See

discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/230667077

Characterization of Chemical Groups and Study

of Antioxidant, Antidiarrhoeal, Antimicrobial
and Cytotoxic activities of ethanolic extract of
bacopa moneri (Family: Ebenaceae) Leave...

Article in Journal of Pharmacy Research · January 2012

Impact Factor: 2.89

READS

677

7 authors, including:

Md. sariful Islam Howlader Maizbha Uddin Ahmed

Khulna University Monash University (Australia)
9 PUBLICATIONS 21 CITATIONS 43 PUBLICATIONS 109 CITATIONS

SEE PROFILE SEE PROFILE

Zubair Labu Mohammad Safiqul Islam

World University of Bangladesh Noakhali Science & Technology University
34 PUBLICATIONS 7 CITATIONS 73 PUBLICATIONS 145 CITATIONS

SEE PROFILE SEE PROFILE

Available from: Mohammad Safiqul Islam

Retrieved on: 15 June 2016
 Sariful Islam Howlader et al. / Journal of Pharmacy Research 2012,5(6),3050-3052
Research Article Available online through
ISSN: 0974-6943 www.jpronline.info
Characterization of Chemical Groups and Study of Antioxidant, Antidiarrhoeal,
Antimicrobial and Cytotoxic activities of ethanolic extract of Diospyros blancoi
(Family: Ebenaceae) Leaves
Md. Sariful Islam Howlader1 , Muhammad Shahdaat Bin Sayeed2 , Maizbha Uddin Ahmed2 ,
Abdul Kader Mohiuddin3 , Zubair Khalid Labu 3 , Sm Faysal Bellah2 , Mohammad Safiqul Islam4 *
1
Department of Pharmacy, University of Khulna, Khulna-9208, Bangladesh
2
Department of Clinical Pharmacy and Pharmacology, University of Dhaka, Dhaka-1000, Bangladesh.
3
Faculty of Pharmacy, University of Dhaka, Dhaka-1000, Bangladesh.
4
Department of Pharmacy, Noakhali Science and Technology University, Sonapur, Noakhali-3802, Bangladesh
Received on:07-03-2012; Revised on: 12-04-2012; Accepted on:16-05-2012

ABSTRACT
The ethanolic extract of leaves of Diospyros blancoi were investigated for potential pharmacological activities. Chemical groups were investigated,
Antioxidant, Antidiarrheal, Antimicrobial and Cytotoxic properties of the extract were investigated by using different suitable reagents; 1, 1-diphenyl-
2-picryl hydrazyl (DPPH) free radical scavenging assay; zone of inhibition method; brine shrimp lethality bioassay and castor oil induced diarrhea
inhibition respectively. There were tannins and alkaloids in the leaves extract. It had statistically significant free radical scavenging activity with an IC50
of 15±0.6 (3.63–86.50) µg mL-1 . In case of antidiarrheal activity test, the extract produced 2.81 (±0.2) h and 3.29 (±0.3) h latent period of diarrheal
induction for 250 mg/kg and 500 mg/kg of leaves extract respectively whereas the positive standard showed 3.88 (± 0.2) h and Control showed 1.30 (±
0.1) h of latent period. The extract also showed significant antimicrobial activities and significant lethality to brine shrimp with LC50 value at 1.56 µg/mL.
Different chemical substances present in the leaves might be responsible for biological activities found in this study. The results of the study support the
traditional uses of Diospyros blancoi and could be the basis of further investigations in future including isolation of novel compounds.

Key words: Diospyros blancoi , Chemical Groups, Antioxidant activity, Antidiarrheal activity,Cytotoxic activity.

INTRODUCTION
In recent times, focus on plant research has increased all over the world and
a large body of evidence has collected to show immense potential of medici-
nal plants used in various traditional systems. More than 13,000 plants
have been studied during the last 5 year period. These small molecules
provide the source of inspiration for the majority of FDA-approved agents
and continue to be one of the major sources of inspiration for drug discov-
ery. In particular, these compounds are important in the treatment of life-
threatening conditions [1].
Among the natural sources, medicinal plants are one of the important con-
tributors to most of the medicinal preparations as raw plant materials,
refined crude extracts and mixtures etc. Several thousands of plants have
been identified containing medicinal values and used to treat different ail-
ments in various cultures worldwide [2]. Even in this modern world, major-
ity of the people are still relying on the traditional medicine for their pri-
mary health care [3].
pointed, base rounded, alternately set on branches. Mature tree has dull
green leaves with silvery undersides, emerging leaves are yellowish green.
Seedlings have glossier and brighter-colored leaves [4,5].
Its traditional use in Southeast Asia includes: Juice of unripe fruit used for
wounds, Oil from seeds used for diarrhea and dysentery, Infusion of fruit
used as gargle in aphthous stomatitis [1]. In Bangladesh its Juice of bark and
leave are used in snakebites, eyewash and in colds, heart problems, hyper-
tension, spider bites, stomach aches, diabetes, eczema [6].
Evidence of antioxidant in Diospyros blancoi leaves is found in some stud-
ies performed in some countries other than Bangladesh [7] and chemical
analysis is done by others [8]. But no study in found regarding investigation
of antioxidant, antidiarrhoeal, antimicrobial and cytotoxic activities of
Diospyros blancoi (Family: Ebenaceae) leaves in Bangladesh. Therefore
this investigation was conducted to find these chemical and biological tests.

