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类风湿性关节炎大鼠模型在昆明山海棠作用下关节炎指数的评分改变目的：探讨昆明山海棠（THH）对胶原诱导性关节炎（CIA）大鼠模型关节炎指数（AI）评分的影响。
方法：建立大鼠CIA动物模型，共选40只Wistar 大鼠，设空白对照组、CIA模型组、地塞米松组和THH组，各10只。
在实验因素干预后对各组大鼠AI评分并进行比较。
结果：在造模最后1 d，CIA模型组、地塞米松组和昆明山海棠组的AI评分均明显高于空白对照组，差异有统计学意义（P＜0.05），说明大鼠造模成功；在治疗最后1 d，昆明山海棠组和地塞米松、对照组的AI值均明显低于CIA模型组，差异有统计学意义（P＜0.05）。
结论：昆明山海棠对CIA大鼠模型有积极的治疗作用。
[Abstract] Objective：To explore the effects of tripterygium hypoglaucum hutch on arthritis index in collagen induced arthritis rats.Method：Establishment of rat animal model of CIA，a total of 40 Wistar rats，compared with blank control group，CIA model group，dexamethasone group and THH group，10 rats in each.The intervention of AI rats in each group were compared in the experimental factors.Result：In the modeling of the last 1 day，the CIA model group，dexamethasone group and Kunming Begonia group AI were significantly higher than that in the blank control group，the difference was statistically significant（P＜0.05），the rats in the treatment ofsuccess；the last 1 day，THH group and dexamethasone group，AI of the control group values obviously lower than in CIA model group，the difference was statistically significant（P＜0.05）.Conclusion：The treatment of THH is exhibited remarkable curative effect in CIA model[Key words] Tripterygium Hypoglaucum Hutch；Collagen induced arthritis；Arthritis index类风湿性关节炎是骨科的常见和多发疾病，是一种以关节滑膜炎症反应为特征的慢性自身免疫性疾病[1]。
3 讨论脂联素亦称A crp30、GBP-28、A d i poQ、APM-1,它是由成熟脂肪组织分泌的一种与细胞外基质相互作用的特异性血清蛋白。
人类脂联素的基因是单拷贝基因,位于染色体3q27,包含3个外显子和2个内含子,而染色体3q27区是 型糖尿病的易感基因位点[4]。
脂联素可以保护血管内皮细胞,其机制与其胰岛素敏感性、抗动脉粥样硬化、抗炎和抗氧化作用有关[2]。
本研究中表1和表2的结果表明冠心病和冠脉造影阳性患者的血清脂联素水平均较相应对照组下降,提示无论冠心病的病变严重程度如何,脂联素可作为冠心病的辅助诊断和判断严重程度的指标之一,与文献报道基本一致[5]。
冠心病是糖尿病的常见并发症之一,据文献报道,冠心病和糖尿病患者的血清脂联素水平均较其相应正常对照组下降[5,6]。
那么糖尿病患者并发冠心病的情况是否与血清脂联素水平有关系呢?本实验表3结果提示糖尿病或糖耐量受损患者中,并发冠心病组的血清脂联素水平明显低于未并发冠心病组,这点未见文献报道。
糖尿病患者糖代谢紊乱可导致脂代谢紊乱,因此易继发血管病变,如冠脉疾病、肾病等,血清脂联素浓度降低可能作为糖尿病患者脂代谢紊乱程度严重,可能并发冠心病的一个新预测指标。
我们前期工作证实,在根据年龄、性别相应匹配对照组后,实验组B M I、腰围、甘油三酯、高密度脂蛋白c、白介素-6 (IL-6)、肿瘤坏死因子- (TN F- )均与脂联素水平相关,提示:脂联素与血脂及动脉硬化相关炎性因子也密切相关[7]。
TNF- 和I L-6及其受体可抑制脂联素表达,脂联素降低和TNF- 、I L-6升高与 型糖尿病正相关[8],这些工作与本文的研究结果是一致的。
冠心病患者和糖尿病或糖耐量受损并发冠心病患者的血清脂联素水平下降提示血清脂联素浓度降低可能作为冠心病的辅助诊断和病变严重程度的新指标;同时它也可能成为糖尿病是否并发冠心病的新预测指标,有潜在的临床应用价值。
参考文献:[1]W ei-Sh i ung Y ang,L ee-M i ng Chuang.H um an gen eti cs of ad i pon ec ti n in t h em etabolic syndrom e[J].J M o lM ed,2006,84:112-21. [2]N an l an Luo,J i an L i u,B.H ong Chung,et a.l M acrophage ad i ponecti n exp ressi on i m proves i nsu li n s en siti vit y and protects agai n st i nfl a mm ati on and at heroscl erosis[J].D i abetes,2010,59:791-9.[3]Ybarra J uan,Pou J oseM,Planas F rancesc,et a.l C orrelati on bet w eeni ns u li n res i s t an ce surrogates and echocard i ograph ic find i ngs i n as y m pto m atic pati en ts w it h m orb id obes it y:a cross-secti ona l study[J].Endo cr Pract, 2007,13:590-600.[4]Francis V asseur,Fr d ri c Lep r tre,C ori nn e L acque m an t,et a.l The gen eti cs of ad i ponecti n[J].Curr D i ab R ep,2003,3:151-8.[5]C hrist oph H.S ael y,Loren z R is ch,Guen ter H oefl e,et a.l Lo w ser um ad i pon ecti n i s i ndependen tly associ ated w it h bot h the m etabo li c synd ro m e and angiograph i call y deter m i ned coron ary atheroscleros i s[J].C lin Chi mA cta,2007,383:97-102.