Bioassay guided fractionation of Cyclea peltata using in vitro RAW 264.7 cell culture, antioxidant assays and isolation of bioactive compound tetrandrine.

BACKGROUND: Cyclea peltata is one of the herbs mentioned in ancient scriptures of Ayurveda and is used in different types of Ayurvedic gritham preparations. Moreover, in traditional/tribal medicine C. peltata is used as digestive, anti-inflammatory, diuretic and to treat jaundice, digestive disorders, etc. OBJECTIVE: Activity guided fractionation of C. peltata and in correlation with the levels of bioactive compound tetrandrine. MATERIALS AND METHODS: Preliminary phytochemical screening, estimation of total alkaloid content, preparation of different extracts of C. peltata (crude extract CP, hexane extract HCP, chloroform extract CCP, methanol extract MCP, alkaloid fraction ACP). In vitro anti-inflammatory studies using RAW 264.7 cells and in vitro antioxidant assays of the different extracts of C. peltata. HPTLC estimation of tetrandrine (TET) was carried out using solvent system toluene: ethyl acetate: diethylamine (7.2: 2: 0.8) and isolation of TET from ACP. RESULTS: Preliminary phytochemical studies of C. peltata showed the presence of alkaloid content in all extracts. Whereas, saponins, steroids and terpenoids were detected in CP and CCP. ACP and TET showed significant in vitro anti-inflammatory and antioxidant activity when compared to other extracts. ACP and TET (100 µg/ml) treatment significantly inhibited the mRNA expression of iNOS, COX-2, TNF-α in LPS treated RAW 264.7 cells. HPTLC estimation of bioactive compound tetrandrine was highest in ACP-228.4 µg/mg followed by CP-29.62 µg/mg, CCP-23.46 µg/mg, MCP-18.82 µg/mg and HCP-1.25 µg/mg. TET has been isolated from ACP. CONCLUSION: The results of the present in vitro assays revealed that the alkaloid fraction (ACP) is the most active fraction when compared to other extracts and has a positive correlation with the levels of bioactive compound tetrandrine.

Protective effect of ethyl acetate fraction of Drynaria quercifolia against CCl4 induced rat liver fibrosis via Nrf2/ARE and NFκB signalling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Drynaria quercifolia rhizome is traditionally used as hepatoprotective drug especially in chronic jaundice. AIM OF THE STUDY: The present study was undertaken to scientifically evaluate the efficacy of D. quercifolia rhizome against liver fibrosis. MATERIALS AND METHODS: D. quercifolia rhizome crude extract (DQ) and its fractions of hexane (HDQ), ethyl acetate (EDQ), butanol (BDQ) were evaluated in vitro using primary hepatocytes and RAW 264.7 cells. In vivo anti-liver fibrotic activity of EDQ was assessed using CCl4 induced liver fibrosis in Wistar rats and serum biochemical parameters (AST, ALT, ALP, SB, cholesterol), MDA, PT, INR, GSH, SOD, CAT, liver glycogen, serum albumin levels were monitored. qRT-PCR analysis of TNF-α, COX-2, iNOS were performed. ELISA method was used to estimate TNF-α, COX-1 & 2. Histopathological studies like H & E, Masson's trichrome, immunohistochemistry staining for α-SMA, TIMP-1, Nrf2 were conducted. LC-Q-TOF-MS analysis of EDQ was conducted. RESULTS: In vitro activity guided fractionation of D. quercifolia revealed EDQ as active fraction when compared to other extracts. EDQ treatment significantly inhibited the expression of α-SMA, TIMP-1, COX-2, TNF-α, iNOS and increased the levels of Nrf2 in rat liver fibrosis. LC-Q-TOF-MS analysis of EDQ confirmed the presence of naringin and naringenin. CONCLUSION: The anti-liver fibrotic activity of EDQ is via inhibition of NFκB signalling pathway, antioxidant response through Nrf2 activation and further inhibition of HSC activation.

Hepatoprotective activity of Oxalis corniculata L. ethanolic extract against paracetamol induced hepatotoxicity in Wistar rats and its in vitro antioxidant effects.

