The Effects of New Zealand Grown Ginseng Fractions on Cytokine Production from Human Monocytic THP-1 Cells.

Pro-inflammatory cytokines and anti-inflammatory cytokines are important mediators that regulate the inflammatory response in inflammation-related diseases. The aim of this study is to evaluate different New Zealand (NZ)-grown ginseng fractions on the productions of pro-inflammatory and anti-inflammatory cytokines in human monocytic THP-1 cells. Four NZ-grown ginseng fractions, including total ginseng extract (TGE), non-ginsenoside fraction extract (NGE), high-polar ginsenoside fraction extract (HPG), and less-polar ginsenoside fraction extract (LPG), were prepared and the ginsenoside compositions of extracts were analyzed by HPLC using 19 ginsenoside reference standards. The THP-1 cells were pre-treated with different concentrations of TGE, NGE, HPG, and LPG, and were then stimulated with lipopolysaccharide (LPS). The levels of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), interleukin-6 (IL-6), interleukin-8 (IL-8), and anti-inflammatory cytokines, such as interleukin-10 (IL-10), and transforming growth factor beta-1 (TGF-ß1), were determined by enzyme-linked immunosorbent assay (ELISA). TGE at 400 µg/mL significantly inhibited LPS-induced TNF-α and IL-6 productions. NGE did not show any effects on inflammatory secretion except inhibited IL-6 production at a high dose. Furthermore, LPG displayed a stronger effect than HPG on inhibiting pro-inflammatory cytokine (TNF-α, IL-1ß, and IL-6) productions. Particularly, 100 µg/mL LPG not only significantly inhibited the production of pro-inflammatory cytokines TNF-α, IL-1ß, and IL-6, but also remarkably enhanced the production of anti-inflammatory cytokine IL-10. NZ-grown ginseng exhibited anti-inflammatory effects in vitro, which is mainly attributed to ginsenoside fractions (particularly less-polar ginsenosides) rather than non-saponin fractions.

Changes of Ginsenoside Composition in the Creation of Black Ginseng Leaf.

Ginseng is an increasingly popular ingredient in supplements for healthcare products and traditional medicine. Heat-processed ginsengs, such as red ginseng or black ginseng, are regarded as more valuable for medicinal use when compared to white ginseng due to some unique less polar ginsenosides that are produced during heat-treatment. Although ginseng leaf contains abundant ginsenosides, attention has mostly focused on ginseng root; relatively few publications have focused on ginseng leaf. Raw ginseng leaf was steamed nine times to make black ginseng leaf using a process that is similar to that used to produce black ginseng root. Sixteen ginsenosides were analyzed during each steaming while using high-performance liquid chromatography (HPLC). The contents of ginsenosides Rd and Re decreased and the less polar ginsenosides (F2, Rg3, Rk2, Rk3, Rh3, Rh4, and protopanaxatriol) enriched during steam treatment. After nine cycles of steaming, the contents of the less polar ginsenosides F2, Rg3, and Rk2 increased by 12.9-fold, 8.6-fold, and 2.6-fold, respectively. Further, we found that the polar protopanaxadiol (PPD) -type ginsenosides are more likely to be converted from ginsenoside Rg3 to ginsenosides Rk1 and Rg5 via dehydration from Rg3, and from ginsenoside Rh2 to ginsenosides Rk2 and Rh3 through losing an H2O molecule than to be completely degraded to the aglycones PPD during the heat process. This study suggests that ginseng leaves can be used to produce less polar ginsenosides through heat processes, such as steaming.

Comparison of Ginsenoside Components of Various Tissues of New Zealand Forest-Grown Asian Ginseng (Panax Ginseng) and American Ginseng (Panax Quinquefolium L.).

