Anticancer effect of the extracts from Polyalthia evecta against human hepatoma cell line (HepG2)

Objective

To investigate the anticancer activity of Polyalthia evecta (P. evecta) (Pierre) Finet & Gagnep against human hepatoma cell line (HepG2).

Methods

The anticancer activity was based on (a) the cytotoxicity against human hepatoma cells (HepG2) assessed using a neutral red assay and (b) apoptosis induction determined by evaluation of nuclei morphological changes after DAPI staining. Preliminary phytochemical analysis of the crude extract was assessed by HPLC analysis.

Results

The 50% ethanol-water crude leaf extract of P. evecta (EW-L) showed greater potential anticancer activity with high cytotoxicity [IC₅₀ = (62.8 ± 7.3)µg/mL] and higher selectivity in HepG2 cells than normal Vero cells [selective index (SI) = 7.9]. The SI of EW-L was higher than the positive control, melphalan (SI = 1.6) and the apoptotic cells (46.4 ± 2.6) % induced by EW-L was higher than the melphalan (41.6 ± 2.1)% (P<0.05). The HPLC chromatogram of the EW-L revealed the presence of various kinds of polyphenolics and flavonoids in it.

Conclusions

P. evecta is a potential plant with anticancer activity. The isolation of pure compounds and determination of the bioactivity of individual compounds will be further performed.     

姜黄素诱导肿瘤细胞凋亡的实验研究

目的:探讨姜黄素诱导HepG2细胞凋亡的作用机制。方法:应用形态学方法、AnnexinV荧光染色和流式细胞仪(FCM)检测HepG2细胞凋亡的发生,应用RT-PCR检测凋亡相关基因P53、bcl-2和Fas的变化。结果:姜黄素对HepG2细胞的生长有明显的抑制作用,并诱导肿瘤细胞发生凋亡。凋亡细胞表现为细胞固缩,核染色质碎裂;流式细胞仪检测凋亡率为11.7%,细胞停在G1和G2期。AnnexinV标记的方法检测凋亡时发现,坏死与凋亡共存。在姜黄素诱导HepG2细胞凋亡过程中,凋亡相关基因Fas转录水平比用药前增强。结论:凋亡为姜黄素抑癌的机制之一,姜黄素诱导HepG2细胞凋亡可能与P53、bcl-2和Fas基因表达有关。     

Bioassay of Eucalyptus extracts for anticancer activity against Ehrlich ascites carcinoma (eac) cells in Swiss albino mice

Objective

To evaluate the antineoplastic activity of Eucalyptus extract (EuE) against Ehrlich ascites carcinoma (EAC) in Swiss albino mice.

Methods

Preliminary examination of four plant extracts (namely Eucalyptus, Costus, Azadirachta, Feronia) has been done by observing the reduction ability of number of EAC cells in previously inoculated Swiss albino mice. Among them as EuE showed maximum capability, the whole study has been conducted with EuE only. Important parameters viz. enhancement of life span, reduction of average tumor weight etc. have been studied. In addition the effects of EuE on hematological parameters in both normal and EAC inoculated mice have been measured. Effect of EuE on normal peritoneal cells has also been studied.

Results

: EuE reduced tumor burden remarkably. It reduced the tumor growth rate and enhanced the life span of EAC bearing mice noticeably. It reversed back the hematological parameters towards normal, reduced the trasplantability of EAC cells and enhanced the immunomodulatory effects in mice. The host toxic effect of EuE in mice is minimum and mostly reversible with time. All such data have been compared with those obtained by running parallel experiments with bleomycin at dose 0.3 mg/kg (i.p.).

Conclusions

The Eucalyptus extract may be considered as a potent anticancer agent for advanced researches.     

Polyphenol extract of Phyllanthus emblica (PEEP) induces inhibition of cell proliferation and triggers apoptosis in cervical cancer cells

Background

The aim of this study is to investigate the effects of polyphenol extract from Phyllanthus emblica (PEEP) on cervical cancer cells and to explore the underlying mechanism.

