RESEARCH ARTICLE

Antioxidant, analgesic, cytotoxic and antidiarrheal activitiesof ethanolic Zizyphus mauritiana bark extract

Shafiur Rahman

Received: 18 September 2011 /Accepted: 25 October 2011 /Published online: 11 November 2011# Institute of Oriental Medicine, Kyung Hee University 2011

Abstract The primary objective of this study was toinvestigate the pharmacological potential of the etha-nolic extract of Zizyphus mauritiana bark. Antioxidant,analgesic, cytotoxic and antidiarrheal activities of theextract were investigated using 1, 1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay, aceticacid induced writhing inhibition, brine shrimp lethalitybioassay and castor oil induced diarrhea inhibitionrespectively. The extract showed remarkable free radicalscavenging activity with an IC50 of 27.47 μg/mL. Theextract produced significant (P<0.001) writhing inhibitionat the doses of 250 mg and 500 mg/kg in acetic acidinduced writhing in mice. With the extract, significantlethality to brine shrimp was found with LC50 value at92.79 μg/mL. In antidiarrheal activity test, the extractproduced 1.25 h latent period of diarrheal induction and75.2% inhibition in diarrheal episodes compared topositive standard which showed 1.3 h of latent periodand 80.55% inhibition in diarrheal episodes. Differentphytoconstituents present in the bark might be responsiblefor biological activities found with this study. The resultsof this study support the traditional uses of this plant partand could form the basis of further investigation includingcompound isolation.

Keywords Zizyphus mauritiana . Antioxidant activity .

Analgesic activity . Cytotoxic activity . Antidiarrhealactivity . Medicinal plant

Introduction

Among the natural sources, medicinal plants are one of theimportant contributors to most of the medicinal prepara-tions as raw plant materials, refined crude extracts andmixtures, etc. Several thousands of plants have beenidentified containing medicinal values and used to treatdifferent ailments in various cultures worldwide (Farnsworthand Soejarto 1991). Even in this modern world, majority ofthe people are still relying on the traditional medicine fortheir primary health care (Bannerman et al. 1986). Zizyphusmauritiana (Family: Rhamnaceae) is a medium size, muchbranched, deciduous tree with spreading crown, dark greyishblack bark having irregular cracks and strong reddishhardwood (Wadekar and Patil 2008). The tree is found indifferent parts of the world including Bangladesh whereit is locally known as ‘Boroi’ and grows wild on theroad-sides and bushes in all parts of the country (Gani2003). The tree is usually planted for its nutritious fruitswhich are usually eaten fresh (Adzu et al. 2001).Traditionally fruits have been used to treat small cuts,wound, fever, ulcers, blood purification, nausea, vomiting,indigestion, dysentery, stomach disorder and pulmonaryailments (Said 1996; Adzu et al. 2001). Fruit containstannins, saponins, flavonoids, mucilage, reducing sugars,vitamins (A, B2, C), organic acids (ascorbic, citric, tartaricacids) and minerals (Chevallier 1996; Wadekar and Patil2008). The seeds are sedative and useful to amelioratenausea, vomiting and abdominal pains associated withpregnancy (Kaaria 1998).

S. Rahman (*)Phytochemistry and Pharmacology Laboratory,Pharmacy Discipline, Khulna University,Khulna 9208, Bangladeshe-mail: shafiur27@yahoo.com

S. RahmanSchool of Pharmacy, Faculty of Health Science,The University of Queensland,Brisbane, Queensland, Australia

Orient Pharm Exp Med (2012) 12:67–73DOI 10.1007/s13596-011-0042-2

Root has useful effects in treating fever, delirium, gout,rheumatism, constipation and skin diseases (Yusuf et al.1994; Adzu et al. 2001; Dahiru et al. 2005, 2006). Theleaves are applied locally as poultices to sores and helpfulin treating fever, asthma and liver disorder (Adzu et al.2001; Michel 2002). The leaf extracts have also beenreported to have hepatoprotective activity against CCl4-induced liver damage in rats and antioxidant activity(Dahiru et al. 2005, 2006; Dahiru and Obidoa 2008).

