Cytochrome P450 Mining for Bufadienolide Diversification.

Bufadienolides are a class of steroids with a distinctive α-pyrone ring at C17, mostly produced by toads and consisting of over 100 orthologues. They exhibit potent cardiotonic and antitumor activities and are active ingredients of the traditional Chinese medicine Chansu and Cinobufacini. Direct extraction from toads is costly, and chemical synthesis is difficult, limiting the accessibility of active bufadienolides with diverse modifications and trace content. In this work, based on the transcriptome and genome analyses, using a yeast-based screening platform, we obtained eight cytochrome P450 (CYP) enzymes from toads, which catalyze the hydroxylation of bufalin and resibufogenin at different sites. Moreover, a reported fungal CYP enzyme Sth10 was found functioning in the modification of bufalin and resibufogenin at multiple sites. A total of 15 bufadienolides were produced and structurally identified, of which six were first discovered. All of the compounds were effective in inhibiting the proliferation of tumor cells, especially 19-hydroxy-bufalin (2) and 1ß-hydroxy-bufalin (3), which were generated from bufalin hydroxylation catalyzed by CYP46A35. The catalytic efficiency of CYP46A35 was improved about six times and its substrate diversity was expanded to progesterone and testosterone, the common precursors for steroid drugs, achieving their efficient and site-specific hydroxylation. These findings elucidate the key modification process in the synthesis of bufadienolides by toads and provide an effective way for the synthesis of unavailable bufadienolides with site-specific modification and active potentials.

Ent-labdane-type diterpene glycosides obtained from Rubus chingii Hu and their inhibitory effects on PDE5A activity.

In this study, 16 new ent-labdane-type diterpene glycosides, designated as goshonosides J1-J16 (1-16), along with nine previously known diterpene glycosides (17-25) were extracted from the fruits of Rubus chingii Hu. The structures of goshonosides J1-J16 were elucidated using various analytical techniques, such as nuclear magnetic resonance, electron capture detector ECD, high-resolution electrospray ionization mass spectrometry HREIMS, single-crystal X-ray diffraction, and hydrolysis. Furthermore, the isolates' efficacy in inhibiting the activity of phosphodiesterase type 5 A was evaluated. Goshonosides J1, J2, and G effectively inhibited the activity of the aforementioned enzyme (IC50 values: 6.15 ± 1.76, 3.27 ± 0.65, and 9.61 ± 2.36 µM, respectively). Our findings highlight the remarkable structural diversity of bioactive compounds in R. chingii Hu and offer insights into the use of this shrub.

Constituents of the fruits of Rubus chingii Hu and their neuroprotective effects on human neuroblastoma SH-SY5Y cells.

Rubi fructus (Rubus chingii Hu) is a fruit of Rubus genus and is used in medicine and food applications. In this study, eight new phenylpropanoids (1-8) and seven known compounds (9-15) were isolated from the dried fruits of Rubus chingii Hu, and their structures were characterized through high-resolution electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. Electronic circular dichroism (ECD) experiments were performed, and the results were compared with ECD spectra. Compound 3 was characterized through extensive single crystal X-ray diffraction. Evaluation of the neuroprotective pharmacological activities revealed that compounds 6, 7, 9, and 14 exerted protective effects against H2O2-induced neurotoxicity by reducing the reactive oxygen species levels at concentrations of 50 and 100 µM. Moreover, the three compounds 6, 9, and 14 significantly inhibited the expression of the Casp3 gene at a concentration of 50 µM. Compounds 7 and 9 effectively repressed the expression of the MYC gene. Compounds 6 and 9 obviously upregulated the ratio of Bcl2/Bax in SH-SY5Y cells and inhibited cell apoptosis. The study results can be used as a reference for the development of R. chingii products to realize their neuroprotective functions in the future.

Nine new sesquiterpenes from Curcuma wenyujin rhizomes.

Nine new sesquiterpenes wenyujinones A-I (1-9), along with ten known ones (10-19), were isolated from the rhizomes of Curcuma wenyujin Y. H. Chen et C. Ling (C. wenyujin). The chemical structures of compounds 1-9 were elucidated by NMR spectroscopic and mass spectrometric data, electronic circular dichroism (ECD) spectra analysis. Furthermore, all compounds were evaluated for the protective effects against hydrogen peroxide (H2O2)-induced injury in human hepatic L02 cells, for the inhibitory effects on nitric oxide (NO) production in RAW 264.7 cells, and for their cytotoxicity against three human cancer cell lines A549, HL60, and MCF7 in vitro. As a result, compounds 2, 4, 14 markedly weakened the oxidative damage induced by H2O2 in L02 cells via strengthening the cell viability.

Review of the traditional uses, phytochemistry, and pharmacology of Curcuma wenyujin Y. H. Chen et C. Ling.