Methods

MTT assay was used to measure inhibition of proliferation of cervical cancer (HeLa) cells after treatment with PEEP at concentrations of 0, 50, 100, 150, and 200 mg/ml for 48 hours. HeLa cells were treated with PEEP (150 mg/ml) for 48 hours in the following analysis. Karyomorphism was assessed by immunofluorescence using DAPI staining, and cell apoptosis and cell cycle were assessed using flow cytometry. Three apoptotic marker proteins, namely, Fas, FasL, and cleaved caspase-8, were assessed by western blotting.

Results

PEEP inhibited the growth of HeLa cells, and the optimum concentration of PEEP was 150 mg/ml. In addition, the karyomorphism of HeLa cells after treatment with PEEP was abnormal. Furthermore, PEEP induced arrest of the HeLa cell cycle at G2/M phase, and triggered apoptosis. PEEP also induced significant Fas and FasL activation, and cleavage of caspase-8.

Conclusions

Our study indicates that PEEP is effective in inhibiting HeLa cell proliferation by inducing cell cycle arrest at G2/M phase and inducing apoptosis.     

Antimicrobial activities of the tissue extracts of Babylonia spirata Linnaeus, 1758 (Mollusca: Gastropoda) from Thazhanguda,southeast coast of India

Objective

To investigate the antimicrobial activity of the tissue extracts of Babylonia spirata (B. spirata) against nine bacterial and three fungal pathogens.

Methods

Crude extract of gastropod was tested for inhibition of bacterial and fungal growth. Antibacterial assay was carried out by disc diffusion method and in vitro antifungal activity was determined against Czapex Dox agar. The antimicrobial activity was measured accordingly based on the inhibition zone around the disc impregnated with gastropod extract. Molecular size of muscle protein was determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). And fourier transform infrared spectroscopy (FTIR) spectro photometry analysis was also studied.

Results

The maximum inhibition zone (12 mm) was observed against Pseudomonas aeruginosa in the crude ethanol extract of B. spirata and the minimum inhibition zone (2 mm) was noticed against Staphylococcus aureus in the crude methanol extract of B. spirata. Water extract of B. spirata showed the highest activity against Vibrio parahaemolyticus, Staphylococcus aureus and Candida albicans. Ethanol, acetone, methanol, chloroform and water extracts showed antimicrobial activity against almost all the bacteria and fungus. Compared with water extracts, ethanol and methanol extracts showed higher activity against all pathogens. The molecular weight of protein of the gastropod sample ranged from 2-110 kDa on SDS-PAGE. FTIR analysis revealed the presence of bioactive compounds signals at different ranges.

Conclusions

The research shows that the great medicinal value of the gastropod muscle of B. spirata may be due to high quality of antimicrobial compounds.     

Cytotoxic (A549) and antimicrobial effects of Methylobacterium sp. isolate (ERI-135) from Nilgiris forest soil,India

Objective

To assess the antimicrobial and cytotoxic effects of Methylobacterium sp. isolated from soil sample of Doddabetta forest, Nilgiris, Western Ghats of Tamil Nadu.

Methods

Isolation of Methylobacterium was performed from soils by serial dilution plate technique. The strain was grown in modified nutrient gulucose agar (MNGA) medium to study the morphology and biochemical characteristics. Methylobacterium sp. was screened for its antimicrobial activity against pathogenic bacteria and fungi. The strain was subjected to 16S rRNA analysis and was identified as Methylobacterium sp. The nucleotide sequence of the 16S rRNA gene of the isolate exhibited close similarity with other Methylobacterium sp. and has been submitted to Genbank. The antibacterial substances were extracted using chloroform and ethyl acetate from MNGA medium in which ERI-135 had grown for 5 d at 30 °C. Cytotoxic effect was also studied. GC-MS analysis was carried out. The antimicrobial activity was assessed using broth micro dilution technique.

