The Content and Principle of the Rare Ginsenosides Produced from Gynostemma pentaphyllum after Heat Treatment.

Ginsenoside Rg3, Rk1, and Rg5, rare ginsenosides from Panax ginseng, have many pharmacological effects, which have attracted extensive attention. They can be obtained through the heat treatment of Gynostemma pentaphyllum. In this study, scanning electron microscopy (SEM) and thermal gravity-differential thermal gravity (TG-DTG) were employed to investigate this process and the content change in ginsenosides was analyzed using liquid chromatography-mass spectrometry (LC-MS). SEM and TG-DTG were used to compare the changes in the ginsenosides before and after treatment. In SEM, the presence of hydrogen bond rearrangement was indicated by the observed deformation of vascular bundles and ducts. The before-and-after changes in the peak patterns and peaks values in TG-DTG indicated that the content of different kinds of compounds produced changes, which all revealed that the formation of new saponins before and after the heat treatment was due to the breakage or rearrangement of chemical bonds. Additionally, the deformation of vascular bundles and vessels indicated the presence of hydrogen bond rearrangement. The glycosidic bond at the 20 positions could be cleaved by ginsenoside Rb3 to form ginsenoside Rd, which, in turn, gave rise to ginsenoside Rg3(S) and Rg3(R). They were further dehydrated to form ginsenoside Rk1 and Rg5. This transformation process occurs in a weak acidic environment provided by G. pentaphyllum itself, without the involvement of endogenous enzymes. In addition, the LC-MS analysis results showed that the content of ginsenoside Rb3 decreased from 2.25 mg/g to 1.80 mg/g, while the contents of ginsenoside Rk1 and Rg5 increased from 0.08 and 0.01 mg/g to 3.36 and 3.35 mg/g, respectively. Ginsenoside Rg3(S) and Rg3(R) were almost not detected in G. pentaphyllum, and the contents of them increased to 0.035 and 0.23 mg/g after heat treatment. Therefore, the rare ginsenosides Rg3(S), Rg3(R), Rk1, and Rg5 can be obtained from G. pentaphyllum via heat treatment.

Liver lipidomics analysis reveals the anti-obesity and lipid-lowering effects of gypnosides from heat-processed Gynostemma pentaphyllum in high-fat diet fed mice.

BACKGROUND: In traditional Chinese medicine, Gynostemma pentaphyllum (G. pentaphyllum) is widely used to treat conditions associated with hyperlipidemia, and its therapeutic potential has been demonstrated in numerous studies. However, the mechanism of lipid metabolism in hyperlipidemic by G. pentaphyllum, especially heat-processed G. pentaphyllum is not yet clear. PURPOSE: The aim of this study was to investigate the therapeutic mechanism of gypenosides from heat-processed G. pentaphyllum (HGyp) in hyperlipidemic mice by means of a lipidomics. METHODS: The content of the major components of HGyp was determined by ultra-performance liquid chromatography-electrospray ionization ion trap mass spectrometry (UPLC-ESI-MS). An animal model of hyperlipidaemia was constructed using C57BL/6J mice fed with high-fat diet. HGyp was also administered at doses of 50, 100 and 200 mg/kg, all for 12 weeks. Serum parameters were measured, histological sections were prepared and liver lipidome analysis using UPLC-MS coupled with multivariate statistical analysis. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were used to analyze the genes and proteins associated with lipid lowering in HGyp. RESULTS: HGyp reduced body weight, serum total cholesterol (TC), triglyceride (TG) and low-density lipoprotein (LDL) and hepatic lipid accumulation in hyperlipidemic obese mice. To explore specific changes in lipid metabolism in relation to HGyp administration, lipid analysis of the liver was performed. Orthogonal partial least squares discriminant analysis (OPLS-DA) score plots showed that HGyp altered lipid metabolism in HFD mice. In particular, fatty acids (FA), triglycerides (DG), TG and ceramides (CER) were significantly altered. Eleven lipids were identified as potential lipid biomarkers, namely TG (18:2/20:5/18:2), TG (18:2/18:3/20:4), DG (18:3/20:0/0:0), Cer (d18:1/19:0), Cer (d16:1/23:0), Ceramide (d18:1/9Z-18:1), PS (19:0/18:3), PS (20:2/0:0), LysoPC (22:5), LysoPE (0:0/18:0), PE (24:0/16:1). Western blot and qRT-PCR analysis showed that these metabolic improvements played a role by down-regulating genes and proteins related to fat production (SREBP1, ACC1, SCD1), up-regulating genes and proteins related to lipid oxidation (CPTA1, PPARα) and lipid transport decomposition in the bile acid pathway (LXRα, PPARγ, FXR, BSEP). CONCLUSION: The lipid-lowering effect of gypenosides from heat-processed G. pentaphyllum is regulate lipid homeostasis and metabolism.