Asian ginseng (Panax ginseng) and American ginseng (Panax quinquefolium L.) are the two most important ginseng species for their medicinal properties. Ginseng is not only popular to consume, but is also increasingly popular to cultivate. In the North Island of New Zealand, Asian ginseng and American ginseng have been grown in Taupo and Rotorua for more than 15 years. There are no publications comparing the chemical constituents between New Zealand-grown Asian ginseng (NZPG) and New Zealand-grown American ginseng (NZPQ). In this study, fourteen ginsenoside reference standards and LC-MS2 technology were employed to analyze the ginsenoside components of various parts (fine root, rhizome, main root, stem, and leaf) from NZPG and NZPQ. Fifty and 43 ginsenosides were identified from various parts of NZPG and NZPQ, respectively, and 29 ginsenosides were found in both ginseng species. Ginsenoside concentrations in different parts of ginsengs were varied. Compared to other tissues, the fine roots contained the most abundant ginsenosides, not only in NZPG (142.49 ± 1.14 mg/g) but also in NZPQ (115.69 ± 3.51 mg/g). For the individual ginsenosides of both NZPG and NZPQ, concentration of Rb1 was highest in the underground parts (fine root, rhizome, and main root), and ginsenoside Re was highest in the aboveground parts (stem and leaf).

Review of Ginseng Anti-Diabetic Studies.

Ginseng is one of the most valuable and commonly used Chinese medicines not only in ancient China but also worldwide. Ginsenosides, also known as saponins or triterpenoids, are thought to be responsible for the beneficial effects of ginseng. In this review, we summarize recent publications on anti-diabetic studies of ginseng extracts and ginsenosides in cells, animals, and humans. It seems that the anti-diabetic effect of ginseng is positive for type 2 diabetic patients but has no significant impact on prediabetes or healthy adults. Regulation of insulin secretion, glucose uptake, anti-oxidative stress, and anti-inflammatory pathways may be the mechanisms involved with ginseng's anti-diabetic effects. Taken together, this summary provides evidence for the anti-diabetes effects of ginseng extracts and ginsenosides as well as the underlying mechanisms of their impact on diabetes.

Analysis of Ginsenoside Content (Panax ginseng) from Different Regions.

Recently Panax ginseng has been grown as a secondary crop under a pine tree canopy in New Zealand (NZ). The aim of the study is to compare the average content of ginsenosides from NZ-grown ginseng and its original native locations (China and Korea) grown ginseng. Ten batches of NZ-grown ginseng were extracted using 70% methanol and analyzed using LC-MS/MS. The average content of ginsenosides from China and Korea grown ginseng were obtained by collecting data from 30 and 17 publications featuring China and Korea grown ginseng, respectively. The average content of total ginsenosides in NZ-grown ginseng was 40.06 ± 3.21 mg/g (n = 14), which showed significantly (p < 0.05) higher concentration than that of China grown ginseng (16.48 ± 1.24 mg/g, n = 113) and Korea grown ginseng (21.05 ± 1.57 mg/g, n = 106). For the individual ginsenosides, except for the ginsenosides Rb2, Rc, and Rd, ginsenosides Rb1, Re, Rf, and Rg1 from NZ-grown ginseng were 2.22, 2.91, 1.65, and 1.27 times higher than that of ginseng grown in China, respectively. Ginsenosides Re and Rg1 in NZ-grown ginseng were also 2.14 and 1.63 times higher than ginseng grown in Korea. From the accumulation of ginsenosides, New Zealand volcanic pumice soil may be more suitable for ginseng growth than its place of origin.

Fermentation of protopanaxadiol type ginsenosides (PD) with probiotic Bifidobacterium lactis and Lactobacillus rhamnosus.