Results

Ethyl acetate extract showed activity against bacteria such as Bacillus subtilis, Klebsiella pneumoniae (K. pneumoniae), Pseudomonas aeruginosa, Salmonella typhimurium, Shigella flexneri, Enterobacter aerogenes, Staphylococcus aureu and Staphylococcus epidermidis (S. epidermidis) and fungi such as, Candida albicans and Trichophyton rubrum. The lowest minimum inhibitory concentrations were: 250 µg/mL against S. epidermidis and 250µg/mL against K. pneumonia. The isolate had the ability to produce enzymes such as protease. The exyract showed cytotoxic effect in human adenocarcinoma cancer cell line (A549). GC-MS analysis showed the presence of isovaleric acid (3.64%), 2-Methylbutanoic acid (5.03%), isobutyramide (5.05%), N,N-oimethylformamide-di-t-butylacetal (9.79%), benzeneacetamide (15.56%), octyl butyl phthalate (3.59%) and diisooctyl phthalate (5.79) in the extract.

Conclusions

Methylobacterium sp. (ERI-135) showed promising antibacterial and cytotoxic activity. This is the first report in the antimicrobial and cytotoxic effect of Methylobacterium sp.     

Production of asiaticoside from centella (Centella asiatica L. Urban) cells in bioreactor

Objective

To investigate the effects of some culture conditions on production of asiaticoside from centella (Centella asiatica L. Urban) cells cultured in 5-L bioreactor.

Methods

The centell cell suspension culture was conducted in 5-L bioreactor to investigate the growth and asiaticoside accumulation under various conditions. Asiaticoside content was determined by HPLC analysis.

Results

The results showed that the cell growth and asiaticoside accumulation peaked after 24 d of culture at an agitation speed of 150 r/min and aeration rate of 2.5 L/min. The cell biomass reached a maximum value of 302.45 g fresh weight (31.45 g dry weight) and growth index of 3.03 with inoculum size of 100 g. However, asiaticoside content was the highest (60.08 mg/g dry weight) when culture was initiated with an inoculum size of 50 g.

Conclusions

The present study found the suitable conditions for growth of centella cells and their asiaticoside production in bioreactor.     

Preliminary phytochemical screening and in vitro anti-Helicobacter pylori activity of acetone and aqueous extracts of the stem bark of Sclerocarya birrea (Anacardiaceae)

Helicobacter pylori antibiotic resistance and other problems associated with combination therapy have generated a considerable interest in the search for alternative therapeutic agents. In order to identify novel sources of such agents, the antimicrobial activity of five solvent extracts of the stem bark of Sclerocarya birrea was investigated against 30 clinical strains of H. pylori and a reference strain NCTC 11638 using standard microbiological techniques. Metronidazole and amoxicillin were included in these experiments as positive control antibiotics. The active phytocomponents were detected by TLC and indirect bioautography. All the extracts exhibited anti-H. pylori activity with zone diameters of inhibition between 0 and 21 mm. The acetone and aqueous extracts showed potent anti-H. pylori activity with minimum inhibitory concentration (MIC(90)) values ranging from 0.06-2.50 mg/mL, whereas those for the control antibiotics ranged from 0.001-5.0 mg/mL. The acetone extract was highly bactericidal at 1.2 mg/mL with complete elimination of the organisms within 18 h. The activity of both acetone and aqueous extracts was better than metronidazole (p<0.05). Most of the active phytocomponents were located in the acetone extract; R(f)≤0.62 with >90% inhibition. These results demonstrate that the acetone and aqueous extracts of S. birrea may contain compounds with therapeutic activity; therefore, they may represent potential sources of new anti-H. pylori regimen.     

Evaluation of anti-resistant activity of Auklandia (Saussurea lappa) root against some human pathogens

Objective

The antimicrobial activity of the ethanol extract of the Auklandia (Saussurea lappa)root plant was investigated to verify its medicinal use in the treatment of microbial infections.

