Chemistry and pharmacology of Withania coagulans:an Ayurvedic remedy Rakesh Maurya, Akanksha and Jayendra Medicinal and Process Chemistry Division, Central Drug Research Institute, CSIR, Lucknow, India
JPP 2010, 62: 153–160 2010 The Authors.
Journal compilation 2010
Royal Pharmaceutical Society of Great Britain
Received May 04, 2009 Accepted November 09, 2009
DOI 10.1211/jpp/62.02.0001 ISSN 0022-3573
Pharmacological Properties
The berries of the plant are used for milk coagulation. It has always had a prominent place in Ayurvedic, Unani, and ancient Indian systems of medicine. A number of reports have revealed that the withanolides isolated from W. coagulans possess interesting biological activities. The fruits of the plant are sweet and have been reported to have sedative, emetic, alterative and diuretic effects. They are useful in chronic complaints of the liver. In some places, they have been used as a blood purifier. They are also used in dyspepsia, flatulent colic and other intestinal infections. These are used for the treatment of asthma, biliousness and stranguary.
Hepatoprotective activity
The aqueous extract of fruits of this plant has been shown to exert hepatoprotective activity. Since the steroidal compounds (glucocorticoids) having anti-inflammatory properties are used in some hepatic disorders, 3-β-hydroxy-2,3 dihydrowithanolide F has been screened for its hepatoprotective effect. It has shown hepatoprotective activity against CCl4-induced hepatotoxicity in adult albino rats of either sex (150–200 g) at 10 mg/kg (i.p.). The protective effect was assessed by observing pentobarbitone (30 mg/kg; i.p.)-induced hypnosis, the determination of serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT) levels, and histopathological examination of hepatic tissues after staining with haematoxylin and eosin solutions. Concomitant treatment of the rats with 10 mg/kg withanolide protected the liver significantly.
Anti-inflammatory activity
The alcoholic extract and total alkaloids showed a significant anti-inflammatory effect in acute inflammation induced with egg albumin, subacute inflammation induced with formalin and granulation tissue formation by the cotton pellet method.
3-β-Hydroxy-2,3-dihydrowithanolide F exhibited a significant anti-inflammatory activity at 10 mg/kg in subacute models of inflammation such as granuloma formation and formalin-induced arthritis in rats. The effect was comparable with that obtained with 50 mg/kg phenylbutazone and 10 mg/kg hydrocortisone. However, it did not show any significant activity in acute models of inflammation.
Anti-hyperglycaemic activity
Administration of an aqueous extract of fruits of W. coagulans (1 g/kg; p.o.) significantly lowered the blood sugar, serum cholesterol, serum lipid peroxide (LPO) and hepatic LPO levels in streptozocin-induced diabetic rats after seven days of treatment (P < 0.001). Such lipid lowering activity in streptozocin-induced diabetic rats may have helped in preventing associated atherogenesis and other secondary complications of diabetes mellitus. Its serum LPO and liver LPO reducing activity suggested that it may prevent lipid peroxidation and may protect tissues from free radicals. It also significantly (P < 0.01) decreased blood glucose level in normal rats (at 1 g/kg; p.o.). Coagulin C (5), 17β-hydroxywithanolide K (3), withanolide F (31), coagulanolide ((17S,20S,22R)-14α,15α,17β,20β-tetrahydroxy-1-oxowitha-2,5,24-trienolide) (30) and coagulin L (14) were evaluated for their antihyperglycaemic activity in the normoglycaemic rat model (SLM) and in the streptozocin-induced diabetic rat model (STZ). Compound 14 was also evaluated in C57BL/KsJ-db/db mice. Compound14 improved glucose tolerance up to 29.8% in SLM and 23.3% in STZ-induced diabetic rats at a dose of 100 mg/kg body weight. Compounds 5, 3, 31 and 30 exhibited significant antihyperglycaemic activity, 22.8, 20.4, 24.9 and 28.1% in SLM and 16.9, 15.8, 18.2 and 19.3% in STZ, models, respectively. Compound 5 (at 50 mg/kg body weight) in db/db mice for 10 consecutive days significantly lowered the postprandial blood glucose level by 22.7% (P < 0.01), whereas metformin decreased the postprandial blood glucose by 18.6% (P < 0.05). For compound 14 in the db/db mice group, the area under the curve (AUC) of blood glucose decreased by approximately 40.5% compared with the vehicle-treated group. Compound 5 (at 50 mg/kg body weight) showed significant improvement in plasma lipid profiles of dyslipidaemic db/db mice after 10 days of consecutive treatment. Increasing oral doses of compound 14 declined the postprandial glucose level, with a calculated ED50 (effective dose 50%) of approximately 25 mg/kg of body weight following oral administration.
