M. Manjunath Setty

Preliminary evaluation of in vitro cytotoxicity and in vivo antitumor activity of Premna herbacea Roxb. in Ehrlich ascites carcinoma model and Dalton’s lymphoma ascites model

In the present study, the root nodules of Premna herbacea Roxb. (PH) was investigated for its in vitro cytotoxicity and in vivo antitumor activity. Two extracts, aqueous and alcoholic; two fractions of alcoholic extract, ethyl acetate and butanol fractions were screened for their in vitro cytotoxicity by brine shrimp lethality (BSL) assay, trypan blue exclusion assay and MTT assay. Alcoholic extract and its ethyl acetate fraction were found to be the most effective in BSL assay, trypan blue exclusion assay. In vivo antitumor activity was screened in the Ehrlich ascites carcinoma (EAC) model and the Dalton lymphoma ascites (DLA) model. The extracts and the fractions were tested at two dosages (250 and 500 mg/kg) by intraperitoneally (i.p.) route on every alternate day upto 13th day. Cisplatin was used as positive control in both studies in single dose (day 1) 3.5 mg/kg by i.p. route. In EAC model, ascites tumor was induced by inoculating 2.5 million of EAC cells i.p. alcoholic extract at 500 mg/kg was the most effective in elevating MST, reduction in body weight in EAC induced tumor. Only the effective extract i.e., alcoholic extract were studied for hematological and antioxidant parameter. It showed a restoring effect on altered hematological parameters and a significant improvement in biochemical parameters at 250 mg/kg dose of alcoholic extract. These results explain the toxicity of 500 mg/kg might be high. In the Dalton lymphoma ascites (DLA) model, solid tumor was developed by i.m. injection of 1 million DLA cells. Both the extracts and the fractions possessed potent antitumor activity against solid tumor models by significantly reducing the solid tumor weight and volume.

Bioactive caffeic acid esters from Glycyrrhiza glabra

Thin layer chromatography bioautography (using DPPH spray reagent) guided fractionation of Glycyrrhiza glabra led to the isolation of two caffeic acid derivative esters, viz. eicosanyl caffeate (1) and docosyl caffeate (2). The two compounds exhibited potent elastase inhibitory activity, with IC50 values of 0.99 µg mL−1 and 1.4 µg mL−1 for 1 and 2, respectively. The compounds also showed moderate antioxidant activity in DPPH and ABTS scavenging assays. The results indicate a possible role of caffeic acid derivatives, in addition to flavonoids in the anti-ulcer properties of G. glabra.

Ethanolic extract of Moringa oleifera Lam. leaves protect the pre-pubertal spermatogonial cells from cyclophosphamide-induced damage

ETHNOPHARMACOLOGICAL RELEVANCE Moringa oleifera Lam. is widely cultivated in Asian and African countries for its medicinal and dietary significance. The leaves are highly nutritious and are known to possess various biological activities. MATERIALS AND METHODS Pre-pubertal Swiss albino male mice were injected with single dose of cyclophosphamide (CP, 200mg/kg body weight) or ethanolic extract of Moringa oleifera leaves (MOE, 100mg/kg body weight) intraperitoneally. In combination group, MOE was administered 24h prior to CP injection. RESULTS CP induced a significant decrease in testicular weight (p<0.01) and depletion of germ cells (p<0.001) and higher level of DNA damage (p<0.001) compared to control. The expression of P53, Bax, Cytochrome C (Cyt C) was increased while there was a decrease in the expression of Bcl2, c-Kit and Oct4. Administration of MOE 24h prior to CP treatment ameliorated the depletion (p<0.001), DNA damage (p<0.001) and apoptosis (p<0.01) of germ cells induced by CP. The mitigating effect of MOE appears to be mediated by up-regulating the expression of c-Kit and Oct4 transcripts in P53-independent manner. CONCLUSION MOE protects the spermatogonial cells from CP-induced damage by modulating the apoptotic response elicited by CP and therefore can be considered as an efficient method of male fertility preservation.