Methods

The antimicrobial activity of the ethanol extract was tested against clinical isolates of some multidrug-resistant bacteria using the agar well diffusion method. Commercial antibiotics were used as positive reference standards to determine the sensitivity of the clinical isolates.

Results

The extracts showed significant inhibitory activity against clinical isolates of methicillin resistant Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumonia, Extended Spectrum Beta-Lactemase, Acinetobacter baumannii. The minimum inhibitory concentration values obtained using the agar dilution test ranged from 2.0 µg/µL-12.0 µg/µL. In the contrary the water extract showed no activity at all against the tested isolates. Furthermore, the results obtained by examining anti-resistant activity of the plant ethanolic extract showed that at higher concentration of the plant extract (12 µg) all tested bacteria isolates were inhibited with variable inhibition zones similar to those obtained when we applied lower extract concentration using the well diffusion assay.

Conclusion

The results demonstrated that the crude ethanolic extract of the Auklandia (Saussurea lappa) root plant has a wide spectrum of activity suggesting that it may be useful in the treatment of infections caused by the above clinical isolates (human pathogens).     

异常黑胆质成熟剂乙酸乙酯萃取物对HepG2细胞生长和凋亡及相关基因调控的研究

目的:观察异常黑胆质成熟剂乙酸乙酯萃取物(ASMq-EtOAc)对人肝癌细胞株(HepG2)生长和凋亡及相关基因调控的作用,探讨其抗癌的物质基础和作用机理。方法:采用四氮甲基唑蓝法(MTT)观察了ASMq-EtOAc对HepG2细胞体外生长的抑制作用;采用琼脂糖凝胶电泳技术和流式细胞术观察ASMq-EtOAc对HepG2细胞凋亡的影响;采用逆转录-多聚酶链式反应技术(RT-PCR)观察ASMq-EtOAc对HepG2细胞凋亡相关基因mRNA表达的影响。结果:ASMq-EtOAc明显抑制HepG2细胞体外生长、诱导细胞凋亡、阻滞细胞周期、明显提高抑癌基因p53、p21基因mRNA的表达,对Bcl-2、Bax基因mR-NA表达不产生明显的影响。结论:ASMq-EtOAc可能是异常黑胆质成熟剂抗癌作用的主要活性部位之一,其抗癌作用可能通过诱导癌细胞凋亡、改变癌细胞周期和促进抑癌基因p53及p21表达来实现。     

Evaluation of phenolic contents and antioxidant activities of brown seaweeds belonging to Turbinaria spp. (Phaeophyta,Sargassaceae) collected from Gulf of Mannar

Objective

To evaluate the antioxidant activities and total phenolic contents of brown seaweeds belonging to Turbinaria spp. [Turbinaria conoides (T. conoides) and Turbinaria ornata (T. ornata) collected from Gulf of Mannar of southeastern coast of India in various in vitro systems.

Methods

The antioxidant activity was evaluated using different in vitro systems, viz., 1, 1-diphenyl-2-picrylhydrazyl (DPPH), 2, 2′-azino-bis-3 ethylbenzothiozoline-6-sulfonic acid diammonium salt (ABTS), H₂O₂/HO radical scavenging, Fe²⁺ ion chelating ability, and reducing potential. Folin-Ciocalteu method was used to determine the total phenolic content of the extracts, and the results were expressed as mg of gallic acid equivalents (GE)/g of the seaweed extracts. Thiobarbituric acid-reactive substances assay was employed to assess the ability of the seaweed extracts to inhibit lipid oxidation.

Results

Ethyl acetate (EtOAc) fraction of T. conoides registered significantly higher phenolic content (105.97 mg GE/g) than that of T. ornata (69.63 mg GE/g). Significantly higher antioxidant potential as determined by DPPH (64.14%) radical scavenging activity was registered in EtOAc fraction of T. ornata. A higher ABTS^•+ radical scavenging (IC₅₀ 3.16 µg/mL), Fe²⁺ chelating (IC₅₀ 0.46 mg/mL), H₂O₂ scavenging (IC₅₀ 4.25 mg/mL), lipid peroxidation inhibitory (TBARS, IC₅₀ 0.21 mg/mL), and reducing abilities (IC₅₀ 52.67 mg/mL) (P<0.05) were realized in EtOAc fraction of T. ornata than other fractions.

