Cytotoxicity, Analgesic and Antidiarrhoeal Activities of Asparagus racemosus
In ayurveda, Asparagus racemosus Willd is known as the queen of herbs because it has a strong rejuvenating, nurturing and stabilizing effect on excessive air, gas, dryness and agitation in the body and mind. Ethanol extracts of Asparagus racemosus (EEAR) belonging to the family Liliaceae was investigated for biological action. The present study was designed to evaluate the cytotoxicity, analgesic and antidiarrhoeal properties of the ethanol extract of whole plant of Asparagus racemosus. The test for analgesic activity of the crude ethanol extract was performed using acetic acid induced writhing model in mice. On the other hand, antidiarrhoeal test of the EEAR was done according to the model of castor oil induced diarrhoea in mice and brine shrimp lethality bioassay was used to determine the cytotoxic activity of ethanol extract of the plant. In acetic acid induced writhing in mice, the ethanol extract exhibited significant inhibition of writhing reflex 67.47% (p<0.01) at the dose of 500 mg kg-1 body weight. The plant extract showed antidiarrhoeal activity in castor oil induced diarrhoea in mice. It increased mean latent period and decreased the frequency of defecation with number of stool count at the dose of 250 and 500 mg kg-1 body weight, respectively comparable to the standard drug Loperamide at the dose of 50 mg kg-1 body weight. In addition to these, the brine shrimp lethality test showed the significant cytotoxic activity of the plant extract (LC50: 10 μg mL-1 and LC90: 47.86 μg mL-1). The obtained results support the traditional uses of the plant and require further investigation to identify the chemical constituent(s) responsible for cytotoxicity, analgesic and antidiarrhoeal activities.
Received: February 16, 2012;
Accepted: March 24, 2012;
Published: June 19, 2012
Asparagus racemosus Willd belonging to the family Liliaceae is a climber
with thin leaves commonly known as Shatamuli. The roots of the plant
are white, long and are tapering at both ends. Small white and fragrant flowers
appear on this plant in the beginning of the rainy season. Fruits in the shape
of small berries appear in the autumn. It grows wild in forest and can also
be planted in gardens in most of the areas (Ghani, 2003).
Asparagus contains steroidal glycosides (asparagosides), bitter glycosides,
asparagine and flavonoids. Asparagine is a strong diuretic. In addition to these,
leaves contain diosgenin and other saponins such as shatavarins I and IV (Ravikumar
et al., 1987). It was also reported that quercetin, rutin and hyperoside
were found in flowers and fruits of the plant. The fruits of Asparagus racemosus
also contain cyanidin-3-glycoside, sitosterol, stigmasterol, sarsasapogenin
and two furostanolic saponins. Tubers and roots contain saccharine matters and
mucilage. An antioxytocic compound, named racemosol (a 9, 10-dihydrophenanthrene
derivative) has been isolated from this plant (Sekine et
The plant also possesses diuretic properties and is effective in the treatment
of cystitis. Ethanol extract of aerial parts possesses anti-cancer properties
whereas, bark shows antibacterial and antifungal activities. Tuberous roots
are used as aphrodisiac, alterative, tonic, demulcent, diuretic and are commonly
prescribed in gastrointestinal disorders like bilious dyspepsia, flatulence,
diarrhea and dysentery. Moreover, the roots play an important role in lactation
of mother and appetite and nourishment in children. It is also used in treating
acidity and as hair tonic. Juice of the roots taken with milk is useful in gonorrhea.
The plant is also used in diabetes, jaundice and other urinary disorders. Aqueous
and ethanol extracts of the plant possess strong molluscicidal property (Ghani,
2003). It is also reported that the ethanol extract of the plant possesses
anti-oxidant activities (Karmakar et al., 2012).
The methanolic root extract of the plant is also used in the treatment of ischemia
and shows cerebroprotective potentials (Nandagopal et
al., 2011). In addition to these, the aqueous root extract of the plant
shows glucose homeostasis in aged rats (Velavan and Begum,
2007a) and acts to alleviate the indices of oxidative stress associated
with stress (Velavan and Begum, 2007b). The objective
of the present study was to identify the cytotoxicity, analgesic and antidiarrhoeal
activities with phytochemical analysis of ethanol extract of Asparagus racemosus.
