[Isolation and functional characterization of (iso)eugenol O-methyltransferase (IEMT) gene in Asarum sieboldii].

Methyleugenol is one of the main active constituents in the volatile oil of the traditional Chinese medicine Asari Radix et Rhizoma. It possesses various pharmacological effects such as analgesic, anesthetic, and anti-inflammatory properties. In biosynthesis, the initial precursor phenylalanine is finally converted into methyleugenol through a series of intermediate compounds including coniferyl acid, courmaryl acid, caffeic acid, ferulic acid/ferulic-CoA, coniferyl aldehyde, conferyl alcohol, cnfiferyl acetate, and eugenol/isoeugenol, which are produced through catalysis of a large number of enzymes. Eugenol O-methyltransferase(EOMT) is one of the key enzymes in the biosynthesis pathway, capable of methylating eugenol on the para-site hydroxyl group of the benzene ring, thereby generating methyleugenol. Here, an(iso)eugenol O-methyltransferase(IEMT) gene was cloned for the first time from Asarum siebo-ldii, holding an open reading frame that consisted of 1 113 bp and encoded a protein containing 370 amino acid residues. Bioinformatics analysis results showed that this protein was equipped with the characteristic structural domains of methyltransferases such as S-adenosylmethionine(SAM) binding sites and dimerization domains. The prokaryotic expression recombinant plasmid pET28a(+)-AsIEMT was constructed, and the candidate protein was induced and purified. In vitro enzyme assays confirmed that AsIEMT had dual functions. The enzyme could catalyze the production either of methyleugenol from eugenol or of methylisoeugenol from isoeugenol, although the latter was more prevalent. When isoeugenol was used as the substrate, the kinetics parameters K_m and V_(max) of catalytic reaction were(0.90±0.06) mmol·L~(-1) and(1.32±0.04)nmol·s~(-1)·mg~(-1), respectively. This study expanded our understandings of critical enzyme genes involved in phenylpropanoid metabolic pathways, and would facilitate the elucidation of quality formation mechanisms of the TCM Asari Radix et Rhizoma.

The genus Broussonetia: a review of its phytochemistry and pharmacology.

Plants belonging to the genus Broussonetia (Moraceae), which grow naturally in Asian and Pacific countries, have long been used in traditional Chinese medicine to treat various conditions, particularly tinea, dysentery, hernia and oedema. Various parts of plants of this genus such as the fruits, bark, leaves, flowers and roots are used medicinally. According to Flora of China, the inner bark fibre is used in making paper, and the wood is used for furniture. Several active compounds including coumarins, polyphenols, alkaloids, flavonoids, glycosides, lignans and diterpenes have been isolated from the plants of this genus in the past few years, and some members of these constituents have been demonstrated to exert antioxidant, antiinflammatory, antiplatelet, antityrosinase, antimicrobial and antinociceptive effects. Antidiabetic and antitumoural properties seem possible, but need further support. Due to the lack of clinical trials, there are few published reports on target-organ toxicity or side effects. Currently, some East Asian nations such as China, South Korea and Japan have shown increased interest in these plants. This article reviews the phytochemical and potential beneficial therapeutic effects of plants of this genus reported since 2000.

A new labdane diterpene from Vitex negundo.