ETHNOPHARMACOLOGICAL RELEVANCE: Curcuma wenyujin is a multifunctional medicinal plant belonging to the ginger family (Zingiberaceae). It has been used to treat blood stasis, promote the flow of qi, dredge the meridians, and relieve pain for more than 1500 years. Its raw rhizomes, steamed rhizomes, and steamed roots constitute three herbal medicines currently listed in the Chinese Pharmacopoeia: pian-jiang-huang (), wen-e-zhu () and wen-yu-jin (), respectively. AIM OF THE REVIEW: The aim of this review was to comprehensively summarize the traditional use, phytochemistry, and pharmacology of C. wenyujin in order to provide theoretical support for its further investigation and utilization. MATERIALS AND METHODS: Multiple databases (Scifinder, CNKI, Web of Science, PubMed, Google Scholar, and Baidu Scholar) were searched. Some information was also obtained from the literatures on traditional Chinese medicine. RESULTS: A total of 169 compounds have been isolated from C. wenyujin so far. Sesquiterpenoids are the major constituents and are crucial chemotaxonomic markers. Modern pharmacological studies and clinical trials have demonstrated that the extracts or active compounds from C. wenyujin have anti-inflammatory, antitumor, antioxidant, antibacterial, antiviral, and hepatoprotective properties. CONCLUSIONS: Until now, significant progress has been witnessed in phytochemistry and pharmacology of C. wenyujin. Some traditional uses of C. wenyujin have been supported by modern pharmacological studies. However, the establishment of quality control standards and additional clinical studies are warranted.

Indole alkaloid glycosides from Isatis tinctoria roots.

Isatindigoside A and B (1 - 2), two new indole alkaloid glycosides along with five known ones (3 - 7) were obtained from the roots of I. tinctoria. Their structures were determined as isatindigoside A (1), isatindigoside B (2), isatindosulfonicacid A 3-O-ß-D-glucopyranoside (3), indole-3-acetonitrile 6-O-ß-D-glucopyranoside (4), isatindigobisindoloside A (5), isatindigobisindoloside B (6) isatindigobisindoloside F (7), by physicochemical properties and spectroscopic methods including 1 D, 2 D NMR, IR, HR-ESI-MS data. Nitric oxide (NO) inhibitory activities of all of the isolated compounds (1 - 7) were also evaluated. Compounds 2 and 7 showed inhibitory effects against LPS-stimulated RAW 264.7 cells with IC50 values of 27.6 µM and 18.8 µM, respectively.

Two new 19-norbufadienolides with cardiotonic activity isolated from the venom of Bufo bufo gargarizans.

Two new 19-norbufadienolides (1 and 2) and one new 14,15-epoxy bufadienolide (3) alongside 16 known bufadienolides (4-19) were isolated from Bufonis Venenum that originated from the skin and parotid venom glands of an Asiatic toad (Bufo bufo gargarizans Cantor). The structures of these bufadienolides were elucidated based on the interpretation of their HRESIMS and NMR data. Compound 1, which had a unique peroxide, was established through extensive single-crystal X-ray diffraction. The two 19-norbufadienolides exhibited more potent cardiotonic activity in the isolated toad heart model and lower cytotoxicity against U87, U251, and LN-18 cell lines than other bufadienolides, such as bufalin and bufotalin. The results suggested that 19-norbufadienolides might be more suitable for developing cardiotonic agents with low cytotoxicity.

Antinociceptive and anti-inflammatory activities of a standardizedextract of bis-iridoids from Pterocephalus hookeri.

ETHNOPHARMACOLOGICAL RELEVANCE: Pterocephalus hookeri (C.B. Clarke) Höeck, one of the most popular Tibetan herbs, has been widely applied in Tibetan medicine prescriptions. Chemical investigations have led to the isolation of many bis-iridoids. However, the pharmacological activities of bis-iridoid constituents of this plant have never been reported before. AIM OF THE STUDY: This study evaluated the anti-inflammatory and analgesic activities of afraction of bis-iridoid constituents of P. hookeri (BCPH) in order to provide experimental evidence for its traditional use, such as for cold, flu, and rheumatoid arthritis. MATERIALS AND METHODS: The analgesic effects of BCPH were investigated using the hot-plate test and acetic acid-induced writhing test. The anti-inflammatory activities were observed using the following models: carrageenin-induced edema of the hind paw of rats and xylene-induced ear edema in mice. The effects of dexamethasone administration were also studied. RESULTS: BCPH significantly increased the hot-platepain threshold and reduced acetic acid-induced writhing response in mice. Moreover, BCPH remarkably inhibited xylene-induced ear edema and reduced the carrageenin-induced rat paw edema perimeter. CONCLUSION: The results reveal that BCPH has central, peripheral analgesic activities as well as anti-inflammatory effects, supporting the traditional application of this herb in treating various diseases associated with inflammation and pain.

