Effects of β-sitosterol on the function of synovial fibroblasts in rheumatoid arthritis and its mechanism / 中国药房

OBJECTIVE To investigate the effects of β-sitosterol on the function of rheumatoid arthritis （RA） fibroblastic synoviocytes MH7A cells and its mechanism. METHODS Network pharmacology was adopted to screen the targets of β-sitosterol and the targets for the treatment of RA. After the intersection of them， topological analysis was performed to find the most critical target in the treatment of RA. MH7A cells were treated with different concentrations （0， 5， 10， 20， 40 μmol/L） of β-sitosterol， and CCK-8 was used to assay cell viability for screening the optimal concentration of β-sitosterol. MH7A cells were induced by 10 ng/mL TNF-α in vitro and treated with β-sitosterol （the optimum concentration）. CCK-8 and EdU were used to detect the ability of cell proliferation. Scratch experiment and Transwell invasion assay were used to analyze cell migration and invasion. The levels of interleukin-1β （IL-1β） and IL-6 in cell supernatant were detected by enzyme-linked immunosorbent assay （ELISA）. The mRNA and protein expressions of peroxisome proliferator-activated receptor α （PPARα） were measured by qRT-PCR and Western blot， respectively. The siRNA targeting PPARα was transfected into MH7A cells， and the effects of β-sitosterol on cell proliferation， migration， invasion， the secretion of inflammatory factors and the expression of PPARα after PPARα knockdown were detected by the above experimental methods. RESULTS PPARα was the most critical target of β-sitosterol in the treatment of RA. The optimal concentration of β-sitosterol was 20 μmol/L. Compared with model group， β-sitosterol decreased the viability of MH7A cells， and the number of proliferating cells also decreased significantly （P＜0.05）； the cell migration rate and the number of cell invasion decreased significantly （P＜0.05）. The levels of IL-1β and IL-6 were also significantly decreased （P＜0.05）， and the mRNA 15 and protein expression levels of PPARα were significantly increased （P＜0.05）. Compared with negative control small interfering RNA group， after PPARα knockdown， the cell viability increased by about 35.6% （P＜0.05）， the number of cell proliferation， the cell migration rate and the number of cell invasion increased significantly （P＜0.05）， and the levels of IL-1β and IL-6 also increased significantly （P＜0.05）. CONCLUSIONS β-sitosterol could effectively inhibit the proliferation， migration， invasion and secretion of inflammatory factors in MH7A cells， the mechanism of which may be associated with activating PPARα pathway.

Molecular mechanism of Ganoderma against gastric cancer based on network pharmacology and experimental test / 中国中药杂志

This study aims to explore the molecular mechanism of Ganoderma against gastric cancer based on network pharmacology, molecular docking, and cell experiment. The active components and targets of Ganoderma were retrieved from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP), and gastric cancer-related targets from GeneCards and Online Mendelian Inheritance in Man(OMIM). The protein-protein interaction(PPI) network of the common targets was constructed with STRING, followed by Gene Ontology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis of the common genes based on Bioconductor and R language. The medicinal-disease-component-target network and medicinal-disease-component-target-pathway network were established by Cytoscape. Molecular docking was performed between β-sitosterol(the key component in Ganoderma) and the top 15 targets in the PPI network. Cell experiment was performed to verify the findings. A total of 14 active components and 28 targets of Ganoderma were retrieved, and the medicinal and the disease shared 25 targets, including caspase-3(CASP3), caspase-8(CASP8), caspase-9(CASP9), and B-cell lymphoma-2(BCL2). The common targets involved 72 signaling pathways and apoptosis and p53 signaling pathway may play a crucial role in the effect of Ganoderma against gastric cancer. β-sitosterol had strong binding activity to the top 15 targets in the PPI network. The in vitro cell experiment demonstrated that β-sitosterol inhibited gastric cancer AGS cell proliferation by inducing cell apoptosis and cell cycle arrest in the S phase, which might be related to the regulation of the p53 pathway. This study shows the multi-component, multi-target, and multi-pathway characteristics of Ganoderma against gastric cancer, which lays a scientific basis for further research on the molecular mechanism.