MATERIALS AND METHODS
Collection of plant materials and preparation of plant extract: The whole plant of Asparagus racemosus was collected from Natore, Bangladesh in the month of May, 2009 and the plant was identified by the taxonomists of Bangladesh National Herbarium, Mirpur, Dhaka (Accession No. DACB 34216). The voucher specimen was deposited at Pharmacy Discipline of Khulna University Bangladesh. The collected plant was dried under shade. After complete drying, the sample was cut into small pieces and then slashed to coarse powder with the help of mechanical grinder. The powder materials were stored in a suitable container. About 500 g of powder was extracted by maceration over 20 days with 1200 mL of 80% ethanol. The whole mixture then underwent a coarse filtration by a piece of clean, white cotton material and then it was filtered through filter paper. The filtrate thus obtained was evaporated by using a rotary evaporator to get a viscous mass which was dried to get a dried ethanol extract (approx. yield value 16%). The extract thus obtained was used for experimental purposes.
Animals: Swiss-Albino mice of either sex (20-25 g body weight) were
collected from animal resources branch of the International Center for Diarrhoeal
Disease Research, Bangladesh (ICDDR, B) and were used for the experiments. The
animals were kept in the standard polypropylene cages and provided with standard
diets formulated by ICDDR, B. The animals were acclimatized in animal house
under standard Laboratory conditions (relative humidity 55-60%, room temperature
25±2°C and 12 h light: dark cycle) for period of 14 days prior to
performing the experiments (Chatterjee, 1993).
Drugs: The standard drugs diclofenac sodium, Loperamide and Chloramphenicol were collected from Beximco Pharmaceuticals Ltd. Dhaka, Bangladesh.
Preliminary phytochemical screening: The crude extract was subjected
to preliminary phytochemical screening for the detection of major functional
groups according to the standard procedures (Trease and Evans,
Determination of analgesic activity: The analgesic activity of EEAR
was studied using acetic acid induced writhing model in mice. Experimental animals
were randomly selected and divided into four groups denoted as control group,
Positive control group and Test group-I and Test group-II consisting of five
mice in each group. Control group received 1% Tween-80 orally at the dose of
10 mg kg-1 body weight whereas, Positive control group received diclofenac
sodium orally at the dose of 25 mg kg-1 body weight. Test group I
and Test group II were given the test sample orally at the dose of 250 and 500
mg kg-1 body weight. A 30 min interval was given to ensure proper
absorption of the administered substances. Then the writhing inducing chemical,
acetic acid solution (0.7%) was administered intra-peritoneally to each of the
animals of a group. Five min was also given for absorption of acetic acid and
number writhing was counted for 15 min. The animals did not always perform full
writhing and the incomplete writhing was taken as half-writhing, so two half-writhing
were taken as one full writhing. This is why total writhing was halved to convert
all writhing to full writhing or real writhing (Whittle,
1964; Ahmed et al., 2004).
Antidiarrhoeal activity: Antidiarrhoeal activity of EEAR was tested
using the model of castor oil induced diarrhoea in mice (Chatterjee,
1993). All the mice were screened initially by giving 0.5 mL of castor oil
and only those showing diarrhea were selected for the experiment. The test animals
were randomly chosen and divided into four groups having five mice in each group.
Control group received 1% Tween-80 at the dose of 10 mg kg-1 body
weight whereas, positive control group received the standard antimotility drug,
loperamide at the dose of 50 mg kg-1 body weight as oral suspension.
Group I and Group II were considered as test groups treated with ethanol extract
of Asparagus racemosus at the oral dose of 250 and 500 mg kg-1
body weight. In this study, the control vehicle and extract were administered
orally 30 min prior to oral administration of castor oil at the dose of 0.5
mL. Individual animal of each group was placed in separate cages having adsorbent
paper beneath and examined for the presence of diarrhea every hour in 5 h study
after the castor oil administration. Number of stool on any fluid material that
stained the adsorbent paper was counted at each successive hour during the experiment.
The latent period of each mouse was also counted. At the beginning of each hour,
new papers were placed for old ones.
Determination of cytotoxic activity: The brine shrimp eggs were hatched
in a conical flask containing brine shrimp medium (300 mL). The flask were well
aerated with the aid of an air pump and kept in a water bath at 29-30°C.
A bright light was left on it. The nauplii hatched within 48 h. The extract
was dissolved in brine shrimp medium with addition of few drops of 5% dimethyl
sulfoxide (DMSO) to obtain a concentration of 5, 10, 20, 40, 60, 80, 160 and
320 μg mL-1. Each preparation was dispensed into clean test
tube in 10 mL volume. For control, same procedure was followed except test samples.
A series of same concentration as of sample was prepared for positive control,
chloramphenicol. After making the test tube properly, 10 living shrimps were
added to each of the test tubes with the help of a Pasteur pipette. The test
tubes containing the sample, control and positive control were then incubated
at 29°C for 24 h in a water bath, after which each test tube was examined
and the surviving brine shrimp counted and recorded. From this, the percentage
of mortality was calculated at each concentration to determine the LC50
and LC90 (Myers, 1982).
