Mechanistic analysis for the origin of diverse diterpenes in Tripterygium wilfordii

Tripterygium wilfordii is a valuable medicinal plant rich in biologically active diterpenoids, but there are few studies on the origins of these diterpenoids in its secondary metabolism. Here, we identified three regions containing tandemly duplicated diterpene synthase genes on chromosomes (Chr) 17 and 21 of T. wilfordii and obtained 11 diterpene synthases with different functions. We further revealed that these diterpene synthases underwent duplication and rearrangement at approximately 2.3-23.7 million years ago (MYA) by whole-genome triplication (WGT), transposon mediation, and tandem duplication, followed by functional divergence. We first demonstrated that four key amino acids in the sequences of TwCPS3, TwCPS5, and TwCPS6 were altered during evolution, leading to their functional divergence and the formation of diterpene secondary metabolites. Then, we demonstrated that the functional divergence of three TwKSLs was driven by mutations in two key amino acids. Finally, we discovered the mechanisms of evolution and pseudogenization of miltiradiene synthases in T. wilfordii and elucidated that the new function in TwMS1/2 from the terpene synthase (TPS)-b subfamily was caused by progressive changes in multiple amino acids after the WGT event. Our results provide key evidence for the formation of diverse diterpenoids during the evolution of secondary metabolites in T. wilfordii.

Cathayanalactone G and other constituents from leaves and twigs of Callicarpa cathayana / 中草药·英文版

Objective: To study the chemical constituents from the leaves and twigs of Callicarpa cathayana. Methods: The chemical constituents were isolated and purified by column chromatography on silica gel, MCI gel CHP 20P/P120, Sephadex LH-20, and HPLC. The structures of the compounds were determined by HR-ESI-MS, 1D and 2D NMR data. Results: A total of 24 compounds were isolated from the 85% methanol extract of leaves and twigs of C. cathayana. They were identified as cathayanalactone G (1), a new diterpene, and 23 known compounds as patagonic acid (2), (-)-16-hydroxycledroda-3,13-dien-16,15-olide-18-oic acid (3), 15-methoxypatagonic acid (4), oleanolic acid (5), ursolic acid (6), siaresinolic acid (7), pomolic acid (8), α-amyrin (9), tormentic acid (10), lupeol (11), 5,7-dihydroxy-3,4′-dimethoxyflavone (12), 5,4′-dihydroxy-3,7,3′-dimethoxyfla-vone (13), 5-hydroxy-3,6,7,4′- tetramethoxyflavone (14), salvigenin (15), kaemferol (16), astragalin (17), pinoresinol 4-O-β-D-glucopyranoside (18), paulownin (19), β-sitosterol (20), β-sitosterol β-D-glucopyranoside (21), 5-hydroxy-coumarin (22), isocopoletin (23), and 4-hydroxycinnamic acid (24). Conclusion: Compound 1 is a new labdane diterpene. Compounds 10, 13, 16 and 17 are isolated from the genus Callicarpa for the first time. Compounds 7, 8, 9, 12, 14, 23 and 24 are reported from C. cathayana for the first time.

Vinegar-processed Euphorbiae Pekinensis Radix improves intestinal flora disorder and reduces colon toxicity / 中国中药杂志

The present study investigated the effect of Euphorbiae Pekinensis Radix(EPR) on intestinal flora structure before and after vinegar processing and explored the detoxification mechanism of vinegar-processed EPR. In this study, the extraction efficiency of casbane diterpenes from EPR with different solvents was investigated, and the optimal solvent was selected to enrich these components. After 14 days of intragastric administration of total diterpene extract of EPR and vinegar-processed EPR, 16 S rDNA sequencing technology was used to detect the structural changes of intestinal flora. The flora related to the intestinal toxicity of EPR was screened out based on the results of intestinal pathological damage by correlation analysis. The results showed that Soxhlet extraction with chloroform as extraction solvent could enrich Casbane diterpenes in EPR. As revealed by 16 S rDNA sequencing results, EPR could significantly change the structure of intestinal flora, which could be reversed by vinegar-processing EPR. Some intestinal flora candidates might be related to detoxification of vinegar processing. The correlation analysis of intestinal flora candidates and indexes related to intestinal mucosal injury showed that compared with EPR, vinegar-processed EPR could down-regulate the abundance of some pathogenic bacteria such as Mucispirillum, Bilophila, and Ruminiclostridium, and up-regulated some probiotics such as Enterorhabdus, Ruminococcaceae_UCG-014, Barnesiella, and Candidatus. The intestinal toxicity caused by EPR may be related to the disturbance of intestinal flora, and vinegar-processed EPR can improve intestinal flora disorder by up-regulating the abundance of probiotics and down-regulating the abundance of pathogenic bacteria to remodel the intestinal mucosal barrier and reduce toxicity.