Preparation and Antidepressant Activity Study of β-sitosterol and Its Derivatives from the Fruit of Sorbus pohuashanensis / 中国药房

OBJECTIVE： To prepare β-sitosterol （shorted fo r “Sit”） and its deri vatives from the fruit of Sorbus pohuashanensis，and to investigte their antidepressant activities. METHODS ：Using the fruit of S. pohuashanensis as raw material ， extracted with 75％ ethanol and 20％KOH solution ，Sit was obtained after extraction and crystallization. C 3 hydroxyl group of Sit was used as the structural modification site ，CH2Cl2 was used as the reaction solvent ，DMAP was used as catalyst ，EDCI was used as dehydrating agent ，4 kinds of organic acids （salicylic acid ，2-tetrahydrofuranic acid ，phenylalanine，sorbic acid ）were added to make the carboxylation reaction to produce ester derivatives Sit-S ，Sit-T，Sit-P and Sit-Sr. The chemical structure of its derivatives were elucidated by IR and NMR. The tail suspension test and the forced swimming model were used to preliminarily explore the antidepressant active components of Sit and 4 kinds of derivatives ；tail suspension test and the spontaneous exercise capacity test were used to screen effective dose of the compounds with antidepressant active. The compound was used alone or in combination with agomelatin [ 40 mg/kg，5-hydroxytryptamine（5-HT）and 5-HT 2C receptor antagonist] ，haloperidol [ 0.2 mg/kg，non-selective D2 receptor antagonist] and bicuculline [ 4 mg/kg，competitive γ-aminobutyric acid（GABA）antagonist]，respectively. Sixty minutes after intraperitoneal injection ，tail suspension test was performed. The levels of 5-HT，dopamine（DA）and GABA in brain tissues of mice were detected by ELISA. The blank control group was given intraperitoneal injection of 10％ propylene glycol solution. RESULTS：By spectrum technology ，the corresponding ester compounds were synthesized by the reaction of Sit with four kinds of organic acids. Among Sit and its four derivatives ，the immobility time of Sit-S group was the shortest in tail suspension test and forced swimming test ，which was significantly shorter than that of blank control group （P＜0.05）. Screening results of Sit-S showed that the effective antidepressant dose was 4 mg/kg， ＊ and it did not affect the spontaneous activity of mice compared with the blank control group （P＞0.05）. With this dose ，Sit-S could significantly shorten the immobility time of mice in tail hua1666@163.com suspension test （P＜0.01），and can s ignificantly increase the ·64levels of 5-HT，DA and GABA in the brain tissue of mice （P＜0.05 or P＜0.01），but its effect can be reversed by agomelatine ， haloperidol and bicuculline to different extent. CONCLUSIONS ：Sit salicylate derivatives Sit-S exhibits good antidepressant activity，and its mechanism of action may be mediated by increasing the levels of 5-HT，DA and GABA.

Response surface methodology-based optimization of ultrasound-assisted extraction of β-sitosterol and lupeol from astragalus atropilosus (roots) and validation by HPTLC method

Objective: To optimize the ultrasonication method for efficient extraction of P-sitosterol and lupeol from the roots of Astragalus atropilosus using Box-Behnken design of response surface methodology (RSM), and its validation by high performance thin layer chromatography (HPTLC) method. Methods: Ultrasonication method was used to extract β-sitosterol and lupeol from Astragalus atropilosus (roots). RSM was used to optimize the different extraction parameters viz. liquid to solid ratio (10-14 mL/g), temperature (60-80 °C) and time (40-60 min) to maximize the yield of β-sitosterol and lupeol. The quantitative estimation of β-sitosterol and lupeol was done in chloroform extract of Astragalus atropilosus by validated HPTLC method on 10 cm × 20 cm glass-backed silica gel 60F

Feasibility of Xiaochaihu Decoction on fever induced by coronavirus disease 2019 (COVID-19) based on network pharmacology / 中草药

