Dammarane triterpenoids with rare skeletons from Gynostemma pentaphyllum and their cytotoxic activities.

Three unreported dammarane-type triterpenoids with rare skeletons (1-3), along with one undescribed gypenoside (4), were isolated from the aerial parts of Gynostemma pentaphyllum using diverse chromatographic materials and pre-HPLC. Their structures were elucidated on the basis of spectroscopic and spectrometric data, while the absolute configurations of 1-3 were assessed via electronic circular dichroism (ECD) analyses. Notably, compounds 1-3 possess a 3,19-hemiketal bridge in the A ring. Saponin 4 possesses an unreported 20,25-oxa structural moiety. Their antiproliferative effects against HepG2, MCF-7, and DU145 cell lines were screened. Compounds 1-3 displayed moderate cytotoxicity with IC50 values ranging from 13.7 ± 0.2 to 32.0 ± 1.7 µM.

Triterpenoids from the genus Gynostemma: Chemistry and pharmacological activities.

ETHNOPHARMACOLOGICAL RELEVANCE: G. pentaphyllum, also known as Jiao-Gu-Lan, has been used traditionally as folk remedies for many diseases, including diabetes mellitus, metabolic syndrome, aging, and neurodegenerative diseases in China and some countries in East and Southeast Asia. It is considered as an "immortality herb" in Guizhou Province, because it was consumed regularly by the elderly native inhabitants. Other species of the same genus Gynostemma such as G. longipes and G. laxum have been used as alternatives to G. pentaphyllum in ethno-medicine in Vietnam and other Asian countries. AIM OF THE REVIEW: The review aims to summarize up-to-date study results on Gynostemma species, including traditional usage, phytochemical profile, pharmacological activities, and toxicological studies, in order to suggest future research orientation and therapeutic applications on acute and chronic diseases. MATERIALS AND METHODS: The relevant literature on the genus Gynostemma was gathered from secondary databases (Web of Science and PubMed), books, and official websites. The latest literature cited in this review was published in February 2020. RESULTS: The genus Gynostemma has been widely used in traditional medicine, mainly for treatment of diabetes, hypertension, obesity, and hepatosteatosis. To date, 328 dammarane-type saponins were isolated and structurally elucidated from Gynostemma species. Crude extracts, saponin-rich fractions (gypenosides), and pure compounds were reported to show a wide range of pharmacological activities in both in vitro and in vivo experiments. The most notable pharmacological effects were anti-cancer, cardioprotective, hepatoprotective, neuroprotective, anti-diabetic, anti-obesity, and anti-inflammatory activities. Toxicological studies were conducted only on G. pentaphyllum, showing that the plant extracts were relatively safe in both acute and long-term toxicity experiments at the given dosage while no toxicological studies were reported for the other species. CONCLUSIONS: The review summarizes current studies on traditional uses, phytochemistry, biological properties, and toxicology of medicinal Gynostemma species. Till now, the majority of publications still focused only on G. pentaphyllum. However, the promising preliminary data of other Gynostemma species indicated the research potential of this genus, both in phytochemical and pharmacological aspects. Furthermore, clinical data are required to evaluate the efficacy and undesired effects of crude extracts, standard saponin fractions, and pure compounds prepared from Gynostemma medicinal plants.

Antiviral activity of furanocoumarins isolated from Angelica dahurica against influenza a viruses H1N1 and H9N2.

