the

distance travelled by the solvent.

Retention Factor�Rf� �Distance travelled by the solute from the origin

Distance travelled by the solvent front from the origin

Column chromatography:

10-20 g of 100-200 mesh size silica gel was first dissolved in methanol, then

stirred to mix the combination thoroughly to remove the air bubble and then slowly

transferred into the glass column, the silica particle settles slowly in the column as

stationary phase. The slurry of activated silica was made in and charged in the column

in small portions with gentle tapping after each addition, in order to ensure uniform

packing. A small quantity of solvent was allowed to remain at the top of the column

in order to avoid the drying of the column. Tapping is necessary to avoid the air

bubble formation in the column during packing which otherwise may interfere in the

separation. The packed column was kept undisturbed overnight.

Once the column is packed with methanol the eluting solvent mixture was

prepared i.e., n-hexane:ethyl acetate (5:4) as mobile phase, mobile was passed

through the column for some time and collected at the bottom. 2g of sample to be

separated was taken and dissolved in known amount of methanol (may be in 1ml of

methanol), and the flow rate was set to 10 ml/min. Sample was loaded on the column

and eluted with mobile phase. The fractions of 10 ml in each tube were collected in a

test tube, about 100 ml of hexane; ethyl acetate phase was eluted following 100 ml of

hexane: ethyl acetate: methanol 30:30:30 was eluted. The column was finally washed

with methanol. The fraction was dried and dissolved in known amount of methanol.

TLC studies were carried out to find the number of compounds present in the eluted

fractions of the column chromatography.

HPLC.

Isolated compound was dissolved uniformly in 100% methanol and 10µl of

sample taken and made up to 1ml in HPLC grade methanol and injected in the HPLC

system.

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HPLC and Mass Spectrometric analysis

The majore peak was obtained at the retention time of 4.58 min. The analysis

T. decandra fraction was done in High Performance Liquid Chromatographic system

(HPLC) equipped with LC8A pump, SPD-M 10 A vp photo array detector in

combination with class LC 10 A software (Agilent). The chromatographic conditions

for the analysis were as follows: mobile phase: Acetonitrile: 0.1% Formic acid (10:90

v/v) column: ODS (Octadecyl silane) Zorbax SB C18 (4.6x250mm) 5µm Detector:

SPD-M 10 A vp photo array detector, wave length: 210 nm, flow rate: 1.0

ml/min, injection volume: 20 µl. HPLC injections for each of the sample was done at

least in duplicate. HPLC chromatogram is shown (Fig. 5.7).

HPLC chromatogram has been detected at absorbance of 210 and 254 nm by DAD.

LCMS/MS

Isolated compound was dissolved in the 100% DMSO, 5µL of sample is

dissolved in 0.5ml methanol and injected in the LCMS/MS system and scanned from

100 -1000 m/z under following chromatographic condition

MS Conditions:

Mobile Phase : Acetonitrile: 0.1%Formic Acid (10:90 v/v)

Mode : Positive

Injection Volume : 5µL

Column : Zorbax SB C18 (4.6x150mm) 3.5µm

Flow rate : 0.5ml/min without splitter

Total run time : 21 mins for HPLC and 1 min for LCMS

LCMS/MS Scan- : 100 amu to 1000 amu

Instrument – Triple Quad-6410 (Agilent Technologies)

NMR

NMR spectra were recorded on Varian 400 MHz instrument in CDCl3 and

DMSO solutions. Chemical shifts are reported with respect to tetramethylsilane

(TMS, δ = 0.0) as internal standard (for 1H NMR) and the central line (77.0 ppm) of

CDCl3 (for 13C NMR) expressed in parts per million (δ) downfield from Me4Si. Spin

multiplicities are given as s (singlet), d (doublet), t (triplet), and m (multiplet) as well

as b (broad). Coupling constants (J) are given in hertz.

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FTIR

Infrared spectra were recorded on a FTIR spectrometer (JASCO Model no: FTIR-

4200).

Results

Preliminary phytochemical analysis:

The present study carried out on the callus extracts of T. decandra, revealed

the presence of medicinally achive metabolites.The phytochemical characters of the

leaf callus investigated are summarized in the table 5.2.