Anticancer activity of Berberis aristata in Ehrlich ascites carcinoma-bearing mice: A preliminary study

Context: Berberis aristata DC (Berberidaceae) is an important medicinal plant with claims of widespread medicinal value in indigenous medicine. It is used by herbal healers to treat oral cancers. Objective: To evaluate the antineoplastic activity of the extracts of Berberis aristata in Ehrlich ascites carcinoma (EAC)-bearing mice with cisplatin as positive control in the advanced stage of tumorigenesis. Materials and methods: Brine shrimp lethality bioassay (BSL) of extracts and effect on the tumor cell viability in vitro were carried out. EAC was induced in Swiss albino mice by injecting 106 cell/mL of tumor cell suspension i.p. Antineoplastic activity of the aqueous and ethanol extracts (100 and 6.5 mg/kg i.p., respectively) was compared with that of cisplatin (3.5 mg/kg i.p.) on the parameters such as percentage increase in weight, median survival time, and hematology. Results: Ethanol extract attenuated percentage increase in weight gain (−6.86 ± 1.50) due to tumor cell proliferation and increased the survival time (19.5 days) when compared to control group (19.10 ± 2.31 and 16 days, respectively). However, the effect was less than that of cisplatin. In vitro cytotoxicity assay as well as BSL test showed the cytotoxic effect of the extracts. Cisplatin and the extracts reversed the tumor-induced alterations in total white blood cell count, differential leukocyte counts, total red blood cell count, and hemoglobin contents. Discussion and conclusion: Of the two extracts, the ethanol extract was observed to be more efficient and the presence of alkaloids and flavonoids may be responsible for the observed anticancer effects.

Bulbophyllum sterile petroleum ether fraction induces apoptosis in vitro and ameliorates tumor progression in vivo

Orchids of the genus Bulbophyllum have been reported to possess antitumor activity. Present study investigated the possible antitumor activity of the active fraction of bulb and root of Bulbophyllum sterile. Alcoholic extract along with petroleum ether, dichloromethane and ethyl acetate fractions were subjected to SRB assay in HCT-116, MDA-MB-231 and A549 cell lines. The active fractions were further evaluated for apoptosis, expression of apoptotic signaling proteins, comet assay and cell cycle analysis. Furthermore, they were assessed for in vivo antitumor activity in Ehrlich ascites carcinoma model. Petroleum fraction of bulbs (PFB) and roots (PFR) was found to be most active in HCT-116 cell lines with IC50 value of 94.2±6.0 and 75.7±9.8, respectively. Apoptosis was evident from acridine orange/ethidium bromide staining along with the expression of phospho-p53 and phospho-Bad. Both PFB and PFR arrested G2/M phase of the cell cycle with 32.6% and 49.4% arrest, respectively compared to 17.5% arrest with control. An increase in mean life span and hepatic antioxidant levels was observed with PFB and PFR treatment in EAC inoculated mice. The results suggested that the active fractions of bulbs and roots possess anticancer activity likely by inducing apoptosis through phospho-p53 dependent pathway.

Characterization of the phenolic compound, gallic acid from sansevieria roxburghiana schult and schult. f. rhizomes and antioxidant and cytotoxic activities evaluation