ETHNOPHARMACOLOGICAL RELEVANCE: Angelica dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav. (Umbelliferae family) is an herbaceous, perennial plant native to northern and eastern Asia. The root of A. dahurica has traditionally been used under the name "Bai Zhi" as a medicinal plant for colds, dizziness, ulcers, and rheumatism. Moreover, it is also an important ingredient of various prescriptions, such as Gumiganghwal-Tang, for the common cold and influenza. AIM OF THE STUDY: Even though various biological activities of the root of A. dahurica have been reported along with its chemical components, the detailed mechanism of how it exerts anti-influenza activity at the compound level has not been studied. Therefore, we investigated the anti-influenza properties of furanocoumarins purified by bioactivity-guided isolation. MATERIALS AND METHODS: Bioactivity-guided isolation from a 70% EtOH extract of the root of A. dahurica was performed to produce four active furanocoumarins. The inhibition of cytopathic effects (CPEs) was evaluated to ascertain the antiviral activity of these compounds against influenza A (H1N1 and H9N2) viruses. The most potent compound was subjected to detailed mechanistic studies such as the inhibition of viral protein synthesis, CPE inhibition in different phases of the viral replication cycle, neuraminidase (NA) inhibition, antiapoptotic activity using flow cytometry, and immunofluorescence. RESULTS: The bioactivity-guided isolation produced four active furanocoumarins, isoimperatorin (1), oxypeucedanin (2), oxypeucedanin hydrate (3) and imperatorin (4) from the n-BuOH fraction. Among them, compound 2 (followed by compounds 1, 4 and 3) showed a significant CPE inhibition effect, which was stronger than that of the positive control ribavirin, against both H1N1 and H9N2 with an EC50 (µM) of 5.98 ± 0.71 and 4.52 ± 0.39, respectively. Compound 2 inhibited the synthesis of NA and nucleoprotein (NP) in a dose-dependent manner. In the time course assays, the cytopathic effects of influenza A-infected MDCK cells were reduced by 80-90% when treated with compound 2 for 1 and 2 h after infection and declined drastically 3 h after infection. The level of viral NA and NP production was markedly reduced to less than 20% for both proteins in compound 2 (20 µM)-treated cells compared to untreated cells at 2 h after infection. In the molecular docking analysis, compound 2 showed a stronger binding affinity for the C-terminus of polymerase acidic protein (PAC; -36.28 kcal/mol) than the other two polymerase subunits. Compound 2 also exerted an antiapoptotic effect on virus infected cells and significantly inhibited the mRNA expression of caspase-3 and Bax. CONCLUSION: Our results suggest that compound 2 might exert anti-influenza A activity via the inhibition of the early phase of the viral replication cycle, not direct neutralization of surface proteins, such as hemagglutinin and NA, and abnormal apoptosis induced by virus infection. Taken together, these findings suggest that furanocoumarins predominant in A. dahurica play a pivotal role in its antiviral activity. These findings can also explain the reasons for the ethnopharmacological uses of this plant as an important ingredient in many antiviral prescriptions in traditional Chinese medicine (TCM).

Antiviral Activities of Compounds Isolated from Pinus densiflora (Pine Tree) against the Influenza A Virus.

Pinus densiflora was screened in an ongoing project to discover anti-influenza candidates from natural products. An extensive phytochemical investigation provided 26 compounds, including two new megastigmane glycosides (1 and 2), 21 diterpenoids (3-23), and three flavonoids (24-26). The chemical structures were elucidated by a series of chemical reactions, including modified Mosher's analysis and various spectroscopic measurements such as LC/MS and 1D- and 2D-NMR. The anti-influenza A activities of all isolates were screened by cytopathic effect (CPE) inhibition assays and neuraminidase (NA) inhibition assays. Ten candidates were selected, and detailed mechanistic studies were performed by various assays, such as Western blot, immunofluorescence, real-time PCR and flow cytometry. Compound 5 exerted its antiviral activity not by direct neutralizing virion surface proteins, such as HA, but by inhibiting the expression of viral mRNA. In contrast, compound 24 showed NA inhibitory activity in a noncompetitive manner with little effect on viral mRNA expression. Interestingly, both compounds 5 and 24 were shown to inhibit nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in a dose-dependent manner. Taken together, these results provide not only the chemical profiling of P. densiflora but also anti-influenza A candidates.

12,23-Dione dammarane triterpenes from Gynostemma longipes and their muscle cell proliferation activities via activation of the AMPK pathway.