Choloroform extracts of the callus showed the presence of saponins,

glycosides, triterpenoids. alkaloids, steroids, flavonoids, tannins, phenolics were

absent in this fraction. Hexane fraction of the callus indicated the presence of

saponins, steroids and glycosides.

Methanol extract of the callus was found to contain phenolics, flavonoids,

alkaloids, steroids, saponins and triterpenoids..

In the ethylacetate fraction almost all the secondary metabolites such as

phenolics, tannins, Havonoids, alkaloids, steroids, saponins, glycosides and

triterpenoids.

From the preliminary phytochemical analysis it is evident that only ethyl

acetate fraction of the callus contains triterpenoid, alkaloid and saponin. Apart from

these, other secondary metabolite constituents are also detected that includes the

tannins and glycosides at very low content. For further charterization of compounds,

ethyl acetate fraction was considered.

172

Table 5.2: Qualitative analysis of the phytochemicals of different fractions of T.

decandra callus extract

Sl

No Test

Hexane

fraction

Chloroform

fraction

Ethyl

acetate

fraction

Methanol

fraction

1 Phenolics a.Shinoda test - - + +

b.Zinc – hydrochloride reduction test

- - + +

c.Ferric chloride test - - + +

2 Tannins

a.Vanillin hydrochloride test _ _ + _

b.Gelatin test _ _ + _

c. Ferric chloride test _ _ + _

3 Flavonoids

a.Alkaline reagent test - - + +

b.Shinoda test - - + +

c.Zinc – hydrochloride reduction test

- - + +

4 Alkaloids

a.Mayer’s reagent test - - + +

b.Wagner’s reagent test - - + +

c.Dragendroff’s reagent test - - + +

5 Steroids

a.Leibermann-Burchardt’s test + _ + +

6 Saponins

a. Foam test + + + +

7 Glycosides

a. Keller Kiliani’s test + + + +

8 Triterpenoids

a. Libermann-Burchard test _ + + _

b. Salkowski’s test _ + + _

173

TLC for ethyl acetate fraction

TLC is used for the separation of mixtures and identification of constituents

using different solvents. Higher the retention speed or low the retention time on TLC

plates, better the solvent would be and vice versa. A number of solvents and solvent

mixtures were tried. Butanol: Acetic acid: Water (4:1:5); Chloroform: Acetone (7:1);

Chloroform: Methanol (9:1); Chloroform: Ethanol (9:1); Chloroform: Acetic acid

(7:1): Chloroform: Methanol: Water (9:0.5:0.5), Chloroform: Methanol: Acetic acid

(9:0.5:0.5), v/ v/ v and finally Methanol : Water : Acetic acid (9:0.5:0.5) was found to

be the best solvent system for separation and from the elution system, three fractions

were separated (Fig. 5.2 & 5.3).

Fig. 5.2: Wave length 366 Fig. 5.3: wave length 254

Fig 5.2 & 5.3: TLC of ethyl acetate fraction

Pigmentation

Stem explants of T. decandra on MS medium supplemented with 2,4-D (0.5 to

1.0 mg/L) and TDZ (0.5 to 2.0 mg/L) produced different intensity of pigmented

callus, but the higher intensity of pigmented callus was observed on the medium

supplemented with 1.0 mg/L 2,4-D and 2.0 mg/L TDZ (Figure 5.4 and Table 5.3).

174

Figure 5.4. Pigmented callus initiation from stem explants on MS medium

supplemented with 1.0 mg/l (2,4-D) and 2.0 mg/l (TDZ) after 8

weeks of culture

Table 5.3: Effect of 2, 4- D and TDZ on pigmented callus formation from stem

explants of T. decandra on MS medium

Growth regulator

(mg/l) Nature of callus Intensity of pigmented callus

formation 2,4-D TDZ

0.5 0.5 Pink, creamy, compact +

1.0 1.0 Pink, creamy, compact +

0.5 1.5 Red, fragile +

1.0 2.0 Dark red, soft +++

Intensity of pigmented callus: + (low), ++ (moderate), +++ (high)

The callus was further subcultured on MS medium supplemented with BAP,

Kn, IBA and adjuvant CW at different concentrations and combinations. The

luxuriant pigmented callus was achieved on MS medium supplemented with BAP

(1.0 mg/l) + CW (20%) as well as IBA (0.5 mg/l) + Kn (0.5 mg/l) + CW (15%) after 8

weeks of subculture (Figure 5.5 and Table 5.4). As the concentration of 2, 4-D and

TDZ increased upto 5 mg/l callogenic potentiality of the explants decreased so also

the pigmentation was reduced. At 1.0 mg/l 2, 4-D and 2.0 mg/l TDZ callus with rich

in betalain pigment was achieved which has medicinal and nutritional importance.

