Promising anti-diabetes mellitus activity in rats of ß-amyrin palmitate isolated from Hemidesmus indicus roots.

While evaluating the toxicity of the tuberous root extracts of Hemidesmus indicus, a traditional medicinal plant, the glucose lowering property of the root was observed by the investigators. Therefore, it was thought of interest to isolate the anti-hyperglycemic principle from the root and determine its utility to develop an anti-diabetes mellitus medicine. The active principle was isolated from H. indicus root extract by anti-hyperglycemic activity guided chromatographic techniques. Glucose tolerance test in rats was used to evaluate the anti-hyperglycenic property. Anti-diabetes mellitus property was evaluated in alloxan-induced diabetic rats as well as streptozotocin-induced (type-2 model) diabetic rats. The active principle was isolated and identified with spectral data as ß-amyrin palmitate. Although it is a known compound, its presence in H. indicus is not known previously. It was observed for the first time that ß-amyrin palmitate has remarkable anti-hyperglycemic activity in orally glucose loaded rats. Further, interestingly, it exhibited excellent anti-diabetes mellitus activity in both alloxan-diabetic and streptozotocin-diabetic rats at a very low concentration (50µg/kg body weight). One of the mechanisms of action of ß-amyrin palmitate appears to be blocking the entry of glucose from the intestine. ß-Amyrin palmitate is very promising to develop a medicine for diabetes for combination therapy and/or mono-therapy.

A novel aphrodisiac compound from an orchid that activates nitric oxide synthases.

Nitric oxide (NO) is known to have roles in several crucial biological functions including vasodilation and penile erection. There are neuronal, endothelial and inducible NO synthases that influence the levels of NO in tissues and blood. NO activates guanylate cyclase and thereby increases the levels of cyclic GMP (cGMP). Viagra (sildenafil), a top selling drug in the world for erectile dysfunction, inhibits phosphodiesterase-5, which hydrolyses cGMP to GMP. Thus, it fosters an NO-mediated increase in the levels of cGMP, which mediates erectile function. Here, we show the aphrodisiac activity of a novel chemical isolate from the flowers of an epiphytic orchid, Vanda tessellata (Roxb.) ex Don, which activates neuronal and endothelial, but not inducible, NO synthases. The aphrodisiac activity is caused by an increase in the level of NO in corpus cavernosum. The drug increases blood levels of NO as early as 30 min after oral administration. The active compound was isolated by column chromatography. Based on the spectral data, the active compound is found to be a new compound, 2,7,7-tri methyl bicyclo [2.2.1] heptane. We anticipate that our findings could lead to the development of a commercially viable and valuable drug for erectile dysfunction.

Induction of cell-specific apoptosis and protection of mice from cancer challenge by a steroid positive compound from Zornia diphylla (L.) Pers.

OBJECTIVES: Zornia diphylla (L.) Pers is an ethnomedical herb. The aim of the study is to scientifically verify the traditional use of Z. diphylla as an anticancer medicine. MATERIALS AND METHODS: Different extracts, fractions, and chemical isolates of the whole plant were screened for cytotoxicity to Dalton's lymphoma ascites (DLA) cells by the Trypan blue exclusion method and MTT assay. Column chromatographic and preparative TLC techniques were used for the isolation of active fraction (AF) and active principle. Cytotoxicity of AF to different cell types was tested. The apoptotic activity of AF was evaluated by morphological observations, nuclear condensation, and comet assay. In vivo antitumor activity of AF was determined in DLA-challenged mice. Short-term (29 days) preliminary toxicity evaluation of AF was done in mice. RESULTS: n-Hexane extract (but not water and ethanol extracts) showed significant cytotoxicity. AF, isolated from n-hexane extract, induced apoptotic cell death (in vitro) to DLA cells, but not to normal thymocytes and macrophages. A steroid positive active principle was isolated which showed 100% cytotoxicity at 5 µg/mL level. Interestingly, AF (50 mg/kg) protected all the mice challenged with one million DLA cells/mouse. AF (up to 10 times higher than the therapeutic dose) did not exhibit any conspicuous adverse toxic symptoms in the toxicity evaluation. CONCLUSION: Z. diphylla (AF) showed promising in vitro and in vivo anticancer activity against DLA cells, and it was devoid of any toxicity to mice in short-term toxicity evaluation. The herb is promising for the development of a valuable anticancer medicine.