[6]Fum i yuk i O ts uka,Se i go Sug i ya m a,Sunao Koji m a,et a.l H ypoad i ponecti ne m i a i s as soci ated w it h i m paired gl u cos e tol eran ce and coronary ar tery d i sease i n non-d i abeti c m en[J].C irc J,2007,71:1703-9.[7]陶红,米树华,陈瑞,等.冠心病患者血清脂联素水平与炎性相关因子的研究[J].中华医学杂志,2006,86(增刊):327.[8]Jong M i n Lee,Soo Ryang K i m,Se J eong Yoo,et a.l The rel ati on s h i p bet w een adipok i nes,m etaboli c para m eters and i n s u li n res i stance in patien t s w ith m etabolic s yndro m e and typ e2d i abetes[J].J I nt M ed Res,2009, 37:1803-12.专题综述RE V I E W ARTI CLES植物ABC转运蛋白及其在A l胁迫下的功能研究进展王康,徐慧妮,李昆志*(昆明理工大学生命科学与技术学院生物工程技术研究中心,云南昆明650224)摘要:ABC(ATP-B i ndi ng C asse tte)转运蛋白家族是目前已知最大、功能最广泛的蛋白家族,能利用水解ATP的能量来参与生物体内多种物质的转运,这一基因家族成员在哺乳动物和微生物中已广泛鉴定,在植物中的研究是一个相对较新的研究领域。
孟迎平，冯杰，魏华，等. 肠道菌群对饮食诱导的高尿酸血症的调控研究进展[J]. 食品工业科技，2022，43（18）：465−473. doi:10.13386/j.issn1002-0306.2021090006MENG Yingping, FENG Jie, WEI Hua, et al. Research Progress in the Regulation of Gut Microbiota on Diet-Induced Hyperuricemia[J]. Science and Technology of Food Industry, 2022, 43(18): 465−473. (in Chinese with English abstract). doi:10.13386/j.issn1002-0306.2021090006· 专题综述 ·肠道菌群对饮食诱导的高尿酸血症的调控研究进展孟迎平1，冯 杰2，魏 华1,3，张志鸿1,3,*（1.南昌大学食品科学与技术国家重点实验室，江西南昌 330047；2.南昌市检验检测中心，江西南昌 330012；3.南昌大学中德联合研究院，江西南昌 330047）摘 要：高尿酸血症是一种人体血尿酸水平异常升高的代谢性疾病，由嘌呤代谢紊乱和/或尿酸排泄障碍导致，其中高果糖和高嘌呤的饮食具有明显的高尿酸诱导效应。
近年来，研究发现高尿酸血症与肠道菌群存在密切关系，患者存在肠道菌群紊乱、有益菌属丰度下降的现象。
合理的膳食及益生菌摄入可有效调节肠道菌群，维持其稳态，并促进肠道嘌呤和尿酸代谢，提示肠道菌群是未来预防高尿酸血症的靶点。
本文概述了高尿酸血症的发病特征和机制、饮食对高尿酸血症的诱导、高尿酸血症与肠道菌群的关联性，以及肠道菌群调控高尿酸血症的策略，以期为未来开发诊治高尿酸血症及痛风的新方法提供参考。
关键词：肠道菌群，饮食，高尿酸血症，尿酸，益生菌本文网刊:中图分类号：TS201.3 文献标识码：A 文章编号：1002−0306（2022）18−0465−09DOI: 10.13386/j.issn1002-0306.2021090006Research Progress in the Regulation of Gut Microbiota on Diet-Induced HyperuricemiaMENG Yingping 1，FENG Jie 2，WEI Hua 1,3，ZHANG Zhihong 1,3, *（1.State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China ；2.Nanchang Inspection and Testing Center, Nanchang 330012, China ；3.Jiangxi-OAI Joint Research Institute, Nanchang Univerisity, Nanchang 330047, China ）Abstract ：Hyperuricemia is a metabolic disease with excessively high level of uric acid in blood. It is triggered by the disorder of purine metabolism and/or uric acid excretion in human, and the purine-rich and fructose-rich diets and purine was confirmed to induce the hyperuricemia. In recent years, some reports have shown that there is a close connection between hyperuricemia and gut microbiota, namely the patients with hyperuricemia have a gut microbiota disorder and decreased abundance of beneficial bacteria. Rational diet and probiotics intake have been confirmed to regulate intestinal microbiota effectively, maintain homeostasis, and promote intestinal purine and uric acid metabolism. Thus, gut microbiota is considered as a target for future preventation of hyperuricemia. In the review, the feature and pathogenesis of hyperuricemia, the induction of diet on hyperuricemia, the association between hyperuricemia and gut microbiota, and the regulation strategies of gut microbiota on hyperuricemia are summarized. This review would provide reference for the diagnosis and treatment of hyperuricemia and gout in the future.Key words ：gut microbiota ；diet ；hyperuricemia ；uric acid ；probiotics收稿日期：2021−09−02基金项目：国家自然科学基金青年科学基金项目（32101915）；江西省自然科学基金资助项目（20212BAB215033）。