Effects of heat-processed Gynostemma pentaphyllum on high-fat diet-fed mice of obesity and functional analysis on network pharmacology and molecular docking strategy.

ETHNOPHARMACOLOGICAL RELEVANCE: Gynostemma pentaphyllum has been used as traditional medicine for many diseases, including metabolic syndrome (Mets), aging, diabetes, neurodegenerative diseases in China, some East Asian and Southeast Asian countries. It was shown that G. pentaphyllum and gypenosides had anti-obesity and cholesterol-lowering effects too. However, its main active ingredients are still unclear. AIMS: The objective of this study was to compare the effects of gypenosides before and after heat-processing on high fat obese mice, and to analyze the function of G. pentaphyllum saponin via network pharmacology and molecular docking. METHODS: The leaves of G. pentaphyllum were heat processed at 120 °C for 3 h to obtain heat-processed G. pentaphyllum. Gypenosides (Gyp) and heat-processed gypenosides (HGyp) were prepared by resin HP-20 chromatography and analyzed using LC-MS from the extracts of G. pentaphyllum before and after heat-processing, respectively. Obesity model was made with high fat diet (HFD). Gyp and HGyp were administrated at 100 mg/kg for 12 weeks in HFD obese mice and the body weight, energy intake, and levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL) were compared. HGyp was administrated at a dose of 50,100,200 mg/kg for 12 weeks in HFD obese mice and the perirenal adipose, epididymal adipose, abdominal adipose, shoulder brown adipose, inguinal adipose were measured. Moreover, the potential targets, hub genes and pathways of damulin A, damulin B, gypenoside L, gypenoside LI for treating Mets were screened out via network pharmacology. According to the results of network pharmacology, core targets of treating Mets were docking with damulin A, gypenoside L, damulin B, gypenoside LI via molecular docking. RESULTS: HGyp showed stronger effects on body weight loss and lipid-lowering in obese mice than Gyp. The contents of gypenoside L, gypenoside LI, damulin A and damulin B of G. pentaphyllum were increased by heat-processing. HGyp significantly decreased the body weight, calorie intake, and levels of TC, TG, LDL, HDL on the obese mice. It up-regulated PPARα and PPARγ in the liver tissues. HGyp reduced significantly the size of adipocytes in inguinal, abdominal, epididymal adipose and increased the proportion of interscapular brown fat. Network pharmacology results showed that 21 potential targets and 12 related-pathways were screened out. HMGCR, ACE, LIPC, LIPG, PPARα PPARδ, PPARγ were the core targets of HGyp against lipid metabolism by molecular docking. The putative functional targets of HGyp may be modulated by AGE-RAGE, TNF, glycerolipid metabolism, lipid and atherosclerosis, cholesterol metabolism, PPAR, fat digestion and absorption, cell adhesion molecules signaling pathway. CONCLUSIONS: Gyp and HGyp are valuable for inhibition obesity, lipid-lowering, metabolic regulation. Especially, the effect of HGyp is better than that of Gyp.

Anatomical and Chemical Characterization of Ulmus Species from South Korea.

Ulmus species (Ulmaceae) are large deciduous trees distributed throughout Korea. Although their root and stem bark have been used to treat gastrointestinal diseases and wounds in folk medicine, commercial products are consumed without any standardization. Therefore, we examined anatomical and chemical differences among five Ulmus species in South Korea. Transverse sections of leaf, stem, and root barks were examined under a microscope to elucidate anatomical differences. Stem and root bark exhibited characteristic medullary ray and secretary canal size. Leaf surface, petiole, and midrib exhibited characteristic inner morphologies including stomatal size, parenchyma, and epidermal cell diameter, as well as ratio of vascular bundle thickness to diameter among the samples. Orthogonal projections to latent structures discriminant analysis of anatomical data efficiently differentiated the five species. To evaluate chemical differences among the five species, we quantified (-)-catechin, (-)-catechin-7-O-ß-D-apiofuranoside, (-)-catechin-7-O-α-L-rhamnopyranoside, (-)-catechin-7-O-ß-D-xylopyranoside, (-)-catechin-7-O-ß-D-glucopyranoside, and (-)-catechin-5-O-ß-D-apiofuranoside using high-performance liquid chromatography with a diode-array detector. (-)-Catechin-7-O-ß-D-apiofuranoside content was the highest among all compounds in all species, and (-)-catechin-7-O-α-L-rhamnopyranoside content was characteristically the highest in Ulmus parvifolia among the five species. Overall, the Ulmus species tested was able to be clearly distinguished on the basis of anatomy and chemical composition, which may be used as scientific criteria for appropriate identification and standard establishment for commercialization of these species.