Protection of immunocompromised mice from fungal infection with a thymus growth-stimulatory component from Selaginella involvens, a fern.

CONTEXT: Recent studies have shown that the water extract of Selaginella involvens (Sw.) Spring, a wild fern, exhibits thymus growth-stimulatory activity in adult mice (reversal of involution of thymus) and remarkable anti-lipid peroxidation activity. Follow-up studies were carried out in the present study. MATERIALS AND METHODS: Activity-guided isolation of the active component (AC) was carried out. The effect of AC on immune function was studied using fungal (Aspergillus fumigatus) challenge in cortisone-treated mice. The in vitro antifungal activity of AC was assayed using disc diffusion assay. In vitro and in vivo effect of AC on DNA synthesis in thymus was studied using (3)H-thymidine incorporation. In in vitro anti-lipid peroxidation, hydroxyl radical scavenging and inhibition of superoxide production were assayed. RESULTS: The active principle/component (AC) was isolated in a chromatographically pure form from the water extract of S. involvens. AC showed positive reaction to glycosides. AC possessed both thymus growth-stimulatory and antioxidant properties. It protected cortisone-treated mice from A. fumigatus challenge. It did not exhibit in vitro antifungal activity. Increased (3)H-thymidine incorporation was observed in the reticuloepithelium of thymus obtained from AC-treated mice. However, in vitro AC treatment to thymus for 5 h did not result in an increase in (3)H-thymidine incorporation. DISCUSSION AND CONCLUSION: AC (named as Selagin), from S. involvens, could reverse involution of thymus to a large extent, exhibit remarkable antioxidant activity, and protect immunocompromised mice from fungal infection. Therefore, it is very promising for the development of a drug to ameliorate old age-related health problems and prolong lifespan.

Hepatoprotective property of Thespesia populnea against carbon tetrachloride induced liver damage in rats.

The present study was carried out to evaluate the possible hepatoprotective activities of the medicinal plant Thespesia populnea in carbon tetrachloride (CCl4)-induced liver injury in rats. The water suspension (500 mg/kg b.wt.) of leaf, flower and stem bark of T. populnea showed varying levels of protective action against CCl4-induced liver damage as evidenced from significant reduction in the activities of serum marker enzymes for liver damage (alanine transaminase, aspartate transaminase, and alkaline phosphatase), and bilirubin levels when compared with CCl4-intoxicated control rats. The stem bark suspension showed maximum hepatoprotection compared with leaf and flower. An ethanol extract of the stem bark was more active than n-hexane and water extracts, showing remarkable protection at a dose of 60 mg/kg b.wt. The hepatoprotective effect of this extract was almost comparable to that of silymarin (100 mg/kg), a reference herbal drug. Thus, the present study indicates that ethanol extract of T. populnea stem bark is promising for further studies leading to hepatoprotective drug development.

Anti-diabetes and hypoglycaemic properties of Hemionitis arifolia (Burm.) Moore in rats.

Hemionitis arifolia, a folklore anti-diabetes fern, was evaluated for its hypoglycaemic and anti-diabetic properties using rats. Glucose lowering effect and anti-diabetes activity were studied using glucose tolerance test in normal rats and alloxan diabetic rats, respectively. When different extracts were tested, the ethanol and, to some extent, the water extracts were found to lower the levels of blood glucose in glucose fed rats. The ethanol extract showed optimum activity at 200 mg/kg. The extract exhibited only marginal hypoglycaemic activity in overnight fasted normal rats and it was devoid of conspicuous toxic symptoms in sub-acute toxicity evaluation in mice. When the alcohol extract was fractionated by sequential solvent extraction, the activity was found in ethyl acetate fraction (50 mg/kg). This fraction containing steroids and coumarins showed anti-diabetes activity in alloxan diabetic rats as judged from serum glucose levels, liver glycogen content and body weight. This fraction is an attractive material for further research vis-à-vis drug development.