肠-脑-皮肤轴与特应性皮炎王晓萌;张玉环;张理涛【摘要】特应性皮炎作为世界范围内常见的皮肤病,病因不明,临床上具有慢性、复发性等特点,严重影响患者的生活质量.目前发现慢性皮肤病和心理疾病共病率逐年上升,且跨学科研究表明肠道、肠道微生物异常以及心理疾病与皮肤疾病之间存在着关联性的通信轴,例如既往已经证实的肠-脑轴、脑-皮肤轴.因此肠道功能的完整性和肠道茵群的平衡状态可能在皮肤炎症和情绪行为中起到中介作用,即存在肠-脑-皮肤轴.本文探讨了肠道茵群和心理因素对特应性皮炎发病及病情发展的影响,以及肠-脑-皮肤轴对特应性皮炎的作用机制,并以中医理论进行阐述,拟利用16SrRNA 测序法从“健脾”的方面观察中医药通过作用该通信轴治疗特应性皮炎远期疗效.【期刊名称】《中国中西医结合皮肤性病学杂志》【年(卷),期】2018(017)001【总页数】4页(P83-86)【关键词】特应性皮炎;肠道菌群;心理疾病;肠-脑-皮肤轴【作者】王晓萌;张玉环;张理涛【作者单位】天津中医药大学,天津300100;天津市中医药研究院附属医院,天津300120;天津市中医药研究院附属医院,天津300120【正文语种】中文【中图分类】R758.3特应性皮炎是一种病程漫长且易复发的常见皮肤病，在高度工业化社会及在社会经济上占优势的阶层中患病率更高。
以湿疹为主要表现，一般有明显的家族史，患者常出现皮肤干燥，以及特应性体质如出现哮喘、过敏性鼻炎等，并且可能出现IgE 介导的系统表现。
特应性皮炎病因众多，包括遗传、食物等，在过去几年中发现日益严重的环境问题对其也有很大影响。
因此治疗难度大，变应原众多难以避免，一般的药物治疗只能够缓解其症状。
在全球范围内影响2%～7%的成年人，近十年内在美国学生中患病率为10%～20%。
其主要患病人群为学生，以瘙痒为主要症状，且常夜间加重从而影响睡眠，严重影响了患者的学习状况和生活质量[1]。
2009 年国外学者[2]提出肠-脑-皮肤轴（Gutbrain-skin axis）的假说。
第34卷第1期 海南师范大学学报（自然科学版）V〇l.34N〇.l 2021 年3 月Journal of Hainan Normal University(Natural Science)Mar.2021Doi ： 10.12051/j.issn. 1674-4942.2021.01.010黑木耳黑色素的研究综述陈雅，徐苗，王欣宜，单欣荷，季琳凯，张拥军*(中国计量大学生命科学学院，浙江杭州310018)摘要：黑木耳是中国一种分布极为广泛且具有独特的营养价值与保健功能的食用菌，含有多 种生物活性化合物，其中黑色素是主要生物活性成分之一，且具有极高的安全性，可进一步开发具 备保健作用的功能色素，该方面的研究已初见成效且应用前景广阔。
文章综述了黑木耳黑色素的 理化性质、分子组成与结构表征、分离制备手段以及其清除自由基、抑菌抗病毒、抗辐射、改善肝损 伤、保护D N A等生物活性的国内外研究现状，分析了限制黑木耳黑色素开发应用的实际问题并提 出展望，并为今后黑木耳黑色素生物活性作用机制的研究及其在食品、药品、化妆品等领域的应用 提供参考。
关键词：黑木耳；黑色素；分离提取；生物合成；分子结构；生物活性中图分类号:0629.1 文献标志码:A文章编号：1674-4942(2021)01-0063-07Summary of Melanin of A u ricu la ria au ricu laCHEN Ya, XU Miao, WANG Xinyi, SHAN Xinhe, Jl Linkai, ZHANG Yongjun' (College of L ife Science,China Jiliang University,Hangzhou 310018, China) Abstract：Auricularia auricula is an edible fungus with a unique nutritional value and healthy function and is widely distributed in China. It contains a variety of bioactive compounds. Melanin is one of the main bioactive components with high safety. It can be further developed into func tional pigments with health care function. The research in this field has broad application prospects. In this paper, the physicochemical properties, molecular composition, structural characterization, separation and prej)aration methods of Auricularia auricula melanin, as well as its biological activities such as scavenging free radicals, antibacterial and antiviral, antiradiation, improving liver injury, protecting DNA and so on, were reviewed. The problems limiting the development and application of Auricularia auricula melanin were analyzed, and the prospects were put forward, which would be helpful for the study of the biological mechanism of Auricularia auricula melanin and its application in food, medicine, cosmetics and other fields.K ey w〇r d s：/lz/m*z//rtrirt ai/rzcw/a;melanin;separation and extraction;biosynthesis;molecular stnicture;biological activity黑木耳(4w r/c a/a r i Y/aur/cu/fl)隶属担子菌亚门（Basi(liomycotina)层菌纲（Hymenomycetes)木耳目（Auricu- I a r i a les)木耳科(A u r i c u1a riaceae)木耳属(yWfcw/rmVz )，为中国珍贵的药用和食用菌，早在19世纪，黑木耳就被 用于民间医药，用于治疗咽喉痛、眼痛、黄疸等病症，并作为收敛剂"_21。
引用格式：王建伟，贺晓岚，王 召，等. 镉胁迫对甘蓝型油菜种子萌发的影响[J]. 湖南农业科学，2023（5）：37-41. DOI:DOI:10.16498/ki.hnnykx.2023.005.009近年来，农田土壤重金属污染已成为威胁我国粮食安全生产的一个严重环境问题。
镉（Cd）是毒性最强的重金属之一，容易在土壤—动植物—人体间富集转移，并且在人体内具有很长的生物半衰期（10~30 a）。
人体内积累过量的镉元素可引发肾损伤、骨质疏松和癌症等多种疾病[1]。
因此，耕地镉污染受到世界各地的广泛关注[2]。
关于镉胁迫对植物的生理毒害作用，国内已经有不少报道，主要集中在小麦、玉米[3]、水稻[4]、谷子、糜子[5]、辣椒[6]和黑麦草[7]等植物上。
有学者指出，种子萌发和苗期生长性状是评价植物重金属耐性的重要指标。
还有研究显示，镉胁迫对作物种子萌发和发芽指数影响不显著，对活力指数和根芽生长抑制作用明显[5,8]。
也有研究认为，油菜品种的根长、根表面积、根体积和根尖数与地上部镉含量及累积量呈显著正相关，根长与镉累积量呈显著正相关[9]。