CONTEXT: Vitex negundo Linn. (Verbenaceae) seeds are pepper substitute and occasionally used as a condiment for edible purposes. The seeds also find use for analgesia, sedation, rheumatism and joint inflammation in folk medicine. OBJECTIVE: To isolate and characterize bioactive constituents from V. negundo seeds. MATERIALS AND METHODS: The ethanol extract of V. negundo seeds was subjected to repeated column chromatography. Chemical structures were elucidated by detailed spectroscopic analyses on the basis of NMR, IR, and MS data. Several pathogenic fungi isolates were employed to evaluate the antifungal activity of the isolated compound. RESULTS: Chemical investigations of the seed extract afforded a new labdane diterpenoid, named negundol (1a + 1b), as an inseparable mixture of two diastereoisomers in a 5:4 ratio. Their structures were identified as (rel 3S, 5S, 8R, 9R, 10S, 13S, 16S)-3-acetoxy-9, 13-epoxy-16-hydroxy-labda-15, 16-olide (1a), and (rel 3S, 5S, 8R, 9R, 10S, 13S, 16R)-3-acetoxy-9, 13-epoxy-16-hydroxy-labda-15, 16-olide (1b). Compound 1 was active as an antifungal agent with MIC80 values in the range of 16-64 µg/mL. DISCUSSION AND CONCLUSION: The presence of compound 1 in V. negundo is of chemotaxonomic significance, since plants under the genus Vitex are chemically characterized with labdane diterpenoids. Compound 1 exhibited potential antifungal activity and may be considered a lead compound for promising antifungal agent.

Antifatigue activity of the liposoluble fraction from Acanthopanax senticosus.

The crude extract of Acanthopanax senticosus (AS) has been used extensively in Russia, China, Korea and Japan as an adaptogenic agent to fight against stress and fatigue. However, whether the liposoluble fraction possesses antifatigue activity or not is still unclear. A liposoluble fraction was administered orally to mice for 9 days. The swimming time to exhaustion was longer in the treatment groups (22.2 ± 3.3, 25.5 ± 4.8 min) than in the control group (13.7 ± 1.2 min, p < 0.05). The plasma TG (triglyceride) and BUN (blood urea nitrogen) levels in the high dose (500 mg/kg) groups were decreased significantly compared with the control group. Plasma lactate dehydrogenase (LDH) was lower in the treatment groups than in the control group. Chemical analysis from GC/MS revealed that the main components of the liposoluble fraction of AS were saturated fatty acid (12.98%), unsaturated fatty acid (33.13%), unsaturated alcohol (27.46%) and diolefine (15.76%). In conclusion, the liposoluble fraction enhanced the forced swimming capacity of mice by decreasing muscle damage, effectively preventing the increase in BUN concentration and increasing fat utilization. It is proposed that the antioxidant effect may be one of the antifatigue mechanisms of the liposoluble fraction of AS.

Hepatotoxicity of kaurene glycosides from Xanthium strumarium L. fruits in mice.

The fruit of Xanthium strumarium L. (Cang-Er-Zi) is a traditional Chinese medicine that is used in curing nasal diseases and headache according to the Chinese Pharmacopoeia. However, clinical utilization of Xanthium strumarium is relatively limited because of its toxicity. The present investigation was carried out to evaluate the toxic effects on acute liver injury in mice of the two kaurene glycosides (atractyloside and carbxyatractyloside), which are main toxic constituents isolated from Fructus Xanthii on acute liver injury in mice. Histopathological examinations revealed that there were not obviously visible injury in lungs, heart, spleen, and the central nervous system in the mice by intraperitoneal injection of atractyloside (ATR, at the doses 50,125 and 200 mg/kg) and carbxyatractyloside (CATR, at the doses 50,100 and 150 mg/kg) for 5 days. However, it revealed extensive liver injuries compared with the normal group. In the determination of enzyme levels in serum, intraperitoneal injection of ATR and CATR resulted in significantly elevated serum alanine aminotransferase (ALT), asparate aminotransferase (AST), alkaline phosphatase (ALP) activities compared to controls. In the hepatic oxidative stress level, antioxidant-related enzyme activity assays showed that ATR and CATR administration significantly increased hepatic malondialdehyde (MDA) concentration, as well as decreased superoxide dismutase (SOD), catalase (CAT) activities and glutathione (GSH) concentration, and this was in good agreement with the results of serum aminotransferase activity and histopathological examinations. Taken together, our results demonstrate that kaurene glycosides induce hepatotoxicity in mice by way of its induction of oxidative stress as lipid peroxidation in liver, which merited further studies. Therefore, these toxic constituents explain, at least in part, the hepatotoxicity of X. strumarium L. in traditional medicine.

Anti-inflammatory diterpenes from the seeds of Vitex negundo.