Oxalis corniculata is well known for its medicinal properties like anti-inflammatory, digestive, diuretic, antibacterial, antiseptic etc. The present study focuses on the ability of O. corniculata to alleviate liver damage caused by over dose of paracetamol. Antioxidant activity of O. corniculata was evaluated using the free radical scavenging activity of 1, 1-diphenyl-2- picrylhydrazyl radicals, total anti oxidant capacity by phosphomolybdenum method and total phenolic content was also evaluated. The ethanolic extract of whole plant of O. corniculata (OC, 500 microg/mL, po) significantly reduced 1, 1-diphenyl-2- picrylhydrazyl radicals. This dose also caused significant reduction (62.67%) in malondialdehyde levels of murine hepatic tissues. The antioxidant capacity of OC was comparable to that of standard ascorbic acid and showed 53.5 microg of phenol/mg OC. Rats pre-treated with OC for 4 days showed significant reduction in the serum enzymes such as glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, alkaline phosphatase, serum bilirubin and showed almost normal histological liver architecture of the treated groups compared to paracetamol induced hepatic damage group, indicating its hepatoprotective and antioxidant potential.

Sesquiterpene lactones isolated from Elephantopus scaber L. inhibits human lymphocyte proliferation and the growth of tumour cell lines and induces apoptosis in vitro.

This study was designed to isolate the compounds responsible for the cytotoxic properties of South Indian Elephantopus scaber L. and further investigate their effects on quiescent and proliferating cells. Bioassay-guided isolation of the whole plant of chloroform extract of South Indian Elephantopus scaber afforded the known sesquiterpene lactone, deoxyelephantopin, and isodeoxyelephantopin whose structures were determined by spectroscopic methods. These compounds caused a dose dependent reduction in the viability of L-929 tumour cells in 72 h culture (IC(50) value of 2.7 µg/mL and 3.3 µg/mL) by the cell viability assay. Both the compounds act selectively on quiescent and PHA-stimulated proliferating human lymphocytes and inhibited tritiated thymidine incorporation into cellular DNA of DLA tumour cells. The compound deoxyelephantopin at a concentration of 3 µg/mL caused maximum apoptotic cells. It also exhibited significant in vivo antitumour efficacy against DLA tumour cells. The results, therefore, indicate that the antiproliferative property of deoxyelephantopin and isodeoxyelephantopin could be used in regimens for treating tumors with extensive proliferative potencies.

Anti-inflammatory and analgesic properties of Drynaria quercifolia (L.) J. Smith.

ETHNOPHARMACOLOGICAL RELEVANCE: Drynaria quercifolia (L.) J. Smith (Polypodiaceae), has been widely used by ethnic groups of India to treat inflammation, rheumatism, headache, bone fracture, jaundice, etc. AIM OF THE STUDY: To evaluate the anti-inflammatory and analgesic properties of the ethanolic extract of rhizome of Drynaria quercifolia (DQ) and its phytochemical profile. MATERIALS AND METHODS: DQ was used to evaluate the anti-inflammatory and analgesic effects using carrageenan-induced paw oedema/cotton pellet-induced granuloma in Wistar rats and acetic acid-induced writhing/formalin-induced paw licking test in Swiss albino mice respectively. RESULTS: Oral administration of DQ produced significant inhibition of carrageenan-induced paw oedema and granuloma formation in rats, almost comparable to that caused by indomethacin. DQ significantly attenuated acute and delayed phases of formalin-induced pain and acetic acid-induced writhing episodes in mice. The analgesia was comparable to that produced by sodium salicylate and aspirin respectively. Phytochemical analysis gave positive tests for catechin, coumarins, flavonoids, phenolics, saponin, steroids, tannins, and triterpenes. The total phenolics in DQ was 244 mg/g and naringin content was 0.048%. CONCLUSION: The results suggest the presence of potent anti-inflammatory and analgesic principles in DQ that justifies its use for alleviating painful inflammatory conditions.

Anti-allergic, anti-inflammatory and anti-lipidperoxidant effects of Cassia occidentalis Linn.

Cassia occidentalis Linn. mast cell degranulation at a dose of 250 mg/kg, showed dose dependent stabilizing activity towards human RBC, with is widely used in traditional medicine of India to treat a number of clinical conditions including allergy and inflammatory manifestations. In the present study anti-allergic, anti-inflammatory and anti-oxidant properties of C. occidentalis whole plant ethanolic extract (CO) was investigated. Effects of CO on rat mast cell degranulation inhibition and human red blood cell (HRBC) membrane stabilization were studied in vitro following standard methods. The anti lipidperoxidant effects of CO were also studied in vitro. Effect of CO on carrageenan-induced mouse paw oedema inhibition was also assessed. CO significantly decreased maximum protection of 80.8% at 15 microg/ml. The extract also caused significant reduction in malondialdehyde (MDA) levels of murine hepatic microsomes at 100 microg/ml (56%) and significantly reduced carrageenan induced inflammation in mice at a dose of 250 mg/kg. Results of the present study indicated that CO inhibited mast cell degranulation, stabilized HRBC membrane thereby alleviating immediate hypersensitivity besides showing anti oxidant activity.