甘蓝型油菜是一种重要的油料和多功能利用作物，其根系发达、分枝多、生物量大，具有重要的经济、科研和生态价值，被认为是修复土壤重金属镉胁迫对甘蓝型油菜种子萌发的影响 王建伟1，贺晓岚2，王 召2，汤 宏1，曾珠亮2（1. 凯里学院理学院，贵州凯里 556011；2. 凯里学院大健康学院，贵州凯里 556011）摘 要：以早熟100天、新都油800、南油868、美国油王88、广源58和丰油9号这6个油菜品种为材料，研究不同浓度镉胁迫（0~200 mg/L）对其种子萌发及根芽生长的影响。
结果表明：高浓度镉胁迫使油菜种子的萌发出现了滞后或抑制现象，主要表现为丧失发芽活力；随着镉浓度的增加，不同品种的发芽势、发芽率、活力指数、发芽指数均呈下降趋势，发芽日数呈增长趋势；其中发芽势、发芽率、活力指数、发芽日数、平均发芽天数受镉胁迫影响较小的品种为广源58和新都油800，其次为美国油王88和南油868，而早熟100天受镉胁迫抑制较为明显；随镉浓度的增加，不同油菜品种芽长、芽鲜重、芽干重、根长、根鲜重、根干重均呈下降趋势；不同浓度Cd胁迫下，早熟100天、丰油9号受到明显抑制，当镉浓度达到120 mg/L时，几乎无可见根；南油868的芽干重降低最小；美国油王88的芽长、芽鲜重、根长、根鲜重和根干重降低幅度较小。
第 33 卷 第 5 期Vol.33，No.5166-1822024 年 5 月草业学报ACTA PRATACULTURAE SINICA 段海霞， 师茜， 康生萍， 等. 丛枝菌根真菌和根瘤菌与植物共生研究进展. 草业学报， 2024， 33（5）： 166−182.DUAN Hai -xia ， SHI Qian ， KANG Sheng -ping ， et al . Advances in research on the interactions among arbuscular mycorrhizal fungi ， rhizobia ， and plants. Acta Prataculturae Sinica ， 2024， 33（5）： 166−182.丛枝菌根真菌和根瘤菌与植物共生研究进展段海霞1，师茜1，康生萍1，苟海青1，罗崇亮2*，熊友才3（1.青海大学省部共建三江源生态与高原农牧业国家重点实验室，青海 西宁 810016；2.中国科学院西北高原生物研究所，中国科学院高原生物适应与进化重点实验室，青海 西宁 810008；3.兰州大学生态学院，草种创新与草地农业生态系统全国重点实验室，甘肃 兰州 730000）摘要：丛枝菌根真菌（AMF ）和根瘤菌可以影响植物生产力、微生物群落结构和土壤质量，是生态系统可持续发展的重要驱动因子。
在长期的进化过程中，AMF 和根瘤菌逐步形成了互惠互利共生关系，充分发挥AMF-根瘤菌-植物共生体的生物固氮和养分吸收等作用，对于减少化学肥料的投入，保障农业可持续发展具有重要意义。
但也有研究表明AMF 和根瘤菌之间存在相互制约的作用，这可能与环境因素密切相关。
因此，需要系统总结AMF-根瘤菌与植物共生相互作用的机理及其影响因素。
本研究通过文献梳理以及定性比较分析，阐明了植物根系通过根系分泌物刺激根瘤菌和AMF 形成结瘤因子和菌根因子，激活后续信号通路，从而使根瘤菌和AMF 与植物建立共生关系的过程和机制；概述了AMF-根瘤菌与植物共生的协同增效和拮抗作用；总结了影响AMF 和根瘤菌与植物共生及其相互作用的生物和非生物因素。
北京普利莱基因技术有限公司电话：************62053186 Email:************************膜再生液(Stripping Solution) P1650描述：膜再生液，也称做抗体剥离液体、一抗二抗去除液。
允许对同一张膜进行多次Western Blot检测。
在室温条件下，将用过的膜浸泡在膜再生液中30分钟，能够选择性清除与膜抗原结合的第一抗体及第二抗体，但不影响电转移到膜上的蛋白。
随后可以使用不同的抗体进行下一轮Western Blot实验，可利用同一张膜进行多次蛋白检测。
不仅适宜用少量样品多次检测多种不同蛋白，即使样品量并不匮乏，采用这种多次检测同一张膜上的蛋白的策略，能省去给药处理、蛋白电泳和膜转移等耗费性步骤。
特点：（1）无毒无味无害，室温保存和使用；（2）可对同一张膜进行10次以上的Western Blot检测。
适用：硝酸纤维素膜或PVDF膜。
使用前膜可以保存在PBS或TBS缓冲液，也可以室温干燥保存数月。
干的PVDF 膜应该先用甲醇泡5分钟。
安全性：无毒无味。
按照普通化学品安全规范进行操作和处置。
储存：室温保存，一年有效。
温度过低可能出现浑浊。
操作步骤：1.将膜充分浸泡于适当体积的再生液中，室温孵育15~30分钟并不时晃动。
孵育时间的长短应参考后面的说明进行优化。
洗脱某些抗体需要较长的时间如30~60分钟。
2.用镊子取出膜，用自备Western Blot洗涤缓冲液或普利莱封闭洗涤缓冲液（B1009）淋洗膜一次，再洗膜5分钟。
3.此时膜上抗体已去除，膜已再生。
用脱脂奶粉或BSA封闭，进行下一轮Western Blot实验。
说明:1. 膜再生或Stripping的实质是在不影响膜上结合的抗原的条件下，将与抗原分子结合的一抗和二抗洗脱下来。
有许多因素影响抗体从膜上的洗脱，如膜的类型、抗体类型和浓度及其与抗原结合特性等。
按下面的说明优化再生液中孵育膜的时间至关重要。
PLEASE SCROLL DOWN FOR ARTICLEThis article was downloaded by:On: 22 January 2011Access details: Access Details: Free Access Publisher Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UKJournal of Asian Natural Products ResearchPublication details, including instructions for authors and subscription information:/smpp/title~content=t713454007Microbial transformation of asiatic acid by Alternaria longipesWen-Ni He ab ; Jun-Gui Dai c ; Min Ye b ; Li-Jun Wu a ; De-An Guo b aShenyang Pharmaceutical University, Shenyang, China b The State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, China c Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing,ChinaOnline publication date: 13 September 2010To cite this Article He, Wen-Ni , Dai, Jun-Gui , Ye, Min , Wu, Li-Jun and Guo, De-An(2010) 'Microbial transformation ofasiatic acid by Alternaria longipes ', Journal of Asian Natural Products Research, 12: 9, 760 — 764To link to this Article: DOI: 10.1080/10286020.2010.501505URL: /10.1080/10286020.2010.501505Full terms and conditions of use: /terms-and-conditions-of-access.pdf This article may be used for research, teaching