Trianthema decandra red-pink pigments are susceptible to temperature and

pigment stability is found at 21±2ºC in the light and 14-16ºC in the dark and pH range

is 5.8. The dried pigments were very stable at 30ºC. they are found to be unstable

beyond 40ºC. The dried pigmented callushave less storage stability (43.2% pigment

175

retention) at 21±2ºC after 12-week storage. At 4ºC there is retention of pigmentation

even upto 10-week-storage and subsequently there is decline in pigmentation.

Plant cells consume the inversion products of sucrose like glucose and

fructose and the process of biosynthesis of betalains may began with the end of

hydrolysis of sucrose and these pigmented calli was initiated from the 12th day of

culture. The biosynthesis of betalains occurred between the 12th and 25th days and

being intensive between the 15th and 20th days and after a period of 20 days the

maximum amount of betalains was produced.

Figure 5.5. Pigmentation in subcultured callus on MS medium supplemented

with BAP (1.0 mg/l) and CM (20%), after 8 weeks of subculture.

176

Table 5.4: Effect of plant growth regulators and an adjuvant on pigmentation of

subcultured red color callus on MS medium

Growth regulators (mg/l) Adjuvant Intensity of pigmentation on

subcultured callus BAP IBA Kn CW (%)

0.5 - - 15 +

1.0 - - 20 +++

1.5 - - 40 ++

2.0 - - 60 +

- 0.5 0.5 15 ++

- 0.5 1.0 30 +++

- 0.5 1.5 45 ++

- 0.5 2.0 60 +

Intensity of pigmentation = + (low), ++ (moderate), +++ (high)

It has been established that sucrose is energetically the most suitable carbon

source for the cultivation of in vitro plant cultures, particularly for the biosynthesis of

secondary metabolites (Su, 1995; Ilieva & Pavlov, 1997). The process of rapid

hydrolysis of sucrose to glucose and fructose was observed on the 6th day. It should be

noted that the process of biosynthesis of betalains began with the end of hydrolysis of

sucrose and correlated with the consumption of glucose and fructose. The pigment so

obtained was subjected to laboratory test using a spectrophotometer confirmed as

betalains. Maximum absorbance of the pigment extract was at 537 nm which indicate

the presence of β-cyanin. Quantification was done according to the reported method

(Sánchez et al., 2006).

Eluent collected from Column Chromatography for ethyl acetate fraction

All the fractions collected through Column Chromatography was evaporated

using rotary evaporator and subjected for TLC. The dried fraction was dissolved in

methanol, spotted on an activated TLC plate, and separated with a solvent

combination of Methanol: Water: Acetic acid (9:0.5:0.5). The TLC plate was kept in

iodine chamber for staining and stained chromatogram plate was photographed.

177

Eluent 13th 14th and 15th have found to be similar in TLC, therefore these were pooled

and categorized as fraction T. decandra (Fig.5.6), and further subjected for spectral

analysis like HPLC, LCMS/MS, FT-IR and NMR.

Fig. 5.6: TLC of T .decandra fraction

The phytochemical analysis of soxhlet extracts using solvents such as

petroleum ether, chloroform, ethyl acetate and methanol successively has shown the

presence of phenols, tannins, flavonoids, alkaloids, steroids, saponins, glycosides and

triterpenoids.

178

Fig. 5.7: HPLC chromatogram of T. decandra fraction.

The HPLC analysis of T. decandra fraction shows that the peak eluted at 4.58

min is predominate showing the absorbance at maximum at 210 nm (Fig.5.7). The

same fraction was then passed to the MS to check the molecular weight of the

compound.