Hypolipidemic activity
The aqueous extract of fruits of W. coagulans (1 g/kg; p.o.) showed 15% reduction in serum cholesterol level in Triton-induced hyperlipidaemic rats in comparison with untreated animals. In rats with a high fat diet-induced hyperlipidaemia, the aqueous extract at the same dose administered for seven weeks showed a significantly reduced body weight, elevated serum cholesterol, triglycerides and lipoprotein levels. The animals treated with aqueous extract of fruits of W. coagulans and the reference drug Navaka guggulu showed less degenerative changes along with micro vesicular fatty changes.
Free radical scavenging activity
The aqueous extract (2 mg/ml) exhibited free radical scavenging activity in an in-vitro system using 1,1-diphenyl-2-picrylhydrazyl (DPPH). This method, used to determine free radical scavenging activity, was based on the reduction of a methanol solution of the colored free radical DPPH. The decrease in absorption of DPPH at its absorption maximum of 517 nm is proportional to the concentration of free radical scavenger added to the DPPH reagent solution. The activity was expressed as the effective concentration at 50%. The presence of free radical scavenging potential might help in protecting against oxidative damage to pancreatic beta cells.
Antimicrobial activity
Antifungal and antibacterial properties have been demonstrated in the withanolides isolated from the ethanolic extract of the whole plant and leaves, respectively. Withaferin A exhibited a significant antibacterial activity against Gram-positive microorganisms at the concentrations 6–100 μg/ml, whereas it was inactive against Gram-negative bacteria or nonfilamentous fungi. The following minimal inhibitory concentrations (mg/ml) have been found for withaferin A: 6.25 for Staphylococcus pyogenes, 12.5 for Sarcinia lutea, 100 for Streptococcus pyogenes, 200 for Streptococcus viridans, 25 for Bacillus subtilis, 50 for Corynebacterium diphtheriae, 6.26 for Bacillus anthracis, 200 for Escherichia coli, and 200 for Pseudomonas aeruginosa. Antifungal activity against Aspergillus niger, Candida albicans and Taenia rubrum at doses of 12.5–50 μg/ml, 100, 150 and 200 μg/ml has been shown. It was found to be more inhibitory to the filamentous fungi than to the yeast group of fungi.The volatile oil obtained from fruits of W. coagulans had antibacterial activity againstStaphylococcus aureus and Vibrio cholerae and was also found to have anthelmintic activity] 17β-Hydroxywithanolide K (3) exhibited antifungal activity against human pathogens Nigrospora oryzae, Aspergillus niger, Curvularia lunata, Stachybotrys atra, Allescheria boydii, Drechslera rostrata, Microsporum canis and Epidermophyton floccosum and plant pathogen Pleurotus ostreatus (minimal inhibitory concentration 300 μg/ml).