and private study purposes. Any substantial or systematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply or distribution in any form to anyone is expressly forbidden.The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss,actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.ORIGINAL ARTICLEMicrobial transformation of asiatic acid by Alternaria longipesWen-Ni He ab ,Jun-Gui Dai c ,Min Ye b *,Li-Jun Wu a and De-An Guo baShenyang Pharmaceutical University,Shenyang 110016,China;b The State Key Laboratory of Natural and Biomimetic Drugs,School of Pharmaceutical Sciences,Peking University,Beijing 100191,China;c Institute of Materia Medica,Chinese Academy of Medical Sciences &PekingUnion Medical College,Beijing 100050,China(Received 21April 2010;final version received 12June 2010)Asiatic acid is a major pentacyclic triterpene isolated from Centella asiatica .It shows a variety of bioactivities.In order to obtain its derivatives,potentially useful for detailed pharmacological studies,the substrate was subjected to incubations with selected micro-organisms.In this work,asiatic acid was converted into three new compounds:2a ,3b ,23,30-tetrahydroxyurs-12-ene-28-oic acid (1),2a ,3b ,22b ,23-tetrahydroxyurs-12-ene-28-oic acid (2),and 2a ,3b ,22b ,23,30-pentahydroxyurs-12-ene-28-oic acid (3)by the fungus Alternaria longipes AS 3.2875.The structures of the three metabolites were determined by 1D and 2D NMR spectral data.Keywords:asiatic acid;Alternaria longipes ;microbial transformation;triterpene1.IntroductionAsiatic acid is a naturally occurring pentacyclic triterpene found in many plants;however,one of the most widely reported sources of it is Centella asiatica [1].Previous investigations have shown that it possessed anti-oxidative,anti-inflammatory,hepatoprotective,anti-Alzheimer’s disease,and anti-depressant activities.Furthermore,it can also protect cardio-cerebral vascular and induce apop-tosis of tumor cells such as human melanoma SK-MEL-2cells [2–6].Because of such properties,the prep-aration of derivatives of this compound for structure–activity relationship studies is valuable.However,because the structures of triterpenoids are complicated,the chemical procedures that can be used had some limitations.Now,microbial trans-formation is an important tool in thestructural modification of organic com-pounds,especially for natural products,due to its significant region-and stereo-selectivities [7,8].There is no report on biotransformation of asiatic acid by fungi.Therefore,the objective of this work was to obtain new derivatives of asiatic acid by the fungus Alternaria longipes AS 3.2875,potentially useful for pharmacological studies.2.Results and discussionAsiatic acid (substrate)showed purity above 95%by HPLC/UV analysis.The 1H NMR spectrum exhibited six methyl signals at d 1.05(9H,s,H-24,26,27),1.18(3H,s,H-25),0.90(3H,d,H-29),and 0.92(3H,d,H-30),whereas the signal at d 5.45(1H,t,J ¼4.0Hz)was attributed to H-12[9].The 13C NMR spectral data are shown in Table 1.ISSN 1028-6020print/ISSN 1477-2213online q 2010Taylor &FrancisDOI:10.1080/10286020.2010.501505*Corresponding author.Email:yemin@Journal of Asian Natural Products Research Vol.12,No.9,September 2010,760–764D o w n l o a d e d A t : 18:20 22 J a n u a r y 2011T a b l e 1.1H N M R (400M H z )a n d13C N M R (100M H z )s p e c t r a l d a t a o f c o m p o