The aging population is growing rapidly around the world and there is also an increase in sarcopenia, which is characterized by decreased muscle mass, strength and function in the elderly population. AMP-activated protein kinase (AMPK) is an essential sensor and regulator of glucose, lipid and energy metabolism throughout the body. Previous studies have shown that AMPK pathway activation by regular exercise and appropriate dietary control have beneficial effects on skeletal muscle. In the process of searching for new AMPK activators from medicinal plants, we isolated and characterized eight new 12,23-dione dammarane triterpenoids (1-3 and 5-9), as well as one known gypentonoside A from Gynostemma longipes. When all isolates were tested for their AMPK activation activities, seven compounds (1 and 3-8) were significantly activated AMPK phosphorylation in mouse C2C12 skeletal muscle cell lines. Since G. longipes contained a significant amount of active compound 1 (over 2.08% per dried raw plant), it suggested the potential of this plant to be developed as a functional food or botanical drug that enhances muscle proliferation by activating AMPK signaling pathways.

Hydroxyoleoside-type seco-iridoids from Symplocos cochinchinensis and their insulin mimetic activity.

As part of an ongoing study of new insulin mimetic agents from medicinal plants, the 70% EtOH extract of Symplocos cochinchinensis was found to have a stimulatory effect on glucose uptake in 3T3-L1 adipocyte cells. The intensive targeted isolation of this active extract resulted in ten new hydroxyoleoside-type compounds conjugated with a phenolic acid and monoterpene (1-6 and 8-11), as well as four known compounds (7 and 12-14). The chemical structures of the new compounds were determined based on spectroscopic data analysis (1H and 13C NMR, HSQC, HMBC, NOESY and MS). The absolute configurations of the isolated compounds were determined by electronic circular dichroism (ECD) analysis of derivatives obtained after a series of reactions, such as those with dirhodium (ІІ) tetrakis (trifluoroacetate) and dimolybdenum (ІІ) tetraacetate. In vitro, compounds 3, 7 and 8 moderately increased the 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG) uptake level in differentiated 3T3-L1 adipocytes. For further studies, we evaluated their effects on the expression of glucose transporter-4 (GLUT4), its translocation, protein tyrosine phosphatase 1B (PTP1B) inhibition and expression of phosphorylated Akt. Our results strongly suggest that the traditional uses of this plant can be described as active constituents by hydroxyoleoside-type compounds.

Insulin Mimetic Activity of 3,4- Seco and Hexanordammarane Triterpenoids Isolated from Gynostemma longipes.

As part of ongoing research to find new antidiabetic agents from medicinal plants, the chemical composition of Gynostemma longipes, an ethnomedicinal plant used to treat type 2 diabetes mellitus by local communities in Vietnam, was investigated. Ten new dammarane triterpenes, including two 3,4- seco-dammarane analogues, secolongipegenins S1 and S2 (1 and 2), a 3,4- seco-hexanordammarane, secolongipegenin S3 (3), two hexanordammarane glycosides, longipenosides ND1 and ND2 (4 and 5), and five other dammarane glycosides, longipenosides GL1-GL5 (6-10), were isolated from a 70% EtOH extract of the whole G. longipes plant. The structures of the new compounds were elucidated using diverse spectroscopic methods. All of the isolates were evaluated for their stimulatory activities on glucose uptake in differentiated 3T3-L1 adipocyte cells using 2-[ N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose as a fluorescent-tagged glucose probe. The stimulant activities on glucose uptake by the test compounds were mediated via the activation of the AMPK pathway using differentiated mouse C2C12 skeletal myoblasts. Consequently, compounds 1, 2, and 4 enhanced glucose uptake and GLUT4 translocation significantly by regulating the AMPK signaling pathway.

Discrimination of different geographic varieties of Gymnema sylvestre, an anti-sweet plant used for the treatment of type 2 diabetes.