Phytochemical investigation of a dichloromethane-soluble extract of Vitexnegundo seeds led to the isolation of five labdane diterpenes, negundoins A-E (1-5), a 9,10-seco-abietane diterpene, negundoin F (6), a sandaracopimara-7,15-diene diterpene, negundoin G (7), and two known diterpene derivatives (8, 9). Their chemical structures were elucidated by detailed spectroscopic analyses on the basis of NMR, IR, and MS data. The anti-inflammatory effects of metabolites 1-7 were also evaluated in vitro. Compounds 3 and 5 were among the most potent inhibitors on nitric oxide production by LPS-stimulated RAW 264.7 macrophages, with IC(50) values of 0.12 and 0.23 microM, respectively. Further studies revealed that compounds 3 and 5 (5 microM) significantly reduced the levels of the iNOS protein to 0.40+/-0.13% and 41.02+/-6.02%, respectively, and COX-2 protein to 2.06+/-0.53% and 26.40+/-7.43%, respectively.

Antinociceptive activities of the liposoluble fraction from Vitex negundo seeds.

The antinociceptive activities of the petroleum ether fraction (PEF) from the aqueous ethanol extract of Vitex negundo Linn. (Verbenaceae) seeds have been evaluated in several nociceptive mouse models. Given orally, the PEF (at doses of 12, 24, and 48 mg/kg body weight) produced significant dose-related inhibitions on chemical nociception induced by intraperitoneal acetic acid and sub-plantar formalin injections and on thermal nociception in the hot-plate test. Naloxone (1 mg/kg bw subcutaneously), a non-selected opioid receptor antagonist, notably reversed the analgesic effect caused by the PEF (48 mg/kg bw) when assessed against the first phase of the formalin test, but this effect was less significant for the PEF in the second phase. Our observations suggest that the PEF probably interacted with the opioid system and may be more effective on inflammatory pain. In addition, potent anti-inflammatory activity of the PEF was observed in the xylene-induced ear edema test, which further indicates that the analgesic effects of the PEF may be partially mediated by its anti-inflammatory activity. Further chemical analysis suggests that the analgesic activities of the PEF could be mostly due to the abundance of fatty acids with synergetic effects in the present work.

Nitric oxide scavenging lignans from Vitex negundo seeds.

A new phenyldihydronaphthalene-type lignan, vitexdoin A (1), a new phenylnaphthalene-type lignan alkaloid, vitedoamine B (2), four new phenylnaphthalene-type lignans, vitexdoins B-E (3-6), and four known lignan derivatives (7-10) were isolated from Vitex negundo seeds. Their structures were elucidated by detailed analyses of NMR, IR, and MS data. The ability of the isolates to prevent nitric oxide (NO) production by LPS-stimulated RAW 264.7 macrophages in a concentration-dependent manner was also studied. Compounds 5, 6, and 9 were among the most potent NO production inhibitors, with IC(50) values of 0.13, 0.15, and 0.11 microM, respectively. The introduction of free hydroxy groups plays a vital role in the potency of these compounds.

[Studies on chemical constituents of fructus broussonetiae].

OBJECTIVE: To study the chemical constituents of Fructus Broussonetiae. METHODS: Column chromatography with silic gel was employed to isolate and purify the 80% alcohol extract of Fructus Broussonetia, and the constituents were identified by spectral methods. RESULTS: Six compounds were isolated from 80% ethanol extract. Their structures were identified as Isoterihanine (1), Chelerythrine (2), Trillin (3), Sucrose (4), beta-sitosterol (5) and Fucosterol (6). CONCLUSION: These compounds are isolated from Fructus Broussonetiae for the first time.

[Cytoxic activities of total alkaloids isolated from fructus broussonetiae in vitro].

OBJECTIVE: To observe the cytoxic activities of total alkaloids isolated from Fructus Broussonetiae on the growth inhibition in five carcinoma cell lines. METHODS: The growth inhibition was analyzed by MTT, Cell colony in the Hela, BEL-7402, A375, SMM1990, Saos-2 cell lines. RESULTS: The growth of all tumor cells were inhibited by total alkaloids isolated from Fructus Broussonetiae. CONCLUSION: The results show that total alkaloids isolated from Fructus Broussonetiae possess the role of antitumors in cell culture.