Mass Spectrometry

The mass analysis of T. decandra was studied in Agilent 6400 QQQ system

equipped with Mass hunter software. The sample was run in ESI positive scan mode

and monitored using single ion monitoring mode. The mobile phase consisted of

Acetonitrile: 0.1% Formic acid: 10:90 v/v, flow rate 0.3 ml/min and mass conditions

optimized for major peaks were as following Fragmentor voltage 135, Delta EMv

350, Gas temperature: 300oC, Gas flow 6 l/min, Nebulizer pressure 15 psi and

capillary voltage 4000V. The sample gave a major peak with a (M+H+) mass of 457.3

(Fig. 5.8).

179

Fig.5.8: LCMS/MS of T. decandra fraction

The MS spectra was scanned from 100 to 1000 amu the major peak of a

(M+H+) 457.3 was observed .

Fig . 5.9: FTIR Spectra of T. decandra

180

Fig

5.1

0:

NM

R a

nd

DE

PT

sp

ectr

a o

f T

. d

eca

nd

ra

181

Fig

5.1

0:

NM

R a

nd

DE

PT

sp

ectr

a o

f T

. d

eca

nd

ra

182

The compound isolated from leaf callus of Trianthema decandra have closely

related structures of Ursolic acid, belongs to the group tritepenoids- a pentacyclic

triterpene, as analysed by means of spectral analysis.

CH3

OH

CH3

CH3

CH3

CH3

O

OHH

H

CH3CH3

• The mass spectra of compound displayed a molecular ion peak atm/z 457.4

corresponding to the molecular formula of C30H48O3

• Its IR spectrum showed characteristic absorption of bands for hydroxyl group at

3354 cm-1 & C-H aliphatic stretching at 2925 cm-1 & 2855 cm-1.

• The 1H-NMR spectrum of the compound exhibited deshielded multiplets at δ 5.32

and δ 3.17 for O-H & C-H of C=CH respectively and for other protons exhibited

between δ 2.26-0.81 ppm. 13C-NMR spectrum displayed its all carbons from δ

13.87-70.85 & at δ 127.70 to 129.66 for alkene carbons and at δ 172.75 for C=O of

COOH.

IR: O-H 3353.6 cm-1, C-H (aliph) 2924.52-2855.1 cm-1, C=O 1737.55 cm-1.

1H-NMR: δ 5.32 (2H, s, br), 3.17 (1H, s), 2.26 (2H, s, br), 1.97 (2H, s, br), 1.88 (3H,

s, alkyl), 1.50 (5H, m, br), 1.23 (27H, s, alkyl), 0.85-0.81 (6H, m, alkyl).

+ESI: m/z 457.4 (M+1).

Discussion

Nature is the best combinatorial chemist and possibly has answers to all

diseases of mankind. Till now, natural products discovered from medicinal plants

have provided numerous clinically useful drugs. In spite of the various challenges

encountered in the medicinal plant-based drug discovery, natural products isolated

from plants will still remain an essential component in the search for new medicines.

183

The fact that only about one-tenth of the flowering species occurring globally are

investigated for their pharmaceutical potential, can be the obvious advantage to

begin with plant/ medicinal plant-based drug discovery programmes.

The present study carried out on the plant callus extracts revealed the presence

of medicinally active constituents. Various solvent extracts of calli were used to

identify for the presence of various phytochemicals. The systematic search for useful

bioactives from the plant callus extracts is now considered to be a rational approach in

nutraceuticals and drug research. The results of phytochemical analysis

comprehensively validate the presence of therapeutically important and valuable

secondary metabolites. The scientific investigation of traditional herbal remedies may

provide valuable tool for the development of alternative drug and therapeutic

strategies. The phytochemical screening and quantitative estimation of the percentage

crude yields of chemical constituents of the plants studied showed that the leaves and

stems were rich in alkaloids, flavonoids, tannins and saponins. They were known to

show medicinal activity as well as exhibiting physiological activity (Sofowora, 1993).

The phytochemical screening and qualitative estimation of the plants studied

showed that the leaf callus was rich in carbohydrates, proteins, amino acids and

sterols in all the extracts. Some extracts showed the presence of alkaloids and

flavonoids too. Steroids were found to be present in almost all the extracts of the

plants. These compounds have significant application against human pathogens and

are reported to have curative properties against several pathogens and therefore could

suggest their use in the treatment of various diseases (Hassan et al., 2004).

Asiatic acid and asiaticoside found in Cantella asiatic showed great promise

in prevention and treatment of cancer either as a plant alone or in combination with

other forms of chemotherapy such as vincristine from Catharanthus rosens

(Brihgman, 2003).