Background: Sansevieria roxburghiana Schult. and Schult. f. (Asparagaceae) grows in India, Indonesia, Sri Lanka, and tropical Africa. Even though the plant has been traditionally used for the treatment of many ailments, the antioxidant and antiproliferative activities of S. roxburghiana methanol extract and its fractions have not yet been explored. Materials and Methods: Quantitative estimation of phenols and different antioxidant assays were performed using standard methods. Anti-proliferative effect of the extract and fractions were evaluated in HCT-116, HeLa, MCF-7, HepG2, and A-549 cancer cell lines by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and sulforhodamine B (SRB) assay methods. High-performance liquid chromatography (HPLC) and high-performance thin layer chromatography (HPTLC) fingerprint profiling were carried out for extract and different fractions. Results: Significant antioxidant and anti-proliferate activity were detected in ethyl acetate fraction. Ethyl acetate fraction showed prominent scavenging activity in 1,1-diphenyl-2-picrylhydrazyl, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, and nitric oxide antioxidant assays with an concentration yielding 50% inhibition (IC50) 15.33 ± 1.45, 45.3 ± 1.93 and 48.43 ± 0.46 μ g/ml, respectively. Cytotoxicity of ethyl acetate fraction was the highest among other fractions against HCT-116, HeLa, and MCF-7cancer cell lines with IC50values 16.55 ± 1.28, 12.38 ± 1.36, and 8.03 ± 1.9 μ g/ml, respectively, by MTT assay and 15.57 ± 0.70, 13.19 ± 0.49, and 10.34 ± 0.9 μ g/ml, respectively, by SRB assay. The presence of gallic acid in the ethyl acetate fraction of S. roxburghiana rhizomes was confirmed by HPLC and HPTLC analysis. Conclusion: Results suggested that ethyl acetate fraction exhibited effective antioxidant and antiproliferative activities. The phenolic compounds identified in ethyl acetate fraction could be responsible for the activities. Abbreviations used: %: Percent, °C: Celsius, μ g: Microgram, μ l-Microlitre, ANOVA: Analysis of variance, DMSO: Dimethyl sulfoxide, g: Grams, IC50: Concentration yielding 50% inhibition, Kg: Kilogram, mg: Milligram, min: Minutes, ml: Milliliter, HPLC: High-performance liquid chromatography, HPTLC: High-performance thin layer chromatography, DPPH: 1,1-diphenyl-2-picrylhydrazyl, ABTS: 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, GAE: Gallic acid equivalents, SRME: Methanol extract of S. roxburghiana, ROS: Reactive oxygen species, SRPE: Petroleum ether fraction of S. roxburghiana, SREA: Ethyl acetate fraction of S. roxburghiana, SRAQ: Aqueous fraction of S. roxburghiana, DMEM: Dulbeccos Minimum Essential Medium, FBS: Fetal bovine serum, OD: Optical density, TPC: Total phenolic content, SRBU: Butanol fraction of S. roxburghiana.

Assessment of the in vitro cytotoxicity and in vivo anti-tumor activity of the alcoholic stem bark extract/fractions of Mimusops elengi Linn.

Various parts of Mimusops elengi Linn. (Sapotaceae) have been used widely in traditional Indian medicine for the treatment of pain, inflammation and wounds. The study was conducted to explore the use of stem bark of M. elengi on pharmacological grounds and to evaluate the scientific basis of cytotoxic and anti-tumor activity. Extract/fractions were prepared and in vitro cytotoxicity was assessed using SRB assay. Most effective fractions were subjected to fluorescence microscopy based acridine orange/ethidium bromide (AO/EB) and Hoechst 33342 staining to determine apoptosis induction and DNA fragmentation assay. Comet and micronuclei assay were performed to assess genotoxicity. Cell cycle analysis was also performed. In vivo anti-tumor potential was evaluated by Ehrlich ascites carcinoma (EAC) model in mice. The alcoholic stem bark extract of M. elengi along with four fractions showed potential in vitro cytotoxicity in SRB assay. Of these, dichloromethane and ethyl acetate fractions were selected for further studies. The fractions revealed apoptosis inducing potential in AO/EB and Hoechst 33342 staining, which was further confirmed by DNA fragmentation assay. Genotoxic potential was revealed by comet and micronuclei assay. Fractions also exhibited specific cell cycle inhibition in G0/G1 phase. In EAC model, ethyl acetate fraction along with the standard (cisplatin) effectively reduced the increase in body weight compared to control and improved mean survival time. Both fractions were able to restore the altered hematological and biochemical parameters. Hence, M. elengi stem bark may be a possible therapeutic candidate having cytotoxic and anti-tumor potential.