Ethnomedicine based evaluation of osteoprotective properties of Tinospora cordifolia on in vitro and in vivo model systems.

Indian ethnomedicine acclaims the use of Tinospora cordifolia (TC) in the treatment of bone fractures and vat rakta (gout). The objective of the study is to investigate the effects of alcoholic extract of Tinospora cordifolia on bone remodeling (involving osteoblastic and osteoclastic actions) in vitro and protect against ovariectomy-induced bone loss in vivo. Human osteoblast-like cells MG-63 and primary osteoblast cells isolated from rat femur were used as osteoblast models and RAW macrophage cell line 264.7 induced to take up osteoclastic lineage using RANK ligand were used as osteoclast models in the current study. Sirius red staining, quantification of osteocalcin, cytopathological analysis by Hematoxylin/eosin staining and semiquantitative reverse transcription PCR (RT-PCR) was carried out to ascertain the effects of T. cordifolia extract on osteoblast cells. MTT assay was perfomed to understand the influence of T. cordifolia extract on osteoclast cells. Adult female Sprague-Dawley rats were used as in vivo models to study the effect of T. cordifolia on ovariectomy induced bone loss. Radiological (DEXA analysis), Biochemical (markers of bone formation and resorption), histopathological (Hematoxylin/eosin staining) and histomorphometric analysis of the bone was carried out. Treatment with T. cordifolia extract resulted in enhanced collagen deposition, increased levels of osteocalcin, increased expression of osteogenic genes all indicative of favourable osteoblastogenesis. Treatment with T. cordifolia extract did not exert any significant influence on the proliferation of osteoclasts. Pretreatment with T. cordifolia extract at a dose of 50mg/kg body wt/day orally for 21days followed by treatment for 12 weeks post ovariectomy was able to prevent ovariectomy-induced bone loss in vivo. Results of the study support the use of T. cordifolia in Indian ethnomedicine for the treatment of bone diseases and fractures.

Purification of Lovastatin from Aspergillus terreus (KM017963) and Evaluation of its Anticancer and Antioxidant Properties.

Cervical cancer is the second most common malignancy in women worldwide and thus one of the leading causes of mortality in women. Lovastatin, a non polar, anticholesterol drug has previously been reported to exert antitumour activity in vitro. In the present study, lovastatin from Aspergillus terreus (KM017963) was purified by adsorption chromatography and evaluated for its anticancer and anti-oxidant properties with a human cervical cancer cell line (HeLa). Growth inhibitory and proapoptotic effects of purified lovastatin on HeLa cells were investigated by determining its influence on cell numbers, mitochondrial membrane potential (MMP), DNA fragmentation and antioxidant properties in terms of hydroxy radical scavenging effects as well as levels of total reduced glutathione. Cell cycle analysis by ow cytometry (propidium iodide staining) confirmed induction of apoptotic cell death and revealed cell cycle arrest in the G0/G1 phase. The results of the study give leads for the anticancer effects of lovastatin and its potential usefulness in the chemotherapy of cervical cancer.

XIAP inhibitor and antiestrogen embelin abrogates metastasis and augments apoptosis in estrogen receptor positive human breast adenocarcinoma cell line MCF-7.

Tamoxifen therapy for the treatment of hormone responsive breast cancer has limitations due to acquired resistance in the case of recurrences. Embelin, a known inhibitor of X-linked inhibitor of apoptosis protein (XIAP) was also reported to exhibit strong antiestrogenic effects in animal models. Dual role of embelin as a proapoptotic and antiestrogenic agent may have potential benefits in the therapy of breast cancer. In this study, the effects of embelin treatment on estrogen receptor positive Human breast adenocarcinoma (MCF-7) cells was investigated to primarily understand if embelin being an antiestrogen and XIAP inhibitor could be a potential alternative to tamoxifen therapy. Results revealed that, embelin at a concentration of 65 µg/ml attenuated proliferation, inhibited metastatic migration, modulated the expression of Bcl2, Caspases and induced apoptosis in MCF-7 cells which was found to be p53 mediated. Hence, chemotherapy with embelin could be a promising strategy to be experimented in hormone responsive breast cancers.

Effects of Tinospora cordifolia (Menispermaceae) on the proliferation, osteogenic differentiation and mineralization of osteoblast model systems in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Ancient Indian ayurvedic literature prescribes Tinospora cordifolia as a remedy to rheumatoid arthritis, inflammatory and allied diseases of musculo skeletal system. AIM: To investigate the effects of the alcoholic extract of Tinospora cordifolia (TC) on the proliferation, differentiation and mineralization of bone like matrix on osteoblast model systems in vitro and hence its possible use as a potential anti-osteoporotic agent. MATERIALS AND METHODS: Two in vitro osteoblast model systems were used in the study viz., human osteoblast-like cells MG-63 and primary osteoblast cells isolated from femur of rats. Cell growth and viability was assessed by standard colorimetric assays like MTT assay. The cell differentiation into osteoblastic lineage was evaluated by the activities of bone marker alkaline phosphatase. The effect of the extract on matrix mineralization was assessed by alizarin red-s staining and Von kossa staining. Cell morphology was studied by phase contrast microscopy and light microscopy (Giemsa/crystal violet staining). RESULTS: Results indicate that the alcoholic extract of TC at a dosage of 25µg/ml stimulated the growth of osteoblasts, increased the differentiation of cells into osteoblastic lineage and increased the mineralization of bone like matrix on both the osteoblast model systems used in the study. Cell morphology studies clearly indicated the increase in cell numbers and absence of adverse change in the cell morphology on treatment with the extract. CONCLUSION: TC extract has a potential influence on osteogenesis and hence its use could be explored as a potential anti-osteoporotic agent.