Ginsenosides are believed to be the principal components behind the pharmacological actions of ginseng, and their bioactive properties are closely related to the type, position, and number of sugar moieties attached to the aglycone; thus, modification of the sugar chains may markedly change their biological activities. In this study, major protopanaxadiol type ginsenosides (PD) Rb1, Rc, and Rb2 were isolated from Panax ginseng and were transformed using two probiotic strains namely Bifidobacterium lactis Bi-07 and Lactobacillus rhamnosus HN001 to obtain specific deglycosylated ginsenosides. It was demonstrated that B. lactis transformed ginsenosides Rb1, Rc, and Rb2 to Rd within 1 h of fermentation and rare ginsenoside F2 by the conversion of Rd after 12-h fermentation. The maximum Rd concentration was 147.52 ± 1.45 µg/mL after 48-h fermentation as compared to 45.85 ± 0.71 µg/mL before fermentation. In contrast, L. rhamnosus transformed Rb1, Rc, and Rb2 into Rd as the final metabolite after 72-h fermentation. B. lactis displayed significantly (p < 0.05) higher ß-glucosidase activity against p-nitrophenyl-ß-glucopyranoside than L. rhamnosus and higher bioconversion efficiency during fermentation. The present study suggests that the fermentation of major PD type ginsenosides with B. lactis Bi-07 may serve as an effective means to afford bioactive deglycosylated ginsenosides and to create novel ginsenoside extracts.

Isolation and characterization of bioactive polyacetylenes Panax ginseng Meyer roots.

Panax ginseng has been studied for its chemo-preventive properties and pharmaceutical potential. Polyacetylenic compounds isolated from Panax ginseng root typically comprised of non-polar C17 compound have been reported to exhibit bioactive properties. The objective of this project is to extract, isolate, and characterize bioactive polyacetylenes from Panax ginseng root using various extraction and separation methods Ginseng was extracted by reflux using methanol, ethanol, hexane, ethyl acetate, methanolic ultrasonication. The extracts were partitioned with hexane to obtain water-soluble portion and hexane-soluble portion. Hexane was subsequently removed under vacuum, and formed a crude polyacetylenes extract (crude PA). Silica gel chromatography and semi-preparative HPLC were utilized to prepare 5 fractions and the polyacetylenes were measure by HPLC and molecular weights confirm my APCI-MS and MNR. The bioactive effect was measured by MTT viability assay using murine 3T3-L1 cells. Extraction with methanol under reflux produced significantly larger amount of polyacetylenes (p<0.05). Liquid-liquid extraction and column chromatography were used to separate polyacetylenic compounds into five different fractions. Major polyacetylenes, panaxynol and panaxydol were found in fraction 1 and 2 respectively. Dose-response relationships were observed in 3T3-L1 cells and LC50 were 13.52±3.05µg/mL (fraction 1), 3.69±1.09µg/mL (fraction 2), 52.88±11.16µg/mL (fraction 3), 85.91±27.37µg/mL (fraction 4) and 135.52±32.91µg/mL (fraction 5). Fraction 2 containing panaxydol was found to have exhibited the greatest anti-proliferative effects on 3T3-L1 preadipocytes. Extraction with methanol under reflux produced significantly more polyacetylenes. Fractions that contain panaxydol was the most cytotoxic.

Distinct Responses of Cytotoxic Ganoderma lucidum Triterpenoids in Human Carcinoma Cells.

The medicinal mushroom Ganoderma lucidum is well recognized for its effective cancer-preventative and therapeutic properties, while specific components responsible for these anticancer effects are not well studied. Six triterpenoids that are ganolucidic acid E, lucidumol A, ganodermanontriol, 7-oxo-ganoderic acid Z, 15-hydroxy-ganoderic acid S, and ganoderic acid DM were isolated and identified from an extract of the mushroom. All compounds reduced cell growth in three human carcinoma cells (Caco-2, HepG2, and HeLa cells) dose dependently with LC50s from 20.87 to 84.36 µM. Moreover, the six compounds induced apoptosis in HeLa cells with a maximum increase (22%) of sub-G1 accumulations and 43.03% apoptotic cells in terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (15-hydroxy-ganoderic acid S treatment). Apoptosis was further confirmed by annexin-V staining. Four of the compounds also caused apoptosis in Caco-2 cells with maximum 9.5% increase of sub-G1 accumulations (7-oxo-ganoderic acid Z treatment) and maximum 29.84% apoptotic cells in TUNEL assay (ganoderic acid DM treatment). Contrarily, none of the compounds induced apoptosis in HepG2 cells. The different responses of the three cell lines following these treatments indicated that the bioactive properties of these compounds may vary from cells of different sites of origin and are likely acting under diverse regulatory mechanisms.