Evaluation of Elephantopus scaber on the inhibition of chemical carcinogenesis and tumor development in mice.

The effect of the active fraction of Elephantopus scaber L. (Asteraceae) (ES) on skin papillomas induced by 7,12-dimethylbenz(a)anthracene (DMBA) as an initiator and croton oil as promoter was studied in mice. The active fraction of E. scaber (100 mg/kg) on topical application delayed the onset of papilloma formation and reduced the mean number of papillomas and the mean weight of papillomas per mouse. The intraperitoneal administration of the active fraction of E. scaber also had a significant effect on subcutaneous injection of 20-methylcholanthrene (20-MCA)-induced soft tissue sarcomas in mice. It inhibited the incidence of sarcomas and reduced the tumor diameter compared to MCA-treated control animals. The subcutaneous administration of the active fraction of E. scaber significantly inhibited the growth of subcutaneously transplanted DLA and EAC solid tumors, delayed the onset of tumor formation, and increased the life span of tumor bearing mice. The present study thus indicates the tumor inhibitory activity of the active fraction of E. scaber against chemically induced tumors and its ability to inhibit the development of solid tumors.

Hepatoprotective effect of three herbal extracts on aflatoxin B1-intoxicated rat liver.

INTRODUCTION: Roots of Ixora coccinea (Rubiaceae), and Rhinacanthus nasuta (Acanthaceae) and whole plants of Spilanthes ciliata (Asteraceae) are extensively used by tribal communities in South India to treat liver diseases. However, the veracity of these tribal claims has not been investigated scientifically using the liver toxin, aflatoxin. This study reports on the protective effects of these three herbal ethanolic extracts on the aflatoxin B1 (AFB1)-intoxicated livers of albino male Wistar rats. METHODS: Biochemical parameters, including serum hepatic enzymes (glutamate oxaloacetate transaminase, glutamate pyruvate transaminase and alkaline phosphatase), were studied. Hepatic tissues were processed for assay of reduced glutathione (GSH) and histological alterations. RESULTS: Pre-treatment of the rats with oral administration of the plant ethanolic extracts, Ixora coccinea (IC), Rhinacanthus nasuta (RN), Spilanthes ciliata (SC), prior to AFB1 was found to provide significant protection against toxin-induced liver damage, determined 72 hours after the AFB1 challenge (1.5 mg/kg, intraperitoneally) as evidenced by a significant lowering of the activity of the serum enzymes and enhanced hepatic reduced GSH status. Pathological examination of the liver tissues supported the biochemical findings. The three plant extracts, IC, RN and SC, showed significant antilipid peroxidant effects in vitro. CONCLUSION: It was concluded that the hepatoprotective effects of the three plant extracts observed in this study might result from their potent antioxidative properties.

Hepatoprotective studies on Sida acuta Burm. f.

ETHNOPHARMACOLOGICAL RELEVANCE: Sida acuta Burm. f. (Malvaceae) is used in Indian traditional medicine to treat liver disorders and is useful in treating nervous and urinary diseases and also disorders of the blood and bile. AIM OF THE STUDY: Evaluation of the hepatoprotective properties of the methanolic extract of the root of Sida acuta (SA) and the phytochemical analysis of SA. MATERIALS AND METHODS: The model of paracetamol-induced hepatotoxicity in Wistar rats, liver histopathological observations, hexobarbitone-induced narcosis and in vitro anti-lipid peroxidation studies were employed to assess the hepatoprotective efficacy of SA. Phytochemical assay of SA was conducted following standard protocols. RESULTS: Significant hepatoprotective effects were obtained against liver damage induced by paracetamol overdose as evident from decreased serum levels of glutamate pyruvate transaminase, glutamate oxaloacetate transaminase, alkaline phosphatase and bilirubin in the SA treated groups (50, 100, 200mg/kg) compared to the intoxicated controls. The hepatoprotective effect was further verified by histopathology of the liver. Pretreatment with Sida acuta extract significantly shortened the duration of hexobarbitone-induced narcosis in mice indicating its hepatoprotective potential. Phytochemical studies confirmed the presence of the phenolic compound, ferulic acid in the root of Sida acuta, which accounts for the significant hepatoprotective effects observed in the present study. CONCLUSION: The present study thus provides a scientific rationale for the traditional use of this plant in the management of liver disorders.

