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ABSTRACT: The objective was to evaluate the in vitro antioxidant, genotoxic, antigenotoxic, and antineoplastic activities of apitoxin produced by the bee Apis mellifera. The antioxidant activity of the apitoxin solution was evaluated using the DPPH (2,2-diphenyl-1-picrilhydrazyl) method. Genotoxic potential of apitoxin was analyzed by comparing the mean DNA damage indices (idDNA) of L929 strain fibroblasts exposed to hydrogen peroxide (H2O2 - genotoxic substance), distilled water, or apitoxin. The antigenotoxic effect of apitoxin was analyzed by assessing the percentage decrease in H2O2-induced genotoxicity in L929 fibroblasts co-treated with three concentrations of the aqueous apitoxin solution and subjected to comet assay. In vitro antineoplastic activity in human tumor cell lines of prostate adenocarcinoma (PC3), hepatocellular carcinoma (HEPGE2), melanoma (MAD-MB435), and astrocytoma (SNB19), were verified by MTT [3- (4) bromide colorimetric method, 5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium]. Apitoxin had no genotoxic effect on L929 cells at concentrations of 30, 10, and 5 µg/mL after 24 hours of exposure. This effect was only evident at 50 µg/mL. Apitoxin promoted a significant reduction in DNA damage index (idDNA) at all concentrations tested. At 30 µg/mL, apitoxin attenuated the genotoxic effects induced by H2O2. Apitoxin also demonstrated in vitro antineoplastic potential, since the cytotoxic effect was observed at concentrations of 50 µg/mL and 25 µg/mL, with significant reduction in viability percentage of PC3 tumor cell lines, HEPGE2, MAD-MB435, and SNB19. The high antioxidant activity associated with the absence of genotoxic effect and the genoprotective and antineoplastic effect demonstrated by apitoxin here provide indications of apitoxin's therapeutic potential.
RESUMO: O objetivo deste estudo foi avaliar as atividades antioxidantes, genotóxicas, antigenotóxicas e antineoplásicas in vitro da apitoxina produzida pela abelha Apis mellifera. A atividade antioxidante da solução da apitoxina foi avaliada pelo método DPPH (2,2-difenil-1-picrilhidrazil). O potencial genotóxico da apitoxina foi analisado através dos índices médios de dano ao DNA (idDNA) dos fibroblastos da linhagem L929 expostos à peróxido de hidrogênio (H2O2 - substância genotóxica), água destilada ou apitoxina. O efeito antigenotóxico da apitoxina foi analisado através da avaliação da diminuição percentual na genotoxicidade induzida por H2O2 nos fibroblastos L929 co-tratados com três concentrações da solução aquosa de apitoxina e submetidos ao ensaio cometa. A atividade antineoplásica in vitro em linhagens celulares tumorais humanas de adenocarcinoma da próstata (PC3), carcinoma hepatocelular (HEPGE2), melanoma (MAD-MB435) e astrocitoma (SNB19), foram verificadas pelo método colorimétrico do brometo de MTT [3- (4), 5-dimetiltiazol -2-il) -2,5-difeniltetrazólio]. A apitoxina não teve efeito genotóxico nas células L929 nas concentrações de 30, 10 e 5 µg / mL após 24 horas de exposição. Este efeito foi apenas evidente a 50 µg / mL. A apitoxina promoveu uma redução significativa no índice de danos ao DNA (idDNA) em todas as concentrações testadas. A 30 µg / mL, a apitoxina atenuou os efeitos genotóxicos induzidos por H2O2. A apitoxina também demonstrou potencial antineoplásico in vitro, uma vez que o efeito citotóxico foi observado em concentrações de 50 µg / mL e 25 µg / mL, com redução significativa na porcentagem de viabilidade das linhagens celulares de PC3, HEPGE2, MAD-MB435 e SNB19. A alta atividade antioxidante associada à ausência de efeito genotóxico e o efeito genoprotetor e antineoplásico demonstrado pela apitoxina aqui fornecem indicações do potencial terapêutico da apitoxina.