Statistical analysis: Students t-test was used to determine significant differences between the control group and test groups. p-values less than or equal to 0.05 were considered statistically significant.
RESULTS AND DISCUSSION
Phytochemical screening: In the preliminary phytochemical screening,
EEAR showed the presence of alkaloids, tannins, saponins, glycosides, flavonoids
and carbohydrates which are shown in Table 1.
Analgesic activity: Analgesic activity of EEAR was tested using acetic acid induced writhing model in mice. The extract produced 52.39% (p<0.05) and 67.47% (p<0.01) writhing inhibition in test mice at the dose of 250 mg kg-1 and 500 mg kg-1 body weight which were comparable to the standard drug diclofenac sodium showing 70.65% (p<0.01) writhing inhibition at the dose of 25 mg kg-1 body weight (Table 2).
Antidiarrhoeal activity: Antidiarrhoeal activity of the EEAR was tested by castor oil induced diarrhoea in mice. The extract caused an increase in latent period (0.326 and 0.574 h) i.e., delayed the onset of diarrhoeal episode at the dose of 250 and 500 mg kg-1 body weight, respectively as compared to the standard antidiarrhoeal agent Loperamide where the mean latent period was 1.57 h (Table 3). The extract also decreased the frequency of defecation at the dose of 250 and 500 mg kg-1 b.wt., respectively where the mean number of stool at the 1st, 2nd, 3rd, 4th and 5th h of study were 2.3, 1.9, 1.7, 1.6, 1.5 and 2.1, 1.8, 1.5, 1.4, 1.3, respectively which were comparable to the standard drug loperamide where the mean number of stool at the 1st, 2nd, 3rd, 4th and 5th h of study were 3.1, 2.2, 1.6, 1.3 and 1.1, respectively (Table 3).
Cytotoxic activity: Brine shrimp lethality bioassay indicates cytotoxicity of the ethanol extract. The extract was found to show lethal activity against brine shrimp nauplii where LC50 and LC90 values were 10 μg mL-1 and 47.86 μg mL-1 (Table 4).
|| Effect of EEAR on acetic acid induced writhing in mice
|Values are expressed as Mean±SEM, SEM = Standard error
of mean, n = No. of mice, *: p < 0.01; **: p < 0.05 vs. Control
|| Effect of EEAR on castor oil induced diarrhea in mice
|Values are expressed as Mean±SEM (n = 5); *: p<0.01;
**: p<0.2 vs. control
|| Results of brine shrimp lethality bioassay of EEAR
To get preliminary idea about the active constituents present in the ethanol
extract of the plant, different chemical tests were performed and the study
results showed the presence of alkaloids, tannins, saponins, glycosides, flavonoids
and carbohydrates. Flavonoids were reported to have a role in analgesic (Zakaria
et al., 2006) and antioxidant activity (Rajnarayana
et al., 2001; Ramesh et al., 1998;
Okwu and Orji, 2007). There are also reports on the
role of tannins in anti-nociceptive activity (Ramprasath
et al., 2006; Rahman et al., 2011).
Besides, alkaloids are well known for their ability to inhibit pain perception
(Uche and Aprioku, 2008). Tannins are also useful in
bacterial infections (Agbafor et al., 2011) and
the antibacterial activities of the ethanol extract of the plant were reported
by Karmakar et al. (2012). Therefore, it is assumed
that these compounds may be responsible for the observed analgesic, antidiarrheal,
antibacterial, antioxidant and cytotoxic activity.
Acetic acid induced writhing model represents pain sensation by triggering
localized inflammatory response. Acetic acid which is used to induce writhing,
causes analgesia by liberation of endogenous substances which in turn excite
the pain nerve endings (Taesotikul et al., 2003).
Increased levels of PGE2 and PGF2á in the peritoneal
fluid have been reported to be responsible for pain sensation caused by intraperitoneal
administration of acetic acid (Deraedt et al., 1980).
Nevertheless, it was found that the intraperitoneal administration of acetic
acid induces the liberation not only of prostaglandins, but also of the sympathetic
nervous system mediators (Hokanson, 1978). The EEAR
produced significant writhing inhibition comparable to the standard drug diclofenac
sodium. Results of the study suggest that the extract might possess the capability
to inhibit prostaglandin synthesis.