Diospyros blancoi A. DC (Family: Ebenaceae) grows well in areas with a
monsoon climate from sea level to 800 m elevation, and on almost any soil.
It has different common names in different languages e.g. Gab (Bengali),
Hong Nhung. (hindi), Mabolo (Philippines), Velvet apple (English), Pommier
velours. (French), Bisbul (Indonesian), Buah mentega ( Malay), Marit (Thai).
It has height of 14 meters or more, has leaves oblong to elliptic, apex
*Corresponding author.
Mohammad Safiqul Islam
Assistant Professor
Department of Pharmacy
Noakhali Science and Technology University
Sonapur, Noakhali-3802,Bangladesh
2. MATERIAL AND METHOD
2.1. Plant materials
For this present investigation the leaves of plant Diospyros blancoi A.DC.
(Family: Ebenaceae) was collected from, Barisal, Bangladesh in February
2010 and was identified by Bangladesh National Herbarium, Mirpur,
Dhaka.(Accession number-34970) and a voucher specimen was also depos-
ited there.
2.2. Chemicals
All the chemical reagents used in the experiment were purchased from
Sigma Chemical Co. Ltd, (St. Louis, MO, USA) and E. Merck (Germany).
2.3. Preparation of plant extract
The plant material was shade-dried with occasional shifting and then pow-
dered with a mechanical grinder, passing through sieve 40 and stored in a