u n d s 1–3a n d a s i a t i c a c i d (p y r i d i n e -d 5,d i n p p m ,J i n H z ).123A s i a t i c a c i dP o s i t i o nd C d H d C d H d C d H d C d H147.91.78,2.30,m 47.92.27,m 47.91.80,2.24,m 47.9268.84.20,d ,J ¼10.068.74.21,d ,J ¼10.068.94.20,d ,J ¼10.068.84.23,d ,J ¼10.0378.24.20,d ,J ¼10.078.24.21,d ,J ¼10.078.34.19,d ,J ¼10.078.24.23,d ,J ¼10.0443.643.643.643.6547.91.78,m 47.81.37,m 48.01.33,m 47.9618.51.62,m 18.61.98,2.69,m 18.71.65,2.73,m 18.5733.21.28,m33.01.70,1.35,m33.01.33,1.68,m33.1840.040.240.240.0948.11.84,m48.01.83,m48.11.85,m48.11038.338.338.338.31123.82.01,2.02,m 23.81.99,m 23.81.98,m 23.812125.65.53,b r s125.55.43,b r s125.85.48,b r s 125.65.45,t ,J ¼4.013139.4139.3139.2139.31442.643.243.242.61528.71.10,1.12,m 28.21.21,2.60,m 28.31.21,2.64,m 28.61625.01.99,m18.51.41,1.70,m18.51.33,2.09,m24.91748.054.154.148.01853.62.73,d ,J ¼11.054.52.64,d ,J ¼11.054.62.73,d ,J ¼11.053.51934.01.99,m 39.01.50,m 33.42.01,m 39.42047.51.28,m 37.61.21,m 45.71.45,m 39.42125.71.99,m 40.11.73,1.98,m 34.92.48,2.29,m 31.02237.51.99,2.19,m 74.44.38,d d ,J ¼4.0,12.074.94.20,d d ,J ¼4.0,12.037.42366.53.70,4.20,d ,J ¼10.866.43.72,4.20,d ,J ¼10.866.93.72,4.20,d ,J ¼10.866.53.72,4.21,d ,J ¼12.02414.41.05,s 14.41.05,s 14.41.06,s 14.41.05,s 2517.51.05,s 17.51.04,s 17.51.05,s 17.51.18,s 2617.61.05,s 17.61.10,s 17.61.12,s 17.51.05,s 2723.91.16,s24.21.16,s24.31.20,s23.91.05,s28179.9179.9178.8179.92917.21.10,d ,J ¼6.417.30.95,d ,J ¼6.417.11.10,d ,J ¼6.417.50.90,d ,J ¼6.43065.23.87,3.96,d ,J ¼8.821.20.93,d ,J ¼6.464.93.95,3.99,m21.40.92,d ,J ¼6.4Journal of Asian Natural Products Research761D o w n l o a d e d A t : 18:20 22 J a n u a r y 2011Compound 1was obtained as a white amorphous powder with mp 211–2128C.The IR spectrum showed the absorption bands at 3363.3(Z OH)and 1689.4(C v C)cm 21.It had a molecular formula C 30H 48O 6established from its HR-ESI-MS at m/z 527.3333[M þNa]þ.Com-pared to the substrate,the 1H NMR spectrum showed two new signals at d 3.87and 3.96integrating for one proton,respectively.Meanwhile,a methyl doub-let signal was absent in 1.Those differences suggested the hydroxylation of a methyl carbon,and this was confirmed by analysis of the 13C NMR and DEPT spectra (Table 1),which showed 10methylenes,instead of nine for the parent compound,as well as the disappearance of a methyl ly,the resonance of C-30(a methyl group at d 21.4in asiatic acid)dis-appeared but another methylene signal appeared at d 65.2in 1.The hydroxy-lation at C-30was further authenticated by the paramagnetic shifts of the neigh-boring carbons C-20(from d 39.4of the substrate to d 47.5),while the signals of C-19and C-21shifted upfield by 5.4and 5.3ppm,respectively.It was also sup-ported by the HMBC spectrum (see Figure 1),which exhibited correlationsof H-30with C-19and C-21,as well as H-29with C-18,C-19,and C-20.Thus,compound 1was determined as 2a ,3b ,23,30-tetrahydroxyurs-12-ene-28-oic acid,which is a new compound.Compound 2was obtained as a white amorphous powder with mp 270–2718C.The IR spectrum showed the absorption bands at 3367.5(Z OH)and 1691.3(C v C)cm 21.Its molecular formula was deter-mined as C 30H 48O 6from its HR-ESI-MS at m/z 503.3375[M 2H]2.The 1H NMR spectrum of the metabolite was similar to that of the substrate displaying six charac-teristic methyl groups.However,it showed a new signal at d 4.38integrating for one proton,indicating