In Vitro and In Vivo Antitumor Effects of n-Butanol Extracts of Pterocephalus hookeri on Hep3B Cancer Cell.

Pterocephalus hookeri is a widely applied Tibetan medicinal prescription for treatment of diseases such as flu, rheumatoid arthritis, and enteritis in China. It has been reported that Pterocephalus hookeri has anti-inflammatory and analgesic actions. However, the antitumor activity of Pterocephalus hookeri remains unknown. In the present study, we demonstrate that n-butanol extracts of Pterocephalus hookeri (YSC-ZDC) has a strong antitumor activity against hepatoma carcinoma cell in vitro and in vivo. YSC-ZDC inhibited proliferation of all cancer cell lines and significantly inhibited Hep3B cells proliferation in a dose- and time-dependant manner. Transmission electron microscopy, hoechst 33258 staining, and flow cytometry analysis revealed that YSC-ZDC induced apoptosis in Hep3B cells. YSC-ZDC treatment dramatically inhibited PDK1 and Akt phosphorylation in Hep3B cells. Moreover, YSC-ZDC increased Bax expression and inhibited Bcl-2 expression. In addition, YSC-ZDC inhibited growth hepatoma xenografts in vivo with no effect on body weight and spleen index. Consistent with results in vitro, YSC-ZDC increased Bax expression and inhibited Bcl-2 expression in tumor tissue. Taken together, this study shows YSC-ZDC with an antitumor activity both in vitro and in vivo. Its mechanism underlying is related to blocking of the Akt pathway and regulation of Bcl-2 family proteins expression.

Meroterpenes from Psoralea corylifolia against Pyricularia oryzae.

Six new meroterpenes, namely, 13-methoxyisobakuchiol (1), 13-ethoxyisobakuchiol (2), 12,13-dihydro-13-hydroxybakuchiol (3), Δ(10)-12,13-dihydro-12-(R,S)-methoxyisobakuchiol (4 and 5), and 15-demetyl-12,13-dihydro-13-ketobakuchiol (6), together with four known ones, namely, Δ(3),2-hydroxybakuchiol (7), Δ(1),3-hydroxybakuchiol (8), bakuchiol (9), and Δ(1,3)-bakuchiol (10), were isolated from the seeds of Psoralea corylifolia. Their structures were identified based on spectral data, including those obtained via 1D and 2D NMR, and MS spectral analyses. Antifungal screening results indicated that all compounds showed moderate inhibitory activities against Pyricularia oryzae.

Humulus scandens exhibits immunosuppressive effects in vitro and in vivo by suppressing CD4(+) T cell activation.

Humulus scandens, rich in flavonoids, is a traditional Chinese medicine. It is widely used in China to treat tuberculosis, dysentery and chronic colitis. In this study, the major active faction of Humulus scandens (H.S) was prepared. Then, its immunosuppressive effects and underlying mechanisms on T cell activation were investigated in vitro and in vivo. Results showed that H.S significantly inhibited the proliferation of splenocytes induced by concanavalin A, lipopolysaccharides, and mixed-lymphocyte reaction in vitro. Additionally, H.S could dramatically suppress the proliferation and interferon-γ (IFN-γ) production from T cells stimulated by anti-CD3 and anti-CD28. Flow cytometric results confirmed that H.S could suppress the differentiation of IFN-γ-producing type 1 helper T cells (Th1). Furthermore, using ovalbumin immunization-induced T cell reaction and CD4(+) T-cell-mediated delayed type hypersensitivity reaction, H.S the immunosuppressive effects of H.S was also demonstrated in vivo. Western blot results showed that H.S could impede the activation of both Erk1/2 and P38 in primary T cells triggered by anti-CD3/28. Collectively, the active fraction of H.S showed promising immunosuppressive activities both in vitro and in vivo.

Four new bis-iridoids isolated from the traditional Tibetan herb Pterocephalus hookeri.

Pterocenoids A-E (1-4), which Pterocenoids A(1) is one novel dimer containing a pyridine monoterpene alkaloid; and Pterocenoids B-E (2-4) are rare arranged non-glycosidic bis-iridoids were isolated from Pterocephlus hookeri. The structures of the compounds were established by 1D and 2D NMR spectroscopy and mass spectrometry. All bis-iridoids isolated from P. hookeri were found to possess secoiridoid/iridoid subtype skeletons. Hence, bis-iridoids can be regarded as the chemotaxonomic markers of P. hookeri. The origins of the new bis-iridoids (1-4) were postulated and their activities of inhibition of the NF-κB pathway were assayed and compounds 1-3 showed moderate activity in inhibiting NF-κB.

[Study on quality standard for Humulus scandens].