Objective: The aim of this article was to study the potential antivirus and fever reducing mechanisms of Xiaochaihu Decoction (XCHD) on novel coronavirus pneumonia based on network pharmacology and molecular docking method. Methods: Firstly, the potential targets and pathways of XCHD on fever were analyzed using network pharmacology. Compounds and potential targets in XCHD were screened using TCMSP and PharmMapper databases. The targets in fever reducing were identified from OMMI and Genecards databases. The protein-protein interaction network was established by String database to analyze key targets. The gene oncology (GO) analysis and Kyoto encyclopedia of genes and genomes (KEGG) analysis of key targets were also conducted to generate the relative pathways based on DAVID and KOBAS 3.0 databases, respectively. The compound-target-pathway network was established using Cytoscape 3.2.7. In addition, we used molecular docking method to identify the crucial compounds with higher connectivity on SARS-CoV-2 and the angiotensin-converting enzyme 2 (ACE2). ACE2 has been identified as the key target of SARS-CoV-2 entering cells. The possible binding sites of compounds on SARS-CoV-2 and ACE2 were predicted. Results: Network pharmacology analysis indicated that 165 active compounds and 168 relative targets were selected. A total of 7006 targets related to fever were identified. In addition, 141 potential targets of XCH on fever were identified. Totally, 292 GO terms of XCHD on fever and 30 pathways were identified using GO and KEGG analysis. Furthermore, molecular docking indicated that main active compounds in XCHD exhibited higher affinity with both SARS-CoV-2 and ACE2. Beta-sitosterol, stigmasterol, 3’-hydroxy-4’-O-methylglabridin were top three candidates with highest affinity. Conclusion: In summary, our study identified the potential mechanisms of XCHD on fever. Besides, Beta-sitosterol, stigmasterol, 3’-hydroxy-4’-O-methylglabridin could be the key compounds to exert anti-viral effects against SARS-CoV-2. Our prediction also provided the research fields to further study the mechanisms of XCH on SARS-CoV-2 infection in future.

Exploring active compounds of Jinhua Qinggan Granules for prevention of COVID-19 based on network pharmacology and molecular docking / 中草药

Objective: To explore the effective chemical constituents of Jinhua Qinggan Granules for treatment of coronavirus disease 2019 (COVID-19). Methods: The compounds and action targets of eleven herbal medicines in Jinhua Qinggan Granules were collected via TCMSP. The genes corresponding to the targets were queried by the UniProt database, then the “herbal medicine-compound-target” network was established by Cytoscape software. The gene ontology (GO) function enrichment analysis and KEGG pathway enrichment analysis were performed by DAVID to predict their mechanism. Molecular docking was used to analyze the binding force of the core effective compounds in the “herbal medicine-compound-target” network with SARS-CoV-2 3CL hydrolase and angiotensin converting enzyme II (ACE2). Results: The “herbal medicine-compound-target” network contained 154 compounds and 276 targets, and the key targets involved PTGS2, HSP90AB1, HSP90AA1, PTGS1, NCOA2, etc. GO function enrichment analysis revealed 278 items, including ATP binding, transcription factor activation and regulation of apoptosis process, etc. KEGG pathway enrichment screened 127 signaling pathways, including TNF, PI3K/Akt and HIF-1 signaling pathways related to lung injury protection. The results of molecular docking showed that formononetin, stigmasterol, beta-sitosterol, anhydroicaritin and other key compounds have a certain degree of affinity with SARS-CoV-2 3CL hydrolase and ACE2. Conclusion: The effective compounds in Jinhua Qinggan Granules regulate multiple signaling pathways via binding ACE2 and acting on targets such as PTGS2, HSP90AB1, HSP90AA1, PTGS1, NCOA2 for the prevention of COVID-19.

