Characterization of a Sesquiterpene Synthase Catalyzing Formation of Cedrol and Two Diastereoisomers of Tricho-Acorenol from Euphorbia fischeriana.

A sesquiterpene synthase gene was identified from the transcriptome of Euphorbia fischeriana Steud, and the function of its product EfTPS12 was characterized by in vitro biochemical experiments and synthetic biology approaches. EfTPS12 catalyzed conversion of farnesyl diphosphate into three products, including cedrol (1) and eupho-acorenols A (2) and B (3) (two diastereoisomers of tricho-acorenol), thereby being named EfCAS herein. The structures of 2 and 3 were determined by spectroscopic methods and comparison of experimental and calculated electronic circular dichroism spectra. EfCAS is the first example of a plant-derived sesquiterpene synthase that is capable of synthesizing acorane-type alcohols. This study also documents that synthetic biology approaches enable large-scale preparation of volatile terpenes and thereby substantially facilitate characterization of corresponding terpene synthases and elucidation of the structures of their products.

Mining of the Catharanthus roseus Genome Leads to Identification of a Biosynthetic Gene Cluster for Fungicidal Sesquiterpenes.

Characterization of cryptic biosynthetic gene clusters (BGCs) from microbial genomes has been proven to be a powerful approach to the discovery of new natural products. However, such a genome mining approach to the discovery of bioactive plant metabolites has been muted. The plant BGCs characterized to date encode pathways for antibiotics important in plant defense against microbial pathogens, providing a means to discover such phytoalexins by mining plant genomes. Here is reported the discovery and characterization of a minimal BGC from the medicinal plant Catharanthus roseus, consisting of an adjacent pair of genes encoding a terpene synthase (CrTPS18) and cytochrome P450 (CYP71D349). These two enzymes act sequentially, with CrTPS18 acting as a sesquiterpene synthase, producing 5-epi-jinkoheremol (1), which CYP71D349 further hydroxylates to debneyol (2). Infection studies with maize revealed that 1 and 2 exhibit more potent fungicidal activity than validamycin. Accordingly, this study demonstrates that characterization of such cryptic plant BGCs is a promising strategy for the discovery of potential agrochemical leads. Moreover, despite the observed absence of 1 and 2 in C. roseus, the observed transcriptional regulation is consistent with their differential fungicidal activity, suggesting that such conditional coexpression may be sufficient to drive BGC assembly in plants.

A Multilayer Ceramic Electrolyte for All-Solid-State Li Batteries.

Despite of the good stability with Li-metal, Li6.75 La3 Zr1.75 Ta0.25 O12 (LLZTO) suffers from large interfacial resistance and severe Li-metal penetration. Herein, a dual layer ceramic electrolyte of Ti-doped LLZTO(Ti-LLZTO)/LLZTO was developed, with the reducible Ti-LLZTO layer contacting Li-metal and the LLZTO layer contacting cathode. The identical crystal structures of Ti-LLZTO and LLZTO enables a seamless contact and a barrierless Li+ transport between them. The densities of Ti-LLZTO pellets are higher than that of LLZTO. With an in situ reduction of Ti-LLZTO by Li-metal, the interfacial wettability was improved and a mixed ion-electron conducting layer was created. Both features help to reduce defects/pores on interface and homogenize the interfacial ionic/electronic flux, facilitating the reduction of interfacial resistance and suppression of dendrites. With the help of Ti-LLZTO layer, long-term stable lithium plating/stripping was reached in an areal capacity of 3.0 mAh cm-2 .

The Electrolysis of Anti-Perovskite Li2 OHCl for Prelithiation of High-Energy-Density Batteries.