Effects of high molecular weight alcohols from sugar cane fed alone or in combination with plant sterols on lipid profile and antioxidant status of Wistar rats.

The effect of feeding a mixture of high molecular weight alcohols derived from sugarcane (SCA), both alone and in combination with phytosterols (PS), on changes in plasma lipids, organ cholesterol accumulation, and antioxidant status of Wistar rats was undertaken. Three separate experiments were conducted and each experiment had 3 subsets. In experiment 1, rats were fed on an AIN-76, semi-synthetic diet supplemented with 0%, 0.5%, and 5% SCA w/w. The second experiment consisted of feeding rats an atherogenic diet (AIN-76+0.5% cholesterol) containing 0%, 0.5%, and 5% SCA w/w. The third experiment consisted of feeding rats an atherogenic diet that contained 2% PS in combination with 0%, 0.5%, and 5% SCA. Rats fed the atherogenic diet exhibited significant elevations in total and low-density lipoprotein cholesterol, and significant reductions in the high-density lipoprotein/total cholesterol ratio, regardless of the presence of 0.5% or 5% SCA mixture. Serum cholesterol increased 29% to 35% in these animals compared with animals fed the nonatherogenic diets. In contrast, animals fed atherogenic diets that contained 2% PS exhibited no difference in serum lipids compared with counterparts fed nonatherogenic diets. The combined presence of SCA with PS had no effect on further lowering plasma cholesterol. No changes in C-reactive protein were observed, but plasma oxygen radical scavenging capacity values significantly (p < 0.05) decreased when rats were fed the atherogenic diets that contained the combination of PS and SCA. This result corresponded to an apparent greater (p < 0.05) susceptibility of red blood cells to oxidative stress.

Ginseng (Panax quinquefolius) Reduces Cell Growth, Lipid Acquisition and Increases Adiponectin Expression in 3T3-L1 Cells.

An American ginseng (Panax quinquefolius) extract (GE) that contained a quantifiable amount of ginsenosides was investigated for the potential to inhibit proliferation, affect the cell cycle, influence lipid acquisition and adiponectin expression in 3T3-L1 cells. Six fingerprint ginsenosides were quantified by high performance liquid chromatography and the respective molecular weights were confirmed by LC-ESI-MS analysis. The extract contained Rg1 (347.3 ± 99.7 µg g(-1), dry weight), Re (8280.4 ± 792.3 µg g(-1)), Rb1 (1585.8 ± 86.8 µg g(-1)), Rc (32.9 ± 8 µg g(-1)), Rb2 (62.6 ± 10.6 µg g(-1)) and Rd (90.4 ± 3.2 µg g(-1)). The GE had a dose-dependent effect on 3T3-L1 cell growth, the LC50 value was determined to be 40.3 ± 5 µg ml(-1). Cell cycle analysis showed modest changes in the cell cycle. No significant changes observed in both G1 and G2/M phases, however there was a significant decrease (P < .05) in the S phase after 24 and 48 h treatment. Apoptotic cells were modest but significantly (P < .05) increased after 48 h (3.2 ± 1.0%) compared to untreated control cells (1.5 ± 0.1%). Lipid acquisition was significantly reduced (P < .05) by 13 and 22% when treated at concentrations of 20.2 and 40.3 µg ml(-1) compared to untreated control cells. In relation to adiponectin activation, western blot analysis showed that the protein expression was significantly (P < .05) increased at concentrations tested. A quantified GE reduced the growth of 3T3-L1 cells, down-regulated the accumulation of lipid and up-regulated the expression of adiponectin in the 3T3-L1 adipocyte cell model.