Antidiarrhoeal activity of the EEAR was tested by castor oil induced diarrhea
in mice. Castor oil mixes with bile and pancreatic enzymes and liberates ricinoleic
acid from the triglycerides upon oral administration. Most of the ricinoleic
acid remains in the intestine and produces anti-absorptive or anti-secretory
effect (Tripathi, 1999). The ricinoleic acid thus liberated
readily forms ricinoleate salts with sodium and potassium in the lumen of intestine.
The salt formed as such behaves like a soap or surfactant within the gut and
at the mucosal surface. Most agreed view is that ricinoleate salts stimulate
the intestinal epithelial cells adenyl cyclase (Racusen
and Binder, 1979) or release prostaglandins which results in an increase
in the net secretion of water and electrolytes in the small intestine (Beubler
and Juan, 1979). The EEAR moderately inhibited and delayed the onset of
diarrhea in mice. The maximum effect was found at 500 mg kg-1 of
body weight. On the basis of these results, it was concluded that the EEAR possessed
moderate antidiarrhoeal activity. Brine shrimp lethality bioassay indicates
cytotoxicity as well as a wide range of pharmacological activities such as antimicrobial,
pesticidal, antitumor, etc. of the compound (Myers, 1982;
Anderson et al., 1988; Alam
et al., 2011). The EEAR was found to show significant activity against
the brine shrimp nauplii; LC50 was found 10 μg mL-1
and LC90 was found 47.86 μg mL-1. However, further
investigations using carcinoma cell line are necessary to isolate the active
compound(s) responsible for the activity.
According to above discussion EEAR contains important chemical constituents that confer upon it as a medicinal agent. It was revealed that the extract containing alkaloids, tannins, saponins, glycosides, flavonoids and carbohydrates might have potential roles in its analgesic, antidiarrhoeal, antimicrobial, cytotoxic and antioxidant activity. This could provide a rationale for traditional uses of this plant and further research is necessary for elucidating the active principles.
The authors are grateful to the authority of International Centre for Diarrhoeal Disease and Research, Bangladesh (ICDDR, B) for providing the experimental mice and bacterial strains. The authors are also grateful to the authority of Beximco Pharmaceuticals Ltd. for providing Diclofenac sodium, Loperamide and Chloramphenicol.
Alam, M.B., M.S. Hossain, N.S. Chowdhury, M.E.H. Mazumder, M.E. Haque and A. Islam, 2011. In vitro
and in vivo
antioxidant and toxicity evaluation of different fractions of Oxalis corniculata
Linn. J. Pharmacol. Toxicol., 6: 337-348.CrossRef | Direct Link |
Agbafor, K.N., E.I. Akubugwo, M.E. Ogbashi, P.M. Ajah and C.C. Ukwandu, 2011.
Chemical and antimicrobial properties of leaf extracts of Zapoteca portoricensis
. Res. J. Med. Plant, 5: 605-612.CrossRef |
Ahmed, F., M.S.T. Selim, A.K. Das and M.S.K. Choudhuri, 2004.
Anti-inflammatory and antinociceptive activities of Lippia nodiflora
Linn. Pharmazie, 59: 329-330.Direct Link |
Beubler, E. and H. Juan, 1979.
Effect of ricinoleic acid and other laxatives in net water flux and prostaglandin e release by the rat colon. J. Pharm. Pharmacol., 31: 681-685.PubMed |
Chatterjee, T.K., 1993.
Handbook of Laboratory Mice and Rats. 1st Edn., Jadavpur University Press, India, pp: 133-139
Deraedt, R., S. Jouquey, F. Delevallee and M. Flahaut, 1980.
Release of prostaglandins E and F in an algogenic reaction and its inhibition. Eur. J. Pharmacol., 61: 17-24.CrossRef | PubMed | Direct Link |
Ghani, A., 2003.
Medicinal Plants of Bangladesh. 2nd Edn., The Asiatic Society of Bangladesh, Dhaka, pp: 114
Karmakar, U.K., S.K. Biswas, A. Chowdhury, S.Z. Raihan, M.A. Akbar, M.A. Muhit and R. Mowla, 2012.
Phytochemical investigation and evaluation of antibacterial and antioxidant potentials of Asparagus racemosus
. Int. J. Pharmacol., 8: 53-57.CrossRef | Direct Link |
Anderson, J.E., C.J. Chang and J.L. McLaughlin, 1988.
Bioactive components of Allamanda schottii
. J. Nat. Prod., 51: 307-308.CrossRef | PubMed | Direct Link |
Phytochemical Methods (A Guide to Modern Techniques to Plant Analysis). 3rd Edn., Champan and Hall, London, pp: 335-337
Nandagopal, M., P. Muralidharan and G. Thirumurugan, 2011.