Anti-perovskite type Li2 OHCl was previously studied as a solid-state Li+ conductor. Here, we report that the Li2 OHCl can be electrolyzed at 3.3 V or 4.0 V, with the creation of O2 /HCl gases and the release of 2 equiv. Li+ via two different decomposition routes, depending on the acidity of electrolyte. In the electrolyte with trace acid, the Li2 OHCl is oxidized at a constant voltage of 3.3 V. In neutral electrolyte, the oxidization of Li2 OHCl starts at 4.0 V, but the produced HCl will increase the acidity of electrolyte and lead to a voltage drop to 3.3 V for the electrolysis of Li2 OHCl. The electrolysis of Li2 OHCl delivers a lithium releasing capacity as high as 810 mAh g-1 , with an equivalent Li-deposition or Li-intercalation on anode, making it a promising candidate as a Li reservoir for prelithiation of anode. Using Li2 OHCl as the lithium source, silicon-carbon (Si@C) composite anode can be effectively prelithiated. The full cells composed of LiNi0.8 Mn0.1 Co0.1 O2 (NMC811) cathode and prelithiated Si@C anode exhibited increased capacities with the increment of prelithiation dosages.

Butylene fipronil induces apoptosis in PC12 murine nervous cells via activation of p16-CDK4/6-cyclin D1 and mitochondrial apoptotic pathway.

Butylene fipronil (BFPN) is a phenylpyrazole insecticide, acting at the γ-aminobutyric acid (GABA) receptor. Here, we show that BFPN inducedcytotoxicity in PC12 murinenervous cells, which lacks GABA receptor. Treatment with BFPN for 48 hours significantly enhanced G0/G1 arrest and induced apoptosis. BFPN decreased the expression of cyclin-dependent kinase (CDK4 and CDK6) and increased P16 and cyclin D1. Simultaneously, Bcl-2 protein was declined while Bax and cytochrome c were significantly enhanced in BFPN-treated groups. The apoptotic enzymes caspase-8, -9, and -3 were also activated by BFPN. Furthermore, treatment with BFPN significantly stimulated reactive oxygen species (ROS) generation, and pretreatment with antioxidant diphenyleneiodonium, substantially reduced cell death. Overall, these results suggest that BFPN is effective to induce G0/G1-phase arrest and apoptosis in PC12 murine nervous cell. Stimulating ROS generation and activation of P16-CDK4/6-cyclin D1 and mitochondrial apoptotic pathway may participate in the cytotoxicity of BFPN.

Characterization of SchTPSs Enables Construction of Yeast for the Bioproduction of α-Cadinol and the Related Sesquiterpenes.

Plant volatile sesquiterpenes (PVSs) play important roles in chemical plant defense. However, it is difficult to isolate sufficient PVSs for deep investigations due to their low contents and chemical and physical properties close to those of other lipids. The extracts of Stellera chamaejasme L. exhibit insecticidal, fungicidal, and allelopathic activities. In this study, we identified three sesquiterpene synthase genes (SchTPS5, SchTPS6, and SchTPS7) from S. chamaejasme L. SchTPS7 is an α-farnesene synthase. SchTPS5 and SchTPS6 are two catalytically promiscuous sesquiterpene synthases, and α-cadinol and τ-muurolol are the predominant products for both of them in Saccharomyces cerevisiae. This study, for the first time, reports plant sesquiterpene synthases capable of producing α-cadinol and/or τ-muurolol in a heterologous host. More intriguingly, seven out of eight products of SchTPS6 in S. cerevisiae possess various insecticidal, fungicidal, and herbicidal activities. Building on this finding, we used SchTPS6 to construct an engineered S. cerevisiae for the production of these sesquiterpenes. The titers of two major products α-cadinol and τ-muurolol, respectively, reached 46.2 ± 4.0 and 11.2 ± 1.4 mg/L in a flask. This study lays a foundation for the development of new agrochemical mixtures.

Manipulation of IME4 expression, a global regulation strategy for metabolic engineering in Saccharomyces cerevisiae.

Metabolic engineering has been widely used for production of natural medicinal molecules. However, engineering high-yield platforms is hindered in large part by limited knowledge of complex regulatory machinery of metabolic network. N6-Methyladenosine (m6A) modification of RNA plays critical roles in regulation of gene expression. Herein, we identify 1470 putatively m6A peaks within 1151 genes from the haploid Saccharomyces cerevisiae strain. Among them, the transcript levels of 94 genes falling into the pathways which are frequently optimized for chemical production, are remarkably altered upon overexpression of IME4 (the yeast m6A methyltransferase). In particular, IME4 overexpression elevates the mRNA levels of the methylated genes in the glycolysis, acetyl-CoA synthesis and shikimate/aromatic amino acid synthesis modules. Furthermore, ACS1 and ADH2, two key genes responsible for acetyl-CoA synthesis, are induced by IME4 overexpression in a transcription factor-mediated manner. Finally, we show IME4 overexpression can significantly increase the titers of isoprenoids and aromatic compounds. Manipulation of m6A therefore adds a new layer of metabolic regulatory machinery and may be broadly used in bioproduction of various medicinal molecules of terpenoid and phenol classes.