Conclusions

This study indicated the potential use of T. conoides and T. ornata as candidate species to be used as food supplements/functional foods to increase shelf-life of food items for human consumption, and nutraceuticals to deter deleterious free radical-induced life-threatening diseases.     

Antiepileptic activity of lobeline isolated from the leaf of Lobelia nicotianaefolia and its effect on brain GABA level in mice

Objective

To investigate the anticonvulsant activity of the lobeline isolated from the Lobelia nicotianaefolia in chemoconvulsant-induced seizures and its biochemical mechanism by investigating relationship between seizure activities and altered gamma amino butyric acid (GABA) in brain of mice in Pentylenetetrazol (PTZ) seizure models.

Methods

The anticonvulsant activity of the isolated lobeline (5, 10, 20 and 30 mg/kg, i.p.) was investigated in PTZ and strychnine induced seizures in mice and the effect of isolated lobeline on brain GABA level in seizures induced by PTZ. Diazepam was used as reference anticonvulsant drugs for comparison.

Results

Isolated lobeline (10, 20 and 30 mg/kg, i.p.) significantly delayed and antagonized (P < 0.050–0.001) the onset of PTZ-induced seizures. It also antagonized strychnine induced seizures. The mortality was also prevented in the test group of animals. In biochemical evaluation, isolated lobeline (5, 10 and 20 mg/kg, i.p.) significantly increased the brain GABA level. And at dose of 30 mg/kg GABA level showed slight decrease in PTZ model.

Conclusions

In our findings, isolated lobeline (20mg/kg) exhibited potent anticonvulsant activity against PTZ induced seizures. Also a biochemical evaluation suggested significant increase in barain GABA level at 20 mg/kg i.p. of isolated lobeline. Hence, we may propose that lobeline reduces epileptic seizures by enhancing the GABA release supporting the GABAergic mechanism.     

Isolation and efficacy of entomopathogenic fungus (Metarhizium anisopliae) for the control of Aedes albopictus Skuse larvae: suspected dengue vector in Pakistan

Objective

To isolate the entomopathogenic fungus Metarhizium anisopliae (M. anisopliae) in the local environment, and evaluate its efficacy against the suspected dengue vector Aedes albopictus in Pakistan.

Methods

According to the standard procedure, M. anisopliae was isolated from the dead mosquitoes which were collected from the field or dead after the collection. Bioassay was performed to determine its efficacy.

Results

The results indicated that M. anisopliae had larvicidal effect with LC₅₀ value 1.09×10⁵ and LC₉₀ value 1.90×10¹³ while it took 45.41 h to kill 50% of tested population.

Conclusions

Taking long time to kill 50% population when compare with the synthetic insecticides, is the only drawback for the use of entomopathogenic fungus but these bio-pesticides are safe for the use.     

Effect of a commercial air ionizer on dust mites Dermatophagoides pteronyssinus and Dermatophagoides farinae (Acari: Pyroglyphidae) in the laboratory

Objective

To investigate the short and long term efficacy of a commercial air ionizer in killing Dermatophagoides pteronyssinus (D. pteronyssinus) and Dermatophagoides farinae (D. farinae) mites.

Methods

The effect of a commercial ionizer on D. pteronyssinus and D. farinae was evaluated in the laboratory, using a specially designed test. Mortality was assessed after 6, 16 and 24 hours for direct exposure and after 24, 36, 48, 60 and 72 hours for exposure in simulated mattress. New batches of mites were used for each exposure time.