Triterpenoids are reported to have useful for antibacterial activity and can be

applied against various bacterial pathogens like S. aureus, Shigella flexneri,

Pasteurella multveida, E. coli, Salmonella etc. (Utami et al., 2011).

Plant tissue culture is a potentially useful technique for the study of the

biosynthesis of secondary metabolites and for the production of commercially

184

important plant natural products (Verpoorte et al., 2002; Noel & Dayrit, 2005).

However, in a number of cases, plant tissue cultures have also produced novel

substances that are not observed in the intact plant. A callus of Trianthema decandra

was established using leaf explants to determine the secondary metabolites, which

would be produced in the tissue culture.

Callus was established for T. decandra on a variety of media. The callus

grown in Murashige and Skoog medium supplemented with 2,4-D and BAP at 1.0

mg/l levels was flesh-colored, friable and fast-growing. It appeared to be

morphologically stable and exhibited a characteristic metabolite profile that

remained unchanged over two years. TLC analysis of extracts showed that under all

culture conditions the secondary metabolites that were present in the ethyl acetate

extract of the intact callus and trace amount were detected in the corresponding

extract from the tissue cultures.

Various factors which affects plant cell culture includes nutrients, light,

incubation period, type and concentration of growth regulators, were found to be

important determinants of secondary metabolite production. This investigation reveals

that the calli proliferation, cell biomass and pigmentation of T. decandra improved

significantly on MS medium containing BAP and NAA. These observations are in

agreement with similar reports for growth of culluses and cell cultures of Carthamus

tinctorius (Hanagata et al., 1992). However, there is decline in cell growth beyond six

weeks and may be due to depletion of some of these medium components. This

finding is supported by the observation of production pattern of a secondary

metabolite which is directly related to cell biomass. The fresh cell biomass revealed

the highest betalains production as compared to the dry biomass. This could be due to

increased oxidation of betalains at high temperature (60ºC) used for drying the

biomass. In addition, the red and pink pigment contents were also found higher in the

cell cultures of T. decandra which is in accordance with the previous reports (Gao et

al., 2000).

It has been established that sucrose is the most suitable carbon source for the

cultivation of in vitro plant cultures for the production of secondary metabolites. The

process of rapid hydrolysis of sucrose to glucose and fructose during the static

cultures of T. decandra and the pigmentation was observed on 12th day after culture.

185

This result is in accordance with the previously reported cultivation of Beta vulgaris

hairy root culture (Ilieva & Pavlov, 1997) who noted that the process of biosynthesis

of betalains began with the end of hydrolysis of sucrose and correlated with the

consumption of glucose and fructose.

Betalains comprise a class of nitrogen-containing plant pigments found in the

cell sap of plants representing most families of the Caryophyllales (Achatocarpaceae,

Aizoaceae, Amaranthaceae, Basellaceae, Cactaceae, Chenopodiaceae, Didiereaceae,

Halophytaceae, Hectorellaceae, Nyctaginaceae, Phytolaccaceae, Portulacaceae and

Stegnospermataceae). Only two families the Caryophyllaceae and Molluginaceae

produce anthocyanins (Mabry, 2001). Betalains are also found in some higher fungi,

belonging to the genera Amanita and Hygrocybe (Zry¨d & Christinet, 2004). Betalain

stability was influenced greatly by light. It has been reported to deteriorate betalain

stability (Herbach et al., 2007). It has been reported that betalain are stable between

pH ranging from pH 3.0 to 7.0 (Stintzing & Carle, 2004). Betalain are readily to be

degraded beyond this range. Color degradation observed at pH 7.0 in the present

study might be due to hydrolytic cleavage of aldimine bond, which yield colorless

Cyclo-Dopa-5-0- β-glucoside (Herbach et al., 2006).