β-Sitosterol treatment attenuates cognitive deficits and prevents amyloid plaque deposition in amyloid protein precursor/presenilin 1 mice

Alzheimer's disease (AD) is the most common neurodegenerative disorder causing dementia worldwide, and is mainly characterized by aggregated β-amyloid (Aβ). Increasing evidence has shown that plant extracts have the potential to delay AD development. The plant sterol β-Sitosterol has a potential role in inhibiting the production of platelet Aβ, suggesting that it may be useful for AD prevention. In the present study, we aimed to investigate the effect and mechanism of β-Sitosterol on deficits in learning and memory in amyloid protein precursor/presenilin 1 (APP/PS1) double transgenic mice. APP/PS1 mice were treated with β-Sitosterol for four weeks, from the age of seven months. Brain Aβ metabolism was evaluated using ELISA and Western blotting. We found that β-Sitosterol treatment can improve spatial learning and recognition memory ability, and reduce plaque load in APP/PS1 mice. β-Sitosterol treatment helped reverse dendritic spine loss in APP/PS1 mice and reversed the decreased hippocampal neuron miniature excitatory postsynaptic current frequency. Our research helps to explain and support the neuroprotective effect of β-Sitosterol, which may offer a novel pharmaceutical agent for the treatment of AD. Taken together, these findings suggest that β-Sitosterol ameliorates memory and learning impairment in APP/PS1 mice and possibly decreases Aβ deposition.

Response surface methodology-based optimization of ultrasound-assisted extraction of β-sitosterol and lupeol from Astragalus atropilosus (roots) and validation by HPTLC method

Objective: To optimize the ultrasonication method for efficient extraction of β-sitosterol and lupeol from the roots of Astragalus atropilosus using Box-Behnken design of response surface methodology (RSM), and its validation by high performance thin layer chromatography (HPTLC) method.Methods: Ultrasonication method was used to extract β-sitosterol and lupeol from Astragalus atropilosus (roots). RSM was used to optimize the different extraction parameters viz. liquid to solid ratio (10–14 mL/g), temperature (60-80 ℃) and time (40–60 min) to maximize the yield of β-sitosterol and lupeol. The quantitative estimation of β-sitosterol and lupeol was done in chloroform extract of Astragalus atropilosus by validated HPTLC method on 10 cm × 20 cm glass-backed silica gel 60F254 plate using hexane and ethyl acetate (8:2, v/v) as mobile phase. Results: A quadratic polynomial model was found to be most appropriate with regard to R1 (yield of total extraction; R2/％ CV = 0.9948/0.28), R2 (β-sitosterol yield; R2/％ CV = 0.9923/0.39) and R3 (lupeol yield; R2/％ CV = 0.9942/0.97). The values of adjusted R2/predicted R2/signal to noise ratio for R1, R2, and R3 were 0.9782/0.9551/48.77, 0.9904/0.9110/31.33, and 0.9927/0.9401/36.08, respectively, indicating a high degree of correlation and adequate signal. The linear correlation plot between the predicted and experimental values for R1, R2, and R3 showed high values of R2 ranging from 0.9905-0.9973. β-sitosterol and lupeol in chloroform extract of Astragalus atropilosus were detected at Rf values of 0.22 and 0.34, respectively, at λ max = 518 nm. The optimized ultrasonic extraction produced 8.462％ w/w of R1, 0.451％ w/w of R2 and 0.172％ w/w of R3 at 13.5 mL/g liquid to solid ratio,78 ℃ of temperature and 60 min of time.Conclusions: The experimental findings of RSM optimized extraction and HPTLC analysis can be further applied for the efficient extraction of β-sitosterol and lupeol in other species of Astragalus.

