Saikosaponin q-1 from Bupleurum chinense

A new saikosaponin was isolated from Bupleurum chinense DC., and its structure was identified as 3β,16α,23,28,30_pentahydroxy_olean_11,13(18)_dien_3_O_β_D_glucopyranosyl(1→6)_[α_L_rhamnopyranosyl (1→4)]_β_D_glucopyranoside on the basis of chemical and spectral evidence, named as saikosaponin q_1. In addition, two known saikosaponins, 3″_O_acetyl_saikosaponin d and 3″_O_acetyl_saikosaponin b 2, were also isolated and identified from this plant for the first time.

The Chemical Constituents from the Roots of Bupleurum chinense DC.

从北柴胡（ＢｕｐｌｅｕｒｕｍｃｈｉｎｅｎｓｅＤＣ．）根的乙醇提取液中分离得到五个化合物，经理化性质及波谱解析分别鉴定为水仙甙（１）、腺苷（２），尿苷（３）、ａ菠甾醇葡萄糖甙（４）及木糖醇（５）。均为首次从北柴胡中分离得到。

Saikosaponin v-2 from Bupleurum chinense

Saikosaponin v-2(1). was isolated li om the roots of the title plant and thc structure was identified on rhs basis of spectral anal? sis. Saikosaponin v-2 is a new compound. which was identified as 3 beta .16 alpha .23.28-tetrahydroxy-olean-11.13(18)-dien-30-oic acid-3-O-beta -D-glucopyranosyl- (1 -->2)glucopyranosyl-(1 -->3)-beta -D-fucopyranosol-30-O-xylitol ester.

A New Chromone Glycoside from Bupleurum Chinense

A new chromone glycoside, saikochromoside A(1), was isolated from the roots of Bupleurum chinense DC. Its structure was determined on the bases of chemical and spectral analyses.

Localization and Dynamic Change of Saikosaponin in Root of Bupleurum chinense

Anatomical, histochemical and phytochemical methods were used to investigate the structure, the localization and content changes of total saikosaponin and saikosaponin-a of the roots of Bupleurum chinense DC. at different developmental stages. Results showed that saikosaponin was mainly distributed in pericycle and primary phloem in the young root; but in the mature root, it was mainly distributed in vascular cambium and secondary phloem. During the whole growth period from the pre-blossom, blossom, fruit, and fruit mature periods until the pre-withering period, it was in the fruit mature period that both the total saikosaponin content and the saikosaponin-a content reached the highest level. So the last 20 d of October was considered as the right collecting season for the drug of B. chinense. In addition, the quality of 1-year-old drug was better than that of 2-year-old drug due to its higher saikosaponin content. On the other hand, judging from the external characteristics of the drug, the one with an acerose taproot and more lateral roots was of better quality. The results offered theoretical bases for selecting medicinal material of high quality and determining the most appropriate harvesting stage and part of B. chinense.

Molecular Cloning and Expression of Squalene Epoxidase from a Medicinal Plant, Bupleurum chinense

Objective In plant, squalene epoxidase （SE） catalyzes the first oxygenation step in the biosynthetic pathway of triterpenoid and phytosterol, representing one of the rate-limiting enzymes in this pathway. Bupleurum chinense is an important medicinal herb with its major active constituents such as triterpenoid saponins and saikosaponins. In order to obtain the series of enzymatic genes involved in saikosaponin biosynthesis, a cDNA of SE, designated BcSEI, was cloned from B. chinense. Methods The BcSEI gene was cloned by homology-based PCR and 5＇/3＇ RACE methods from the adventitious roots of B. chinense. The physical and chemical parameters of BcSE1 protein were predicted by protparam. In order to discover hints in amino acid sequences on the dominant functions in the biosynthesis of saponin or phytosterol, sequences of SE from other plants were downloaded from NCBI for sequences alignment and phylogenetic analysis. BcSEI was cloned into a yeast mutant KLNI （MATa, ergl.＇：URA3, leu2, ura3, and trpl） to verify the enzyme activity of BcSE1. Additionally, the tissue-specific expression and methyl jasmonate （MeJA） inducibility of BcSEI were investigated using quantitative real-time PCR. Results The predicted protein of BcSE1 is highly similar to SEs from other plants sharing amino acid sequence identities of up to 88%. The BcSEI can functionally complement with yeast SE gene （ERGI） when expressed in the KLNI mutant （MATa, ergl：：URA3, leu2, ura3, and trpl）. Using as controls with ^-amyrin synthase （G-AS） which is presumed to catalyze the first committed step in saikosaponin biosynthesis and a cycloartenol synthase （CAS） relating to the phytosterol biosynthesis, the transcript of BcSE1 was significantly elevated by MeJA in adventitious roots of B. chinenseand the transcript of BcSElwas most abundant in the fruits and flowers of plants, followed by that in the leaves and roots, and least in stems. Conclusion It is the first time to illustrate the molecular information of SE in B. chinense and to clone the full-length SEgene in plants of genus Bupleurum L.