Modulating effect of Leptadenia reticulata (Retz) Wight & arn against chromate (VI)-induced immunosuppression and oxidative stress on mouse splenic lymphocytes and bone marrow derived macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: Leptadenia reticulata (Retz) Wight & arn is mentioned in the ancient ayurvedic literature as an immune booster and rejuvenator. AIMS OF THE STUDY: To investigate, the effects of different forms of the extract of Leptadenia reticulata [Aqueous extract (JAE), Padavashesha kashaya (JPK) and Tarpana kashaya (JTK)] to alleviate the experimental immunosuppression induced by the immunotoxicant chromate (VI) in vitro. MATERIALS AND METHODS: Standard cell proliferation and cytotoxicity assays like MTT assay, trypan blue dye exclusion test, neutral red dye uptake test, NBT reduction test, determination of percentage cell survival and estimation of markers of oxidative stress were performed in the study. The study was conducted on primary cultures of mouse splenic lymphocytes and bone marrow derived macrophages. RESULTS AND CONCLUSIONS: Treatment with all the three forms of the extract used in the study offered protection against chromate (VI)-induced immunosuppression and the overall protective effect was found to be superior in the case of the aqueous extract of Leptadenia reticulata (JAE). These results confirm that Leptadenia reticulata acts as a modulator and alleviates the immunosuppressive conditions induced by chromate (VI).

Protective effect of Premna tomentosa extract (L. verbanacae) on acetaminophen-induced mitochondrial dysfunction in rats.

Allurement of herbs as health beneficial foods (physiologically functional foods) and as a source material for the development of new drugs, has led to greater furtherance in the study of herbal medicines during recent years. Plant extracts are being utilized to treat a wide variety of diseases like hepatotoxicity. Premna tomentosa is one such medicinal plant used widely in Indian ayurvedic medicine for the treatment of liver disorders. This study appraised the effectiveness of P. tomentosa leaf extract in protecting the liver against mitochondrial damage induced by acetaminophen, since mitochondrial injury has been investigated as a potential initiator of hepatotoxicity. Normal Wistar strain rats were pre-treated with P. tomentosa extract (750 mg/kg, orally) for 15 days and then intoxicated with acetaminophen (640 mg/kg, orally). Mitochondria were isolated from liver of experimental animals and assessed for the levels of lipid peroxide products, GSH and mitochondrial enzymes (isocitrate dehydrogenase, alpha-keto glutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, NADH dehydrogenase and cytochrome-C-oxidase). The levels of Lipid peroxidation products were increased and the levels of the other assessed parameters were significantly decreased in hepatotoxicity induced animals. Whereas, the levels were brought back to normal in P. tomentosa pre-treated rats, which shows the protective effect of the extract against mitochondrial damage. Presence of anti-oxidant compound D-limonene (58%) in P. tomentosa leaves, which is known to enhance conjugation of toxic metabolites by maintaining liver GSH concentrations may explain the hepatoprotective property of the extract.

Chemopreventive effects of embelin and curcumin against N-nitrosodiethylamine/phenobarbital-induced hepatocarcinogenesis in Wistar rats.

The effects of embelin (50 mg/kg/day), a benzoquinone derivative of Embelia ribes, and the effects of curcumin (100 mg/kg/day), the active principle of Curcuma longa, against N-nitrosodiethylamine (DENA)-initiated and phenobarbital (PB)-promoted hepatocarcinogenesis were studied in Wistar rats. They were able to prevent the induction of hepatic hyper plastic nodules, body weight loss, increase in the levels of hepatic diagnostic markers, and hypoproteinemia induced by DENA/PB treatment. Hence, results of our study suggest the possible chemopreventive effects of embelin (EMB) and curcumin (CUR) against DENA/PB-induced hepatocarcinogenesis in Wistar rats.

Protective effect of Premna tomentosa (L. Verbenaceae) extract on membrane-bound phosphatases and inorganic cations transport in acetaminophen-induced hepatotoxicity rats.