Journal of Pharmacy Research Vol.5 Issue 6.June 2012 3050-3052

 Sariful Islam Howlader et al. / Journal of Pharmacy Research 2012,5(6),3050-3052
tight container. The dried powder material (1.5 kg) was refluxed with etha-
nol for three hours. The total filtrate was concentrated to dryness, in vacuo
at 40°C to render the ethanol extract for investigation.
2.4. Animal
For the experiment, twenty swiss albino mice of either sex, 3-4 weeks of
age, weighing between 20-25 g, were collected from the animal research
branch of the International Center for Diarrheal Disease and Research,
Bangladesh (icddr,b). Animals were maintained under standard environ-
mental conditions (temperature: (24.0±1.0°C), relative humidity: 55-65%
and 12 h light/ dark cycle) and had free access to feed and water ad libitum.
The animals were acclimatized to laboratory condition for one week prior
to experiments. All protocols for animal experiment were approved by the
institutional animal ethical committee.
2.5. Phytochemical screening
The freshly prepared crude extract was qualitatively tested for the pres-
ence of chemical constituents. Phytochemical screening of the extract was
performed using the following reagents and chemicals: Alkaloids with
Dragendorff’s reagent, flavonoids with the use of Mg and HCl; tannins
with ferric chloride and potassium dichromate solutions and saponins with
ability to produce stable foam and steroids with Libermann- Burchard
reagent. Reducing sugars with Benedict’s reagent. These were identified by
characteristic color changes using standard procedures[9].
2.6. Antioxidant activity
2.6.1. Qualitative assay:
Suitably diluted stock solutions were spotted on pre-coated silica gel TLC
plates and the plates were developed in different solvent systems (polar,
medium polar and non-polar) to resolve the components of the extract. The
plates were dried at room temperature and were sprayed with 0.02% 1,1-
Diphenyl-2-picryl hydrazyl (DPPH) in ethanol. Bleaching of DPPH by
the resolved bands was observed for 10 min and the color changes (yellow
on purple background) were noted [10].
2.6.2. Quantitative assay:
Quantitative assay was performed on the basis of a modified method [11].
Samples of different concentrations such as, 1, 5, 10, 50, 100 and 500 µg/
mL were prepared from the ethanol stock solution of the extract (10 mg/
mL). Diluted solution (2 mL) from each concentration mentioned was added
to 2 mL of 0.004% ethanol solution of DPPH, mixed and allowed to stand
for 30 min for the complete reaction. The absorbance was determined at
517 nm for each concentration while ascorbic acid was used as positive
control. Then with the percent inhibition, IC50 was calculated.
tion gradient. Dried and sterilized filter paper discs (6 mm diameter) con-
taining the test samples of known amounts (250 µg/disc and 500 µg/disc)
are placed on nutrient agar medium uniformly seeded with the test microor-
ganisms. Standard antibiotic (Erythromycin) discs and blank discs were
used as positive and negative control. These plates were kept at low tem-
perature (4°C) for 24 hours to allow maximum diffusion of the test materi-
als to the surrounding media [15]. The plates were then inverted and incu-
bated at 37°C for 24 hours for optimum growth of the organisms. The test
materials having antimicrobial property inhibit microbial growth in the
media surrounding the discs and thereby yield a clear, distinct area defined
as zone of inhibition. The antimicrobial activity of the test agent was then
determined by measuring the diameter of zone of inhibition expressed in
millimeter [15].
2.9. Cytotoxic activity
The cytotoxic property of the extract was evaluated using brine shrimp
lethality test [16, 17].
2.9.1. Brine shrimp:
The investigation was done on Artemia salina (Brine shrimp). One spoon
of cyst was hatched for 48 h in saline water, prepared by dissolving 20 g
pure NaCl and 18 g normal edible NaCl into 1 L water. The cyst in turn
became living nauplii.
2.8.2. Lethality bioassay:
In the test, different concentrations of the extract were prepared using
dimethyl sulfoxide as solvent. For the test, different concentrations of
extract prepared were added to test tubes, each containing 10 shrimps in
saline water and finally volume was adjusted by saline water. Control
(blank) group with 10 shrimps were kept in saline water under the same
condition and positive control group, with 10 shrimps in saline water, was
treated with a known drug chloramphenicol (200 µg/mL). All the test tubes
were kept in rest for 24 h and then counted for living and dead nauplii.
Finally, the percent mortality produced by each concentration was used to
determine LC50 (lethal concentration) of the extract.
3. RESULT
3.1. Phytochemical screening
Phytochemical analyses of the crude extract of leaves of Diospyros blancoi
A. DC. (Family: Ebenaceae) indicated the presence of alkaloid and tannin.
The results are shown in Table-1.
Table- 1: Phytochemical screening of extracts
SerialNo. Chemical Constituents Test Extract Resul

2.7. Antidiarrhoeal Activity Screening 1. Test for Reducing Sugar Benedict’s Test Ethanolic -
Antidiarrheal test of the extract was conducted using castor oil induced Fehling’s Test Ethanolic -
Alpha Napthol Solution TestEthanolic -
diarrhea in mice [12]. For the test fresh female mice were randomly chosen 2. Test for Tannins Ferric Chloride Test Ethanolic +
from the animal laboratory of icddr,b, Dhaka, Bangladesh, and divided into Potassium dichromate Test Ethanolic +
four groups having five mice in each. Of the experimental groups, Group-I 3. Test for Flavonoids Hydrochloric Acid Test Ethanolic -
4. Test for Saponins Foam Test Ethanolic -
or the Control received only distilled water containing distilled water. Group- 5. Test for Gums Molisch Test Ethanolic -
II or the positive control received standard anti-motility drug, loperamide 6. Test for Steroids Libermann-Burchard Test Ethanolic -
at a dose of 3mg/kg-body weight as oral suspension. The test groups (Group- Sulphuric acid Test Ethanolic -
7. Test for Alkaloids Mayer’s Test Ethanolic +
III and Group- IV) were treated with suspension of leaves extract of Wagner’s Test Ethanolic +
Diospyros blancoi A. DC at the oral dose of 250 mg/kg-body weight and Dragendroff’s Test Ethanolic +
500 mg/kg-body weight. The mice were fed with the samples 1 hour prior Hager’s Test Ethanolic +
to the oral administration of castor oil at a dose of 0.5ml per mouse respec- + Present, - Absent
tively for individual animal of each group placed in separate cages having
adsorbent paper beneath. Then the animals were examined for the presence 3.2. Antioxidant activity
of diarrhoea every hour for 6 hours study after the castor oil administra- With the antioxidant test performed reacting DPPH with extract, bleaching
tion. followed by color change (yellow on purple background) of DPPH was
observed on the TLC plates in qualitative experiment. In quantitative test,
2.8. Antimicrobial activity the extract showed remarkable antioxidant activity with an IC50 of 15±0.6
For the evaluation of antimicrobial activity widely acceptable disc diffu- (3.63–86.50) µg mL-1 against DPPH free radical, whereas, standard, ascor-
sion method [13, 14] was used. In this classical method, antibiotics diffuse bic acid produced IC50 at 8±0.11 µg mL-1 (9.68–91) µg/mL . The result is
from a confined source through the nutrient agar gel and create a concentra shown in the Figure- 1.