Optimization of processing technology of Momordicae Semen cream by Box- Behnken design-response surface methodology / 中草药

Objective To optimize the optimum processing technology of Momordicae Semen cream using Box-Behnken design-response surface method (BBD-RSM). Methods The overall desirability (OD) of indexes of the content of 3-O-β-D- glucuronic acid methyl ester, β-sitosterol, and oleanolic acid and the content of fatty oil in gypsogenin was comprehensively evaluated, and BBD-RSM with three factors and three levels was used to study the effect of baking temperature and time, and pressing time on the processing technology of Momordicae Semen cream. Results According to the experiment, the optimum processing conditions were optimized: the baking time 1.68 h, the baking temperature 80 ℃, the pressing time 30 min, and the OD value was 0.777. Considering the actual situation, the optimum processing technology of Momordicae Semen was obtained by fine-tuning the baking time (A). That was, the baking time was 1.7 h, the baking temperature was 80 ℃, and the pressing time was 30 min. Also, the calculated OD values were 0.779, 0.783, 0.766, and its RSD was 1.15% through the obtained conditions in parallel with three batches of samples. Conclusion The processing technology optimized by Box-Behnken design-response surface method has the advantages of stable, good model prediction effect and a new processing technology for Momordicae Semen cream production.

Network pharmacological analysis of Chaihu Guizhi Decoction for treating different diseases with same method on gastric ulcer and epilepsy / 中草药

Objective: Under the guidance of traditional Chinese medicine theory, this paper used network pharmacology model to explore the effective components and mechanism of “treating different diseases with same method” of Chaihu Guizhi Decoction (CHGZD) in treating gastric ulcer and epilepsy, which provided modern medical evidence for the theory of “treating different diseases with same method” of traditional Chinese medicine. Methods: The chemical constituents and potential targets of CHGZD were collected by TCMSP and TCMID databases. Disease targets of gastric ulcer and epilepsy were obtained in PubMed, CTD, and OMIM databases. Cytoscape 3.6.0 software was used to map CHGZD for gastric ulcer and epilepsy. The pharmacodynamic basis and molecular mechanism of the “treating different diseases with same method” network, and the functional and pathway enrichment analysis of gene targets was analyzed by DAVID 6.8 and KOBAS 3.0. Results: A total of 198 kinds of chemical constituents, 417 target targets, 114 gastric ulcer disease targets, and 461 epileptic disease targets were collected from CHGZD. Network analysis showed that 152 active components such as quercetin, beta-sitosterol, wogonin, kaempferol, and saikosaponin a in CHGZD played a role in the treatment of gastric ulcers and epilepsy through 17 common targets including PTGS2, VEGFA, TP53, IL6, and TNF, and 62 pathways such as pathways in cancer, and proteoglycans in cancer. Conclusion: The similar efficacy network composed of 17 common targets in gastric ulcer and epilepsy was the basis for CHGZD to play the role of “different disease and common treatment”. A total of 152 components work together through 62 pathways and 17 pathways to achieve the effect of “treating different diseases with same method”, which provides modern medical evidence for the traditional Chinese medicine to treat gastric ulcer and epilepsy from the liver and spleen.

Efficacy network and mechanism prediction of Chaihu Longgu Muli Decoction in treatment of essential hypertension / 中草药

Objective: To study the efficacy network and potential mechanisms of effects of Chaihu Longgu Muli Decoction in the treatment of essential hypertension by using network pharmacology methods. Methods: TCMSP, TCMID, and Stitch were used to obtain the components and their corresponding targets. PubMed, CTD, TTD, OMIM, and DrugBank were used to search disease targets of essential hypertension. The common targets between components and disease targets were screened and builded the “compound - target” efficacy network and the protein-protein interaction network by STRING and Cytoscape. The key components and core targets of Chaihu Longgu Muli Decoction in the treatment of hypertension were screened in these networks. Finally, relevant software was applied to GO analysis and pathway analysis of core targets to predict potential mechanisms. Results: A total of 137 components of Chaihu Longgu Muli Decoction and 168 targets of essential hypertension were screened. According to the analysis, quercetin, β-sitosterol, kaempferol, and stigmasterol were found as the four key components and 12 core targets such as IL-6, AKT1, and MAPK8 were found involving the Chaihu Longgu Muli Decoction-induced treatment of essential hypertension. The result of GO analysis and KEGG pathway analysis showed that the mechanisms of Chaihu Longgu Muli Decoction for treating essential hypertension were related to pathways such as activation of AP-1 family transcription factors, interleukin-10 signaling, interleukin-4 signaling, interleukin-13 signaling, the intrinsic pathway of apoptosis, and MAPK targeting/by MAP kinase-mediated nuclear events. Conclusion: The mechanisms of the effect of Chaihu Longgu Muli Decoction in the treatment of essential hypertension were through the above-mentioned “multi-components-multi-targets-multi-pathways”. This study provides a foundation for further investigation of the effective compound and specific mechanisms of Chaihu Longgu Muli Decoction in the treatment of essential hypertension.