Hepatic injury elicits intracellular stress that leads to peroxidation of membrane lipids accompanied by alteration of structural and functional characteristics of membrane, which affect the activities of membrane-bound ATPases. The present study appraised the membrane protective effect of Premna tomentosa, a hepatoprotective drug used in Indian traditional medicine. Wistar strain rats were pre-treated with Premna tomentosa extract (750 mg/kg, orally) for 15 days, 24 h prior to administration of acetaminophen (640 mg/kg, orally). During acetaminophen intoxication, the levels of membrane-bound enzymes were significantly decreased, total ATPase (1.63-fold), Mg(2+)ATPase (1.9-fold), Ca(2+)ATPase (1.33-fold) and Na(+)K(+)ATPase (1.73-fold) which was accompanied by changes in the levels of inorganic cations N+, K+ and Ca2+. These alterations were prevented by Premna tomentosa extract pre-treatment, which shows that Premna tomentosa supplementation could exert a beneficial effect against liver injury-induced membrane damage. The potential of the plant might be credited to the presence of antioxidant compound limonene in the plant.

Antinociceptive and hypnotic effects of Premna tomentosa L. (Verbenaceae) in experimental animals.

Medicinal plants are believed to be an important source of new chemical substances with potential therapeutic effects. The research into plants with alleged folklore use as pain relievers should therefore be viewed as a fruitful and logical research strategy in the search of new analgesic drugs. In the present inquiry, antinociceptive effects of Premna tomentosa (PT) leaf extract (in methanol) were explored in experimental animals by acetic acid-induced writhing, tail flick and tail clip tests. Oral administration of PT extract at different doses (100, 200, 400 and 500 mg/kg) led to significant antinociceptive effects. The extract was also tested for hypnotic effects. Treatment with extracts at different doses (100, 200, 400 and 500 mg/kg) decreased the locomotor activity and potentiated the pentobarbitone-induced sleep time. The responses were dose-dependent. On the basis of the present finding, we can conclude that PT possesses antinociceptive and hypnotic activities.

Immunomodulatory effects of Premna tomentosa extract against Cr (VI) induced toxicity in splenic lymphocytes--an in vitro study.

Premna tomentosa (L. Verbanacae) is a widely used medicinal plant. Our earlier studies show that the extract of P. tomentosa leaves prevents acetaminophen-induced hepatotoxicity owing to its antioxidant property. In the present study, we have investigated the immunomodulatory effects of P. tomentosa extract against Chromium (VI) induced immunosuppression in splenic lymphocytes. Chromium (Cr) addition at a concentration of 5 microg showed an increase in cytotoxicity, apoptosis and reactive oxygen species (ROS) and a decrease in lymphocyte proliferation and antioxidant levels, whereas pre-treatment of the cells with P. tomentosa extract (at 500 microg concentration) resulted in decreased cytotoxicity and ROS levels. Further, the drug treatment also maintained antioxidant levels and restored lymphocyte proliferation similar to that of control cells. The results indicated that the leaf extract of P. tomentosa has cytoprotective and immunomodulatory activities.

Effect of Indigofera tinctoria Linn on liver antioxidant defense system during D-galactosamine/endotoxin-induced acute hepatitis in rodents.

Effects of pre-treatment with the alcoholic extract of I. tinctoria (500 mg/kg body wt/day, p.o. for 21 days) on liver antioxidant defense system during acute hepatitis induced by D-galactosamine (D-GalN)/endotoxin (LPS extracted by phenol water method from E. coli serotype 0111.B4; 300 mg and 30 micrograms/kg body wt/day, i.p., 18 hr before the assay) were investigated on the activities of enzymic antioxidants such as superoxide dismutase, catalase, glutathione peroxidase and glutathione-s-transferase, and levels of total reduced glutathione in the liver of normal and experimental groups of male albino rats. Since lipid peroxidation and associated membrane damage is a key feature of D-galN/LPS-induced liver injury, the levels of lipid peroxides, was estimated and used as an index of oxidative stress. D-GalN/endotoxin-induced hepatic damage was manifested by a significant decrease in the activities of antioxidant enzymes, decreased glutathione levels and increased levels of lipid peroxides. I. tinctoria pre-treated rats showed considerable protection against D-galN/endotoxin, induced oxidative stress as evidenced by a significant increase in the activities of all the antioxidant enzymes studied and significant decrease in the levels of lipid peroxides. Results indicate that pretreatment with I. tinctoria extract in rats is very effective in reducing D-GalN/endotoxin-induced oxidative stress suggesting an antioxidant effect.

Protective effects of Indigofera tinctoria L. against D-Galactosamine and carbon tetrachloride challenge on 'in situ' perfused rat liver.

The effect of pre-treatment with Indigofera tinctoria (IT) extract against the toxicity of D-Galactosamine (D-GalN) and carbon tetrachloride (CCl4) during 'in situ' perfusion of the liver for 2 hr was studied in rats. Release of LDH and levels of urea in the liver effluent perfusate, was studied and the rate of bile flow was monitored. Perfusion with D-Galactosamine (5 mM) or carbon tetrachloride (0.5 mM) resulted in increased LDH leakage, decreased urea levels in the liver effluent and reduction in bile flow. IT pretreatment (500 mg/kg body weight) in vivo ameliorated D-GalN and CCl4 induced adverse changes towards near normalcy and thereby indicates its hepatoprotective effects in rats.