Reconstitution of biosynthetic pathway for mushroom-derived cyathane diterpenes in yeast and generation of new "non-natural" analogues

Mushroom-derived cyathane-type diterpenes possess unusual chemical skeleton and diverse bioactivities. To efficiently supply bioactive cyathanes for deep studies and explore their structural diversity,

A review: biosynthesis of plant-derived labdane-related diterpenoids / 中国天然药物

Plant-derived labdane-related diterpenoids (LRDs) represent a large group of terpenoids. LRDs possess either a labdane-type bicyclic core structure or more complex ring systems derived from labdane-type skeletons, such as abietane, pimarane, kaurane, etc. Due to their various pharmaceutical activities and unique properties, many of LRDs have been widely used in pharmaceutical, food and perfume industries. Biosynthesis of various LRDs has been extensively studied, leading to characterization of a large number of new biosynthetic enzymes. The biosynthetic pathways of important LRDs and the relevant enzymes (especially diterpene synthases and cytochrome P450 enzymes) were summarized in this review.

One new cembranoid diterpene from gum resin of Boswellia carterii / 中国中药杂志

This study aims to study the chemical components from the gum resin of Boswellia carterii. Five cembranoid diterpenes were isolated from the gum resin of B. carterii by various of column chromatographies including silica gel, Sephadex LH-20, and semi-preparative HPLC. Their structures were identified on the basis of physicochemical properties, mass spectrometry(MS), nuclear magnetic resonance(NMR), Ultraviolet(UV) and infrared(IR) spectroscopic data. These compounds were identified as(1S,2E,4R,5S,7E,11E)-4-methoxy-5-hydroxycembrane(1),(1R~*,4R~*,5E,8E,12E,15E)-4-hydroxycembra-5,8,12,15-tetraene(2), cembrene A(3),(3S,4S,7R)-4-hydroxycembrane(4), and pavidolide D(5). Compound 1 was a new compound. Compounds 2, 4, and 5 were obtained from the gum resin of B. carterii for the first time. Compound 2 showed weak inhibition on the human liver cancer cell line HepG2.

Anti-acetylcholinesterase Active Ingredients from Aconitum tanguticum by UPLC-Q-TOF-MS/MS / 中国实验方剂学杂志

Objective:To identify the anti-acetylcholinesterase active ingredients in <italic>Aconitum tanguticum</italic>， so as to lay the foundation for finding new anti-Alzheimer's disease （AD） drugs. Method:The anti-acetylcholinesterase active fractions of <italic>A. tanguticum</italic> were screened by the modified Ellman's method， and the chemical composition of the active fraction was analyzed by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry （UPLC-Q-TOF-MS/MS）. The chromatographic separation was performed on an ACQUITY UPLC BEH C₁₈ column （2.1 mm×50 mm， 1.7 μm） with acetonitrile （A）-0.4% ammonia aqueous solution （B） as mobile phase for gradient elution， and the column temperature was set at 30 ℃ with the flow rate of 0.4 mL·min^-1. Phase A of the dichloromethane fraction changed with time as follows：0-3 min， 5%A； 3-7 min， 5%-20%A； 7-11.5 min， 20%-33%A； 11.5-15.5 min， 33%-50%A； 15.5-20.5 min， 50%-80%A； 20.5-23 min， 80%-85%A； 23-25 min， 85%-95%A. Phase A of the <italic>n</italic>-butanol fraction changed with time as follows：0-2 min， 5%A； 2-8 min， 5%-20%A； 8-11 min， 20%-33%A； 11-15 min， 33%-95%A. Mass spectrometry was performed on electrospray ionization， data were collected in positive ion mode， and the detection range was <italic>m</italic>/<italic>z</italic> 100-1 500. Result:Both the dichloromethane and <italic>n</italic>-butanol fractions had a certain inhibitory effect on acetylcholinesterase， their half inhibitory concentration （IC₅₀） values were （64±4.4） mg·L^-1 and （85.7±3.8） mg·L^-1， respectively. By UPLC-Q-TOF-MS/MS analysis， a total of 21 alkaloids were identified from the dichloromethane fraction， and 11 alkaloids were identified from <italic>n</italic>-butanol fraction. Guan-fu base Ⅰ， found in both fractions， was first discovered in <italic>A. tanguticum</italic>. Conclusion:Diterpene alkaloids are the main anti-acetylcholinesterase substances of <italic>A. tanguticum</italic>， which is worth further exploration.