Cerebroprotective effect of root extract of Asparagus racemosus
Willd. in global cerebral ischemia in rats. J. Pharmacol. Toxicol., 6: 49-61.CrossRef | Direct Link |
Okwu, D.E. and B.O. Orji, 2007.
Phytochemical composition and nutritional quality of Glycine max
and Vigna unguiculata
(L.) Walp. Am. J. Food Technol., 2: 512-520.CrossRef | Direct Link |
Racusen, L.C. and H.J. Binder, 1979.
Ricinoleic acid stimulation of active anion secretion in colonic mucosa of the rat. J. Clin. Invest., 63: 743-749.CrossRef | PubMed |
Rahman, M.A., E. Haque, M. Hasanuzzaman and I.Z. Shahid, 2011.
Antinociceptive, antiinflammatory and antibacterial properties of Tamarix indica
roots. Int. J. Pharmacol., 7: 527-531.CrossRef |
Rajnarayana, K., M.S. Reddy, M.R. Chaluvadi and D.R. Krishna, 2001.
Biflavonoids classification, pharmacological, biochemical effects and therapeutic potential. Ind. J. Pharmacol., 33: 2-16.Direct Link |
Ramesh, M., Y.N. Rao, A.V.N.A. Rao, M.C. Prabhakar, C.S. Rao, N. Muralidhar and B.M. Reddy, 1998.
Antinociceptive and anti-inflammatory activity of a flavonoid isolated from Caralluma attenuata
. J. Ethnopharmacol., 62: 63-66.CrossRef | Direct Link |
Ravikumar, P.R., R. Soman, G.L. Chetty, R.C. Pandey and D. Sukh, 1987.
Chemistry of ayrvedic crude drugs: Part 6-(shatavari-1): Structure of Shatavarin-4. Indian J. Chem., 26: 1012-1017.
Sekine, T., N. Fukasawa, I. Murakoshi and N. Ruangrungsi, 1997.
A 9, 10 dihydrophenanthrene from asparagus racemosus. Phytochemistry, 44: 763-764.CrossRef |
Taesotikul, T., A. Panthong, D. Kanjanapothi, R. Verpoorte and J.J.C. Scheffer, 2003.
Anti-inflammatory, antipyretic and antinociceptive activities of Tabernaemontana pandacaqui
poir. J. Ethnopharmacol., 84: 31-35.CrossRef | PubMed |
Trease, G.E. and W.C. Evans, 1989.
Trease and Evan's Textbook of Pharmacognosy. 13th Edn., Cambridge University Press, London, Pages: 546
Tripathi, K.D., 1999.
Essentials of Medical Pharmacology. 4th Edn., Jaypee Brothers Medical Publishers (P) Ltd., New Delhi, India, ISBN: 81-7179-633-8, Pages: 432
Uche, F.I. and J.S. Aprioku, 2008.
The phytochemical constituents, analgesic and anti-inflammatory effects of methanol extract of Jatropha curcas
sp leaves in mice and Wister albino rats. J. Applied Sci. Environ. Manage., 12: 99-102.Direct Link |
Ramprasath, V.R., P. Shanthi and P. Sachdanandam, 2006.
Immunomodulatory and anti-inflammatory effects of Semecarpus anacardium
Linn. Nut milk extract in experimental inflammatory conditions. Biol. Pharm. Bull., 29: 693-700.CrossRef | Direct Link |
Velavan, S. and V.M.H. Begum, 2007.
Modulatory role of Asparagus racemosus
on glucose homeostasis in aged rats. Int. J. Pharmacol., 3: 149-154.CrossRef | Direct Link |
Velavan, S. and V.M.H. Begum, 2007.
Restorative effect of Asparagus racemosus
on age related oxidative damage in heart lysosome of aged rats. Int. J. Pharmacol., 3: 48-54.CrossRef | Direct Link |
Whittle, B.A., 1964.
The use of changes in capillary permeability in mice to distinguish between narcotic and nonnarcotic alalgesics. Br. J. Pharmacol. Chemother., 22: 246-253.PubMed | Direct Link |
Zakaria, Z.A., R.N.S.R.M. Nor, M.R. Sulaiman, Z.D.F.A. Ghani, G.H. Kumar and C.A. Fatimah, 2006.
Antinociceptive and anti-inflammatory properties of Melastoma malabathricum
leaves chloroform extract in experimental animals. J. Pharmacol. Toxicol., 1: 337-345.CrossRef | Direct Link |
Hokanson, G.C., 1978.
Acetic acid for analgesic screening. J. Natl. Prod., 41: 497-498.