Chamaejasnoids A-E, a 2,3-seco-guaiane sesquiterpenoid with a 5/6/7 bridged ring system and related metabolites from Stellera chamaejasme L.

Sixteen guaiane-type sesquiterpenoids were isolated from Stellera chamaejasme L. Among them, chamaejasnoids A-F (1-5) are new compounds. 1 represents the first example of 2,3-seco-guaiane sesquiterpenoid with a 5/6/7 bridged ring system. 2 is a unique 2-nor-guaiane sesquiterpenoid. A plausible biosynthetic pathway for 1 was proposed, involving a Baeyer-Villiger oxidation and a non-enzymatic intramolecular transesterification. 5 exhibited a selective cytotoxicity against HCT8 cell line with an IC50 of 11.82 ± 2.89 µM.

Rationally engineering santalene synthase to readjust the component ratio of sandalwood oil.

Plant essential oils (PEOs) are widely used in cosmetic and nutraceutical industries. The component ratios of PEOs determine their qualities. Controlling the component ratios is challenging in construction of PEO biotechnological platforms. Here, we explore the catalytic reaction pathways of both product-promiscuous and product-specific santalene synthases (i.e., SaSSy and SanSyn) by multiscale simulations. F441 of SanSyn is found as a key residue restricting the conformational dynamics of the intermediates, and thereby the direct deprotonation by the general base T298 dominantly produce α-santalene. The subsequent mutagenesis of this plastic residue leads to generation of a mutant enzyme SanSynF441V which can produce both α- and ß-santalenes. Through metabolic engineering efforts, the santalene/santalol titer reaches 704.2 mg/L and the component ratio well matches the ISO 3518:2002 standard. This study represents a paradigm of constructing biotechnological platforms of PEOs with desirable component ratios by the combination of metabolic and enzymatic engineering.

Efficacy of ginseng supplements on disease-related fatigue: A systematic review and meta-analysis.

BACKGROUND: Ginseng has been believed to provide energy, physical health, and well-being to patients for hundreds of years. Fatigue is a multidimensional symptom with unknown etiology and varying severity, and lots of patients suffer from fatigue. METHODS: We search for research of ginseng treatment of disease-related fatigue in adult patients in Pubmed, Embase, Medline, and Cochrane library. Two independent reviewers assessed included studies and met to develop consensus on included studies. And we used Review Manager 5.3 software to evaluate the risk of bias. RESULTS: The present meta-analysis included 12 randomized controlled trial containing 1298 patients. In the fixed-effect meta-analysis of 12 randomized controlled trial, ginseng supplements had a statistically significant efficacy on disease-related fatigue reduction (standardized mean difference = 0.33, 95% confidence interval = 0.44-0.22). CONCLUSIONS: The use of ginseng supplements is benefit for patients to reduce disease-related fatigue.

A review: biosynthesis of plant-derived labdane-related diterpenoids.

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.

Chemical structure and anti-inflammatory activity of a branched polysaccharide isolated from Phellinus baumii.

Phellinus baumii is used to treat inflammatory bowel disease (IBD) and gastroenteritis. In this study, a 46 kDa heteropolysaccharide SHPS-1 was isolated from fruiting bodies of P. baumii. SHPS-1 consisted of arabinose, mannose, glucose, and galactose at a molar ratio of 2.2:15.7:49.3:32.8. SHPS-1 had a backbone containing 1,3-linked ß-D-Glcp and 1,6-linked α-D-Galp residues, and Araf, Manp and Galp units were attached as oligosaccharidic side chains to the backbone at C-6 of some glucopyranoses. SHPS-1 decreased phosphorylation level of STAT-1 and expression levels of STAT-1 targeted genes such as iNOS and TNF-α in lipopolysaccharide-stimulated macrophage RAW 264.7 cells. Furthermore, SHPS-1 promoted the expression of IL-10 and macrophage mannose receptor CD 206, markers of tissue repairing macrophages. SHPS-1 alleviated ulcerative colitis in mice by decreasing pro-inflammatory genes and increasing anti-inflammatory and tissue repairing genes. Collectively, SHPS-1 polysaccharide from P. baumii had anti-inflammatory activity and can potentially treat IBD.