A new cembranoid diterpene from Olibanum / 中国中药杂志

Five compounds were isolated from the alcohol extract of Olibanum by MCI, silica gel, ODS, and Sephadex LH-20 column chromatographies and preparative high-performance liquid chromatography(HPLC). On the basis of spectral data and literature data, the compounds were identified as:(1S,3R,4S,7R,11S,12R)-1:12,4:7-diepoxisonane-8(19)-ene-3,11-diol(1), boscartin A(2),(+)-resinolin(3),(+)-5-hydroxy-3,4-dimethyl-5-pentylfuran-2(5H)-one(4), and acerogenin A(5). Compound 1 is a new compound, and compounds 3-5 were isolated from Olibanum for the first time. The structure of compound 1 was determined by spectroscopic analysis and single-crystal X-ray diffraction. Compounds 1 and 2 were tested for PC12 neurotoxicity, and the results showed that they were both safe compounds.

Effect of ginkgo diterpene lactones on hypoxia-induced apoptosis and angiogenesis of human umbilical vein endothelial cells and its mechanisms / 第二军医大学学报

Objective To investigate the effect of ginkgo diterpene lactones on hypoxia-induced apoptosis and angiogenesis of human umbilical vein endothelial cells (HUVECs) and the related molecular mechanisms. Methods HUVECs were cultured under hypoxia for 24 h, and then treated with ginkgo diterpene lactones (low-dose: 6.25 mg/L and high-dose: 25.00 mg/L). MTT assay was used to detect the cell activity. Flow cytometry was used to detect the apoptosis of HUVECs. Transwell assay was employed to detect the migration of HUVECs. Quantitative real-time polymerase chain reaction and Western blotting were used to test the expression levels of mRNA and protein of hypoxia-inducible factor 1α (HIF-1α), apoptosis-related genes (B-cell lymphoma 2［Bcl-2］and B-cell lymphoma 2-related X protein［Bax］), and angiogenesis-related genes (vascular endothelial growth factor［VEGF］and transforming growth factor β［TGF-β］). Results Compared with the normal group, the HUVEC activity was significantly decreased after exposed to hypoxia for 24 h (P＜0.05), and the apoptosis rate of HUVECs and mRNA and protein expression levels of HIF-1α were significantly higher (all P＜0.05). The cell activity and migration ability of HUVECs were significantly higher in the low- and high-dose ginkgo diterpene lactones groups than those in the hypoxia group (all P＜0.05), and the apoptosis rates of HUVECs were significantly lower than those in the hypoxia group (both P＜0.05). Meanwhile, the cell activity and migration ability were significantly higher in the high-dose group than those in the low-dose group (both P＜0.05), and the apoptosis rate was significantly lower than that in the low-dose group (P＜0.05). The mRNA and protein expression levels of HIF-1α and Bax were lower in the low- and high-dose ginkgo diterpene lactones groups than those in the hypoxia group, and the mRNA and protein expression levels of Bcl-2, VEGF and TGF-β were higher than those in the hypoxia group; and the expression changes of the above genes were more significant in the high-dose group. Conclusion Ginkgo diterpene lactones can improve hypoxia-induced apoptosis and angiogenesis of HUVECs by regulating the expression levels of HIF-1α and apoptosis- and angiogenesis-related genes, and it might be used as a new agent to treat anoxic vascular diseases.