Journal of Pharmacy Research Vol.5 Issue 6.June 2012 3050-3052

 Sariful Islam Howlader et al. / Journal of Pharmacy Research 2012,5(6),3050-3052
DPPH radical scavenging activity Table 3: Antimicrobial in-vitro antibacterial activity of crude extract of Diospyros blancoi A.DC.
100
90 Serial Bacterial Strains Strains* Diameter of Zone of Inhibition in mm
80 Blank Erythromycin Crude extract of Crude
No (30 µg/disc) D blancoi A.DC extract of
70 (250 µg/disc) D blancoi A.DC
% inhibition

60 (500 µg/disc)
50 D. blancoi 1 Staphylococcus aureus (+) 0 13.0 8.0 9.0
40 2 Staphylococcus epidermidis (+) 0 27.5 6.5 7.5
Ascorbic acid
30 3 Streptococcus pyogenes (+) 0 9.5 6.5 8.5
20 4 Enterococcus faecalis (+) 0 32.5 7.0 10.0
5 Shigella sonnei (-) 0 30.0 7.5 9.0
10 6Shigella boydii (-) 0 7.5 7.0 8.5
0 7Shigella dysenteriae (-) 0 21.5 8.0 9.0
1 µg/ml 5 µg/ml 10 25 50 100 200 500 8Shigella flexneri (-) 0 23.5 5.0 8.0
9Enterococcus coli (-) 0 10.0 6.5 8.5
µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml
Concentration (µg/ml) * (-):-Gram Negative Bacteria; (+):-Gram Positive Bacteria
Figure-1. Percent inhibition of crude extract of Diospyros blancoi A. DC compared with Table 4: Result of Brine shrimp Lethality Bio-assay of Diospyros blancoi
ascorbic acid (Standard).
Concentration Number of Number of % of mortality LC 50 (µ/ml)
3.3. Anti diarrhoeal activities (µ/ml) nauplii live
The ethanolic extract of Diospyros blancoi has antidiahoeal properties in
100 10 00 100 1.56
dose dependent manner. It affects on latent period and number of stools in 50 10 00 100
first four hours after what number of stools depletes. The result of 25 10 00 100
antidiarrhoeal activities is shown in Table- 2. 12.5 10 01 90
6.25 10 02 80
Table 2: Experimental profile to observe the effect plant of extract of Diospyros blancoi on 3.125 10 03 70
castor oil induced diarrhoea in mice. 1.5625 10 05 50
0.78125 10 06 40
Animal Dose (/kg, Latent period Period of No. of stools Total no. of 0.3906 10 07 30
group/Treatment per oral) (h) study (h) (Mean±SE) stools(Mean±SE) 0.1953 10 08 20