[Studies on chemical constituents of Liriope platyphylla].

Seven chemical constituents were isolated from the CHCl3 fraction and n-BuOH fraction from EtOH ext. of Liriope platyphylla Wang et Tang. Their structures were elucidated as lupenone (I), lupeol (II), ursolic acid (III), beta-sitosterol (IV), diosgenin (V), LP-A (VI) and LP-B (VII) respectively. Compound I, II and V were isolated from this genus of Liriope for the first time. Compound III and IV were isolated from this plant for the first time.

[Effect of Liriope platyphylla total saponin on learning, memory and metabolites in aging mice induced by D-galactose].

OBJECTIVE: To investigate the effects of Liriope platyphylla total saponin (LPTS) on learning, memory, neuromediators and metabolites in aging mice induced by D-galactose. METHODS: Ninety Kunming mice were randomly divided into nine groups: normal saline (NS)-treated group, untreated group, high- (100 mg/kg), medium- (50 mg/kg) and low-dose (10 mg/kg) LPTS-treated groups, Shuxuening-treated group, jiaogulanosidi-treated group, flunarizine-treated group and vitamin E-treated group. The Kunming mice in the NS-treated group were administered with NS by intraperitoneal injection, while the aging mice in the other eight groups were administered with D-galactose by intraperitoneal injection. At the same time, the aging mice in different groups were fed with corresponding drugs for 42 days, and the aging items of the mice in different groups were measured, respectively. RESULTS: LPTS could improve the memory of aging mice induced by D-galactose, promote its body weight, and increase the thymus and spleen indexes of the aging mice. LPTS could decrease the levels of MDA and lipofuscin, inhibit MAO activity and increase SOD activity and GSH-Px level. CONCLUSION: LPTS may improve the ability of learning and memory and delay aging.

Antiosteoporotic activity of Er-Xian Decoction, a traditional Chinese herbal formula, in ovariectomized rats.

The antiosteoporotic effect of a herbal formula, Er-Xian Decoction (EXD), in ovariectomized (OVX) rats model of osteoporosis was investigated. The rats were divided into Sham and OVX groups. The OVX rats were further sub-divided into four groups administered orally with water, nylestriol (1 mg/kg, weekly) or EXD (300, 600 mg/kg, daily) for 12 weeks. In OVX rats, the increases of body weight, serum BGP and ALP were significantly decreased by EXD treatment. In OVX rats, atrophy of uterus and descent of BMD were suppressed by treatment with EXD and nylestriol. In addition, EXD completely corrected the decreased concentration of calcium, phosphorus, and estradiol in serum observed in OVX rats. EXD also significantly increased biomechanical strength comparable to the Sham group. This was also confirmed by histological results that showed its protective action. The findings assessed on the basis of biochemical, bone mineral density, biomechanical, and histopathological parameters strongly suggested that EXD had a definite antiosteoporotic effect, which is similar to estrogen.

[Antioxidation activities of Fructus Broussonetia haematochrome in vitro].

OBJECTIVE: To investigate the antioxidative effects of Fructus Broussonetia Haematochrome. METHODS: NADH-PMS-NBT system was used to produce supetoxide free radical (O2-*), Fe(2+) -H2O2 system to generate hydroxyl free radical (*OH), H2O2 to stimulate oxidative hemolysis of erythrocytes of mice. MDA production in liver homogenate induced by auto-oxidation was measured by MDA kits method. RESULTS: FBH not only scavenged O2-* and *OH produced by the experimental systems directly, but also inhibited H2O2 stimulated oxidative hemolysis of erythrocytes of mice, depressed MDA production in mice liver homogenate by auto-oxidation and hepatic mitochondria expanded induced by Vit C-Fe2+ system. CONCLUSION: FBH possess an antioxidative activity.