Analysis of Solubilization of Fatty Oil from Euphorbiae Semen on Four Euphorbia Factors Based on Thinking of Self-assembled Micelles in vivo / 中国实验方剂学杂志

Objective: The solubilization of 4 compounds(Euphorbia factor L1,L2,L3 and L8),caused by mixed micelles self-assembled from fatty oil of Euphorbiae Semen and bile salt of intestinal juice,was researched in the simulated human intestinal environment. Method: The mixed micelles were prepared with different amounts of fatty oil of Euphorbiae Semen.The transmission electron microscope(TEM) was used to observe the morphology of the micelles.Particle size detector was used to determine the particle size and Zeta potential.HPLC was used to assay the solubility of these 4 compounds.The variation tendency of the total dissolution of these 4 compounds with the change of standing time was observed. Result: Particle size of the mixed micelles was uniform and its morphology was spherical.The absolute values of Zeta potential were less than 20 mV.When the amount of sodium deoxycholate was fixed 4.96 g·L-1,the solubility of these 4 compounds with the concentration of fatty oil at 0.1-4 g·L-1 were significantly greater than that at the dosage of 0 g·L-1.The solubility of these 4 compounds in the micelles formed by fatty oil was 1.3 to 4 times as much as the micelles without fatty oil.The micelles was stable for 36 h. Conclusion: The micelles self-assembled from fatty oil of Euphorbiae Semen and bile salt of intestinal juice,have significant solubilization effect on Euphorbia factor L1,L2,L3 and L8.This research can lay the foundation for clarifying the detoxification mechanism of removing fatty oil and making frostlike powder from the perspective of pharmaceutics.

Quality Evaluation of Andrographis Herba Based On Electronic-eye Technique / 中国实验方剂学杂志

Objective: Taking electronic-eye (visual analyzer) technique,based on the powder color of Andrographis Herba,to investigate the applicability of electronic-eye technique and evaluate the quality of Andrographis Herba with different commercial specifications. Method: HPLC was employed to determine contents of andrographolide,dehydroandrographolide,14-deoxyandrographolide,neoandrographolide in 50 batches of Andrographis Herba with different commercial specifications(stems,leaves and aerial parts).Color of these samples were measured by electronic-eye technique.The data were analyzed by principal component analysis(PCA) and Pearson correlation analysis.The ability of electronic-eye to distinguish the different commercial specifications of Andrographis Herba was investigated and the correlation of chroma space system parameters (L*,a*,b*) with active components was investigated. Result: There was remarkable difference in contents of 4 diterpenoids in Andrographis Herba from different parts,their contents in leaves was the highest,followed by the aerial parts(mixture of stems and leaves),and their contents in stems was the lowest.The results of PCA was divided into two classes,namely the stem part,leaf and aerial parts,indicating that electronic-eye could be used to distinguish the quality of Andrographis Herba.The correlation results showed that there were significant negative correlation(PL*(lightness value) and the contents of andrographolide,dehydroandrographolide,14-deoxyandrographolide,neoandrographolide and the total content of these 4 components.In addition,L* of samples that did not conform to the lower limit of determination in the 2015 edition of Chinese Pharmacopeia was ≥ 69.5,and the L* of more than 90% of the samples in accordance with the requirements was Conclusion: Electronic-eye technique provides a new method and idea for the quality evaluation of Andrographis Herba.

Chemical constituents from rhizome of Tripterygium hypoglaucum / 中草药

Objective: To study the chemical constituents from the rhizome of Tripterygium hypoglaucum. Methods: The concrete exacted by 95% ethanol from the rhizome of T. hypoglaucum was isolated and purified by chromatography on silica gel column chromatography, gel column chromatography, MPLC, etc. The structures of the chemical constituents were elucidated by means of NMR, MS, etc. Results: Twelve compounds were isolated and identified as 3β-hydroxy-4-hydroxymethyl-abieta-8,11,13-trien-7-one (1), 3β-acetoxy oleanolic acid (2), physcion (3), canophyllal (4), chrysophenol (5), quinone 21 (6), tripterifordin (7), triptobenzene B (8), 3,4-dimethoxyphenol (9), integracin A (10), celastrol (11), and β-sitosterol (12). Conclusion: Compound 1 is a new diterpene named tripterone, compounds 8-10 are isolated from this plant for the first time.