I - Control 0 1.30 ± 0.1 1 2.6±0.5 19±0.1

2 3.0±0.6
3 3.9±0.5
4 4.0±0.4
5 3.1±0.2
6 2.2±0.3
II - Loperamide** 3 3.88 ± 0.2 1 0.4 ±0.1 4.2±0.3
2 0.6 ±0.2
3 0.7±0.3
4 1.1±0.1
5 1.0 ±0.2
6 0.9 ±0.1
III - 250 mg/kg extract 250 2.81 ±0.2 1 1.7± 0.2 12.03±0.2
of D. blancoi) ** 2 1.8± 0.4
3 1.9± 0.2
4 2.1±0.1
5 1.8±0.4
6 1.7±0.2
IV - 500 mg/kg extract 500 3.29 ±0.3 1 1.6±0.1 10.1 ±0.4
of D. blancoi) ** 2 1.8±0.1
3 1.8 ± 0.3
4 1.9±0.4 Figure 2: Brine shrimp lethality bioassay of Diospyros blancoi
5 1.7± 0.1
6 1.6± 0.3
4. DISCUSSION
**P<0.01 and therefore statistically significant. Total Number of animal is 5 in each group.
Medicinal plants are an important element of indigenous medical systems
and have a long history of serving people in many regions of the world.
3.4. Antimicrobial activity
The antimicrobial activities of leaves extracts from Diospyros blancoi showed Report shows that about 80% of the world population still uses plants for
various medical purposes [18, 19].
antimicrobial activity against both gram positive and negative bacteria. The
zones of inhibition produced by the crude ethanolic extract of D. blancoi
were microorganism specific and ranged from 9.00-12.00 mm (approxi- In our study with antioxidant test performed using DPPH, the qualitative
experiment primarily indicated the antioxidant potential of the extract. In
mately) compared with standard erythromycin (7.50- 32.50 mm) at a con-
the quantitative test, the extract showed remarkable antioxidant activity
centration of 250 µg/disc and 500 µg/disc. The results are given in Table 3.
with an IC50 which was close to value the standard drug ascorbic acid
produced. Antioxidant effect of this study could be attributed to the pres-
3.5. Cytotoxic effect
ence of tannin found with the extract. However, further studies using other
In the bioassay, the ethanolic extract showed lethality indicating the bio-
logical activity of the compound present in the extract. Test samples showed models such as lipid per-oxidation inhibition, xanthin oxidase inhibition,
erythrocytic membrane stability and others may be suggested to further
different mortality rate at different concentrations. The LC50 value for the
characterize the extract.
extract was obtained from the Table 4.
Many people in the developing countries still rely on the treatment system
Plot of percent of mortality versus log concentration on the graph produced
employing medicinal plants [20]. World Health Organization has given
an approximate linear correlation between them. From the graph (Figure-
2) the concentration at which 50% mortality (LC50 ) of brine shrimp nauplii special focus on studies of traditional medical practices directing to the
prevention and treatment of diarrheal diseases [21]. In our study, castor oil,
occurred can be obtained by extrapolation.
used to induce diarrhoea, is hydrolysed in the upper small intestine to