Daphnane-type diterpenoids from Euphorbia fischeriana Steud and their cytotoxic activities.

Two new daphnane-type diterpenoids fischerianin A (1) and fischerianin B (2), as well as two known ones langduin A (3) and langduin A6 (4), were isolated from the extracts of Euphorbia fischeriana Steud dry roots. Their structures including the absolute stereochemistry were determined by various spectroscopic methods and comparing their experimental and calculated CD spectra. 1 and 2 harbor a ketal group and a 9,13-oxide bridge in their C ring which is rare in daphnane-type diterpenoids. In cytotoxic assays, moderately inhibitory activities of 1-4 against human cancer cell lines (human malignant melanoma cell line, A375; human liver carcinoma cell line, HepG2; human promyelocytic leukemia cell line, HL-60; human Caucasian chronic myelogenous leukemia cell line, K562; human cervix epithelioid carcinoma cell line, HeLa) were observed, with IC50 values ranging from 5.31 to 21.46 µM.

Aqueous extracts of Sanghuangporus vaninii induce S-phase arrest and apoptosis in human melanoma A375 cells.

Sanghuangporus vaninii, also called 'Sanghuang' mushroom in Chinese, has various medicinal uses, but its effects on human melanoma cells have not been reported. The present study investigated the inhibitory ability and potential anticancer mechanism of the aqueous extracts of S. vaninii (SH). The results revealed that SH inhibited the proliferation of A375 human melanoma cells in a dose-dependent manner, and flow cytometry analysis suggested that SH induced A375 cell cycle arrest at S phase and apoptosis. Reverse transcription-quantitative PCR, western blotting and immunofluorescence analyses indicated that SH induced S-phase arrest by upregulating p21 expression, and p21 inhibited the expression of cyclin-cyclin-dependent kinases complexes at both the RNA and protein levels. In addition, SH induced apoptosis of A375 cells by inhibiting the expression levels of the anti-apoptosis gene Bcl-2. Therefore, the results suggested that SH may be a potential candidate for the treatment of human melanoma, thus providing new ideas for developing drugs that target melanoma.

Phellinus gilvus­derived protocatechualdehyde induces G0/G1 phase arrest and apoptosis in murine B16­F10 cells.

Protocatechualdehyde (PCA) is considered to be the main phenolic component of Phellinus gilvus responsible for its anticancer properties. Previous studies have demonstrated that PCA can have an anticancer effect on multiple cancer types, but little is known about the effect of PCA on melanoma cells. The present study investigated the inhibitory abilities and potential anticancer mechanisms of PCA on B16­F10 cells using MTT assay. Cell apoptosis and cell cycle were assessed by flow cytometry using Annexin V­FITC and propidium iodide staining. Whole­transcriptome analysis was used to investigate the effects of PCA on gene expression. PCA significantly decreased cell viability, induced cell cycle arrest at G0/G1 phase and promoted apoptosis of B16­F10 cells, suggesting that PCA could have anticancer effects against melanoma cells. Whole­transcriptome analysis indicated that PCA treatment upregulated genes involved in histone modification and decreased the transcription of genes involved in DNA repair and replication. Kyoto Encyclopedia of Genes and Genomes analysis showed that PCA treatment enhanced the complement and coagulation cascades, and the p53 signaling pathway. The present results indicated that PCA could act as an antitumor agent in melanoma cells, which may provide experimental support for the development of novel therapies to treat melanoma.

Enhance production of diterpenoids in yeast by overexpression of the fused enzyme of ERG20 and its mutant mERG20.