Gymnema sylvestre (Retz.) R.Br. ex Sm. (Asclepiadaceae) is a well-known Ayurvedic anti-sweet plant for the treatment of type 2 diabetes mellitus. Although it was previously proposed that G. sylvestre exhibits chemical variation based on geography, most research on G. sylvestre has used material originating from India. Morphological and anatomical descriptions, ITS1-5.8S-ITS2 DNA sequencing, and acid hydrolysis analyses showed that G. sylvestre samples from Vietnam are distinguishable from those of Indian origin and thus suggest a dissimilarity among G. sylvestre samples with different geographic distributions. An LC-MS-guided strategy targeting 3ß-glucuronide oleane-triterpenes in the Vietnamese G. sylvestre variety led to the isolation of four known compounds and nine previously undescribed compounds, named gymnemosides ND1-ND9. None of the isolated compounds were reported in the Indian sample, further supporting the geo-diversity of G. sylvestre. Three compounds, gymnemosides ND7-9, exerted significant stimulatory effects on the uptake of 2-NBDG in 3T3-L1 adipocyte cells and thus have potential as lead molecules for anti-diabetes agents.

Anti-influenza effect of the major flavonoids from Salvia plebeia R.Br. via inhibition of influenza H1N1 virus neuraminidase.

To determine the compounds responsible for its anti-influenza activities, we isolated the three flavonoids, 6-hydroxyluteolin 7-O-ß-d-glucoside (1), nepitrin (2), homoplantaginin (3) from the MeOH extract of Salvia plebeia R.Br. and identified them by comparing the spectroscopic data with that reported in the literature. The contents of the three flavonoids in the whole extract were 108.74 ± 0.95, 46.26 ± 2.19, and 69.35 ± 1.22 mg/g for 6-hydroxyluteolin 7-O-ß-d-glucoside, nepitrin, and homoplantaginin, respectively, which demonstrates that they are the major constituents of this plant. The three flavonoids were evaluated for their inhibitory activities against influenza virus H1N1 A/PR/9/34 neuraminidase and H1N1-induced cytopathic effect (CPE) on Madin-Darby canine kidney (MDCK) cells. Our results demonstrated the following arrangement for their anti-influenza activities: nepitrin (2) > 6-hydroxyluteolin 7-O-ß-d-glucoside (1) > homoplantaginin (3). The potent inhibitory activities of these flavonoids against influenza suggested their potential to be developed as novel anti-influenza drugs in the future.

PTP1B inhibitors from the seeds of Iris sanguinea and their insulin mimetic activities via AMPK and ACC phosphorylation.

To find PTP1B inhibitors from natural products, two new compounds (1 and 2), along with nine known compounds (3-11), were isolated from a methanol-soluble extract of Iris sanguinea seeds. The structures of compounds 1 and 2 were determined based on extensive spectroscopic data analysis including UV, IR, NMR, and MS. The IC50 value of compound 5 on protein tyrosine phosphatase 1B (PTP1B) inhibitory activity is 7.30±0.88µM with a little activity compared to the IC50 values of the tested positive compound. Compound 5 significantly enhanced glucose uptake and activation of pACC, pAMPK and partially Erk1/2 signaling. These results suggest that compound 5 from Iris sanguinea seeds are utilized as both PTP1B inhibitors and regulators of glucose uptake. These beneficial effects could be applied to treat metabolic diseases such as diabetes and obesity.

Antiviral escin derivatives from the seeds of Aesculus turbinata Blume (Japanese horse chestnut).

Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea and high fatality of piglets, influencing the swine industry. Japanese horse chestnut (seed of Aesculus turbinata) contains many saponin mixtures, called escins, and has been used for a long time as a traditional medicinal plant. Structure-activity relationship (SAR) studies on escins have revealed that acylations at C-21 and C-22 with angeloyl or tigloyl groups were important for their cytotoxic effects. However, the strong cytotoxicity of escins makes them hard to utilize for other diseases and to develop as nutraceuticals. In this research, we investigated whether escin derivatives 1-7 (including new compounds 2, 3, 5 and 6), without the angeloyl or tigloyl groups and with modified glycosidic linkages by hydrolysis, have PEDV inhibitory effects with less cytotoxicity. Compounds 1-7 had no cytotoxicity at 20µM on VERO cells, while compounds 8-10 showed strong cytotoxicity at similar concentrations on PEDV. Our results suggest that escin derivatives showed strong inhibitory activities on PEDV replication with lowered cytotoxicity. These studies propose a method to utilize Japanese horse chestnut for treating PEDV and to increase the diversity of its bioactive compounds.