Bioassay-guided isolation of saikosaponins with agonistic activity on 5-hydroxytryptamine 2C receptor from Bupleurum chinense and their potential use for the treatment of obesity

5-Hydroxytryptamine 2C(5-HT2C) receptor is one of the major targets of anti-obesity agents, due to its role in regulation of appetite. In the present study, the 70% EtO H extract of the roots of Bupleurum chinense was revealed to have agonistic activity on 5-HT2 C receptor, and the subsequent bioassay-guided isolation led to identification of several saikosaponins as the active constituents with 5-HT2 C receptor agonistic activity in vitro and anti-obesity activity in vivo. The new compound, 22-oxosaikosaponin d(1), was determined by extensive spectroscopic analyses(HR-ESI-MS, IR, and 1D and 2D NMR). The primary structure-activity relationship study suggested that the intramolecular ether bond between C-13 and C-28 and the number of sugars at C-3 position were closely related to the 5-HT2 C receptor agonistic activity. Saikosaponin a(3), the main saponin in B. chinense, showed obviously agonistic activity on 5-HT2 C receptor with an EC50 value of 21.08 ± 0.33 μmol×L^(–1) in vitro and could reduce food intake by 39.1% and 69.2%, and weight gain by 13.6% and 16.4%, respectively, at 3.0 and 6.0 mg×kg^(–1) in vivo. This investigation provided valuable information for the potential use of B. chinense as anti-obesity agent.

Factors Affecting Embryogenic Callus Production and Plant Regeneration in Anther Culture of Bupleurum chinense

Objective To evaluate the influences of the genotypes,anther developmental stages,and cultural conditions on the efficiency of embryogenic callus induction and plant regeneration in the anthers culture of Bupleurum chinense.Methods The different effects such as four genotypes,plant growth regulators,and temperature condition were compared in the experiments.The histological study was performed with the process of the anther culture.Results The highest inducing rate of embryogenic calli were achieved for the genotypes Zhongcaiyihao(ZCYH),Z4,and Z5 at the early-to middle-uninucleate stages,except for genotype ZPM1 at the tetrad stage.Cold pretreatment increased the production of the embryogenic callus,in which 4-day cold pretreatment improved the production of embryogenic callus from 0% to 2.2% and 5.0% for genotypes ZPM1 and ZCYH,respectively.No embryogenic callus was induced in the medium containing less than 0.75 mg/L 2,4-dichlorophenoxyacetic acid(2,4-D) .The highest regeneration rate(34.6%) was obtained in 1/2 MS salts regeneration medium supplemented with 0.1 mg/L 6-benzylmaminopurine(BA) .The low concentration of BA was able to promote the embryogenic callus formation and subsequent plantlet regeneration via somatic embryogenesis.Chromosome counting of regenerated plantlets showed mostly diploid plant(2n = 12) with only one haploid plant(n = 6) .Because of the low rate of microspore embryo formation,we only tracked the process of embryogenesis from the connective tissue,instead of microspore by histological observations.Conclusion This study establishes an efficient system for embryogenic callus induction and plant regeneration system.This is the first report on the haploid plantlet through the anther culture of B.chinense.