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AIM To optimize the aqueous extraction for polysaccharides from Astragali Radix and to evaluate the in vitro antitumor activity.METHODS With extraction temperature,extraction time and solid-liquid ratio as influencing factors,extraction rate of polysaccharides as an evaluation index,the extraction was optimized by uniform design.The effect of polysaccharides on the proliferation of non-small cell lung cancer NCI-H460 cells,the apoptosis rate and cell cycle of NCI-H460 cells,and the expressions of Caspase-3,Bax and Bcl-2 were detected by MTT assay,flow cytometry and Western blot,respectively.RESULTS The optimal conditions were determined to be 100 ℃ for extraction temperature,1 h for extraction time,and 1 ∶ 35 for solid-liquid ratio,the extraction rate of polysaccharides was 3.62％.Compared with the control group,the proliferation of NCI-H460 cells was significandy inhibited in a dose-dependent manner (P ＜ 0.01),the S phase ratio,early apoptosis rate,late apoptosis rate and total apoptosis rate were markedly increased (P ＜ 0.01),and the Caspase-3 expression and Bax/Bcl-2 ratio were also obviously increased (P ＜ 0.01) in the polysaccharides group.CONCLUSION This fast,stable and reliable method can be used for the aqueous extraction for polysaccharides from Astragali Radix,which can significantly inhibit the proliferation of NCI-H460 cells and induce apoptosis of NCI-H460 cells.
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AIM To optimize the aqueous extraction for polysaccharides from Astragali Radix and to evaluate the in vitro antitumor activity.METHODS With extraction temperature,extraction time and solid-liquid ratio as influencing factors,extraction rate of polysaccharides as an evaluation index,the extraction was optimized by uniform design.The effect of polysaccharides on the proliferation of non-small cell lung cancer NCI-H460 cells,the apoptosis rate and cell cycle of NCI-H460 cells,and the expressions of Caspase-3,Bax and Bcl-2 were detected by MTT assay,flow cytometry and Western blot,respectively.RESULTS The optimal conditions were determined to be 100 ℃ for extraction temperature,1 h for extraction time,and 1 ∶ 35 for solid-liquid ratio,the extraction rate of polysaccharides was 3.62％.Compared with the control group,the proliferation of NCI-H460 cells was significandy inhibited in a dose-dependent manner (P ＜ 0.01),the S phase ratio,early apoptosis rate,late apoptosis rate and total apoptosis rate were markedly increased (P ＜ 0.01),and the Caspase-3 expression and Bax/Bcl-2 ratio were also obviously increased (P ＜ 0.01) in the polysaccharides group.CONCLUSION This fast,stable and reliable method can be used for the aqueous extraction for polysaccharides from Astragali Radix,which can significantly inhibit the proliferation of NCI-H460 cells and induce apoptosis of NCI-H460 cells.
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AIM To optimize the supercritical CO2 extraction of sauchinone and to evaluate the in vitro antitumor activity of Saururi Herba supercritical extract.METHODS With extraction pressure,extraction temperature,extraction time,entrainer (ethanol) concentration and entrainer amount as influencing factors,together with extraction rate of sauchinone as an evaluation index,orthogonal test was used for optimizing the extraction.Then MTT was applied to determining the extract's inhibitory effect on human multidrug-resistant hepatocellular carcinoma cell line (7721/Adm).RESULTS The optimal conditions were determined to be 30 MPa for extraction pressure,50 ℃ for extraction temperature,2 h for extraction time,95％ for ethanol concentration,and one time for ethanol amount,the average extraction rate of sauchinone was 0.173％.The obtained extract significantly inhibited the proliferation of 7721/Adm cells (IC50 =50.08 μg/mL),demonstrating a stronger activity than that of ethanol extract (ICs0 =150.59 μg/mL).CONCLUSION This stable and feasible method is appropriate for sauchinone extraction,and the supercritical CO2 extract from Saururi Herba shows a strong in vitro antitumor activity.
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The dried whole plant of Pteris dispar were milled and extracted with 95% EtOH. The resulting dried extract was isolated by kinds of chromatographic column, including polyamide, Sephadex LH-20, preparative HPLC. As a result, ten diterpenes were isolated from the plant. By analyzing of ESI-MS and NMR spectroscopic data, the structures were established as geopyxin B(1), geopyxin E(2), ent-11α-hydroxy-18-acetoxykaur-16-ene(3), ent-14β-hydroxy-18-acetoxykaur-16-ene(4), neolaxiflorin L(5), ent-3β,19-dihydroxy-kaur-16-ene(6), ent-3β-hydroxy-kaur-16-ene(7), 7β,17-dihydroxy-16α-ent-kauran-19-oic acid 19-O-β-D-glucopyranoside ester(8), crotonkinin C(9)and crotonkinin C(10). Compounds 1-10 were obtained from P. dispar for the first time. Compounds 1 and 2 showed moderate activities against Bel-7402 with IC₅₀ values of 7.50 and 10.60 μmol•L⁻¹, and against HepG2 with IC₅₀ values of 6.68,11.80 μmol•L⁻¹, respectively.