Enhancement in the absorption of water and electrolytes from rat intestine by Hemidesmus indicus R. Br. root (water extract).

Hemidesmus indicus root in the form of suspension in water (10 mg/ml) containing 15.5 mM NaCl, 3 mM KCl and 12 mM glucose, when injected into the ligated jejunal sac (1 ml/sac) of rat, increased the absorption of water, Na(+) and K(+) (but not glucose) from the sac. This bioactivity was present in the water extract (5 or 10 mg/sac) of the root and not in the hexane extract. In contrast, the ethanol extract decreased the absorption of water and electrolytes from the jejunal sac. The effect of water extract was not affected by heat at 100 degrees C for 30 min. Intraperitoneal administration of the water extract (50 to 200 mg/kg) was devoid of any significant effect on the jejunal absorption. Neither the root suspension nor the water extract (125-500 mg/kg) showed any significant anti-ulcer and diuretic activities in rats. The intestinal motility was also not influenced by the root (water extract) when tested in mice. The present study indicates that H. indicus root powder or its water extract can be incorporated in oral rehydrating salt solution (ORS) for increasing its anti-diarrhoeal efficacy.

Further studies on the antihepatotoxic activity of Phyllanthus maderaspatensis Linn.

Phyllanthus maderaspatensis (whole plant extracts) was evaluated for its antihepatotoxic and choleretic activities in rats. The plant extracts (200 mg/kg, n-hexane, ethyl alcohol or water) showed a remarkable hepatoprotective activity against acetaminophen-induced hepatotoxicity as judged from the serum marker enzymes. The water and ethyl alcohol extracts showed moderate activity compared to the n-hexane extract, which showed activity at a dose as low as 1.5 mg/kg. The antihepatotoxicity of the hexane extract was found to be better than silymarin, a standard hepatoprotective herbal drug. The effect of n-hexane extract was found to be concentration-dependent. This extract also exhibited choleretic activity in normal rats, and in vitro hydroxyl radical scavenging activity and inhibition of lipid peroxidation.

Antitumour property and toxicity of Barringtonia racemosa Roxb seed extract in mice.

Ethnomedical survey has shown that the seeds of Barringtonia racemosa Roxb are traditionally used in certain remote villages of Kerala (India) to treat cancer like diseases. So the seed extracts were tested for their antitumour activity and toxicity. Intraperitoneal (i.p.) daily administration of 50% methanol extract of this seed to mice challenged with 1 million Dalton's Lymphoma Ascitic (DLA) cells resulted in remarkable dose dependent anti-DLA activity in mice. The optimum dose was found to be 6 mg/kg. This dose protected all the animals challenged with the tumour cells. The efficacy of the drug was found to be better than that of a standard drug, vincristine in this tumour model. However, the oral administration showed only marginal activity compared to i.p. administration. The extract was found to be devoid of conspicuous acute and short-term toxicity to mice, when administered daily, (i.p.) for 14 days up to a dose of 12 mg/kg (which was double the concentration of optimum therapeutic dose). The treated mice showed conspicuous toxic symptoms only at 24 mg/kg. The LD(50) to male mice for a single i.p. dose was found to be 36 mg/kg. The seed extract is an attractive material for further studies leading to drug development.

Inhibition of antigen-induced degranulation of sensitized mast cells by Trichopus zeylanicus in mice and rats.