Establishment of QAMS method with dehydroandrographolide as internal reference substance for quality control of Andrographis Herba / 中国中药杂志

To solve the problems of the poor resolution of chromatographic separation,the weak durability of the relative correction factors,and the low accuracy of content determination results in the quantitative analysis of multi-components by single-marker( QAMS) method with andrographolide as the internal reference substance in the existing research of Andrographis Herba,a new QAMS method using dehydroandrographolide as the internal reference substance was established for the first time in this study. This new method can be used to simultaneously determine four diterpene lactones,including andrographolide( A),neoandrographolide( B),14-deoxyandrographolide( C),and dehydroandrographolide( S) through the optimization of chromatographic conditions and systematic investigation of methodology. At the present HPLC chromatographic conditions,four components could be well separated( R > 1. 5),and the methodology validations could satisfy the requirement of quantitative analysis. The relative correction factors( RCFs) of fA/S,fB/S,fC/S were determined as 0. 65,0. 54,0. 78,respectively. The relative standard deviations( RSDs) of their RCFs ranged between 1. 3%-5. 1%,0. 25%-0. 33%,0. 070%-0. 15%,0. 070%-0. 22%,respectively with three brands of HPLC instruments,five brands of C18 column,different flow rates( 0. 9,1. 0,1. 1 m L·min~(-1)),and different column temperatures( 25,30,35 ℃),indicating good durability of the RCFs. The relative retention value( RRV) method was used to locate the chromatographic peak of the components to be determined.The RRVs of rA/S,rB/S,and rC/Swere 0. 44,0. 86,0. 97,respectively. The RSDs of the RRVs ranged between 0. 030%-1. 6% with different HPLC instruments and columns,showing accurate peak location. The present QAMS method and the external standard method( ESM)were both used to determine the contents of four diterpene lactones from Andrographis Herba( 6 batches of medicinal materials and 18 batches of cut crude drugs). The relative errors of the determined content results between two methods were less than 2. 0%. It demonstrated that there was no significant difference in content results between these two methods,indicating good accuracy of the present QAMS method. Therefore,in this study,an accurate and highly durable QAMS method using dehydroandrographolide as the internal reference substance was established for simultaneous determination of four diterpene lactones. This method could be used to effectively control the quality of Andrographis Herba and provide technical basis for the formulation of traditional Chinese medicine industry standard and improvement of the Chinese Pharmacopoeia standard of Andrographis Herba.

The efficacy and safety of diterpene ginkgolides meglumine injection in stroke:a Meta-analysis / 药学实践杂志

Objective Diterpene ginkgolides meglumine injection (DGMI) is widely used in patients with stroke, but its efficacy and safety are not consistent.We performed a Meta-analysis to comprehensively evaluate the efficacy and safety of DG-MI in acute ischemic stroke and recovered stroke.Methods The wanfang, VIP, CNKI and PubMed were searched, the randomized controlled trials (RCTs) were enrolled.Data collection and quality evaluation of the included RCTs were performed according to Cochrane systematic evaluation method.Meta-analysis was performed by using Stata software.Results 9RCTs involving 1 129subjects were included with 706subjects in DGMI treatment group and 423subjects in control group. (1) For acute ischemic stroke, DGMI group had superior effective rate compared to conventional therapy group (RR=1.19, 95％CI:1.09, 1.31, P＜0.000 1), improvement of neurologic impairments (SMD=3.23, 95％CI:2.87, 3.60, P＜0.000 1) and improvement of living quality (SMD=3.23, 95％CI:2.87, 3.60, P＜0.000 1). (2) For recovered stroke, DGMI group had better effective rate than Shuxuening injection group (RR=1.17, 95％CI:1.05, 1.30, P＜0.05) and improvement of neurologic impairments (SMD=-0.69, 95％CI:-0.88, -0.49, P＜0.000 1).There was no significant difference in adverse events between DGMI and control groups (P＞0.05).Conclusion DGMI had superior efficacy over control group for both acute ischemic stroke and recovered stroke.There was no significant difference in adverse events between these two groups.However, we still need better quality RCTs to confirm these results.