LC/MS-based Analysis of Bioactive Compounds from the Bark of Betula platyphylla var. japonica and Their Effects on Regulation of Adipocyte and Osteoblast Differentiation

Betula platyphylla var. japonica (Betulaceae), also known as Asian white birch, is an endemic medicinal tree, the bark of which has been used in Chinese traditional medicine for the treatment of various inflammatory diseases. In our continuing search for bioactive compounds from Korean natural resources, a phytochemical investigation of the bark of B. platyphylla var. japonica led to the isolation of 7-oxo-β-sitosterol (1) and soyacerebroside I (2) from its ethanol extract as main components by liquid chromatography (LC)/mass spectrometry (MS)-based analysis. The structures of isolates were identified by comparison of ¹H and ¹³C nuclear magnetic resonance spectroscopic data and physical data with the previously reported values and LC/MS analyses. To the best of our knowledge, this is the first study to demonstrate that the isolated compounds, 7-oxo-β-sitosterol and soyacerebroside I, were isolated in B. platyphylla var. japonica. We examined the effects of the isolates on the regulation of adipocytes and osteoblast differentiation. These isolates (1 and 2) produced fewer lipid droplets compared to the untreated negative control in Oil Red O staining of the mouse mesenchymal stem cell line without altering the amount of alkaline phosphatase staining. The results demonstrated that both compounds showed marginal inhibitory effects on adipocyte differentiation but did not affect osteoblast differentiation.

Effects of Different Drying Technologies and Slicing on the Quality of Tetrastigma hemsleyanum by Multi-in-dexes Evaluation / 中国药房

OBJECTIVE:To study the effects of different drying technologies and slicing on the quality of Tetrastigma hemsley-anum,and optimize the drying methods for T. hemsleyanum. METHODS:2 treatment methods(slicing and no slicing)and 5 dry-ing methods(drying in the shade,drying in the sunlight,hot-air drying,microwave drying and freeze drying)were respectively ad-opted for the T. hemsleyanum root. After drying for 3.5-213.0 h,using the total flavonoids,total polyphenols,polysaccharides andβ-sitosterol as indexes,effects of different drying technologies on the quality of T. hemsleyanum were comparatively analyzed. RE-SULTS:Compared with no slicing,sliced T. hemsleyanum can shorten the drying time and reduce the loss of active ingredients. In the 5 drying methods,freeze drying was the best for keeping the active ingredients in T. hemsleyanum. After drying,the contents of total flavonoids,polysaccharides,total polyphenols and β-sitosterol were 18.5 mg/g,92.7 mg/g,9.19 mg/g and 0.344 mg/g respec-tively,followed by microwave drying,hot-air drying,drying in the shade and drying in the sunlight. The contents of active ingredi-ents had statistical significance by each drying methods (P<0.05 or P<0.01). CONCLUSIONS:Different drying technologies have obvious effects on the quality of T. hemsleyanum. Slicing and hot-air drying at 60 ℃ were suggested as suitable method for T. hemsleyanum in terms of cost,content of active ingredients and practicability.