Sesquiterpenoids with Various Carbocyclic Skeletons from the Flowers of Chrysanthemum indicum.

A phytochemical investigation of the flowers of Chrysanthemum indicum yielded sesquiterpenoids 1-25 with various carbocyclic skeletons, including 10 new (1-10) and 15 known (11-25) analogues. The structures were elucidated via their physical data, while the absolute configuration of compounds 6, 8, and 10 was assessed via electronic circular dichroism analysis. The evaluation of the effect of sesquiterpenoids on porcine epidemic diarrhea virus (PEDV) replication showed that compounds 1-5, 12, 14, 16, 17, 19, and 21 increased cell viability against cell death in PEDV-injected cells. Compounds 2, 12, and 17 were selected and investigated for their inhibition of proteins required for PEDV replication. Compounds 2 and 17 significantly reduced PEDV nucleocapsid and spike protein synthesis compared with azauridin as a positive control.

Anthraquinones from Morinda longissima and their insulin mimetic activities via AMP-activated protein kinase (AMPK) activation.

AMP-activated protein kinase (AMPK) activators are known to increase energy metabolism and to reduce body weight, as well as to improve glucose uptake. During for searching AMPK activators, a new anthraquinone, modasima A (10), along with eighteen known analogues (1-9 and 11-19) were isolated from an ethanol extract of the roots of Morinda longissima Y. Z. Ruan (Rubiaceae). Using the fluorescent tagged glucose analogues, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxy-D-glucose (2-NBDG), insulin mimetics were screened with compounds 1-19 in 3T3-L1 adipocytes. Among them, compounds 2, 8 and 10 enhanced significantly glucose uptake into adipocytes and up-regulated the phosphorylated AMPK (Thr172) whereas the glucose uptake enhancing activities of compounds 2, 8 and 10 were abrogated by treatment of compound C, an AMPK inhibitor. Taken together, these anthraquinones showed the potential action as insulin mimetic to improve glucose uptake via activation of AMPK.

Protein Tyrosine Phosphatase 1B Inhibitors from the Stems of Akebia quinata.

PTP1B deficiency in mouse mammary tumor virus (MMTV)-NeuNT transgenic mice inhibited the onset of MMTV-NeuNT-evoked breast cancer, while its overexpression was observed in breast cancer. Thus, PTP1B inhibitors are considered chemopreventative agents for breast cancer. As part of our program to find PTP1B inhibitors, one new diterpene glycoside (1) and 13 known compounds (2-14) were isolated from the methanol extract of the stems of Akebia quinata. All isolates were identified based on extensive spectroscopic data analysis, including UV, IR, NMR and MS. Compounds 2, 3, 6, 8 and 11 showed significant inhibitory effects on the PTP1B enzyme, with IC50 values ranging from 4.08 ± 1.09 to 21.80 ± 4.74 µM. PTP1B inhibitors also had concentration-dependent cytotoxic effects on breast cancer cell lines, such as MCF7, MDA-MB-231 and tamoxifen-resistant MCF7 (MCF7/TAMR) (IC50 values ranging from 0.84 ± 0.04 to 7.91 ± 0.39 µM). These results indicate that compounds 6 and 8 from Akebia quinata may be lead compounds acting as anti-breast cancer agents.

Antiviral phenolics from the leaves of Cleistocalyx operculatus.