[Simultaneous quantitative analysis of nine saponins in Gynostemma pentaphyllum before and after heat processing based on UPLC-Q-Trap-MS].

Heat-processed Gynostemma pentaphyllum has strong biological activity, and saponins are the main components. To investigate the changes of saponins in G. pentaphyllum before and after heat processing, the present study determined and analyzed the content of nine saponins in G. pentaphyllum from Zhangzhou of Fujian and Jinxiu of Guangxi by ultra-high performance liquid chromatography with quadrupole ion-trap mass spectrometry(UPLC-Q-Trap-MS). The separation of the analytes was performed on an ACQUITY UPLC BEH C_(18) column(2.1 mm×50 mm, 1.7 µm) at 30 ℃, with acetonitrile and 0.1% formic acid in water as the mobile phase by gradient elution, and the flow rate was 0.3 mL·min~(-1). Quantitative analysis was performed using electrospray ionization source(ESI) in the multiple reaction-monitoring(MRM) mode. The results showed that the content of saponins with biological activities increased after heat processing. Specifically, gypenoside L, gypenoside LI, damulin A, damulin B, ginsenoside Rg_3(S), and ginsenoside Rg_3(R) in G. pentaphyllum produced in Zhangzhou of Fujian increased by 7.369, 8.289, 12.155, 7.587, 0.929, and 1.068 µg·g~(-1), respectively, while the content of ginsenoside Rd, gypenoside LVI, and gypenoside XLVI, which were abundant in the raw materials, decreased by 0.779, 19.37, and 9.19 µg·g~(-1), respectively. The content of gypenoside L, gypenoside LI, damulin A, damulin B, ginsenoside Rg_3(S), and ginsenoside Rg_3(R) in G. pentaphyllum produced in Jinxiu of Guangxi increased by 0.100, 0.161, 0.317, 0.228, 3.280, and 3.395 µg·g~(-1), respectively, while the content of ginsenoside Rd, gypenoside LVI, and gypenoside XLVI in the raw materials was reduced by 1.661, 0.014, and 0.010 µg·g~(-1), respectively. The results suggest that heat processing is an effective way to transform rare gypenosides. Furthermore, it is found that there are great differences in the content of gypenosides in different regions.

Uncovering the anti-NSCLC effects and mechanisms of gypenosides by metabolomics and network pharmacology analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Lung cancer is the chief reason of cancer death worldwide, and non-small cell lung cancer (NSCLC) make up the majority of lung cancers. Gypenosides are the main active constituents from Gynostemma pentaphyllum. Previous studies showed that they were used to remedy many cancers. The effect of gypenosides on NSCLC has never been studied from the perspective of network pharmacology and metabolomics. The mechanism is still not clear and remains to be explored. AIM OF THE STUDY: To explore the anti-NSCLC activity and mechanism of gypenosides in A549 cells. MATERIAL/METHODS: Gypenosides of G. pentaphyllum were detected by HPLC-MS. The cytotoxicity was detected by MTT assay. The migration, cell cycle and apoptosis of gypenosides were studied by wound healing assay, JC-1 assay and flow cytometry. The mechanism of gypenosides on NSCLC was studied by metabolomics and network pharmacology. Some key proteins and pathways were further confirmed by Western blot. RESULTS: Eleven gypenosides were detected by HPLC-MS. Gypenosides could suppress the proliferation of A549 cells, inhibit the migration of A549 cells, induce apoptosis and arrest cell cycle in G0/G1 phase. Metabolomics and network pharmacology approach revealed that gypenosides might affect 17 metabolite related proteins by acting on 9 candidate targets (STAT3, VEGFA, EGFR, MMP9, IL2, TYMS, FGF2, HPSE, LGALS3), thus resulting in the changes of two metabolites (uridine 5'-monophosphate, D-4'-Phosphopantothenate) and two metabolic pathways (pyrimidine metabolism; pantothenate and CoA biosynthesis). Western blotting indicated that gypenosides might inhibit A549 cells through MMP9, STAT3 and TYMS to indirectly affect the pathways of pyrimidine metabolism, pantothenate and CoA biosynthesis. CONCLUSIONS: This study revealed that metabolomics combined with network pharmacology was conducive to understand the anti-NSCLC mechanism of gypenosides.