Gastric antisecretory and antiulcer activities of Cyclea peltata (Lam.) Hook. f. & Thoms. in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Cyclea peltata (Lam.) Hook. f. & Thoms. (Menispermaceae), locally called 'Padathaali/Padakizhangu' is used in Ayurvedic medicine to treat peptic ulcer. AIM OF THE STUDY: To evaluate the gastric antisecretory and antiulcer activity of Cyclea peltata. MATERIALS AND METHODS: The ethanolic extract of Cyclea peltata root was used to evaluate its gastric antisecretory and antiulcer effect in the pylorus-ligated rat model and gastric lesions induced by ethanol or ethanol and indomethacin respectively in rats. The levels of gastric wall mucus, non-protein sulfhydryl groups (NP-SH), malondialdehyde, protein and catalase activity in the stomach samples of Cyclea peltata treated and control groups of rats were also quantified. RESULTS: The ethanolic extract of Cyclea peltata roots showed significant antisecretory activity as evidenced by decreased pepsin secretion, gastric juice volume and acid output in pylorus-ligated rats. Pretreatment with Cyclea peltata extract provided significant protection against the peptic ulceration caused by ethanol administered individually, or in combination with indomethacin. Our studies also revealed that pretreatment with Cyclea peltata significantly increased the gastric protein and catalase concentration of ethanol treated rats. Further, it showed significant gastroprotective effects on the stomach wall of ethanol or ethanol and indomethacin treated rats by decreasing malondialdehyde level, increasing the gastric wall mucus and non-protein sulfhydryl groups. CONCLUSION: The present findings demonstrate that Cyclea peltata ethanolic extract has potent antisecretory and antiulcer effects and justify the traditional/ethnic usage of this herb to treat peptic ulcers and consequent stomach ache.

Hepatoprotective effect of Hibiscus hispidissimus Griffith, ethanolic extract in paracetamol and CCl4 induced hepatotoxicity in Wistar rats.

Hibiscus hispidissimus Griff. is used in tribal medicine of Kerala, the southern most state of India, to treat liver diseases. In the present study, the effect of the ethanolic extract of Hibiscus hispidissimus whole plant on paracetamol (PCM)-induced and carbon tetrachloride (CCl4)-induced liver damage in healthy Wistar albino rats was studied. The results showed that significant hepatoprotective effects were obtained against liver damage induced by PCM and CCl4 as evidenced by decreased levels of serum enzymes, glutamate oxaloacetate transaminase (SGOT), glutamate pyruvate transaminase (SGPT), serum alkaline phosphatase (SAKP), serum bilirubin (SB) and an almost normal histological architecture of the liver of the treated groups compared to the toxin controls. The extract also showed significant antilipid peroxidant effects in vitro, besides exhibiting significant activity in quenching 1, 1-diphenyl-2-picryl hydrazyl (DPPH) radical, indicating its potent antioxidant effects.

Hepatoprotective effects of Pittosporum neelgherrense Wight&Arn., a popular Indian ethnomedicine.

The stem bark of Pittosporum neelgherrense Wight&Arn. is used by the Kani and Malapandaram tribes of Kerala as an effective antidote to snake bite and for the treatment of various hepatic disorders. In the present study, the effect of the methanolic extract of the stem bark of Pittosporum neelgherrense was studied against carbon tetrachloride (CCl(4))-, d-galactosamine (D-GalN)- and acetaminophen (APAP)-induced acute hepatotoxicity in Wistar rats. Significant hepatoprotective effects were obtained against liver damage induced by all the three liver toxins, as evident from decreased levels of serum enzymes, glutamate oxaloacetate transaminase (SGOT), glutamate pyruvate transaminase (SGPT) and an almost normal architecture of the liver in the treated groups, compared to the toxin controls. Thus the present study provides a scientific rationale for the traditional use of this plant in the management of liver diseases.

Hepatoprotective studies on Hedyotis corymbosa (L.) Lam.

Hedyotis corymbosa is used in traditional medicine of India and China to treat various hepatic disorders. In the present study, the hepatoprotective effect of the methanolic extract of the whole plant of Hedyotis corymbosa against paracetamol overdose-induced liver damage in Wistar rats was studied. The methanolic extract of the plant produced significant hepatoprotective effects as evidenced by decreased serum enzyme activities, SGPT, SGOT, SAKP and serum bilirubin and an almost normal histological architecture of the liver, in treated groups, compared to the controls. Hedyotis corymbosa shortened hexobarbitone-induced sleeping time in mice, besides showing significant antilipid peroxidant effect in vitro. The results thus support the use of Hedyotis corymbosa as a hepatoprotective agent.