A comparative study on different multiple cropping patterns of Bupleurum chinense

Bupleurum chinense is a genuine medicinal material in Shanxi Province,and planting B.chinense is one of the main sources for local farmers to increase their income.In B.chinense cultivation,wheat-B.chinense,maize-B.chinense and soybean-B.chinense are commonly used as multiple cropping patterns.In the present study,we studied the effects of three different multiple cropping patterns on N,P and K levels of soil,photosynthetic parameters,agronomic characteristics,saikosaponin A,C and D contents,yield and economic benefits of B.chinense.The results showed that the soybean-B.chinense multiple cropping pattern was beneficial to the enrichment of N in the soil,thus promoting photosynthetic parameters,growth,saikosaponin accumulation,yield and economic benefits of B.chinense.It is suggested that soybean-B.chinense multiple cropping was an appropriate multiple cropping pattern for local cultivation of B.chinense.

Effects of Different Treatments on Seed Germination of Bupleurum chinensis

In order to solve the problem of low emergence rate of Bupleurum chinense seeds, and screen out the best germination conditions, the seeds of Bupleurum chinense L. were treated with different soaking time, germination temperature and exogenous regulating substances to improve the germination rate. The results showed that the germination rate of fresh water at 12 h was significantly higher than that at 8 h and 36 h; The germination rate of seeds treated with constant temperature 15℃ and variable temperature 25 ℃/15 ℃ （day/night） was significantly higher than that of other temperature treatments. Seed soaking time and culture temperature also influenced seed germination start day and peak day. Different concentrations of regulatory substances could promote seeds germination. The germination rate of seeds induced by 0.50 mg/L 6-BA was the highest, the start day and peak day were 4 days and 5 days earlier than control check （CK） respectively.

促根肥料对柴胡苗期生长的影响

为了提高柴胡(Bupleurum chinense DC.)地下部分的产量,使用9种促根肥料对柴胡幼苗进行灌溉施肥处理,研究不同促根肥料对柴胡株高、根干重、叶绿素含量等18个农艺性状以及柴胡皂苷含量和产量的影响。结果表明,微补精力提高了地上部干重;根多乐抑制株高增长趋势,延缓了叶绿素含量的衰减和增长趋势;海博氏促长精华增加了株高,但抑制了株高的增长趋势;其中5种肥料降低了柴胡皂苷的产量。柴胡生产栽培中,合理地施肥抑制地上器官生长,促进根系生长和柴胡皂苷合成积累极为重要。

水分胁迫对北柴胡生理特性和柴胡皂苷含量的影响

以北柴胡(Bupleurum chinense DC.)二年生幼苗为试验材料,采用盆栽称重控水法,研究4种土壤水分处理(田间持水量的30%、50%、70%和90%)对北柴胡生理特性和柴胡皂苷含量的影响。结果表明,轻度干旱胁迫显著增加柴胡根冠比、柴胡皂苷a和总皂苷的含量,重度干旱胁迫显著增加柴胡体内可溶性糖、脯氨酸、丙二醛、柴胡皂苷d和总皂苷的含量,可溶性蛋白质和FW/BW(自由水/结合水)随着干旱胁迫程度的增加逐渐减小。综合分析认为,柴胡具有一定的耐旱性;轻度干旱胁迫有利于柴胡根冠比的提高和柴胡皂苷的积累,而土壤水分过多则不利于柴胡根系生长和柴胡皂苷的积累。因此,人工栽培柴胡时应采用科学合理的控水方式,避免低洼易积水地块,在保障植物正常生长时也能够促进柴胡有效成分的积累,提高柴胡的产量和品质。

星点设计-响应面法优化柴胡皂苷提取工艺研究

在单因素试验基础上,分析提取次数对柴胡(Bupleurum chinense)皂苷类成分提取的影响。采用Central composite design(CCD)响应面法优化柴胡皂苷类成分的提取工艺,考察提取时间、提取温度、液料比对柴胡皂苷类成分提取的影响。结果表明,柴胡皂苷类成分的最佳提取工艺参数为提取温度80℃、提取时间60 min、液料比8∶1(mL∶g),提取2次。在此优选条件下柴胡皂苷类成分提取含量为1.80 mg/g,与预测值1.81 mg/g有良好的拟合性,提取工艺可行,预测性良好。