[A novel dammarane-type saponin from Gynostemma pentaphyllum and its neuroprotective effect].

One new and two known dammarane-type saponins were isolated from the leaves of Gynostemma pentaphyllum using various chromatographic methods. Their structures were identified by HR-ESI-MS,~( 1)H-NMR, ~(13)C-NMR, 2 D-NMR spectra as 2α,3ß,12ß,20,24(S)-tetrahdroxydammar-25-en-3-O-[ß-D-glucopyranosyl(1→2)-ß-D-glucopyranosyl]-20-O-ß-D-xylopyranosyl(1→6)-ß-D-glucopyranoside(1, a new compound, namely gypenoside J5) and 2α,3ß,12ß,20,24(R)-tetrahdroxydammar-25-en-3-O-[ß-D-glucopyranosyl(1→2)-ß-D-glucopyranosyl]-20-O-ß-D-xylopyranosyl(1→6)-ß-D-glucopyranoside(2) and 2α,3ß,12ß,20-tetrahydroxy-25-hydroperoxy-dammar-23-en-3-O-[ß-D-glucopyranosyl(1→2)][ß-D-glucopyranosyl]-20-O-[ß-D-xylopyranosyl(1→6)]-ß-D-glucopy-ranoside(3), respectively. Compounds 1 and 2 were a pair of C-24 epimers. All compounds showed weak cytotoxicity agxinst H1299, HepG2, PC-3, SH-SY5 Y cancer cell lines. However, they exerted protective effect against SH-SY5 Y cellular damage induced by H_2O_2 dose-dependently, of which compound 1 displayed the strongest antioxidant effect. The present study suggested that G. pentaphyllum has antioxidative potential and the saponins from G. pentaphyllum are considered as the active compounds with neuroprotecitve effect.

Gypenoside LI arrests the cell cycle of breast cancer in G0/G1 phase by down-regulating E2F1.

ETHNOPHARMACOLOGICAL RELEVANCE: Gynostemma pentaphyllum (Thunb.) Makino, a traditional medicine in China, has been widely used for the treatment of various diseases. Gypenoside LI (Gyp LI) is a major constituent from steamed G. pentaphyllum. Previous studies have shown that gypnenoside LI possess inhibitory effect on the growth of many cancer cells. However, its pharmacological effect in breast cancer and the mechanism have not been reported yet. AIM OF THE STUDY: To investigate the anti-breast cancer activity of gypenoside LI and underlying mechanisms of gypenoside LI in MDA-MB-231 and MCF-7 cells. MATERIAL/METHODS: The cytotoxicity of gypenoside LI was determined by MTT, colony-formation and three-dimensional spheroid assay. The migration, cell apoptosis and the cell cycle were investigated through cell morphology observation, flow cytometry analysis and key proteins detection. The anticancer mechanisms of gypenoside LI were detected by RNA sequencing (RNA-seq) and Gene Set Enrichment Analysis (GSEA) transcriptome analysis. RESULTS: Gypenoside LI inhibited cell proliferation, migration, induced cell apoptosis and cell cycle arrest. Gypenoside LI arrested cell cycle at G0/G1 phase by regulating E2F1. It also inhibited tumor proliferation by regulating the expression of ERCC6L. Interestingly, we found that E2F1 siRNA also down-regulated the expression of ERCC6L. Gypenoside LI showed potential anti-breast cancer cells activity, especially on triple-negative breast cancer cells. CONCLUSIONS: These data indicate that gypenoside LI could inhibit human breast cancer cells through inhibiting proliferation and migration, inducing apoptosis, arresting cell cycle at G0/G1 phase by regulating E2F1. It could be used as potential multi-target chemopreventive agents for cancer.