Momordica charantia seed extract reduces pre-adipocyte viability, affects lactate dehydrogenase release, and lipid accumulation in 3T3-L1 cells.

A triterpenoid containing bitter melon (Momordica charantia) seed (BMS) extract was found to reduce cultured 3T3-L1 cell viability. The 50% lethal concentration values were determined to be 0.78 ± 0.01 mg/mL at 24 hours, 0.69 ± 0.01 mg/mL at 48 hours, and 0.56 ± 0.02 mg/mL at 72 hours. 3T3-L1 cells were utilized as models of pre-adipocyte to adipocyte differentiation. BMS extract also caused a G(2)/M arrest in the cell cycle reducing cells by 23.9%, 37.7%, and 34.7% compared with the control after 72 hours of treatment at concentrations of 0.4, 0.5, and 0.6 mg/mL respectively. BMS extract did not increase the release of lactate dehydrogenase from 3T3-L1 cells, which was unexpected. Furthermore, BMS extract reduced lipid accumulation during differentiation from pre-adipocyte to adipocyte corresponding to reduction in overall triglyceride of 32.4% after 72 hours compared with untreated control cells. BMS is an underutilized agricultural commodity that may have potential for nutraceutical and functional food development.

A quantified ginseng (Panax ginseng C.A. Meyer) extract influences lipid acquisition and increases adiponectin expression in 3T3-L1 cells.

A Panax ginseng extract (PGE) with a quantified amount of ginsenosides was utilized to investigate its potential to inhibit proliferation, influence lipid acquisition and adiponectin expression in 3T3-L1 cells. Seven fingerprint ginsenosides were quantified using high performance liquid chromatography and their respective molecular weights were further confirmed via LC-ESI-MS analysis from four different extraction methods. Extraction using methanol under reflux produced significantly higher amounts of ginsenosides. The methanol extract consisted of Rg1 (47.40 ± 4.28 mg/g, dry weight of extract), Re (61.62 ± 5.10 mg/g), Rf (6.14 ± 0.28 mg/g), Rb1 (21.73 ± 1.29 mg/g), Rc (78.79 ± 4.15 mg/g), Rb2 (56.80 ± 3.79 mg/g), Rd (5.90 ± 0.41 mg/g). MTT analysis showed that PGE had a concentration-dependent cytotoxic effect on 3T3-L1 preadipocyte and the LC(50) value was calculated to be 18.2 ± 5 µg/mL. Cell cycle analysis showed minimal changes in all four phases. Differentiating adipocytes treated with ginseng extract had a visible decrease in lipid droplets formation measured by Oil red O staining. Consequently, triglycerides levels in media significantly (P < 0.05) decreased by 39.5% and 46.1% when treated at concentrations of 1 µg/mL and 10 µg/mL compared to untreated control cells. Western blot analysis showed that the adiponectin protein expression was significantly (P < 0.05) increased at 10 µg/mL, but not at 1 µg/mL. A quantified PGE reduced the growth of 3T3-L1 cells, down-regulated lipid accumulation and up-regulated adiponectin expression in the 3T3-L1 adipocyte cell model.

Ginseng (Panax quinquefolius) and Licorice (Glycyrrhiza uralensis) Root Extract Combinations Increase Hepatocarcinoma Cell (Hep-G2) Viability.