Chemical Constituents of Catharanthus roseus / 中国药学杂志

OBJECTIVE: To study the chemical constituents of Catharanthus roseus. METHODS: Various column chromatograghic methods on silica gel, Rp-18, and Sephadex LH-20 were applied for the isolation and purification of the 95% ethanol extract. The structures were elucidated by their physicochemical properties and spectral data. RESULTS: Twenty-two compounds were obtained and identified as ursolic acid (1), daucesterol (2), tetrahydroalstonine (3), 7α-hydroxy-β-sitosterol (4), vindoline (5), β-sitosterol (6), aurantiamide acetate (7), lochnericine (8), oleanolic acid (9), ajmalicine (10), (22E, 24R)-ergosta-7, 22-dien-3β, 5α, 6β-triol (11), betulinic acid (12), stigmasterol (13), quercetin (14), keampferol (15), vindorosine (16), catharanthine (17), diaaurantiamide acetate (18), reserpine (19), panarine (20), serpentine (21), and 16R-E-isositsirikine (22). CONCLUSION: Compounds 4, 7, 11, 13, 18, and 20 are isolated from C. roseus for the first time.

Triterpenes and sterols from Hoya diversifolia Blume.

Chemical investigation of the dichloromethane extracts of Hoya diversifolia Blume led to the isolation of β-amyrin cinnamate (1), squalene (2), β-sitosterol (3), a mixture of β-amyrin (4a), α-amyrin (4b) and lupeol (4c) in a 4:2:1 ratio and saturated hydrocarbons from the leaves; and 2, taraxerol (5), lupeol cinnamate (6), and a mixture of 3 and stigmasterol (7) in a 2:1 ratio from the stems. The structures of 1-7 were identified by comparison of their NMR data with those reported in the literature.

Chemical constituents from Alpinia coriacea / 中草药

Objective: To study the chemical constituents of Alpinia coriacea. Methods: The chemical constituents were separated and purified by silica gel, ODS, Sephadex LH-20 column chromatographies, and preparative HPLC. Their structures were determined by physicochemical properties and spectral data analyses. Results: Fifteen compounds were isolated from A. coriacea, and identified as 4-hydroxybenzaldehyde (1), anisic acid (2), pinocembrin (3), apigenin (4), izalpinin (5), kaempferol (6), 5,7,3',4'-tetrahydroxy-flavanone (7), 3,5-dihydroxy-7,4'-dimethoxyflavone (8), α-tocospiro A (9), stigmast-4-ene-6α-ol-3-one (10), stigmast-4-en-3-one (11), 7-keto-β-sitosterol (12), (22E,24R)-5α, 8α-epidioxyergosta-6,22-dien-3β-ol (13), stigmasterol (14), and β-sitosterol (15). Conclusion: All compounds are isolated from A. coriacea for the first time, among which compounds 2,7, and 9-13 are isolated from the plants of Alpinia L. for the first time.

Study on mechanism of regulating synthesis of β-sitosterol by SiSQS1 and SiSQS2 in Saussurea involucrata cells / 中草药

Objective: To explore the synthetic mechanism of β-sitosterol by comparing the locus mutation, prokaryotic expression, expression level of SiSQS1 and SiSQS2, and the content of β-sitosterol in three color types of cells. Methods: Firstly, we preformed the cloning and bioinformatic analysis of SiSQS1 and SiSQS2 which were key enzymes involved in the biosynthesis of β-sitosterol in Saussurea involucrata cells. Secondly, we compared the differences of prokaryotic expression between SiSQS1 and SiSQS2, then optimized the expression conditions. Finally, we compared the expression levels of SiSQS1 and SiSQS2 by Real-time PCR and the content of β-sitosterol by GC-MS in three color types of cells, and made the correlative analysis on the expression level and the content of β-sitosterol. Results: There was a locus mutation of amino acid residues in 242E/D between SiSQS1 and SiSQS2. The results of prokaryotic expression analysis and conditions optimization showed that both target proteins had been expressed successfully, but the optimal prokaryotic expression system was different. The results of expression level and quantitative analysis showed a positive correlation to the expression levels of SiSQS1 and SiSQS2 and the content of β-sitosterol, the correlation coefficients were 0.92 and 0.89, respectively. Conclusion: A locus mutation of amino acid residues in 242E/D between SiSQS1 and SiSQS2 may influence the expression of SiSQS, and there may exist the functional differences in catalytic activity and the accumulation of β-sitosterol. The study will provide technical support and lay a theoretical foundation for studying the accumulated mechanism of β-sitosterol regulated by SQS in S. involucrata cells.