Bark has been reported to contain tannin, leucocyani-dine, leucopalargonidine, betulinic acid, ziziphinic acid,resin, zizogenin, peptide alkaloids and different allergens(Srivastava and Srivastava 1979; Devi et al. 1987; Said1996; Lee et al. 2004; Wadekar and Patil 2008). Recentreports show that the tree has enormous potential in provingpharmacological benefit such as anti-steroidogenic activity,antidiabetic activity and antioxidant activity (Gupta et al.2004; Jarald et al. 2009; Bhatia and Mishra 2010; Bhuiyanand Hoque 2010).

However, bark is traditionally used for different ailmentsfor instance as astringent and remedy for diarrhea, woundsand ulcers in different localities of Bangladesh (Gani 2003;Yusuf et al. 1994). No prominent scientific research on thebiological activity of this plant part has been reported. Incontinuation of effort to verify the efficacy and scientificvalue of traditional medicine, the present study was aimedto investigate the antioxidant, analgesic, cytotoxic andantidiarrheal activities of the bark of this plant.

Materials and method

Plant

The bark of Zizyphus mauritiana, collected from Rangpurdistrict of Bangladesh, was identified by the taxonomydivision of Botany Department of Carmichael UniversityCollege, Rangpur. The specimen sample was preserved inthe Phytochemistry Laboratory of Khulna University.

Extraction

The bark was shade dried ground and macerated in 80%ethanol. The solvent was filtered and evaporated to getdried extract (yield value, 12.5%).

Animals

Male and female mice Swiss-webstar strain, 20–25 g, bredin the animal house of the Department of Pharmacy, KhulnaUniversity, were collected from animal resources branch ofthe International Centre for Diarrheal Disease and Re-search, Bangladesh (ICDDR, B) and used for the experi-

ments. The animals were kept in animal house understandard laboratory conditions (relative humidity 55–65%,room temperature 25.0±2°C and 12 h light/dark cycle) andfed with standard diets (ICDDR, B formulated) with freeaccess to tap water.

Preliminary phytochemical analysis

The crude extract was subjected to preliminary phytochem-ical screening for the detection of major chemical groups(Evans 1989). In each test 10% (w/v) solution of the extractwas taken unless otherwise mentioned in individual test.

Acute toxicity test

Test mice were divided into different groups containing sixanimals in each. The groups received the extract orally atthe doses of 62.5, 125, 250, 500, 1,000, 2,000 and4,000 mg/kg body weight, whereas, the control groupreceived distilled water. General sign and symptoms oftoxicity and mortality were recorded for 24 h (Lork 1983).

Antioxidant activity

Antioxidant activity of the extract was determined on the basisof its scavenging activity of the stable DPPH free radical.

Qualitative assay: Suitably diluted stock solutions werespotted on pre-coated silica gel TLC plates and the plateswere developed in different solvent systems (polar, mediumpolar and non-polar) to resolve the components of theextract. The plates were dried at room temperature and weresprayed with 0.02% DPPH in ethanol. Bleaching of DPPHby the resolved bands was observed for 10 min and thecolor changes (yellow on purple background) were noted(Sadhu et al. 2003).

Quantitative assay: Quantitative assay was performed onthe basis of a modified method (Gupta et al. 2003). Samplesof different concentrations such as, 1, 5, 10, 50, 100 and500 μg/mL were prepared from the ethanol stock solutionof the extract (10 mg/mL). Diluted solution (2 mL) fromeach concentration mentioned was added to 2 mL of0.004% ethanol solution of DPPH, mixed and allowed tostand for 30 min for the complete reaction. The absorbancewas determined at 517 nm for each concentration whileascorbic acid was used as positive control. Then with thepercent inhibition, IC50 was calculated. The experiment wasperformed in triplicate.

Analgesic activity

Analgesic activity of the extract was tested using the aceticacid induced writhing model in mice (Whittle 1964; Ahmedet al. 2001). The experimental animals were randomly

68 S. Rahman

divided into four groups, each consisting of eight animals.Control group I received 1% (v/v) Tween-80 solution inwater, group II, as positive standard, was given diclofenacsodium at dose of 25 mg/kg of body weight, test groups IIIand IV were treated with the extract at the doses of 250 and500 mg/kg, respectively. Control vehicle, standard drug andextracts were administered orally 30 min prior to acetic acid(0.7%) injection. After 15 min interval, the numbers ofwrithes (squirms) were counted for 5 min.