Evaluation of hepatoprotective effects of Helminthostachys zeylanica (L.) Hook against carbon tetrachloride-induced liver damage in Wistar rats.

The rhizomes of Helminthostachys zeylanica (L.) are used by the Kattunaikan tribe of Kerala, for the treatment of various hepatic disorders. In the present study, the effect of the methanolic extract of Helminthostachys zeylanica rhizomes on carbon tetrachloride (CCl4)-induced liver damage in Wistar rats was studied. The results showed that significant hepatoprotective effect was obtained against CCl4-induced liver damage, by oral administration of Helminthostachys zeylanica methanolic extract as evident from decreased levels of serum enzymes and an almost normal architecture of the liver, in the treated groups, compared to the controls. The extract was effective in increasing the choleretic activity of anaesthetised normal rats. It also shortened hexobarbitone-induced sleeping time in mice, which was increased by CCl4 treatment, besides showing significant antilipid peroxidant effect in vitro. Thus, the present study provides a scientific rationale for the traditional use of this plant in the management of liver diseases.

Chemoprotective effect of Ixora coccinea L. flowers on cisplatin induced toxicity in mice.

The active fraction from Ixora coccinea flowers prevented a decrease in body weight, haemoglobin levels and leucocyte counts of mice treated with cisplatin. It also significantly prolonged the life span of cisplatin treated mice and maintained their blood urea nitrogen levels in the near normal range, indicating its chemoprotective effects.

Immunomodulatory and antitumour properties of Psoralea corylifolia seeds.

Psoralea corylifolia seed extract has been found to stimulate the immune system in mice. Administration of the extract was found to inhibit EAC ascitic tumour growth and stimulate natural killer cell activity, antibody-dependent cellular cytotoxicity, antibody-forming cells and the antibody complement-mediated cytotoxicity during tumour development.

Modulatory effects of ixora coccinea flower on cyclophosphamide-induced toxicity in mice.

The active fraction of Ixora coccinea flowers showed chemoprotective effects on cyclophosphamide - induced toxicity by increasing the life span of treated mice, preventing body weight loss and maintaining near normal leucocyte and haemoglobin levels compared with cyclophosphamide treated controls. Decreased serum glutamate pyruvate transaminase (SGPT) and serum alkaline phosphatase (SAKP) levels in the Ixora coccinea treated groups indicated protection against hepatic toxicity. The active fraction was identified by phytochemical methods as the triterpenoid, ursolic acid.

Inhibition of chemical carcinogenesis by Psoralea corylifolia seeds.

Psoralea corylifolia L. (Fabaceae) seeds are extensively used in Ayurvedic medicine. Topical application of 100 mg/kg body weight of the active fraction (AF) of P. corylifolia seeds inhibited the growth and delayed the onset of papilloma formation in mice, initiated with 7,12-dimethyl benz(a) anthracene and promoted using croton oil. The AF at the same dose, when administered orally, inhibited the growth of subcutaneously injected 20-methylcholanthrene (MCA)--induced soft tissue fibrosarcomas significantly. The AF has been shown by gas chromatography analysis to be composed of a mixture of glycerides of fatty acids.

Cytotoxic and antitumour principles from Ixora coccinea flowers.

The antitumour activity of Ixora coccinea L. (Rubiaceae) flowers was studied in comparison to intraperitoneally transplanted Dalton's lymphoma (ascitic and solid tumours) and Ehrlich ascites carcinoma (EAC) tumours in mice. Intraperitoneal administration of 200 mg/kg of the active fraction (AF) of the I. coccinea flower increased the life-span of DLA and EAC ascitic tumour-bearing mice by 113 and 68%, respectively. The AF showed less activity against solid tumours (DLA) as compared to ascitic tumours. The same active fraction showed 50% cytotoxicity to DLA, EAC and Sarcoma-180 (S-180) cells in vitro at concentrations of 18, 60 and 25 microg/ml, respectively. It was not toxic to normal lymphocytes, whereas it was toxic to transformed lymphocytes from leukaemic patients, acute lymphoblastic leukaemia (ALL) and chronic myelogenous leukaemia (CML) and K-562 suspension cell cultures. The AF inhibited tritiated thymidine incorporation in cellular DNA.

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.