Treatment of mice with Trichopus zeylanicus leaf resulted in inhibition of antigen-induced degranulation of sensitized peritoneal mast cells. Further, it reduced the ratio of mast cells in the peritoneal exudate cells. The plant drug treatment did not protect mice from E. coli-induced abdominal sepsis. Studies in rats using mesenteric mast cells confirmed the above mast cell-stabilizing property of T. zeylanicus. This activity was found in the butanol fraction of methanol extract of T. zeylanicus leaf. The treatment with this fraction also reduced the number of rat mesenteric mast cells. However, the in vitro treatment of the mast cells with the butanol fraction did not inhibit antigen-induced degranulation of the mast cells.

Hepatoprotective activity of Trichopus zeylanicus extract against paracetamol-induced hepatic damage in rats.

Trichopus zeylanicus extract has been evaluated for its antihepatotoxic and choleretic activities in rats. The plant leaf suspension (1000 mg/kg; wet weight) as well as its methanol extract (100 mg/kg) showed a remarkable hepatoprotective activity against paracetamol-induced hepatotoxicity as judged from the serum marker enzymes, liver histology and levels of lipid peroxides in liver. The effect of the methanol extract was found to be concentration dependent. The water and hexane extracts were almost inactive. The methanol extract (100 mg/kg) also exhibited choleretic activity in anesthetized normal rats. In short, we report here for the first time the antihepatotoxic as well as choleretic properties of T. zeylanicus.

Aphrodisiac property of Trichopus zeylanicus extract in male mice.

Administration of Trichopus zeylanicus leaf (ethanol extract) to male mice stimulated their sexual behaviour as evidenced by an increase in number of mounts and mating performance. This activity of the ethanol extract was concentration dependent and destroyed by heat treatment at 100 degrees C for 15 min. Although oral administration of a single dose (200 mg/kg) was effective, daily administration of the extract for 6 days was found to be more effective. The pups fathered by the drug treated mice were found to be normal with reference to foetal growth, litter size and sex ratio. The water as well as n-hexane extracts of the plant leaf were inactive. The present study reveals for the first time the aphrodisiac activity of Trichopus zeylanicus, an endemic herb of India.

Antitumour activity of mitoxantrone-loaded chitosan microspheres against Ehrlich ascites carcinoma.

Glutaraldehyde cross-linked chitosan microspheres containing the antineoplastic agent mitoxantrone were prepared and the antitumour activity was evaluated against Ehrlich ascites carcinoma in mice by intraperitoneal injections. The tumour inhibitory effect was followed by monitoring animal survival time and change in body weight for a period of 60 days. While the mean survival time of animals which received 2 mg and 1 mg of free mitoxantrone intraperitoneally was 2.1 and 4.6 days, respectively, animals which received 2 mg mitoxantrone via microspheres showed a mean survival time of 50 days. Five out of 8 animals treated using microspheres lived beyond 60 days. The percentage ratio mean survival time of the treated group divided by the mean survival time of the untreated group for animals treated using mitoxantrone-loaded chitosan microspheres containing 2 mg of the drug was 290 compared with 12.2 for those which received 2 mg of the free drug. The antitumour effect of mitoxantrone-loaded microspheres against Ehrlich ascites carcinoma was much higher than that of doxorubicin-loaded microspheres reported by previous workers. Our data demonstrate the potential of mitoxantrone-loaded chitosan microspheres for sustained drug delivery to minimize drug toxicity and maximize therapeutic efficacy.

Effects of Artemisia pallens Wall. on blood glucose levels in normal and alloxan-induced diabetic rats.

Oral administration of the methanol extract of the aerial parts of Artemisia pallens Wall. (used in Indian folk medicine for the treatment of diabetes mellitus) led to significant blood glucose lowering effect in glucose-fed hyperglycaemic and alloxan-induced diabetic rats. This effect of the extract was dose dependent and significant at 100 mg/kg level in glucose-fed rats. In fasted normal rats, the extract caused a moderate hypoglycaemic effect at a higher dose (1000 mg/kg). The water extract (1000 mg/kg) was inactive.

Influence of hexachlorocyclohexane on phosphoinositides in rat erythrocyte membranes and brain.