Effects of ginkgo diterpene lactone meglumine on learning and memory in old mice and its mecha-nisms / 中华行为医学与脑科学杂志

Objective To study the effects of diterpene ginkgolides meglumine injection ( DGMI) on memory impairment, activation of microglia and astrocytes and inflammatory cytokines in aged mice. Methods Twenty aged mice (22 months old) were randomly divided into two groups:aged mouse group(n=10) and DGMI group(n=10). Another 10 mice (2 months old) were selected as young mouse control group. The mice in DGMI group were received 5 mg/kg DGMI per day by tail vain injection for 4 weeks. The mice in the other two groups were received the same amount normal saline for 4 weeks. The Morris water maze was used to evaluate the function of spacial learning and memory after administration of drugs. The ex-pression of CD11b,GFAP,IL-1β,IL-6,TNF-α and NFκB in mice brain hippocampus were detected by West-ern blot. Results (1) The escape latency time of aged mouse group was significantly longer than that of young mouse control group from the 2nd day to the 7th day(P<0. 01). The times of platform crossing,time and distance in target quadrant of aged mouse group were significantly shorter than those of young mouse group (all P<0. 01). Compared with aged mouse group,DGMI significantly reduced the escape latency time of DGMI group (P<0. 01). DGMI increased the times of platform crossing,time and distance in target quad-rant of aged mouse group (P<0. 01). (2) The expressions of CD11b,GFAP in young mouse control group, aged mouse group and DGMI group were as follows respectively:CD11b:(1. 036±0. 023),(1. 757±0. 046), (1. 214±0. 024);GFAP:(1. 022±0. 071),(1. 344±0. 021),(1. 086±0. 073). DGMI reduced the expres-sion of CD11b and GFAP in hippocampus compared with aged mouse group ( t=5. 556,P<0. 01;t=5. 484, P<0. 01). (3) The expressions of IL-1β,IL-6,TNF-α and NFκB in young mouse control group,aged mouse group and DGMI group were as follows respectively:IL-1β:( 1. 003 ± 0. 057),( 2. 062± 0. 105),( 1. 182± 0. 084);IL-6:(1. 018±0. 024),(1. 583± 0. 052),( 1. 152± 0. 031); TNF-α:( 1. 021± 0. 054),(1. 449± 0. 053),(1. 211±0. 036);p-NFκB:(1. 052±0. 034),(1. 782± 0. 113),( 1. 158± 0. 066). DGMI reduced the expression of p-NFκB(t=6. 547,P<0. 01) and pro-inflammatory cytokines including IL-1β(t=8. 513,P<0. 01),IL-6(t=3. 421,P<0. 01) and TNF-α( t=5. 562,P<0. 01) in hippocampus compared with aged mouse group. Conclusion DGMI can improve the ability of learning and memory in aged mice. The mecha-nism may be related with inhibiting activity of microgliosis,astrocytosis,NFκB and neuroinflammaton.

Antiproliferative potential of solidagenone isolated of Solidago chilensis

ABSTRACT Plants are considered among the main sources of biologically active chemicals. The species Solidago chilensis Meyen, Asteraceae, is native to the southern parts of South America, where the aerial parts of the plant are commonly used for the treatment of inflammatory conditions. However, the effects of S. chilensis on human cancer cells remain to be elucidated. In this study, we evaluated the antiproliferative effects of the hydroalcoholic and dichloromethane extracts of S. chilensis, as well as their chemical constituents quercitrin and solidagenone against the five human tumor cell lines in vitro. The dichloromethane extract showed a promisor antiproliferative effects in vitro, especially against glioma cell line. Besides, the hydroalcoholic extract and quercitrin were inactive. The diterpene solidagenone showed highly potent antiproliferative effects against breast (MCF-7), kidney (786-0), and prostate cancer (PC-3) cells (total growth inhibition: TGI < 6.25 µg/ml). Solidagenone meets the theoretical physico-chemical criteria for bioavailability of drugs, according to the "Rule of Five" and, by theorical studies, the observed biological effects were probably related to the interaction of the molecule with nuclear receptors and as an enzymatic inhibitor. This study contributes to chemical study and to the identification of antiproliferative molecules in S. chilensis.