Chemical constituents of ethyl acetate extract of Empetrum nigrum var. japonicum and their effect on alcoholic fatty liver of rats / 中草药

Objective: To study the chemical constituents from the ethyl acetate extract fraction of 70% ethanol extract in the aerial parts of northeastern crowberry (Empetrum nigrum var. japonicum) and their effect on the alcohol fatty liver (AFL) of rats, and to provide the scientific basis for the use and development of the northeastern crowberry. Methods: Chromatography separation technology and spectral analysis technology were used for the separation, purification, structure analysis, and identification. AFL model of SD rats was induced by 45% ethanol. To observe the effect of ethyl acetate extract in northeastern crowberry on the liver index, serology, and liver tissue homogenates in AFL of rats. Results: Eight compounds were isolated and identified as β-sitosterol (1), ursolic acid (2), uvaol (3), 2',4'-dihydroxy dihydrochalcone (4), 2',4'-dihydroxy chalcone (5), 7-hydroxy flavone (6), 4'-methoxy-2'-hydroxyl-dihydroxy chalcone (7), and isoliquiritigenin (8). Compared with the model group, giving the ethyl acetate extract of northeastern crowberry could decrease the liver index of rats, reduce serum lipid metabolism of TG, TC, reduce the serum enzyme ALT, AST, liver homogenate in oxidative stress, and increase the activities of SOD and GSH. Conclusion: Compounds 1-4, and 6-8 are isolated from E. nigrum var. japonicum for the first time. The effect of ethyl acetate extract in E. nigrum var. japonicum on AFL of rats is obvious.

Chemical Constituents of Hoya buotii Kloppenb.

Chemical investigation of the dichloromethane extracts of Hoya buotii Kloppenb. afforded taraxerone (1), taraxerol (2), a mixture of β-sitosterol (3a) and stigmasterol (3b) in about 2:1 ratio, and a mixture of α-amyrin cinnamate (4a) and β-amyrin cinnamate (4b) in about 1:2 ratio from the stems; 1, 2, and 3a from the roots; a mixture of 4a and 4b in about 3:2 ratio from the flowers; and 3a, squalene (5) and saturated hydrocarbons from the leaves. The structures of 1-5 were identified by comparison of their NMR data with those reported in the literature.

Cytotoxic Triterpenes and Sterols from Pipturus arborescens (Link) C.B. Rob.

The triterpenes, squalene (1), friedelin (2) and a mixture of ursolic acid (3a) and oleanolic acid (3b) in a 2:3 ratio, and a mixture of β-sitosterol (4a) and stigmasterol (4b) in a 2:1 ratio, obtained from the dichloromethane extract of Pipturus arborescens (Link) C.B. Rob., were evaluated for their anti-proliferative activities against three human cancer cell lines, breast (MCF-7) and colon (HT-29 and HCT-116), and a normal cell line, human dermal fibroblast- neonatal (HDFn) using the in vitro PrestoBlue® cell viability assay. The HCT-116 cell line was most susceptible to the compounds and mixtures tested. Triterpene 1 was most cytotoxic against HCT-116 and MCF-7 with IC50 values of 4.21 and 5.92 μg/mL, respectively. Triterpene 2 and the mixture of 3a and 3b were highly anti-proliferative against HCT-116 cells (IC50 of 1.22 and 1.66 μg/mL, respectively) and moderately inhibitory against MCF-7 cells (IC50 of 16.51 and 23.97 μg/mL, respectively). The mixture of 4a and 4b exhibited high cytotoxicity against HCT-116 cells (IC50 of 1.14 μg/mL). Compounds 1-4b showed the least activity against HT-29 cells (IC50 of 11.97 to 52.52 μg/mL). Cytotoxic effect was not observed against HDFn cells (>100 μg/mL). Comparing the effects of 1-4b on the two colon cancer cell lines, the IC50 values of 1-4b against HCT-116 were lower than those of HT-29.