Light affects betalain biosynthesis in all types of plant in vitro systems. Leaf

discs from Beta vulgaris (Wohlpart & Black, 1973) and seedlings of Amaranthus

caudatus L. cv. Pendula (Bianco-Colomas, 1980) grown in vitro reportedly

accumulate more betacyanins and amaranthin, respectively, when they are cultivated

in light rather than in the dark. When green callus culture of Beta vulgaris cv. Bikores

Monogerm was exposed to light, it started to form redcolored segments (Girod &

Zryd, 1987). It should be noted that in some cases the effects of light depends on its

wavelength. For example, white light enhances the accumulation of betacyanins by

nodal segments of Alternanthera brasiliana L. cv. Kuntze more strongly than UV-A

light (Silva et al., 2005). Betalain biosynthesis can be induced by exposure to blue

and UV light in callus cultures of Portulaca sp. Jewel (Kishima et al., 1995). It has

also been suggested that betacyanin formation in suspension cultures of Chenopodium

album L. is regulated by a mechanism that can be induced by UV-light (Rudat &

Go¨ring, 1995). Maximum levels of betalains are accumulated by hairy root cultures

of Beta vulgaris L. when they are grown under continuous illumination (Mukundan et

al., 1999). Moreover, when hairy roots of Beta vulgaris cv. Detroit Dark Red are

186

grown under a combination of blue and far red radiation, the accumulation of both

betacyanins and betaxanthins is enhanced (Shin et al., 2003).

Phytochemicals have significant application against human pathogens, and are

reported to have curative properties against several pathogens and therefore could

suggest their use in the treatment of various diseases (Hassan et al., 2004). As

phytochemicals often play an important role in plant defense against prey,

microorganism, stress as well as interspecies protection, these plant components have

been used as drugs for millennia and hence, screening of phytochemicals serves as the

initial step in predicting the types of potential active compounds from plants (Chew et

al., 2011). The presence of phenolic compounds are thought to be toxic to

microorganisms, inhibiting the enzymes which are essential for the growth of

microorganisms (Khanamadi et al., 2010).

All these phytochemicals possess good antioxidant activities and has been

reported to exhibit multiple biological effects including anti-inflammatory, anti tumor

activities. These results are in support with the observation made by Bigoniya and

Rana (2009). Presence of saponin, flavonoids and tannins in the extract of T.

decandra has been correlated to possess good haemolytic, in vitro antioxidant activity

and as free radical scavangers as reported by Pracheta et al., (2011b). Plant phenolics

are the widest spread secondary metabolite in plant kingdom. These compounds have

received much attention as potential natural anti-oxidant in terms of their ability to act

as both efficient radical scavengers and metal chelator. The importance of the

antioxidant constituents of plant materials in the maintenance of health and protection

is also raising interest among scientists, food manufacturers and consumens (Kumaran

& Karunakaran, 2007). The phytochemical screening and qualitative estimation of the

callus of T. decandra studied showed to be rich in steroids, saponins and glycosides in

most of the extracts. It should be noted that steroidal compounds are of importance

and of interest in pharmacy due to their relationship with such sex hormones (Edeoga

et al., 2005b). The presence of glycosides in the extract of T. decandra leaf callus

correlates its usage in Indian medicinal system (Ayoola et al., 2008). The

phytochemical screening shows T. decandra are rich in alkaloids, flavonoids, …. are

popular phytochemical constituents. Hence the study has provided some biochemical

basis for ethnopharmacological claims of this plant in the treatment and prevention of

187

various diseases and disorders including the preventing or slowing the progress of

ageing.

It is suggested that the methanol extracts of leaf and its callus revealed a

significant scope to develop a novel broad spectrum used to carry out

pharmacological evaluation to be used as antibacterial drugs (Arumugam et al., 2011).

The results of the present study showed that, leaf callus extracts of T. decandra

especially ethyl acetate extract possess bioactive compounds with antibacterial

activity (previous chapter) against many pathogens and can be used to carry out

further pharmacological evaluation.

Various factors affecting plant cell culture, such as concentration of essential

nutrients, stress factors, light, incubation period and concentration of growth

regulators, were found to be important determinants of secondary metabolite

production.

With reference to physico-chemical parameters, the stability of pigments is

better at 21±2ºC and pH range is 5.8 and the pigment retention is only upto 10 weeks,

contrarily Cai et al., (1998) have shown that betalains in Amaranthus are susceptible

at lower temperatures (≤ 14ºC) and being more stable at pH 5.6 and at 40ºC, so also

storage stability (95.6% pigment retention) even after 20 week storage at 22ºC and

significantly increased pigment retention (76.2%) after 20 week storage.