Cytotoxic activity

The cytotoxic property of the extract was evaluated using brineshrimp lethality test (Meyer et al. 1982; McLauglin et al. 1993).

Brine shrimp The investigation was done on Artemiasalina (Brine shrimp). One spoon of cyst was hatched for48 h in saline water, prepared by dissolving 20 g pure NaCland 18 g normal edible NaCl into 1 L water. The cyst inturn became living nauplii.

Lethality bioassay In the test, different concentrations ofthe extract were prepared using dimethyl sulfoxide assolvent. For the test, different concentrations of extractprepared were added to test tubes, each containing 10shrimps in saline water and finally volume was adjusted bysaline water. Control (blank) group with 10 shrimps werekept in saline water under the same condition and positivecontrol group, with 10 shrimps in saline water, was treatedwith a known drug chloramphenicol (200 μg/mL). All thetest tubes were kept in rest for 24 h and then counted forliving and dead nauplii. Finally, the percent mortalityproduced by each concentration was used to determineLC50 (lethal concentration) of the extract.

Antidiarrheal activity by castor oil challenge

Antidiarrheal test of the extract was conducted using castoroil induced diarrhea in mice (Nwodo and Alumanah 1991).Three groups of Swiss albino mice, each group consistingof eight animals, were taken for the study. Group I (control)was treated with only saline, while group II (positivestandard) and III (test) were treated with loperamide (4mg/kg)and extract (500 mg/kg) respectively. Both loperamide andextract were administered orally 1 h before the administration

of diarrhea-induced agent (castor oil, 0.1 mL/mice, orally).The animals were kept individually in the cages on filter paperafter treating castor oil. The mice were examined for theoccurrence of diarrhea each hour for 5 h and filter paperwas changed every hour. The latent period (onset ofdiarrhea) and number of diarrheic drops or faces (diarrhealepisodes) were measured.

Results

The phytochemical investigation showed the presence ofalkaloids, tannins, reducing sugars, steroids, flavonoids andgums (Table 1). In the acute toxicity test, the animals (mice)exhibited decreased mobility but no convulsions or loss ofrighting reflex and mortality were observed even at thehighest dose of 4,000 mg/kg tested.

With the antioxidant test performed reacting DPPH withextract, bleaching followed by color change (yellow on purplebackground) of DPPH was observed on the TLC plates inqualitative experiment. In quantitative test, the extract showedremarkable antioxidant activity with an IC50 of 27.47 (20–34.94) μg/mL against DPPH free radical, whereas, standarddrug, ascorbic acid produced IC50 at 18.63(10–27.26) μg/mL(Table 2). In the analgesic activity performed using aceticacid-induced writhing model in mice, the extract producedabout 42.92% and 59.67% writhing inhibition at the doses of250 and 500 mg per kg body weight respectively, when thepositive standard diclofenac sodium inhibited about 77.71%at the dose of 25 mg/per kg (Table 3).

With the lethality bioassay performed with freshly hatchednauplii, the extract produced 100% of mortality at 320 μg/mLconcentration, compared to the positive control chloramphen-icol producing the same effect at 100 μg/mL. LC50 of theextract was calculated to be 92.79 (77.3–108.3) μg/mL(Table 4). In castor oil induced diarrheal test, the extractdelayed the onset of diarrhea by 1.25 h and inhibited 75.2%of the diarrheal episodes (number of stool), compared to thepositive standard producing 1.3 h of latent period and80.55% inhibition in diarrheal episodes (Table 5).

Discussion

Medicinal plants are an important element of indigenousmedical systems and have a long history of serving people

Table 1 Phytochemical groups test of the extract

Plant Extract Alkaloids Reducing Sugars Taninns Gums Flavonoids Saponins Steroids

Ethanolic extract + + + + + – +

+ = Positive result; - = Negative result

Antioxidant, analgesic, cytotoxic and antidiarrheal activities 69

in many regions of the world. Report shows that about 80% ofthe world population still use plants for various medicalpurposes (Schulz et al. 2002; Kong et al. 2003). In the presentstudy, the phytochemical investigation, with Zizyphus maur-itiana bark extract, showed the presence of differentchemical groups including some important chemicals suchas alkaloids, tannins, steroids and flavonoids (Evans 1989).The presence of some of these components was also reportedwith the bark and other parts of this tree (Chevallier 1996;Said 1996). In the acute toxicity test (Lork 1983), the crudeextract appeared to be safe for oral use up to an extendeddose, as the animals (mice) exhibited only decreasedmobility with no mortality. The extract probably did notcontain any toxic components to kill the animals.