Journal of Pharmacy Research Vol.5 Issue 6.June 2012 3050-3052

 Sariful Islam Howlader et al. / Journal of Pharmacy Research 2012,5(6),3050-3052
ricinoleic acid [22]. Ricinoleic acid is believed to act by irritating the gas-
trointestinal tract mucosa and reducing sodium ion and chloride ion perme-
ability, resulting in increased intestinal motility followed by diarrhea [23].
However, the extract delayed the onset of diarrhea and inhibited the diar-
rheal episodes (number of stool) indicating the antidiarrheal potentials of
the leaves. Since castor oil induces diarrhoea by preventing fluid and elec-
trolyte absorption [24], one of the probable mechanisms the extract might
have is its ability to enhance fluid and electrolyte absorption through the
gastrointestinal tract. This finding can be attributed to support the tradi-
tional use of this plant part especially in diarrhea [6]. Considering the
impact of diarrheal disease in the developing countries [25], this plant part
could play a potential role in finding new molecule.
Antimicrobial activities showed by the leaves extract of Diospyros blancoi
indicates its potential use in infectious diseases. Brine shrimp lethality
bioassay indicates cytotoxicity as well as a wide range of pharmacological
activities, such as pesticidal and antitumor activities of the compounds
obtained from Diospyros blancoi leaves extract [16, 17]. With the assay
performed with freshly hatched nauplii, the extract appeared to be signifi-
cantly lethal. However, further investigations using tumour cell line or
others along this line could be suggested.
5. REFERENCES
[1] Bensky D, Clavey S, Stoger E and Gamble A Chinese Herbal Medi-
cine. Materia Medica 2004.pp 7-8.
[2] Farnsworth NR, Soejarto DD. Global importance of medicinal plants.
In: Akerele O, Heywood V, Synge H (eds) The conservation of
medicinal plants. Cambridge University Press, Cambridge, 1991,
25–51.
[3] Bannerman PGC, Mirsky R, Jessen KR, Timpl R, Duance VC, Light
microscopic immunolocalization of laminin, type IV collagen,
nidogen, heparan sulphate proteoglycan and fibronectin in the en-
teric nervous system of rat and guinea pig. J Neurocytol, 1986,
15:432–443.
[4] Perry LM, Medicinal Plants of East and South East Asia , Attributed
properties and Uses, IT Press, London. 1980. Pp 23-24
[5] Yusuf M, Chowdhury JU, Wahab MA, Begum J, Medicinal plants of
Bangladesh. BCSIR, Dhaka, 1994, 17–266.
[6] Gani A, Medicinal plants of Bangladesh. Chemical Constituents and
Uses. Dhaka, Asiatic Society of Bangladesh, 2003, 434-55.
[7] Leea MH, Jiangb CB, Juanc SH, Lind RD, Hou WC, Antioxidant and
heme oxygenase-1 (HO-1)-induced effects of selected Taiwanese
plants. Fitoterapia , 2006, 77(2):109-115.
[8] Ragasaa CY, Punoa MRA, Sengsona JMAP, Shenb CC, Rideoutc JA,
Ragad DD;Bioactive triterpenes from Diospyros blancoi. Natural
Product Research, 2009, 23 (13): 1252-1258.
Source of support: Nil, Conflict of interest: None Declared
Journal of Pharmacy Research Vol.5 Issue 6.June 2012
[9] Evans WC, Trease and Evan’s textbook of pharmacognosy, 13th
edn. Cambridge University Press, London 1989.
[10] Sadhu SK, Okuyama E, Fujimoto H, Ishibashi M, Separation of Leucas
aspera , a medicinal plant of Bangladesh, guided by prostaglandin
inhibitory and antioxidant activities. Chem Pharm Bull ,2003,
51(5):595–598.
[11] Gupta M, Mazumdar UK, Sivahkumar T, Vamis MLM, Karki S,
Sambathkumar R, Manikandan L Antioxidant and anti-inflamma-
tory activities of Acalypha fruticosa . Nig J Nat Prod Med ,2003,
07:25–29.
[12] Nwodo OFC, Alumanah EO, Studies on Abrus Precatorius seeds II:
Antidiarrheal activity. J Ethnopharmacol ,1991, 31(3):391–395.
[13] Bayer AW, Kirby WMM, Sherris JC and Turck M. Antibiotic
susceptibility testing by a standardized single disc method. Am. J.
Clin. Pathol. 1966, 45; 493-496.
[14] Satheesh LS and Murugan K. (2011) Antimicrobial activity of
protease inhibitor from leaves of Coccinia grandis (L.) voight. In-
dian Journal of Experimental Biology, 49: 366-374.
[15] Barry A.L.,.Principle & practice of Microbiology. 3 rd Ed., Lea &
Fabager, Philadelphia. 1976.
[16] Meyer BN, Ferrigni NR, Putnam JE, Jawbson LB, Nicholas DE,
McLaughlin JL, Brine shrimp: a convenient general bioassay for
active plant constituents. Planta Med, 1982, 45:31–34.
[17] McLauglin JL, Chang CJ, Smith DL, Simple bench-top bioassays
(brine shrimp and potato discs) for the discovery of plant antitumour
compounds. In: Human Medicinal Agents from Plants. Kinghorn,
A. D. And Balandrin, M. F. (Eds.), ACS Symposium 534, Am Chem
Soc, Washington DC: , 1993, 112–137.
[18] Schulz V, Hänsel R, Tyler VE, Fitoterapia racional: Um Guia de
Fitoterapia para as Ciências da Saúde, 4th edn. Barueri, Manole,
2002, 386.
[19] Kong JM, Goh NK, Chia LS, Chia TF, Recent advances in traditional
plant drugs and orchids. Acta Pharmacol Sin, 2003, 24:7–21.
[20] Ojewole JAO, Evaluation of antidiarrheal, anti-inflammatory and
antidiabetic properties of Sclerocarya birrea (A. Rich.) Hochst. stem
bark aqueous extract in mice and rats. Phytotherapy Res , 2004,
18:601–608.
[21] Atta AH, Mouneir SM, Antidiarrheal activity of some Egyptian
medicinal plant extracts. J Ethnopharmacol, 2004, 92:303–09.
[22] Altman DF, Drugs used in gastrointestinal diseases. In: Katzung BG
(ed) Basic and clinical pharmacology, 8th edn. McGraw Hill, San
Francisco, 2001, 1070–1071.
[23] Zavala MA, Perez S, Perez C, Vargal R, Perez RM, Antidiarrhoeal
activity of Waltheria americana, Commelins cuelestris and
Alternanthra repens. J Ethnopharmacol (1998) 61:41–47.
[24] Goodman A, Gillman A (Editors): The Pharmacological Basis of
Therapeutics. (9 th Edition), Macmillan Publishers, New York. 1996,
924–926.
[25] Das AK, Mandal SC, Banerjee SK, Sinha S, Das J, Saha BP, Pal M,
Studies of antidiarrheal activity of Punica granatum seed extracts. J
Ethnopharmacol, 1999, 68:205–208.
3050-3052