Results

LT₅₀ for direct exposure of ionizer was 10 hours for D. pteronyssinus and 18 hours for D. farinae. The LT₅₀ for exposure in simulated mattress was 132 hours or 5.5 days for D. pteronyssinus and 72 hours or 3 days for D. farinae. LT₉₅ for direct exposure of ionizer was 36 hours for D. pteronyssinus and D. farinae. Meanwhile, the LT₉₅ for exposure in simulated mattress was 956 hours or 39.8 days for D. pteronyssinus and 403 hours or 16.8 days for D. farinae.

Conclusions

This study demonstrates the increasing mite mortalities with increasing exposure time of a commercial ionizer and suggests that negative ions produced by an ionizer kill dust mites and can be used to reduce natural mite populations on exposed surfaces such as floors, clothes, curtains, etc. However, there is reduced efficacy on mites inside stuffed materials as in mattresses and furniture.     

In vitro inhibition activity of polyphenol-rich extracts from Syzygium aromaticum (L.) Merr. & Perry (Clove) buds against carbohydrate hydrolyzing enzymes linked to type 2 diabetes and Fe2+-induced lipid peroxidation in rat pancreas

Objective

To investigate and compare the inhibitory properties of free and bound phenolic extracts of clove bud against carbohydrate hydrolyzing enzymes (alpha-amylase & alpha-glucosidase) and Fe²⁺-induced lipid peroxidation in rat pancreas in vitro.

Methods

The free phenolics were extracted with 80% (v/v) acetone, while bound phenolics were extracted from the alkaline and acid hydrolyzed residue with ethyl acetate. Then, the interaction of the extracts with alpha-amylase and alpha-glucosidase was subsequently assessed. Thereafter, the total phenolic contents and antioxidant activities of the extracts were determined.

Results

The result revealed that both extracts inhibited alpha-amylase and alpha-glucosidase in a dose-dependent manner. However, the alpha-glucosidase inhibitory activity of the extracts were significantly (P<0.05) higher than their alpha-amylase inhibitory activity. The free phenolics (31.67 mg/g) and flavonoid (17.28 mg/g) contents were significantly (P<0.05) higher than bound phenolic (23.52 mg/g) and flavonoid (13.70 mg/g) contents. Both extracts also exhibited high antioxidant activities as typified by their high reducing power, 1,1 diphenyl-2- picrylhydrazyl (DPPH) and 2, 2-azinobis-3-ethylbenzo-thiazoline-6-sulfonate (ABTS) radical scavenging abilities, as well as inhibition of Fe²⁺-induced lipid peroxidation in rat pancreas in vitro.

Conclusions

This study provides a biochemical rationale by which clove elicits therapeutic effect on type 2 diabetes.     

Effects of the Extract of Ammopiptanthus mongolicus cheng f. （JA1） on Induction of Apoptosis of HepG2 in vitro and Its Molecular Mechanisms

Objective To study the effects and the mechanisms of extract from a leguminous plant （Ammopiptanthus mongolicus cheng f.） （JAl ） in northwest China on inducing apoptosis and inhibiting proliferation of HepG2 hepatocarcinoma cell in vitro. Methods The HepG2 cell line was used as target cells. The effect of 3A 1 on HepG2 cell growth was detected by microculture tetrazolium assay （MTr）, flow cytometry assay, DNA agarose gel electrophoresis and transmission electronic microscopy. The expressive effect of the wt-p53 in HepG2 cells was analyzed with p53 protein test-reagent. Results JAl not only had significant anti-proliferative effects depending upon time and dosage, but also induced apoptosis of HepG2 cells. Apoptotic typical morphological changes were observed in JAl-treated HepG2 cells under transmission electronic microscope, ＂Sub-G 1＂ phase peak occurred in flow cytometry and DNA ＂ladder＂ was found in DNA agarose gel electrophoresis. The expression of the wt-p53 increased in vitro, and 3Al-treated HepG2 and the positive cell percentage of the wt-p53 protein also increased. Conclusions JAl could obviously induce apoptosis and inhibit proliferation of HepG2 cells in vitro, and these effects are closely related with the increase of wt-p53 expression. JAl can be used as a good source of medicinal plant for the treatment of hepatocarcinoma.