Free radical related injury now appears to be thefundamental mechanism covering a range of disorders suchas inflammation, viral infections, autoimmune pathologies,digestive system disorders including gastrointestinal inflam-mation and ulcer, atherosclerosis, ischaemic heart disease,ageing, diabetes, immunosuppression, neurodegenerative dis-eases, cancer and others (Jadhav and Bhutani 2002; Gulcin etal. 2002; Aruoma 2003). Plants are an important source ofnatural antioxidants and in order to survive it producesvarious compounds with scavenging potentials (Lu and Foo

1995). In recent years much attention, therefore, has beenfocused on to the plant derived natural antioxidants and theirassociation with health benefits (Arnous et al. 2001). In ourstudy with antioxidant test performed using DPPH, thequalitative experiment primarily indicated the antioxidantpotential of the extract. In quantitative test, the extractshowed remarkable antioxidant activity with an IC50 whichwas close to value the standard drug ascorbic acid produced.Antioxidant effect of plant products is usually believed to bemainly due to the presence of phenolic compounds such asflavonoids, polyphenols, tannins, and phenolic terpenes(Rahman and Moon 2007; Robaki and Glyglewski 1988;Birs et al. 1991). Therefore, antioxidant effect of this studycould be attributed to the presence of flavonoids and tanninfound with the extract. However, further studies using othermodels such as lipid per-oxidation inhibition, xanthinoxidase inhibition, erythrocytic membrane stability andothers may be suggested to further characterize the extract.

In the analgesic activity performed using acetic acid-induced writhing model in mice, the writhing inhibitionincreased as the concentration of extract was increased. Theextract at higher dose tested (500 mg per kg body weight)produced remarkable writhing inhibition which was compa-rable to the positive standard diclofenac sodium. Acetic acid,which is used to induce writhing, causes algesia by liberatingendogenous substances which then excite the pain nerveendings (Vyklicky 1979; Taesotikul et al. 2003). Increasedlevels of PGE2 and PGF2α in the peritoneal fluid have beenreported to be responsible for pain sensation caused by intra-peritoneal administration of acetic acid (Derardt et al. 1980;Franzotti et al. 2002; Sawadogo et al. 2006). On the basis ofthis result, it can be assumed that the mode of action mightinvolve a peripheral mechanism. The results of phytochem-icals investigation of the extract support the antinociceptiveproperty since, alkaloid, flavonoids and saponins have beenfound responsible for analgesic and anti–inflammatoryproperties (Wagner 1989; Xu 1996; Fernanda et al. 2002;Onasanwo and Elegbe 2006). The presence of steroids mayalso partly contribute to the analgesic effects in animalmodels of nociception (Calixto et al. 2000).

Brine shrimp lethality bioassay indicates cytotoxicity aswell as a wide range of pharmacological activities, such as

Table 2 Antioxidant activity of the extract against DPPH free radicals

Sample Conc. (μg/mL) % inhibition IC50 (μg/mL)

Ethanolic extract 1 1±0.05 27.47(20–34.94)5 8±0.1

10 34±0.2

50 85±0.2

100 86±0.1

200 89±0.05

Ascorbic acid 1 16±0.1 18.63(10–27.26)5 29±0.01

10 46±0.05

50 96±0.1

100 97±0.2

200 97±0.1

Values are expressed as mean ± stdv of three replicates

Table 3 Effect of the extract on acetic acid induced writhing in mice

Animal Group/Treatment Number of writhes (% writhing) n Inhibition (%)

Control (1% tween-80 solution in water, p.o.) 30.5±0.10 (100) –

Positive control (Diclofenac sodium 25 mg/kg, p.o.) 6.8±0.217 ** (22.29) 77.71

Test group I (Et. extract 250 mg/kg, p.o.) 17.41±0.32** (57.08) 42.92

Test group II (Et. extract 500 mg/kg, p.o.) 12.3±0.23** (40.33) 59.67

Values are expressed as mean ± S.E.M of three replicates; **P<0.001 vs. control Students t-test;

Et. Ethanolic,% = percentage, p.o. per oral, n (number of animals)=8

70 S. Rahman

antimicrobial, pesticidal and antitumor activities of thecompounds (Meyer et al. 1982; McLaughlin et al. 1988).With the assay performed with freshly hatched nauplii, theextract appeared to be significantly lethal. However, furtherinvestigations using tumour cell line or others along thisline could be suggested.