Mechanism of gypenosides of Gynostemma pentaphyllum inducing apoptosis of renal cell carcinoma by PI3K/AKT/mTOR pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Gynostemma pentaphyllum (Thunb.) Makino is a traditional medicine commonly used in China, East Asia and Southeast Asia. In clinic, it is mainly used for hyperlipidemia and antitumor. Its antitumor activity was first recorded in "Illustrated Catalogue of Plants". Gypenosides were the main active ingredients of G. pentaphyllum. The anticancer activity of gypenosides in vivo and in vitro had been widely reported. However, the mechanism of gypenosides in renal cell carcinoma (RCC) still unclear. AIM OF THE STUDY: In this study, we tried to investigate the active constituents from G. pentaphyllum and potential mechanisms in RCC treatment through network pharmacology and in vitro experiments. MATERIAL/METHODS: Active compounds and their targets were evaluated and screened through TCMSP and Swiss Target Prediction database. Notably, nine preliminary screened components obtained from database were identified by LC-MS and LC-MS/MS. The targets associated with RCC were obtained from OMIM, TTD and GeneCards database. The PPI network and active component/target/pathway networks were constructed to identify the potential drug targets using String database and Cytoscape software. The functions and pathways of targets were analyzed through DAVID database. Finally, AutoDockTools 1.5.6 was used for molecular docking to assess the binding ability between compounds and targets. To support our prediction, we then explore the antitumor effect and mechanism of gypenosides by vitro experiments. CCK8 and flow cytometry were performed to evaluate cell death treated with gypenosides. Quantitative real-time PCR and Western blot were conducted to detect the changes of PI3K/AKT/mTOR signaling pathway. RESULTS: Nine saponins and 68 targets have been screened. The hub targets covered PIK3CA, VEGFA, STAT3, JAK2, CCND1 and MAPK3. Enrichment analysis showed that the pathways mainly contained PI3K/Akt/mTOR, HIF-1, TNF, JAK-STAT and MAPK signaling pathways. Gypenosides extracted from G. pentaphyllum showed strong activity against 786-O and Caki-1 cells, and cell apoptosis were detected through Annexin V/PI dual staining assay. RT-qPCR showed that gypenosides downregulated the levels of PIK3CA, Akt and mTOR in Caki-1 and 786-O cells. Mechanistically, gypenosides induced apoptosis of RCC cells through regulating PI3K/Akt/mTOR signaling pathway which was implemented though decreasing the phosphorylation level of Akt and mTOR. CONCLUSIONS: Gypenosides induced apoptosis of RCC cells by modulating PI3K/Akt/mTOR signaling pathway.

Monomer gypenoside LI from Gynostemma pentaphyllum inhibits cell proliferation and upregulates expression of miR-128-3p in melanoma cells.

Gypenosides have anticancer activity against many cancers. Gypenoside LI is a gypenoside monomer from Gynostemma pentaphyllum, its pharmacological functions in melanoma have not been reported. In this study, we found that gypenoside LI had a potent cytotoxic effect on melanoma cells. Gypenoside LI can induce intrinsic apoptosis along with S phase arrest. Furthermore, gypenoside LI inhibited the colony formation ability of melanoma through inhibition of the Wnt/ß-catenin signaling pathway. Interestingly, we also found that gypenoside LI can induce the upregulation of the tumor suppressor miR-128-3p during melanoma apoptosis. In contrast, gypenoside LI induced apoptosis, cell cycle arrest, and inhibition of the Wnt/ß-catenin signaling pathway, which were abolished by overexpression of the miR-128-3p inhibitor in A375 cells. Taken together, these results showed that gypenoside LI could inhibit human melanoma cells through inducing apoptosis, arresting cell cycle at the S phase and suppressing the Wnt/ß-catenin signaling pathway in a miR-128-3p dependent manner.

Novel dammarane-type saponins from Gynostemma pentaphyllum and their neuroprotective effect.