Yeast has been widely used for large-scale production of terpenoids. In yeast, modifications of terpenoid biosynthetic pathways have been intensively studied. tHMG1 (encoding the catalytic domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase of yeast) and UPC2-1 (the G888D mutant of UPC2 encoding a transcription factor) were integrated into yeast chromosome, and ERG9 (the squalene synthase gene of yeast) was knocked down to yield the chassis strain DH02. A F96C mutation in ERG20 (farnesyl diphosphate synthase of yeast) was conducted to obtain mERG20 which can function as a geranylgeranyl diphosphate synthase (GGPS). Then, three fused genes, including BTS1 (the yeast innate GGPS)-ERG20, ERG20-mERG20 and mERG20-ERG20, were constructed, and expressed either by the pESC-based plasmids in DH02, or by being integrated into DH02 chromosome. The highest geranylgeraniol (GGOH) content was observed in the extracts of DH12 integrated with ERG20-mERG20, corresponding to 3.2 and 2.3 folds of those of the strains integrated with BTS1 and mERG20, respectively. Finally, three genes encoding nor-copalyl diphosphate synthase (nor-CPS), ent-CPS and syn-CPS were integrated into the chromosome of DH12, respectively, to construct yeasts for producing corresponding copalyl diphosphates (CPPs). Thus, a yeast-based platform was built for characterizing all types of diterpene synthases using GGPP or various CPPs as their substrates.

Impact of Phellinus gilvus mycelia on growth, immunity and fecal microbiota in weaned piglets.

BACKGROUND: Antibiotics are the most commonly used growth-promoting additives in pig feed especially for weaned piglets. But in recent years their use has been restricted because of bacterial resistance. Phellinus, a genus of medicinal fungi, is widely used in Asia to treat gastroenteric dysfunction, hemrrhage, and tumors. Phellinus is reported to improve body weight on mice with colitis. Therefore, we hypothesize that it could benefit the health and growth of piglets, and could be used as an alternative to antibiotic. Here, the effect of Phellinus gilvus mycelia (SH) and antibiotic growth promoter (ATB) were investigated on weaned piglets. METHODS: A total of 72 crossbred piglets were randomly assigned to three dietary treatment groups (n = 4 pens per treatment group with six piglets per pen). The control group was fed basal diet; the SH treatment group was fed basal diet containing 5 g/kg SH; the ATB treatment group was feed basal diet containing 75 mg/kg aureomycin and 20 mg/kg kitasamycin. The experiment period was 28 days. Average daily gain (ADG), average daily feed intake (ADFI), and feed intake to gain ratio were calculated. The concentrations of immunoglobulin G (IgG), interleukin-1ß (IL-1ß), tumor necrosis factor (TNF)-α and myeloperoxidase (MPO) in serum were assessed. Viable plate counts of Escherichia coli in feces were measured. Fecal microbiota was analyzed via the 16S rRNA gene sequencing method. RESULTS: The ADG (1-28 day) of piglets was significantly higher in SH and ATB treatment groups (P < 0.05) compared to the control, and the ADG did not show significant difference between SH and ATB treatment groups (P > 0.05). Both SH and ATB treatments increased the MPO, IL-1ß, and TNF-α levels in serum compared to the control (P < 0.05), but the levels in SH group were all significantly higher than in the ATB group (P < 0.05). Fecal microbiological analysis showed that viable E. coli counts were dramatically decreased by SH and ATB. The 16S rRNA gene sequencing analysis showed that ATB shifted the microbiota structure drastically, and significantly increased the relative abundance of Prevotella, Megasphaera, and Faecalibacterium genera. But SH slightly influenced the microbiota structure, and only increased the relative abundance of Alloprevotella genus. CONCLUSION: Our work demonstrated that though SH slightly influenced the microbiota structure, it markedly reduced the fecal E. coli population, and improved growth and innate immunity in piglets. Our finding suggested that SH could be an alternative to ATB in piglet feed.

Reconstruction of the Biosynthetic Pathway of Santalols under Control of the GAL Regulatory System in Yeast.