To control the quality of Humulus scandens, the quality standard was established in this study. According to the method recorded in the Appendix of Chinese Pharmacopoeia (2010 Edition) , the water and ash inspections were carried out. The component luteoloside and cosmosiin in Humulus scandens were identified and assayed by TLC and HPLC. The results showed a strong characteristics microscopic of Humulus scandens, and trichoromethane-methanol-formic acid (10: 3: 0. 3) as the mobile phase of TLC, the spots at 365 nm with a UV lamp was clear. The 16 batches of samples were analyzed by HPLC with a gradient elution of acetonitrile and phosphate solution (0.2%) at a flow rate of 1.0 mL · min(-1) and detected at 350 nm. The content of luteoloside was 0.015%- 0.651% (average 0.148%); the content of cosmosiin was 0.003%-0.118% (average 0.036%). The linear calibration curve of luteoloside and cosmosiin was acquired in the ranges of 0.011-0.364 g · L(-1) (r = 1.000 0) and 0.003-0.096 g · L(-1) (r = 1.000 0), respectively. The average recovery was 100.5% and 98.5%, respectively. The methods are convenient and reliable, which can be ap- plied for quality assessment of Humulus scandens.

Effects of ß-glucosidase hydrolyzed products of harpagide and harpagoside on cyclooxygenase-2 (COX-2) in vitro.

Harpagide (1) and harpagoside (2) are two iridoid glycosides existing in many medicinal plants. Although they are believed to be the main bioactive compounds related to the anti-inflammatory efficacy of these plants, the mechanisms of their anti-inflammatory activities remain unclear. The results of our present study showed that 1 and 2 had no effects on inhibitions of cyclooxygenase (COX)-1/2 enzyme activity, tumor necrosis factor-α (TNF-α) release, and nitric oxide (NO) production in vitro. However, the hydrolyzed products of 1 and 2 with ß-glucosidase treatment showed a significant inhibitory effect on COX-2 activity at 2.5-100 µM in a concentration-dependent manner. Our further study revealed that the hydrolyzed 2 product was structurally the same as the hydrolyzed 1 product (H-harpagide (3)). The structure of 3 was 2-(formylmethyl)-2,3,5-trihydroxy-5-methylcyclopentane carbaldehyde, with a backbone similar to prostaglandins and COX-2 inhibitors such as celecoxib. All of them have a pentatomic ring with two adjacent side chains. The result of molecular modeling and docking study showed that 3 could bind to the COX-2 active domain well through hydrophobic and hydrogen-bonding interactions, whereas 1 and 2 could not, implying that the hydrolysis of the glycosidic bond of 1 and 2 is a pre-requisite step for their COX-2 inhibitory activity.

Hypoglycemic activities of A- and B-type procyanidin oligomer-rich extracts from different Cinnamon barks.

Procyanidin oligomers in Cinnamon are thought to be responsible for the biological activity in the treatment of diabetes mellitus (DM). To clarify types of procyanidin oligomers in different Cinnamon species and investigate their different effects, the present study investigated procyanidin oligomers in polyphenolic oligomer-rich extracts of three Cinnamon samples by LC-MS methods, and their hypoglycemic activities were detected in vivo and in vitro. The results showed that two of the three samples from Cinnamomum cassia were rich in B-type procyanidin oligomers, and the other sample was rich in A-type procyanidin oligomers. The Cinnamon extracts were administered at doses of 200 and 300 mg/kg body wt. in high-fat diet-fed and low-dose streptozotocin (STZ)-induced diabetic mice for 14 days. The results showed that blood glucose concentrations were significantly decreased in all Cinnamon extract groups compared with the control group (p<0.05). Administration of the Cinnamon extracts significantly increased the consumption of extracellular glucose in insulin-resistant HepG2 cells and normal HepG2 cells compared with the control group. These results suggest that both A- and B-type procyanidin oligomers in different Cinnamon species have hypoglycemic activities and may improve insulin sensitivity in type 2 DM.

Novel flavonoids from the leaves of Actinidia valvata Dunn: structural elucidation and antioxidant activity.

Two novel flavonoids, kaempferol 3- O- α-L-rhamnopyranosyl (1-3) (2,4-di- O-acetyl-α-L-rhamnopyranosyl) (1-6) ß-D-galactopyranoside (1) and kaempferol 3- O-α-L-rhamnopyranosyl (1-3) (4-O-acetyl-α-L-rhamnopyranosyl) (1-6) ß-D-galactopyranoside (2), along with three known ones, kaempferol-3- O-ß-D-galactopyranoside (3), kaempferol (4), and 7-hydroxychromone (5), have been isolated from the leaves of Actinidia valvata Dunn, and their structures were elucidated based on spectroscopic methods. Compounds 1-3 exhibited dose-dependent activity in scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals, superoxide anion radicals, and hydroxyl radicals, and inhibited lipid peroxidation of mouse liver homogenate IN VITRO.