Single exposure of rats to hexachlorocyclohexane (100 mg/kg) did not cause any significant change in phosphoinositide levels in rat erythrocyte membrane and cerebrum (fore brain) 2 or 24 h after exposure. However, the phosphoinositide turnover and generation of second messengers from phosphoinositides were increased in the treated erythrocyte membranes as judged from a marked increase in the incorporation of [2-3H]inositol into phosphoinositides 24 h after the treatment. A significant decrease in phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) was observed in the erythrocyte membrane and cerebrum of rats repeatedly exposed to the pesticide for 3 or 6 months. This drastic reduction in phosphoinositide levels suggests adverse effects on vital membrane and cell functions modulated by phosphoinositides.

Dibutyltin dilaurate induced thymic atrophy and modulation of phosphoinositide pathway of cell signalling in thymocytes of rats.

A marked dose dependent reduction in thymus weight and its nucleated cell counts with histological alterations was observed in rats exposed to oral dibutyltin dilaurate (DBTL) for 2 weeks at 2, 4, 8 or 16 mg/kg body weight. The incorporation of [3H]-inositol into all the three major phosphoinositides was drastically reduced in thymocytes in a dose dependent manner. Furthermore, the basal and the mitogen (Con A) stimulated [3H]-inositol phosphates generation was diminished significantly in 8 mg DBTL group. However, in vitro incubation of DBTL with thymocytes failed to evoke any change in phosphoinositide hydrolysis. Similarly, a time and dose dependent inhibition in phosphoinositide synthesis with as high as 80% by 10 microM DBTL was exhibited under in vitro conditions. A 130% and 600% enhancement of protein kinase C (PKC) activity in thymocytes was seen in 4 mg and 8 mg DBTL group, respectively. Addition of DBTL to the cell free assay system of thymocytes resulted in a concentration dependent activation of the enzyme activity. A dose dependent increase in intracellular calcium was also evident when DBTL was added to thymocytes under in vitro conditions. These results are of significance and may bear close relationship to the observed thymic atrophy by DBTL.

Influence of mancozeb on mitogenically responsive lipids in rat cerebrum and liver.

Mancozeb, a commonly used fungicide, has been shown to induce tumours in mouse skin and maneb, unit constituent of mancozeb, is reported to induce tumours in rats. The mechanism by which mancozeb induced tumorigenicity is not known. Since the levels of inositol phospholipids and phosphatidic acid have roles in the regulation of cell proliferation, the effects of mancozeb on the levels of these lipids were studied in rats. Daily oral administration of commercial grade mancozeb at a concentration of 50 mg/kg body wt for 30 days (5 days a week) caused no significant change in the levels of inositol phospholipids and phosphatidic acid (PA) in both cerebrum and liver, while at high concentration (250 mg/kg body wt) under the same treatment schedule mancozeb increased the levels of these lipids. In cerebrum, the levels of phosphatidylinositol (PI) and PA were increased by 36 and 43% respectively without affecting the levels of polyphosphoinositides, whereas in liver the levels of not only PI (50%) and PA (49%) but also those of polyphosphoinositides were increased. These results show that mancozeb influences the levels of PA and inositol phospholipids, involved in phospholipase C-pathway of signalling.

Reduction of phosphoinositides and diacylglycerol levels in repeatedly dibutyltin-dilaurate-treated rat brain.

Oral administration of a single dose of dibutyltin dilaurate (DBTL, 80 mg/kg body wt.), 2 or 24 h after treatment, caused no significant change in the levels of diacylglycerol and phosphoinositides in rat cerebrum (forebrain), whereas daily administration of DBTL (40 or 80 mg/kg body wt.) for 3 days (24 h after the final treatment) decreased the levels of diacylglycerol and phosphoinositides (phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate) in a concentration-dependent manner without influencing the levels of phosphatidylcholine in rat cerebrum. These studies indicate impairment of the phosphoinositide messenger system in rat cerebrum following repeated exposure to DBTL.