The combined cytoactive effects of American ginseng (Panax quinquefolius) and licorice (Glycyrrhiza uralensis) root extracts were investigated in a hepatocarcinoma cell line (Hep-G2). An isobolographic analysis was utilized to express the possibility of synergistic, additive or antagonistic interaction between the two extracts. Both ginseng and licorice roots are widely utilized in traditional Chinese medicine preparations to treat a variety of ailments. However, the effect of the herbs in combination is currently unknown in cultured Hep-G2 cells. Ginseng (GE) and licorice (LE) extracts were both able to reduce cell viability. The LC50 values, after 72 h, were found to be 0.64 ± 0.02 mg/mL (GE) and 0.53 ± 0.02 mg/mL (LE). An isobologram was plotted, which included five theoretical LC50s calculated, based on the fixed fraction method of combination ginseng to licorice extracts to establish a line of additivity. All combinations of GE to LE (1/5, 1/3, 1/2, 2/3, 4/5) produced an effect on Hep-G2 cell viability but they were all found to be antagonistic. The LC50 of fractions 1/3, 1/2, 2/3 were 23%, 21% and 18% above the theoretical LC50. Lactate dehydrogenase release indicated that as the proportion of GE to LE increased beyond 50%, the influence on membrane permeability increased. Cell-cycle analysis showed a slight but significant arrest at the G1 phase of cell cycle for LE. Both GE and LE reduced Hep-G2 viability independently; however, the combinations of both extracts were found to have an antagonistic effect on cell viability and increased cultured Hep-G2 survival.

Lovastatin interacts with natural products to influence cultured hepatocarcinoma cell (hep-g2) growth.

OBJECTIVE: To assess the interaction of the hydroxy-3-methylglutaryl-coezyme A reductase inhibitor lovastatin individually and in combination with 3 natural plant compounds on hepatocarcinoma cell growth. METHODS: The cytotoxic effects of lovastatin in combination with epigallocatechin gallate (EGCG), capsaicin, and curcumin were investigated in cultured hepatocarcinoma cells (Hep-G2), and the interactions were depicted using an isobolographical analysis. RESULTS: All compounds tested reduced Hep-G2 cell growth to various degrees. In terms of individual cytotoxicity LC50 values, curcumin (55.5 ± 7.6 µmol/L) was found to be the most cytotoxic and had the lowest LC50, followed by lovastatin (62.3 ± 5.34 µmol/L), EGCG (82.1 ± 15.1 µmol/L), and capsaicin (199.5 ± 11.72 µmol/L). The individual LC50 values were used in a fix-fraction isobolographical analysis to predict the effect of combining lovastatin and the individual compounds. Experimentally derived LC50 values of 5 fractions containing fixed proportions of lovastatin to each of the 3 natural compounds allowed comparison of the experimentally derived LC50 to the predicted values depicted on the isobologram. CONCLUSION: Lovastatin in combination with capsaicin was found to be synergistic at all concentrations tested, and EGCG combinations produced both synergistic and additive results. Unexpectedly, lovastatin in combination with curcumin produced an antagonistic effect on cell growth, resulting in a greater concentration required than if the compounds were used individually.

Bog bilberry (Vaccinium uliginosum L.) extract reduces cultured Hep-G2, Caco-2, and 3T3-L1 cell viability, affects cell cycle progression, and has variable effects on membrane permeability.

Bog bilberry (Vaccinium uliginosum L.) is a blue-pigmented edible berry related to bilberry (Vaccinium myrtillus L.) and the common blueberry (Vaccinium corymbosum). The objective of this study was to investigate the effect of a bog bilberry anthocyanin extract (BBAE) on cell growth, membrane permeability, and cell cycle of 2 malignant cancer cell lines, Caco-2 and Hep-G2, and a nonmalignant murine 3T3-L1 cell line. BBAE contained 3 identified anthocyanins. The most abundant anthocyanin was cyanidin-3-glucoside (140.9 +/- 2.6 microg/mg of dry weight), followed by malvidin-3-glucoside (10.3 +/- 0.3 microg/mg) and malvidin-3-galactoside (8.1 +/- 0.4 microg/mg). Hep-G2 LC50 was calculated to be 0.563 +/- 0.04 mg/mL, Caco-2 LC50 was 0.390 +/- 0.30 mg/mL and 0.214 +/- 0.02 mg/mL for 3T3-L1 cells. LDH release, a marker of membrane permeability, was significantly increased in Hep-G2 cells and Caco-2 cells after 48 and 72 h compared to 24 h. The increase was 21% at 48 h and 57% at 72 h in Caco-2 cells and 66% and 139% in Hep-G2 cells compared to 24 h. However, 3T3-L1 cells showed an unexpected significant lower LDH activity (P < or = 0.05) after 72 h of exposure corresponding to a 21% reduction in LDH release. BBAE treatment increased sub-G1 in all 3 cell lines without influencing cells in the G2/M phase. BBAE treatment reduced the growth and increased the accumulation of sub-G1 cells in 2 malignant and 1 nonmalignant cell line; however, the effect on membrane permeability differs considerably between the malignant and nonmalignant cells and may in part be due to differences in cellular membrane composition.