It has been established that sucrose is the most suitable carbon source for the

cultivation of in vitro plant cultures for the production of secondary metabolites. The

process of rapid hydrolysis of sucrose to glucose and fructose during the static

cultures of T. decandra and the pigmentation was observed on 12th day after culture.

This result is in accordance with the previously reported cultivation of Beta vulgaris

hairy root culture (Ilieva & Pavlov, 1997), who noted that the process of biosynthesis

of betalains began with the end of hydrolysis of sucrose and correlated with the

consumption of glucose and fructose. The results obtained from the growth of T.

decandra callus culture, biosynthesis of betalains and the uptake of the main nutrients

showed that it was distinguished by certain physiological peculiarities, which is

related to the biosynthesis of betalains. Betalains exhibit anti cancer activity and

antioxidant properties (Gentile et al., 2004).

188

Frighetto et al., (2008) isolated UA from apple peels by high speed counter-

current chromatography where they were able to isolated UA in all the solvents used

including ethyl acetate. But in the present investigation, UA was the major component

in the ethyl acetate extract only, and was not found in the other solvent used. Ethyl

acetate extraction has been reported as an effective process, when made by successive

supersonication followed by defatting with petroleum ether and could be purified by

conventional chromatographic method (Ma et al., 2005). The UA sample obtained

from HPLC, LCMS/MS experiments from the leaf callus of T. decandra extract was

compared with the commercial sample of the acid in TLC and GC and showed IR

spectrum identical to the commercial sample.

The chemical analysis of ethyl acetate fraction of T. decandra leaf callus

revealed UA as its main component and in the CHCl3 fraction this triterpenoid is

found to be absent. In contrast, the UA was more concentrated in the chloroform

extract of Salvia officinalis leaves. Furthermore, UA, as a component of S. officinalis,

exhibited strong anti-inflammatory properties (Baricevic et al., 2001).

Certain cultures produce novel secondary metabolites not found in vivo (Bohm et al.,

1980). In the present investigation from the cultures of T. decandra a de novo

pharmaceutical compound UA has been identified which is absent in vivo leaf extract.

Currently UA is in human clinical trial for treating cancer, tumor and skin

wrinkles. UA, a pentacyclic triterpenoid derived from a variety of medicinal plants,

exhibits potent anticancer activity against many types of cancer cells. However, the

anticancer mechanism of UA is not clearly understood (Sultana, 2011).

Triterpenoids are an interesting group of compounds in nature. OA and UA

are triterpenoid compounds that exist widely in food, medicinal herbs and

other plants. During the last two decades, pharmacological studies of OA and UA

indicate that these two triterpenoids have many beneficial effects, notably

hepatoprotection, antiinflammation, antitumor-promotion and antihyperlipidemia.

These two triterpenoids are relatively non-toxic, and oleanolic acid has been marketed

in China for human hepatitis (Liu, 1995; Silva et al., 2008; Yu et al., 2010). In the

future, more mechanistic-oriented basic research is needed to elucidate the

mechanisms of action.

189

To conclude, the scientific investigation of traditional herbal remedies may

provide valuable tool for the development of alternative drug and therapeutic

strategies while the actual compounds isolated from the plant frequently may not

serve as the drugs, they provide leads for the development of potential novel agents.

The UA may be considered as a novel compound in the callus extract of T. decandra

and as promosing lead compound for developing new anti-cancerous and anti-

inflammatory drugs.

The coloring properties and desirable biological activities of betalains have

aroused strong scientific interest in the in vitro production of these important food

colorants. Although no large-scale processes have been developed yet, several highly

productive plant in vitro systems, including cell suspentions, hairy root cultures and

bioreactors have been reported. These considerations clearly indicate the large-scale

production of betalains is technically and commercially feasible.

The presence of biologically important phytochemicals in the calli of T.

decandra, as tested for in this study, contribute to their medicinal value, and therefore,

point to potential sources for useful drugs.

These findings provide, at least in part, some scientific basis for the

ethnomedicinal use of this species. This study also supports the view that the ethno-

directed bio-rational approach is the best search strategy for discovering bioactive

extracts of medicinal plants with an increased hit rate. Above all, these studies form a

good preliminary basis for the selection of plant species for further in-depth

phytochemical and pharmacological investigations.