Phenolic acids in Fructus Xanthii and determination of contents of total phenolic acids in different species and populations of Xanthium in China.

OBJECTIVE: To study the chemical constituents of Fructus Xanthii and to determine the contents of total phenolic acids (TPA) in fruits of Xanthium from different populations for evaluating the quality of them. METHODS: Components in Fructus Xanthii were isolated and purified by various column chromatographies and the contents of TPA were determined by ultraviolet spectrophotometry with chlorogenic acid (CHA) as reference substance. RESULTS: Six caffeoylquinic acids along with caffeic acid and ferulic acid were isolated from Fructus Xanthii. The contents of TPA of the samples collected from 29 populations in China varied from 0.31% to 1.44%. Among the samples originated from two species and 1 variety of Xanthium, the contents of TPA in X. sibiricum var. subinerme samples with an average of 0.36% were relatively lower than those in other 2 species. While the content of TPA in Sample 3 collected from Shanghai was 1.44% and the highest among all the samples, and that in Sample 12 from Xinjian of Jiangxi Province was 0.38% and the lowest among the X. sibiricum samples. CONCLUSION: 5-O-caffeoylquinic acid, 1,4-di-O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid were isolated from Xanthium plant for the first time. The difference of contents of TPA in samples from different species and different populations in China was relatively significant. Fructus Xanthii in Shanghai and Sanming of Fujian Province were considered high quality if contents of TPA were used as reference for quality evaluating.

Traditional Chinese medicine formulas for the treatment of osteoporosis: Implication for antiosteoporotic drug discovery.

ETHNOPHARMACOLOGICAL RELEVANCE: Osteoporosis is a chronic epidemic which can leads to enhanced bone fragility and consequent an increase in fracture risk. Traditional Chinese medicine (TCM) formulas have a long history of use in the prevention and treatment of osteoporosis. Antiosteoporotic TCM formulas have conspicuous advantage over single drugs. Systematic data mining of the existing antiosteoporotic TCM formulas database can certainly help the drug discovery processes and help the identification of safe candidates with synergistic formulations. In this review, the authors summarize the clinical use and animal experiments of TCM formulas and their mechanism of action, and discuss the potential antiosteoporotic activity and the active constituents of commonly used herbs in TCM formulas for the therapy of osteoporosis. MATERIALS AND METHODS: The literature was searched from Medline, Pubmed, ScienceDirect, Spring Link, Web of Science, CNKI and VIP database from 1989 to 2015, and also collected from Chinese traditional books and Chinese Pharmacopoeia with key words such as osteoporosis, osteoblast, osteoclast, traditional Chinese medicine formulas to identify studies on the antiosteoporotic effects of TCM formulas, herbs and chemical constituents, and also their possible mechanisms. RESULTS: Thirty-three TCM formulas were commonly used to treat osteoporosis, and showed significant antiosteoporotic effects in human and animal. The herb medicines and their chemical constituents in TCM formulas were summarized, the pharmacological effects and chemical constituents of commonly used herbs in TCM formulas were described in detail. The action mechanisms of TCM formulas and their chemical constituents were described. Finally, the implication for the discovery of antiosteoporotic leads and combinatory ingredients from TCM formulas were prospectively discussed. CONCLUSIONS: Clinical practice and animal experiments indicate that TCM formulas provide a definite therapeutic effect on osteoporosis. The active constituents in TCM formulas are diverse in chemical structure, and include flavonoids, lignans, saponins and iridoid glycosides. Antiosteoporotic mechanism of TCM formulas and herbs involves multi regulatory pathways, such as Wnt/ß-catenin, BMP/Smad, MAPK pathway and RANKL/OPG system. Phytochemicals from TCM formulas and their compositional herb medicines offer great potential for the development of novel antiosteoporotic drugs. The active ingredients in TCM formulas can be developed in combination as potent drugs, which may exhibit better antiosteoporotic effects compared to the individual compound.

Deep Sequencing Reveals the Effect of MeJA on Scutellarin Biosynthesis in Erigeron breviscapus.