Cardiovascular effects
An alcoholic solution of 3β-hydroxy-2,3-dihydrowithanolide F (26) 5 mg/kg exhibited a moderate fall of blood pressure (34 ± 2.1 mmHg) in mongrel dogs (weight 12–15 kg). The hypotensive response was blocked by atropine (2 mg/kg) but not by mepyramine (2 mg/kg) and propranolol (1 mg/kg). At the same dose, the hypotensive response was less with a suspension of the withanolides. On administration of a 10 mg/kg bolus dose in alcohol, a depression of the S-T segment was caused in ECG studies of dog. A 2 mg dose in suspension produced a positive inotropic and chronotropic effect in perfused frog heart. The heart rate increased from 61.2 ± 1.39 to 77 ± 1.94 beats/min (P < 0.01). In rabbit Langendorff preparations 2 mg withanolide produced negative inotropic and chronotropic effects. The heart rate decreased from 71 ± 2.4 to 19 ± 0.28 beats/min. In rat limb preparation, 1 mg withanolide caused insignificant (P > 0.05) vasoconstriction. On administration of 5 mg/kg it increased the rate and depth of respiration. The rate of respiration increased from 18 ± 1.4 to 65 ± 5.3 breaths/min in dogs, which was insignificant (P < 0.01).
Central nervous system depressant activity and acute toxicity
The total extract of W. coagulans fruit has been reported to have central nervous system (CNS) depressant activity in mice, rabbits and dogs. The extract was hypotensive in animals and had respiratory stimulant and smooth muscle relaxant activity. Alcoholic extract, total alkaloids and aqueous extract at doses of 1 g/kg, 200–400 mg/kg and 5 mg/100 g exhibited CNS depression in albino rats characterized by sedation, reduced exploratory, spontaneous activity and hypothermia. At the same doses but administered 30 min before a hypnotic, they potentiated pentobarbitone sleeping time in rats. They did not show any analgesic and diuretic activity in albino rats. Alcoholic extract (1 g/kg) and total alkaloids (200–400 mg/kg) did not protect against convulsions induced by pentylenetetrazol (70 mg/kg). They increased the lethal effect of amphetamine in aggregated mice. 3β-Hydroxy-2,3-dihydrowithanolide F (26) was tested for its CNS depressant activity. It was found nonlethal to mice up to a dose of 625 mg/kg (i.p.). It did not show analgesic, hypothermic or local anesthetic activity.
Immunomodulating activity
Withaferin A (32) has been reported in various studies to possess both immune activating and immunosuppressive properties, even at a low dose of 10 mg/kg for six consecutive days. Withaferin A was found also to impart immune activation by specifically inducing proliferation of peritoneal macrophages in mice but not in splenocytes, resulting in regression of tumor cells in a mouse carcinoma model, which was persistent even after passive transfer of the serum or macrophages of the treated mice into another model. Withaferin A had specific immunosuppressive effects on human B and T lymphocytes as well as on mice thymocytes. It inhibited E rosettes and EAC rosette formation by normal human T and B lymphocytes at very low concentrations. It was demonstrated to affect the functional activity of normal human T lymphocytes as assessed by a local xenogeneic graft versus host reaction. It had specific action on antigen recognition as well as proliferative capacity of T lymphocytes and B-lymphocytes.
5,20α(R)-dihydroxy-6α,7α-epoxy-1-oxo-(5α)-witha-2,24-dienolide is known to show immunosuppressant activity in spleen cell culture. On administration of doses above 1 μg/ml, it inhibited proliferation of murine spleen cell cultures. A solution of 5,20α(R)-dihydroxy-6α,7α-epoxy-1-oxo-(5α)-witha-2,24-dienolide in dimethyl sulfoxide was mixed with RPMI 1640 medium to achieve a fine suspension (0.37% dimethyl sulfoxide).
Coagulin H (10) exhibited effects on the immune response, including an inhibitory effect on lymphocyte proliferation, and expression of interleukin-2 (IL-2) cytokine. A complete suppression of phytohaemagglutinin-activated T-cells was observed at ≥2.5 μg/ml coagulin H and this suppression activity was similar to that of prednisolone, a commonly used immune modulating drug. Coagulin H also significantly inhibited IL-2 production by 80%. Docking studies predicted that coagulin H bound to the receptor binding site of IL-2 more effectively than prednisolone. Based on the computational and the experimental results, coagulin H was identified as a potential immunosuppressive candidate.