Sandalwood oil has been widely used in perfumery industries and aromatherapy. Santalols are its major components. Herein, we attempted to construct santalol-producing yeasts. To alter flux from predominant triterpenoid/steroid biosynthesis to sesquiterpenoid production, expression of ERG9 (encoding yeast squalene synthase) was depressed by replacing its innate promotor with PHXT1 and fermenting the resulting strains in galactose-rich media. And the genes related to santalol biosynthesis were overexpressed under control of GAL promotors, which linked santalol biosynthesis to GAL regulatory system. GAL4 (a transcriptional activator of GAL promotors) and PGM2 (a yeast phosphoglucomutase) were overexpressed to overall promote this artificial santalol biosynthetic pathway and enhance galactose uptake. 1.3 g/L santalols and 1.2 g/L Z-α-santalol were achieved in the strain WL17 expressing SaSS (α-santalene synthase from Santalum album) and WL19 expressing SanSyn (α-santalene synthase from Clausena lansium) by fed-batch fermentation, respectively. This study constructed the microbial santalol-producing platform for the first time.

The Gut Microbiota-Produced Indole-3-Propionic Acid Confers the Antihyperlipidemic Effect of Mulberry-Derived 1-Deoxynojirimycin.

Hyperlipidemia is a worldwide epidemic with an obvious gender disparity in incidence. Modulations on gut microbiota by traditional Chinese medicines (TCM) are emerging as a potential rationale governing the profitable effects of drugs on hyperlipidemia. However, it is unclear how gut microbes regulate the progression of hyperlipidemia. Here, we found that mulberry leaf extract (MLE) and its active component 1-deoxynojirimycin (DNJ) diminished hyperglycemia and hypertriglyceridemia with similar efficacy in male and female mice but preferentially alleviated hypercholesterolemia in female mice. Further investigations showed that DNJ sex-specifically downregulated the expression of lipogenic genes, especially cholesterol-biosynthetic genes. Oral administration of DNJ imposed more profound modulation on gut microbiota in female mice than in male ones, as estimated by 16S rRNA metatranscriptomic analysis. DNJ markedly enriched Akkermansia and Clostridium group XIVa and promoted the production of indole-3-propionic acid (IPA) in a sexually dimorphic way. Importantly, IPA tightly associates with the antihyperlipidemic effect of DNJ and exhibited a potent lipid-lowering effect both in vitro and in vivo Together, our results have established a regulatory mechanism by which DNJ sex-specifically improves hyperlipidemia, offering an in-depth theoretical basis for therapeutic exploitation of DNJ as a sex-specific intervention against hyperlipidemia.IMPORTANCE Hyperlipidemia has been intensively focused on by researchers around the world owing to its major contribution to cardiovascular diseases. Various evidence reveals that women are more susceptible than male counterparts to dyslipidemia, making sex-dependent therapeutic strategies and drugs urgently needed. In the present work, we demonstrate that DNJ, the main active component of mulberry leaves, exerts an obvious female-preferential antihyperlipidemic effect through specifically enriching Akkermansia and Clostridium XIVa and elevating an active microbial metabolite, indole-3-propionic acid (IPA), in female mice. Moreover, we have corroborated the potent lipid-lowering efficacy of IPA both in vitro and in vivo These findings not only indicate a potential mechanism by which gut microbes and their metabolites confer the beneficial role of DNJ in ameliorating hyperlipidemia but also provide an in-depth theoretical basis for therapeutic exploitation of DNJ as a female-specific intervention against hyperlipidemia.

Safety evaluation of aqueous extracts of Sanghuangporus vaninii fruiting body in Sprague-Dawley rats.

Sanghuangporus vaninii, called "Sanghuang," is orally used for health care, tumor, and inflammation treatment in Asia. However, the safety of S. vaninii has not been evaluated. The major compounds analysis showed that aqueous extracts of S. vaninii fruiting body were rich in polysaccharides, nucleotides, and polyphenols. Then, the aqueous was given orally to Sprague-Dawley rats for toxical test. In acute toxicity study, the maximum tolerated dose was 21 g/kg. In 17-week repeated dose toxicity experiment, all rats had no abnormal reaction among control group, low dose group (0.15 g/kg) and middle dose group (1.00 g/kg). At high dose group (6.00 g/kg), the feces began to darken on 16th day (D16), and turned to drug stained stool on D21, all rats recovered on the 3rd day (D92) of recovery period. During the whole experiment, there were no animal death and no treatment-related changes in any of the parameters under the all doses. These results indicated the No-Observed Adverse Effect Level of aqueous extract of S. vaninii fruiting body was 1.0 g/kg.