Cytotoxic activity of abietane diterpenoids from roots of Salvia sahendica by HPLC-based activity profiling

ABSTRACT Screening of medicinal plants from Iranian flora against human cancer cell-lines have shown that an hexane extract from roots of Salvia sahendica Boiss. & Buhse, Lamiaceae, is active against human cervical cancer (HeLa) and colorectal adenocarcinoma (Caco-2) cell-lines at the test concentration of 100 µg/ml (100% inhibition). Cytotoxicity of the extract was localized with the aid of HPLC-time-based activity profiling adapted to the tetrazolium colorimetric bioassay. Four abietane-type diterpenoids in active time-windows were identified as cytotoxic compounds namely: sahandone (1), sahandol (2), 12-deoxy-salvipisone (3) and sahandinone (4). Compound 1 showed the highest toxicity against HeLa cells (IC50 = 5.6 ± 0.1 µg/ml), which was comparable with betulinic acid (IC50 = 4.3 ± 1.2 µg/ml), the positive control. Compound 2 was active against the HeLa cells (IC50 = 8.9 ± 0.7 µg/ml) but not the Caco-2 cell-line. Compounds 1, 3 and 4 exhibited moderate activity (IC50 = 22.9-41.4 µg/ml) against the Caco-2 cells. This study reveals that the HeLa cells are more sensitive to all tested compounds than the Caco-2 cells. In silico molecular docking study showed a rigid binding of the compounds to tyrosine kinase pp60src, and proved their cytotoxic activity.

Induction effects of rabdosin B on differentiation of human promyelocytic leukemia HL-60 cells / 中草药

Objective To investigate the characteristics and mechanism of spironolactone ent-kaurane diterpene type diterpene rabdosin B (RB) induced HL-60 differentiation into mature granulocytes. Methods The effects of RB on proliferation, cell cycle, NBT reducing power, phagocytosis, and cell surface antigen CD11b expression of HL-60 were detected by trypan blue staining, Giemsa staining, NBT reducing power assay, and flow cytometry, respectively. Results RB inhibited the proliferation of HL-60 cells at the concentration of 1.0, 3.0, and 5.0 μmol/L, which resulted in the arrest of G0/G1 phase, increased the ratio of renal nucleated and lobate nucleus, and markedly enhanced the NBT reducing power and phagocytic capacity and the significant increase of CD11b positive cell rate. Further tests showed that the active oxygen scavenger NAC (300 μmol/L), NADPH oxidase inhibitor APO (100 μmol/L), and DPI (0.1 μmol/L) not only significantly inhibited the intracellular ROS upregulation induced by RB, but also inhibited various features of RB induced HL-60 cell differentiation. Conclusion RB can increase the activity of NADPH oxidase in HL-60 cells, increase the intracellular ROS concentration, and induce the differentiation of HL-60 cells to mature granulocytes.

A new cembranoid diterpene from twigs of Euonymus alatus / 中草药

Objective To study the chemical constituents from the twigs of Euonymus alatus. Methods Compounds were isolated and purified by a combination of various chromatographic techniques, and their structures were elucidated by physiochemical property and spectral analysis. Results Three compounds were obtained from 95% ethanol extract of the twigs of E. alatus and identified as (1S,2S,7E,11R,12S)-2,11-dihydroxy-1,12-oxidocembra-4(18),7(8)-diene (1), hemerocallal A (2), and betulinic acid methyl ester (3). Conclusion Compound 1 is a new cembranoid diterpene and named euonymuditerpene A, and compounds 2 and 3 are isolated from the twigs of Euonymus alatus for the first time.

A new norclerodan diterpene from twigs and leaves of Croton euryphyllus / 中草药

To study the diterpenoids from the twigs and leaves of Croton euryphyllus. Methods Compounds were isolated and purified by column chromatography on silica gel, MCI, and semi-preparative HPLC, and their structures were elucidated by spectroscopic analysis and comparisons with published literature values. Results Four compounds were obtained from the petroleum ether fraction of 95% alcoholic extract of the twigs and leaves of C. euryphyllus, and structures were identified as crotoeurin D (1), mallotucin C (2), mallotucin D (3), and plaunotol (4), respectively. Conclusion Compound 1 is a new norclerodan diterpene. Compounds 2-4 are isolated from this plant for the first time.