Three novel dammarane-type saponins, 2α,3ß,12ß,20(S),24(S)-pentahydroxydammar-25-ene-3-O-ß-D-glucopyranosyl-(1→2)-ß-D-glucopyranosyl-20-O-ß-D-glucopyranoside (1, namely gypenoside J1), 2α,3ß,12ß,20(S),25-pentahydroxydammar-23-ene-3-O-ß-D-glucopyranosyl-(1→2)-ß-D-glucopyranosyl-20-O-ß-D-glucopyranoside (2, namely gypenoside J2) and 2α,3ß,12ß,20(S)-tetrahydroxydammar-25-en-24-one-3-O-ß-D-glucopyranosyl-(1→2)-ß-D-glucopyranosyl-20-O-ß-D-xylopyranosyl-(1→6)-ß-D-glucopyranoside (3, namely gypenoside J3) along with one known gypenoside (gypenoside LVII) were isolated from the aerial parts of G. pentaphyllum using various chromatographic methods. Their structures were elucidated on the basis of IR, 1D- (1H and 13C), 2D-NMR spectroscopy (HSQC, HMBC and COSY), and mass spectrometry (ESI-MS/MS). Their activity was tested using CCK-8 assay. These four compounds showed little anti-cancer activity with IC50 values more than 100 µM against four types of human cancer lines. The effects of them against H2O2-induced oxidative stress in human neuroblastoma SH-SY5Y cells were evaluated and they all showed potential neuroprotective effects with 3.64-18.16% higher cell viability than the H2O2-induced model group.

[Protective effects of flavonoids from Gynostemma pentaphyllum on oxidative damage in LLC-PK1 cells].

Four flavonoids were isolated from Gynostemma pentaphyllum by chromatography methods and their structures were identified by MS and NMR spectra data as quercetin-3-O-( 2″,6″-di-α-L-rhamnosyl)-ß-D-galactopyranoside( 1),quercetin-3-O-( 2″,6″-di-α-L-rhamnosyl)-ß-D-glucopyranoside( 2),quercetin-3-O-( 2″-α-L-rhamnosyl)-ß-D-galactopyranoside( 3),and quercetin-3-O-( 2″-α-L-rhamnosyl)-ß-D-glucopyranoside( 4). Among them,compounds 1-3 were obtained from the Cucurbitaceae family for the first time.The four flavonoids showed potent antioxidant effects against the DPPH,·OH and ■radicals in vitro,especially for DPPH radical scavenging activity with the IC50 values of 71. 4,29. 5,48. 3 and 79. 2 µmol·L~(-1),respectively. Moreover,the four flavonoids displayed strong cytoprotection against AAPH-induced oxidative damage in LLC-PK1 cells by suppressing the increase of malondialdehyde( MDA) and the decrease of the superoxide dismutase( SOD) and glutathione( GSH). Since further research is needed to prove its efficacy in vivo and clinical trial,the study may provide four potential antioxidants from G. pentaphyllum.

Inhibitory Effect of Damulin B from Gynostemma pentaphyllum on Human Lung Cancer Cells.

Damulin B, a dammarane-type saponin from steamed Gynostemma pentaphyllum, exhibits the strongest activity against human lung carcinoma A549 cells among the isolated active saponins. In this study, the structure-activity relationship of a series of saponin compounds was discussed. The inhibitory effect of damulin B on human lung cancer A549 and H1299 cells was investigated from apoptosis, cell cycle, and migration aspects. In vitro, human lung cancer cells were more susceptible to damulin B treatment than human normal fibroblasts. Damulin B exhibited a strong cytotoxic effect, as evidenced by the increase of apoptosis rate, reduction of mitochondrial membrane potential (MMP), generation of reactive oxygen species, and G0/G1 phase arrest. Furthermore, damulin B activated the following: both intrinsic and extrinsic apoptosis pathways along with early G1 phase arrest via the upregulation of the Bax, Bid, tBid, cleaved caspase-8, and p53 expression levels; downregulation of the procaspase-8/-9, CDK4, CDK6, and cyclin D1 expression levels; and more release of cytochrome c in the cytoplasm. In addition, antimigratory activities and suppressive effects on metastasis-related factors, such as MMP-2 and MMP-9, accompanied by the upregulation of IL-24 were revealed. Altogether, the results proved that damulin B could inhibit human lung cancer cells by inducing apoptosis, blocking the cell cycle at early G0/G1 phase and suppressing the migration. Hence, damulin B has potential therapeutic efficacy against lung cancer.

[Effects of flavonoids from Gynostemma pentaphyllum on A549 cells damaged by hydrogen peroxide].