BACKGROUND: Erigeron breviscapus, a well-known traditional Chinese medicinal herb, is broadly used in the treatment of cerebrovascular disease. Scutellarin, a kind of flavonoids, is considered as the material base of the pharmaceutical activities in E. breviscapus. The stable and high content of scutellarin is critical for the quality and efficiency of E. breviscapus in the clinical use. Therefore, understanding the molecular mechanism of scutellarin biosynthesis is crucial for metabolic engineering to increase the content of the active compound. However, there is virtually no study available yet concerning the genetic research of scutellarin biosynthesis in E. breviscapus. RESULTS: Using Illumina sequencing technology, we obtained over three billion bases of high-quality sequence data and conducted de novo assembly and annotation without prior genome information. A total of 182,527 unigenes (mean length = 738 bp) were found. 63,059 unigenes were functionally annotated with a cut-off E-value of 10(-5). Next, a total of 238 (200 up-regulated and 38 down-regulated genes) and 513 (375 up-regulated and 138 down-regulated genes) differentially expressed genes were identified at different time points after methyl jasmonate (MeJA) treatment, which fell into categories of 'metabolic process' and 'cellular process' using GO database, suggesting that MeJA-induced activities of signal pathway in plant mainly led to re-programming of metabolism and cell activity. In addition, 13 predicted genes that might participate in the metabolism of flavonoids were found by two co-expression analyses in E. breviscapus. CONCLUSIONS: Our study is the first to provide a transcriptome sequence resource for E. breviscapus plants after MeJA treatment and it reveals transcriptome re-programming upon elicitation. As the result, several putative unknown genes involved in the metabolism of flavonoids were predicted. These data provide a valuable resource for the genetic and genomic studies of special flavonoids metabolism and further metabolic engineering in E. breviscapus.

Chemical constituents from an endophytic fungus Chaetomium globosum Z1.

A new ergosterol, 15beta-hydroxyl-(22E,24R)-ergosta-3, 5, 8, 22-tetraen-one (1), along with three known ergosterols, two known cytochalasins, and two known azapholines were isolated from Chaetomium globosum Z1. The structures of these compounds were elucidated on the basis of spectroscopic methods (HR-ESI-MS, 1D NMR, and 2D NMR). Compound 6 showed significant cytotoxic activity against A-549 and MG-63 cell lines with IC50 values of 6.96 and 1.73 microg/mL, respectively.

Illicium verum: a review on its botany, traditional use, chemistry and pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: The fruit of Illicium verum Hook. f. (Chinese star anise) has long been used in traditional Chinese medicine and food industry with the actions of dispelling cold, regulating the flow of Qi and relieving pain. MATERIALS AND METHODS: A bibliographic investigation was carried out by analyzing recognized books including Chinese herbal classic, and worldwide accepted scientific databases (Pubmed, SciFinder, Scopus and Web of Science) were searched for the available information on I. verum. RESULTS: I. verum is an aromatic evergreen tree of the family Illiciaceae. It is sometimes contaminated with highly toxic Japanese star anise (I. anisatum L.) and poisonous star anise (I. lanceolatum A. C. Smith), which contain several neurotoxic sesquiterpenes. Traditional uses of I. verum are recorded throughout Asia and Northern America, where it has been used for more than 10 types of disorders. Numerous compounds including volatiles, seco-prezizaane-type sesquiterpenes, phenylpropanoids, lignans, flavonoids and other constituents have been identified from I. verum. Modern pharmacology studies demonstrated that its crude extracts and active compounds possess wide pharmacological actions, especially in antimicrobial, antioxidant, insecticidal, analgesic, sedative and convulsive activities. In addition, it is the major source of shikimic acid, a primary ingredient in the antiflu drug (Tamiflu). AIM OF THE REVIEW: This review summarizes the up-to-date and comprehensive information concerning the botany, traditional use, phytochemistry and pharmacology of I. verum together with the toxicology, and discusses the possible trend and scope for future research of I. verum.