Antitumour activity
Withaferin A showed marked tumour-inhibitory activity when tested in-vitro against cells derived from human carcinoma of the nasopharynx (KB). Withaferin A inhibited RNA synthesis of Sarcoma-180 ascites tumour cells. At 40 μg/ml, within 30 min of incubation it showed inhibition of RNA synthesis of more than 50%. It inhibited protein synthesis of Sarcoma-180 cells. In this way, withaferin A inhibited transcription and translation processes of these cells. At concentrations of 0.01–0.5% it showed inhibition on the growth of roots of Allium cepa by arresting the cell division at metaphase after 2-h treatment. Withaferin A acted as a mitotic poison via arresting the division of cultured human larynx carcinoma cells at metaphase. It showed a similar, but less marked effect on HeLa and embryonal chicken fibroblast cells. Withaferin A inhibited human umbilical vein endothelial cell (HUVEC) sprouting in three-dimensional collagen-I matrix at doses which were relevant to nuclear factor-kappa B-inhibitory activity. Withaferin A inhibited cell proliferation in HUVECs at doses that were significantly lower than those required for tumour cell lines through a process associated with the inhibition of cyclin D1 expression.
Cytotoxic activity
In-vitro effects of Withaferin A on P388 cells have been studied. The cytotoxicity was calculated from the utilization of precursors in protein and nucleic acid synthesis and from capacity to suppress cell proliferation. Withaferin A stopped cell proliferation and, at the same time, killed the cells. Cytotoxicity was found to be due to a double bond at position C2–3; on dissociating this bond the cytotoxicity markedly decreased. A dissociation of the double bond at C24–25 or a removal of OH group from C27 did not cause any significant changes in the biological effects. An addition of a carbonyl group at C4 increased the effect. As withaferin A promptly reacted with l-cysteine, it was presumed that one of the possible target sites in the cell might be the SH groups of enzymes, which react with the lactones and epoxide groups of the agent.
Conclusions
The use of herbal drugs is increasing worldwide as they has fewer or no side effects as compared with synthetic drugs. Ayurveda claims therapeutic potentials of various plants. In India, two species of the genus Withania are found, W. somnifera and W. coagulans. W. somnifera is known by the name ‘Ashwagandhain’ in Hindi and ‘Indian ginseng’ in English. Both species closely resemble each other. Though withanolides are the principal compounds found in both species, there are some withanolides specific to each of them. Withaferin A is a major compound found in W. somnifera, whereas, coagulin L has been found in major amounts in W. coagulans. Where antihyperglycaemic leads from W. coagulans have been identified, it is still to be determined in W. somnifera. A unique thio-dimer of withanolide named Ashwagandhanolide has been found in W. somnifera. Withanolides containing a 14,20-epoxide bridge are specific to W. coagulans. W. somnifera has been used as an antioxidant, adaptogen, aphrodisiac, liver tonic, anti-inflammatory agent and astringent and more recently as an antibacterial, anti-hyperglycemic, hypolipidaemic and antitumoral, as well as to treat ulcers and senile dementia. Hepatoprotective, anti-inflammatory, anti-hyperglycemic, hypolipidaemic, free radical scavenging, antimicrobial, cardiovascular, central nervous system depressant, immune modulating, antitumor and cytotoxic activities have been studied in W. coagulans. W. coagulans had the greater therapeutic value overall. The variety of activities reported for the extracts, fractions and withanolides isolated from W. coagulans provide promising evidence for future research. Withanolides could achieve an important place in the world of modern drugs. Isolation on a large scale, chemical transformations and synthesis of the active compounds will definitely enhance their pharmacological value. The pharmacophores of various pharmacologically active withanolides have not yet been identified. Clinical trials using the active compounds for a variety of conditions need conducting. All these advantages prove the significance of W. coagulans in natural product research.