the possible hydroxy-lation at the methylene group.It was confirmed by the analysis of the 13C NMR and DEPT spectra (Table 1),which showed nine methylenes,instead of 10for the parent compound.The resonance of C-22(a methylene group at d 37.4)in the substrate was absent,but another methine signal was displayed at d 74.4in 2.The hydroxylation at C-22was further confirmed by the paramagnetic shifts of the neighboring carbons C-21(from d 31.0of substrate to d 40.1)and C-17(from d 48.0of the substrate to d 54.1).It was also supported by the HMBC spectrum (see Figure 1),3Figure 1.Key HMBC correlations of compounds 1–3and the structure of asiatic acid.W.-N.He et al.762D o w n l o a d e d A t : 18:20 22 J a n u a r y 2011which exhibited correlations of H-16and H-21with C-22.The stereochemistry at C-22was deduced from the multiplicity of the corresponding C Z H signal at d 4.38in the 1H NMR spectrum.It was a double doublet (J ¼4.0,12.0Hz)signal consistent with an axial (a )position for that proton.Therefore,the biotransformation product was 2a ,3b ,22b ,23-tetrahydroxyurs-12-ene-28-oic acid,which is a new pound 3was obtained as a white amorphous powder with mp 231–2328C.The IR spectrum showed the absorption bands at 3340.9(Z OH)and 1716.7(C v C)cm 21.Its molecular formula,C 30H 48O 7,was determined according to HR-ESI-MS at m/z 519.3339[M 2H]paring the 1H NMR spectrum of the metabolite 3with that of compound 2,it has been found that two new signals were displayed at d 3.95and 3.99integrating for one proton,respect-ively.However,a methyl doublet signal was absent in 3.These were confirmed by the analysis of the 13C NMR and DEPT spectra (Table 1),which showed only five methyl signals,instead of six.The methyl signal of C-30at d 21.4in compound 2disappeared,but a new methylene signal at d 64.9was exhibited.The hydroxylation at C-30was further authenticated by the paramagnetic shifts of the neighboring carbons C-20(from d 37.6of compound 2to d 45.7),while the signals of C-19and C-21shifted upfield to d 33.4and 34.9,respectively.It was also proved by the HMBC spectrum (see Figure 1),which exhibited the corre-lation of H-30with C-21.As with compound 2,the stereochemistry at C-22was deduced from the multiplicity of the corresponding C Z H signal in the 1H NMR spectrum at d 4.20.It was a double doublet (J ¼4.0,12.0Hz)peak consistent with an axial (a )position for that proton.Therefore,the biotransformation product was 2a ,3b ,22b ,23,30-pentahydroxyurs-12-ene-28-oic acid,which is a new compound.3.Experimental3.1General experimental procedure Melting points were determined with the Electrothermal melting point apparatus and are uncorrected.The IR spectrum were recorded with a Nicolet Avatar spectrometer.NMR spectra were recorded in pyridine-d 5with a Bruker Avance III 400spectrometer.HR-ESI-MS was detected on a Bruker APEX IV FT-MS spectrometer.Silica gel (200–300mesh;Qingdao Haiyang Chemical Company,Qingdao,China)was used for column chromatography.Other normal reagents were purchased from Beijing Chemical Corporation (Beijing,China).Semi-HPLC (Alltech 426HPLC Pump and LINEAR UVis 200Detector)and Agilent Zorbax SB-C 18column (Agilent Technologies,Inc.,Santa Clara,CA,USA)were used for purification.3.2Chemicals and micro-organismsAsiatic acid was purchased from Nanjing Zelang Medical Technological Co.Ltd (Nanjing,China).All