This study focuses on the therapeutical effect of flavonoids from Gynostemma pentaphyllum on human lung carcinoma A549 cells induced by H2O2 oxidative stress and its possible mechanisms. The oxidative damage model was established using different concentrations H2O2 to induce A549 cell for different hours, and then treated with the flavonoids for 10 hours. The effects of flavonoids from G. pentaphyllum on cell viability of A549 cell damaged by H2O2 were detected by MTT assay. The contents of ROS were detected by DCFH-DA fluorescent probe method via flow cytometer. The contents of MDA, SOD and GSH were detected by TBA，NBT and DTNB-linked colorimetry assay, respectively. Expressions levels of Nrf2, NQO1 and HO-1 in A549 cells were evaluated by Western blot. The results showed that the cell activity was decreasing with the rise of H2O2 concentration within the range of 200-700 µmol·L⁻¹. The cell viability was 60.4% after treated with 500 µmol·L⁻¹H2O2 for 10 h, so it was chosen to be as an oxidant stress model. Compared with normal group，the contents of SOD, GSH and HO-1 expressions were lower after damaged with H2O2. On the contrary, the contents of ROS and MDA expressions were increased. Compared with model group, the contents of SOD, GSH and the expressions of Nrf2, NQO1 and HO-1 were increased after treated with flavonoids from G. pentaphyllum. The above results demonstrate that flavonoids from G. pentaphyllum may attenuate the effect of H2O2-induced oxidative stress on A549 cell by resisting oxidation. The finding may provide a biological evidence for the application of the G. pentaphyllum to fight the oxidative stress related diseases.

The inhibitory effect of gypenoside stereoisomers, gypenoside L and gypenoside LI, isolated from Gynostemma pentaphyllum on the growth of human lung cancer A549 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Gypenosides are major constituents in Gynostemma pentaphyllum (Thunb.) Makino. Previous studies have shown that gypenosides isolated from G. pentaphyllum possess inhibitory effect on the growth of cancer cells, especially A549 cells, with structure-activity relationship (SAR). However, the underlying mechanism of gypenoside-induced A549 cell death remains to be clarified. AIM OF THE STUDY: To further investigate SAR and the underlying mechanism of gypenosides in A549 cells. MATERIALS AND METHODS: Gypenosides were isolated from G. pentaphyllum using chromatography methods and identified using MS and NMR data. The cytotoxicity was determined with CCK-8 assay. The effects of gypenosides on apoptosis, cell cycle and migration were investigated through cell morphology observation, flow cytometry analysis and key proteins detection. RESULTS: Three gypenosides, 2α,3ß,12ß,20(S)-tetrahydroxydammar-24-ene-3-O-ß-D-glucopyranoside-20-O-ß-D-glucopyranoside, gypenoside L and gypenoside LI were isolated from G. pentaphyllum. Gypenoside stereoisomers, gypenoside L (S configuration at C20) and gypenoside LI (R configuration at C20) showed stronger activity against A549 cells. Furthermore, both induced A549 cell apoptosis through intrinsic and extrinsic pathways evidenced by reducing mitochondrial membrane potential (MMP), generating reactive oxygen species (ROS), releasing more cytochrome c and down-regulating procaspase 8. However, gypenoside L blocked A549 cells in G0/G1, while gypenoside LI induced G2/M arrest, which was further verified by different expression of CDK1, CDK2 and CDK4. In addition, both inhibited A549 cell migration, which was evidenced by down-regulation of MMP-2/9 as well as scratch wound assay and transwell assay. CONCLUSION: C20 of gypenoside played an important role in A549 cell cytotoxicity and gypenoside stereoisomers could be used as potential multi-target chemopreventive agents for cancer.

Determination of eurycomanone in rat plasma using hydrophilic interaction liquid chromatography-tandem mass spectrometry for pharmacokinetic study.

In this study, a rapid, sensitive, and reliable hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method for the determination of eurycomanone in rat plasma was developed and validated. Plasma samples were pretreated with a protein precipitation method and quercitrin was used as an internal standard (IS). A HILIC silica column (2.1 × 100 mm, 3 µm) was used for hydrophilic-based chromatographic separation, using the mobile phase of 0.1% formic acid with acetonitrile in gradient elution at a flow rate of 0.25 mL/min. Precursor-product ion pairs for multiple-reaction monitoring were m/z 409.1 → 391.0 for eurycomanone and m/z 449.1 → 303.0 for IS. The linear range was 2-120 ng/mL. The intra- and inter-day accuracies were between 95.5 and 103.4% with a precision of <4.2%. The developed method was successfully applied to the pharmacokinetic analysis of eurycomanone in rat plasma after oral dosing with pure compound and E. longifolia extract. The Cmax and AUC0-t , respectively, were 40.43 ± 16.08 ng/mL and 161.09 ± 37.63 ng h/mL for 10 mg/kg eurycomanone, and 9.90 ± 3.97 ng/mL and 37.15 ± 6.80 ng h/mL for E. longifolia extract (2 mg/kg as eurycomanone). The pharmacokinetic results were comparable with each other, based on the dose as eurycomanone.