Group B oleanane triterpenoid extract containing soyasaponins I and III from soy flour induces apoptosis in Hep-G2 cells.

Soyasaponins I and III are monodesmodic oleanane triterpenoids that are thought to be one of the main potentially bioactive saponins found in soy ( Glycine max ) and related products. An extract that contained a majority of the soyasaponins as I (62%) and III (29%) was studied in a hepatocarcinoma cell line (Hep-G2) to determine the potential cellular bioactivity. The extract was found to inhibit the growth of Hep-G2 cells measured by the MTT viability assay (LC50 0.389 +/- 0.02 mg/mL) dose-dependently. Cell death was determined using three distinct flow cytometry assays for apoptosis (sub-G1 cell-cycle analysis and multi-caspase and TUNEL assays), and cellular morphological images were acquired by confocal laser scanning microscopy. All of the apoptotic detection assays were positive for apoptotic cell accumulation. The TUNEL detection assay after 72 h of treatment showed the greatest apoptotic cell accumulation (40.45 +/- 4.95%), followed by the multi-caspase assay for 48 h treated cells (6.97 +/- 0.14% mid-apoptotic and 12.87 +/- 0.81% late apoptotic) and sub-G1 accumulation (17.67 +/- 0.42%). The multi-caspase assay indicated that the cellular protein expression of the caspase family of enzymes was the main apoptotic pathway trigger. Confocal laser scanning microscopy further confirmed the induction of apoptosis in Hep-G2 cells. Soyasaponins I and III may contribute to the reported bioactive and chemopreventative properties of soy by the induction of apoptosis.

Bitter melon (Momordica charantia) triterpenoid extract reduces preadipocyte viability, lipid accumulation and adiponectin expression in 3T3-L1 cells.

Bitter melon (Momordica charantia) contains a variety of potentially bioactive compounds which includes two classes of saponins known as cucurbitane and oleanane-type triterpenoids. A bitter melon (BM) extract was investigated for the potential to reduce cell viability, reduce lipid accumulation in differentiating 3T3-L1 cells and affect subsequent adiponectin expression. BM extract contained at least five different triterpenoids and reduced preadipocyte viability with an LC50 concentration after 24h determined to be 0.402+/-0.04 mg/mL, 0.314+/-0.01 mg/mL for 48 h and 0.310+/-0.01 mg/mL for 72 h. BM treatment increased the release of lactate dehydrogenase (LDH) from cells and significantly (p<0.05) increased cells in the G2/M while reducing cells in the G1 phase of the cell cycle. BM treatment of 3T3-L1 cells resulted in a decreased in lipid accumulation and significantly (p<0.05) decreased intracellular triglyceride amount compared to untreated control cells. PPARgamma expression was significantly (p<0.05) down-regulated. Adiponectin expression was significantly (p<0.05) decreased after 12 and 24h of treatment and was increased in response to troglitazone, a positive control. BM extract reduced preadipocyte proliferation by a G2/M arrest of the cell cycle and reduced lipid accumulation of the adipocyte.

Bioactive responses of Hep-G2 cells to soyasaponin extracts differs with respect to extraction conditions.