Diarrheal diseases are one of the prominent causes ofmorbidity and mortality found especially in developingcountries (Carlos and Saniel 1990). Many people in thedeveloping countries still rely on the treatment systememploying medicinal plants (Ojewole 2004). World HealthOrganization has given special focus on studies oftraditional medical practices directing to the preventionand treatment of diarrheal diseases (Atta and Mouneir2004). In our study, castor oil, used to induce diarrhoea, ishydrolysed in the upper small intestine to ricinoleic acid(Altman 2001). Ricinoleic acid is believed to act byirritating the gastrointestinal tract mucosa and reducingsodium ion and chloride ion permeability, resulting in

increased intestinal motility followed by diarrhoea (Zavalaet al. 1998; Gaginella and Phillips 1975). However, theextract delayed the onset of diarrhea and inhibited thediarrheal episodes (number of stool) indicating the anti-diarrheal potentials of the bark. Since castor oil inducesdiarrhoea by preventing fluid and electrolyte absorption(Goodman and Gillman 1996), one of the probablemechanisms the extract might have is its ability to enhancefluid and electrolyte absorption through the gastrointestinaltract. The extract might contain certain components that actas agonist to opioid receptors present in the GI mucosa andthus relieves diarrhoea (Goodman and Gillman 1996). Thisfinding can be attributed to support the traditional use ofthis plant part especially in diarrhea (Gani 2003; Yusuf etal. 1994). Considering the impact of diarrheal disease in thedeveloping countries (Das et al. 1999), this plant part couldplay a potential role in finding new molecule.

Overall, the findings of this research support some of thetraditional uses of this plant part in different ailments and

Table 4 Effect of the extract in Brine shrimp lethality bioassay

Sample Conc. of extract Number of shrimpstaken

Number of shrimpsalive (avg)

Number of shrimpsdied (avg)

% Mortality (avg)

Ethanolic extract 5 μg/mL 10 10 0 0

10 μg/mL 10 10 0 0

20 μg/mL 10 9 1 10

40 μg/mL 10 07 03 30

80 μg/mL 10 05 05 50

160 μg/mL 10 02 08 80

320 μg/mL 10 0 10 100

Chloramphenicol 100 μg/mL 10 0 10 100

LC50 92.79 (77.3 – 108.3) μg/mL (Values represent mean of three replicates)

Table 5 Activity of the extract against castor oil induced diarrhea

Animal group/Treatment Dose(/kg, per oral)

Avg. latentperiod (h)

Period ofstudy (h)

No. ofstools (avg)

Total no. ofstools (avg)

% inhibition

Control (1% Tween-80) n=8 1 2.6±0.5

30 mL 0.69±0.1 2 4.8±0.7 14.9±0.6 –3 3.8±0.7

4 3.7±0.8

Positive standard(Loperamide) n=8

1 0.4±0.2

50 1.3±0.1 2 0.8±0.4 2.9±0.3** 80.553 1.4±0.4

4 1.1±0.2

Test group (extract ofZizyphus mauritiana) n=8

1 0.8±0.4

500 1.25±0.1 2 0.9±0.4 3.7±0.3** 75.23 1.2±0.3

4 0.8±0.4

Values are expressed as mean ± S.E.M of three replicates; **P<0.01 vs. control Students t-test;

% = percentage, n (number of animals)=8

Antioxidant, analgesic, cytotoxic and antidiarrheal activities 71

could form the basis to lead the study up to isolation ofuseful compounds and compound specific pharmacologicalinvestigations of this plant part which bears enormouspotential.

Conclusion

Ethanolic extract of Zizyphus mauritiana bark has beenfound to possess antioxidant, analgesic, cytotoxic andantidiarrheal activities. The results of this study supportand justify some of its traditional uses.

Declaration of Interest The author thank Pharmacy Discipline,Khulna University for necessary funding to conduct this research. Theauthor also thank Department of Botany, Carmichael UniversityCollege, Rangpur to help identify the plant.

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