A new dammarane-type saponin from Gynostemma pentaphyllum induces apoptosis in A549 human lung carcinoma cells.

Gynostemma pentaphyllum has been widely used as a traditional herb for its antioxidant and immunostimulatory activities. We have previously reported several useful dammarane-type saponins with cytotoxicity against A549 human lung cancer cells from heat-processed G. pentaphyllum. In this study, a new dammarane-type saponin, 20(S)-2α,3ß,12ß-tetrahydroxydammar-3-O-ß-d-glucopyranoside (namely gypenoside Jh1), was isolated from the ethanol extract of heat-processed G. pentaphyllum using column chromatography and semi-preparative HPLC. Gypenoside Jh1 exhibited strong cytotoxicity against A549 cells in a concentration-dependent manner, which was associated with apoptotic cell death characterized by morphological changes, Hoechst 33258 nuclear staining, Annexin V and propidium iodide binding and mitochondrial potentials assay. Quantitative analysis using flow cytometry also showed that the proportion of apoptotic cells was increased after gypenoside Jh1 treatment. These findings indicated that gypenoside Jh1 showed antiproliferative effects on A549 cells and mitochondrial-dependent pathway is involved in gypenoside Jh1-induced apoptosis.

Metabolite profiling of gypenoside LVI in rat after oral and intravenous administration.

Gypenoside LVI, one of the major bioactive triterpene saponins in Gynostemma pentaphyllum, has been regarded as a potential and promising lead drug for anti-tumor strategy. To better understand the pharmacological activities of the component, an investigation of its in vivo metabolism is important and necessary. In the present study, a liquid chromatography-ion trap time of flight tandem mass spectrometry has been utilized to discover and identify the metabolites of gypenoside LVI in rat urine after oral and intravenous administration. Negative electrospray ionisation mass spectrometry was used to discern gypenoside LVI and its possible metabolites in urine samples. As a result, after oral and intravenous administration, eight and six metabolites together with gypenoside LVI were detected and identified in rat urine, respectively. As metabolites of gypenoside LVI, they have never been reported before. Deglycosylation and dehydration were found to be the major metabolic processes of gypenoside LVI in rat.

Protective effects of resveratrol oligomers from Vitis amurensis against sodium nitroprusside-induced neurotoxicity in human neuroblastoma SH-SY5Y cells.

Nitric oxide (NO) induces apoptosis in neuronal cells, and has been implicating in a variety of neuronal pathological process. Thus, there is much interest in identifying natural substances which have protective effects against damage induced by nitrosative stress. The roots of Vitis amurensis have been used as traditional medicine and contain structurally diverse resveratrol oligomers with various biological activities. However, there have been few studies on the protective effect of resveratrol oligomers against neurotoxic reactive nitrogen species. In this study, we evaluated the protective effects of two resveratrol oligomers from V. amurensis, vitisin A and heyneanol A, against NO-induced toxicity. Additionally, their antioxidant activities were determined by measuring NO and hydroxyl radical scavenging ability. Both vitisin A and heyneanol A reduced cell death and DNA fragmentation induced by sodium nitroprusside in SH-SY5Y cells. The present study indicates that radical scavenging activities of vitisin A and heyneanol A contribute to protecting neuronal cells against nitrosative stress.

Metabolic profiling of Gynostemma pentaphyllum extract in rat serum, urine and faeces after oral administration.

Folk drug Gynostemma pentaphyllum (Thunb.) Makino contains many biologically active phytochemicals which have been demonstrated to be effective against chronic diseases. As in vivo anti-tumor experiments of G. pentaphyllum extract (GP) show much stronger antitumor activities than in vitro, it is important and necessary to understand the metabolic study of GP. A sensitive and specific U-HPLC-MS method was utilized for the first time to rapidly identify gypenosides and its possible metabolites in rat serum, urine, and faeces after oral administration. Solid phase extraction was utilized in the sample preparation. Negative Electrospray ionisation (ESI) mass spectrometry was used to discern gypenosides and its possible metabolites in rat samples. As a result, after oral administration, a total of seven metabolites of G. pentaphyllum extract were assigned, two from the rat serum and seven both from the rat urine and faeces. As metabolites of G. pentaphyllum extract, all of them have never been reported before.