Soyasaponins are bioactive oleanane triterpenoids found in soy and other legumes. The effect of two methanolic extractions of soy flour, room temperature (RT) and reflux (RE) extractions on composition and bioactive properties in hepatocarcinoma cells (Hep-G2) were investigated. A greater amount of 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) conjugated soyasaponins betag was measured in RT and a greater amount of the structurally related non-DDMP soyasaponins I and III were detected in RE. MTT cell viability yielded an LC50 of 0.926+/-0.08 mg/mL for RT and 0.546+/-0.06 mg/mL for RE. ViaCount viability assay showed similar results for RE as the MTT assay however, RT treatment produced no difference compared with the control. Analysis using TUNEL and cell cycle analysis revealed that RE treatment induced apoptosis and flow cytometry forward side scatter and morphological assessment of RT showed evidence of Hep-G2 differentiation after 72 h. Differences in the bioactivities may be attributed to the different concentration of DDMP conjugated soyasaponin betag recovered in RT and RE extracts.

Evaluation of viability assays for anthocyanins in cultured cells.

The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay is a widely accepted cytotoxicity assay which can produce inaccurate results due to possible interference with the antioxidant property of anthocyanins. Alternative methods to the MTT assay, such as BrdU (DNA-based) and CellTiter-Glo (ATP-based) assays were evaluated to assess anthocyanin cytotoxicity, derived from blackberry in LNCaP, MCF-7 and MDA-MB-453 cell lines. The standard cell counting method was the reference assay. Greater correlation of cell viability values following anthocyanin exposure was obtained from multiple cell lines with the alternative assays when compared with cell counting. MTT and cell counting results were not always correlated, albeit this was a function of cell type. In particular, poor correlations between cell counting and MTT procedures used to assess cytotoxicity of anthocyanins were observed in the MDA-MB-453 cell lines. Comparison of cytotoxicity derived from alternative assays and the MTT assays with the cell counting method was dependent on the assay procedure and the cell type. The LC(50) of blackberry crude extract ranged from 0.4 to 9.4 mg/mL between assays and across all cell lines, whereas a semi-purified anthocyanin extract was not cytotoxic. Cytotoxicity evaluation of polyphenolic-rich extracts using BrdU and CellTiter-Glo assays as alternatives to the MTT method is recommended.

Effect of soyasapogenol A and soyasapogenol B concentrated extracts on HEP-G2 cell proliferation and apoptosis.

The growth inhibition and the induction of apoptosis brought about by soyasaponins extracted from soy flour ( Glycine max (L.)) and concentrated for soyasapogenols A and B formed by hydrolysis were tested for cytoactivity in the human hepatocellular carcinoma cell line Hep-G2. Concentrated soyasapogenol A (SG-A) and soyasapogenol B (SG-B) extracts contained approximately 69.3% and 46.2% of their respective aglycones (soyasapogenols) assessed by HPLC and ESI-MS, while the soyasaponin extract (TS), derived from crude methanol extraction, did not contain any detectable amounts of SG-A or SG-B. An MTT viability assay showed that all three extracts had an effect on Hep-G2 proliferation in a dose-response manner with 72 h LC50 values of 0.594+/-0.021 mg/mL for TS, 0.052+/-0.011 mg/mL for SG-A, and 0.128+/-0.005 mg/mL for SG-B. Apoptotic cells were determined by flow cytometry cell cycle analysis and confocal laser scanning microscopy (CLSM). Cell cycle analysis indicated a significant ( P< 0.05) greater sub-G1 buildup of apoptotic cells at 24 h (25.63+/-2.1%) and 72 h (47.1+/-3.5%) for the SG-A extract compared to SG-B, whereas the TS extract produced only a minor buildup of sub-G1 cells. CLSM confirmed a morphological change of all treatments after 24 h, at the respective LC50 concentrations. These results show that the samples that contained mainly soyasapogenols A and B showed a greater ability to inhibit proliferation of cultured Hep-G2 when compared to a total soyasaponin extract that did not contain any soyasapogenols.