micro-organisms screened in our experiments were obtained from the China General Microbiology Culture Center (Beijing,China).Stock cultures of the fungi were stored on potato dextrose agar slants at 48C.3.3Biotransformation proceduresScreening scale biotransformation of asia-tic acid was carried out in 250ml Erlenmeyer flasks containing 80ml of medium.The flasks were placed in a rotary shaker operating at 180rpm at 258C.The substrate was dissolved in methanol to reach a concentration of 20mg/ml.After 48h of pre-culture,2mg of asiatic acid was added into each flask and these flasks were maintained under fermentation con-ditions for 4days.Culture controls consisted of fermentation blanks in which micro-organisms were grown for 48h,and then fed with the same amount of methanolJournal of Asian Natural Products Research763D o w n l o a d e d A t : 18:20 22 J a n u a r y 2011without containing the substrate.Substrate controls contained the sterile medium with the same amount of the substrate and were incubated under the above conditions.Preparative scale biotransformation of asiatic acid was carried out in 1000ml Erlenmeyer flasks containing 350ml of medium by A.longipes (Ellis et Everhart).A substrate of 20mg with 1ml methanol was added to each flask containing the pre-culture medium.In total,200mg of the substrate were used.Incubation conditions were the same as described above.3.4Extraction and isolationThe culture was filtered and the filtrate was extracted with the same volume of EtOAc three times.The organic phase was collected and concentrated in vacuo .The residue (460mg)was applied to a silica gel column and eluted with chloroform–methanol (in a gradient from 20:1to 1:1),which yielded fractions 1–3.Fraction 2(100mg)was purified by an ODS column (MeOH–H 2O:20–100%)and semi-preparative HPLC (MeOH–H 2O:58%)to give compounds 1(10mg)and 2(22mg).Fraction 3(15mg)was purified by a silica gel column repeatedly to afford compound 3(5mg).3.4.1Compound 1White amorphous powder (10mg),mp:211–2128C.IR (KBr)n max (cm 21):3363.3,1689.4cm 21;1H and 13C NMR spectral data,see Table 1;HR-ESI-MS m/z :527.3333[M þNa]þ(calcd for C 30H 48O 6Na,527.3343).3.4.2Compound 2White amorphous powder (22mg),mp:270–2718C.IR (KBr)n max (cm 21):3367.5,1691.3cm 21;1H and 13C NMR spectral data,see Table 1;HR-ESI-MS m/z :503.3375[M 2H]2(calcd for C 30H 47O 6,503.3378).3.4.3Compound 3White amorphous powder (5mg),mp:231–2328C.IR (KBr)n max (cm 21):3340.9,1716.7cm 21;1H and 13C NMR spectral data,see Table 1;HR-ESI-MS m/z :519.3339[M 2H]2(calcd for C 30H 47O 7,519.3329).AcknowledgementsThis work was financially supported by the Beijing Natural Science Foundation (No.7102103)and the Beijing NOVA Program (No.2009B02).References[1]L.P.Qin,W.G.Zhuang,H.C.Zheng,andR.X.Ding,World Notes:Phytomed.12,154(1997).[2]Y.L.Hsu,P.L.Kuo,L.T.Lin,and C.C.Lin,Pharm.Exp.Ther.313,333(2004).[3]B.C.Park,K.O.Bosire,E.S.Lee,Y.S.Lee,and J.A.Kim,Cancer Lett.218,81(2005).[4]J.Chen,W.Y.Hua,and H.B.Sun,Chin.Tradit.Herb.Drugs 37,458(2006).[5]H.F.Zhang,Master’s dissertation,Jiang SuUniversity,2008,p.12.[6]K.F.Ma,Y.Y.Zhang,D.C.Zhu,and Y.J.Lou,Eur.J.Pharmacol.603,98(2009).[7]A.M.Clark and C.D.Hufford,Med.Res.Rev.11,473(1991).[8]R.Azerad,Adv.Biochem.Eng.Biotechnol.63,169(1999).[9]Y.Liu and Y.Q.Zhao,Mod.Chin.Med.10,7(2008).W.-N.He et al.764D o w n l o a d e d A t : 18:20 22 J a n u a r y 2011。