During the screening program for anti-influenza agents from medicinal plants, the ethanolic extract of Cleistocalyx operculatus leaves was found to exhibit potential neuraminidase (NA) inhibitory activity. Bioassay-directed fractionation led to the isolation of two new acetophenones (1 and 2) and one new flavanone (3), along with six known compounds (4-9). The structures of all isolated compounds were elucidated using various spectroscopic methods and through comparison with the previous literature. Compounds 6 and 8 exhibited strong enzymatic inhibition on various neuraminidases from different influenza viruses, including H1N1, H9N2, novel H1N1, and oseltamivir-resistant novel H1N1 (H274Y mutation) expressed in HEK293 cells (IC50 values ranging from 5.07 ± 0.94 µM to 9.34 ± 2.52 µM, respectively). Kinetic experiments revealed the non-competitive inhibitory mode of both compounds 6 and 8. Furthermore, these flavonoids reduced the cytopathic effect of the H1N1 virus in MDCK cells. The present study suggests the potential of two flavonoids (6 and 8) as new lead compounds for the development of novel NA inhibitors in the future.

In vitro antituberculosis activity of diterpenoids from the Vietnamese medicinal plant Croton tonkinensis.

Diterpenoids from the Vietnamese medicinal plant Croton tonkinensis are rich in ent-kaurane, kaurane and the grayanane class and are valuable intermediate plant metabolites with different bioactivities. In this study, we report the antituberculosis activity of these diterpenoids against both susceptible and resistant strains of M. tuberculosis for the first time. All of the ent-kaurane, kaurane and grayanane diterpenoids showed high to moderate activity against Mycobacterium. The highest antituberculosis activity was observed for ent-1ß,7α,14ß-triacetoxykaur-16-en-15-one (cpp604), with MIC values of 0.78, 1.56 and 3.12-12.5 µg/ml against H37Ra, H37Rv and all other resistant strains of Mycobacterium tuberculosis examined. In addition, other ent-kaurane-type diterpenoids also showed very high activities against mycobacterium, including cpp609 (1.56 µg/ml), cpp610 (1.56 µg/ml), cpp601 (3.12-6.25 µg/ml), cpp602 (3.12-6.25 µg/ml), cpp607 (3.12-6.25 µg/ml) and cpp608 (3.12-6.25 µg/ml). From the structure-activity relationship, functional groups R3 and R5 of the ent-kaurane skeleton were found to modulate the antimycobacterial activity.

Oleanane triterpenes from the flowers of Camellia japonica inhibit porcine epidemic diarrhea virus (PEDV) replication.

Porcine epidemic diarrhea virus (PEDV) infections have resulted in a severe economic loss in the swine industry in many countries due to no effective treatment approach. Fifteen oleanane triterpenes (1-15), including nine new ones (1-4 and 10-14), were isolated from the flowers of Camellia japonica, and their molecular structures were determined by extensive spectroscopic methods. These compounds were evaluated for their antiviral activity against PEDV replication, and the structure-activity relationships (SARs) were discussed. Compounds 6, 9, 11, and 13 showed most potent inhibitory effects on PEDV replication. They were found to inhibit PEDV genes encoding GP6 nucleocapsid, GP2 spike, and GP5 membrane protein synthesis based on RT-PCR data. Western blot analysis also demonstrated their inhibitory effects on PEDV GP6 nucleocapsid and GP2 spike protein synthesis during viral replication. The present study suggested the potential of compounds 6, 9, 11, and 13 as promising scaffolds for treating PEDV infection via inhibiting viral replication.

Dammarane triterpenes as potential SIRT1 activators from the leaves of Panax ginseng.

During a search for SIRT1 activators originating in nature, three new dammarane triterpenes, 6α,20(S)-dihydroxydammar-3,12-dione-24-ene (1), 6α,20(S),24(S)-trihydroxydammar-3,12-dione-25-ene (2), and 6α,20(S),25-trihydroxydammar-3,12-dione-23-ene (3), as well as two known triterpenes, dammar-20(22),24-diene-3ß,6α,12ß-triol (4) and 20(S)-ginsenoside Rg3 (5), were isolated from Panax ginseng leaves. Compounds 1 and 3-5 showed potential as SIRT1 activators, as analyzed by in vitro enzyme-based SIRT1-NAD/NADH and SIRT1-p53 luciferase cell-based assays. They were also found to increase the level of NAD(+)/NADH ratio in HEK293 cells. This study presents a new class of chemical entities that may be able to be developed as SIRT1 activators for antiaging and treatment of age-associated diseases.