Advances in Research of Resources and Cultivation Techniques of Chinese Medicinal Material Radix Bupleuri

At present,with the vigorous development of traditional Chinese medicines,wild Bupleurum chinense resources become increasingly depleted,and its cultivars have become the mainstream of the market.However,due to factors such as a large number of original plants,irregular planting techniques,and poor growth environment,the quality of cultivated B.chinense is uneven and it is difficult to achieve the expected efficacy.In order to protect the wild resources of B.chinense and expand the source of medicinal materials,this paper summarized the research advances of the main resource producing areas and cultivation techniques of B.chinense in combination with relevant documents published at home and abroad,and discussed the future development of B.chinense,to provide a reference for research and sustainable development of B.chinense resources.

Simultaneous determination of Saikosaponin a, c and d in different parts of wild bupleurum from Shanxi Province

As a traditional Chinese herbal medicine exhibiting analgesic,fever-reducing and anti-inflammatory effects,Radix Bupleuri（Chai-Hu） is commonly used for the treatment of influenza,which is derived from the dried roots of Bupleurum chinense DC.and Bupleurum scorzonerifolium Willd.Among of diverse chemical components,saikosaponins are the key active components of the herb medicine.In the present study,we established a method of high performance liquid chromatography（HPLC） coupled with evaporative light scattering detection（ELSD） for simultaneous determination of saikosaponin a,c and d in root,stem,leaf and flower of Bupleurum chinense（B chinense） collected from different areas of Shanxi Province,China.The results from 16 samples of root,stem,leaf and flower of B chinense demonstrated that the total contents of the three saikosaponins in the root of B chinense collected from Dongshan Taiyuan,Xishan,Tianlongshan and Pangquangou were 4.26 mg/g,3.22 mg/g,4.23 mg/g and 3.05 mg/g,respectively.However,there was scarcely any saikosaponins in the stem,leaf and flower of B chinense collected from above-mentioned areas.The method of HPLC coupled with ELSD was suitable for quality control of Radix Bupleuri.The result also confirmed that the root of B chinense was the best medicinal part.

Development of a C3c-based ELISA method for the determination of anti-complementary potency of Bupleurum polysaccharides

Traditionally, determination of inhibitory potency of complement inhibitors is performed by the hemolytic assay. However, this assay is not applicable to the lectin pathway, thus impeding the understanding of complement inhibitors against the overall function of the complement system. The main objective of our study was to develop a specific enzyme-linked immunosoihent assay (ELISA) as an alternative method to assess the anti-complement activity, particularly against the lectin pathway. By using respective coating substrates against different activation pathways, followed by capturing the stable C3c fragments, our ELBA method can be used to screen complement inhibitors against the classical pathway and the lectin pathway. The inhibitory effect of sununin on the classical pathway, as measured by our hemolytic assay is consistent with previous reports. Further assessment of suramin and Bupleurum polysaccharides against the lectin pathway showed a good reproducibility of the method. Comparison of the lectin pathway IC5is between Ruplearum.smithii var, purviPliam polysaccharides (1.055 ingtmE) and Buplcurann chinense polysaccharides (0.98 ing/mL) showed that similar to the classical and alterative pathway, these two Bupleurum polysaccharides had comparable anti complementary properties against the lectin pathway. The results demonstrate that the described EIASA assay can compensate for he shortcomings of the hemolytic assay in lectin pathway. (C) 2015 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Chromatographic fingerprint analysis of Bupleuri Radix by HPLC-ELSD

To facilitate the species identification and quality assessment of Chaihu （Bupleuri Radix）, a simple and valid chromatographic fingerprint method was developed. The method uses high-performance liquid chromatography coupled with evaporative light scattering detector （HPLC-ELSD） and the data analysis is assisted by professional analytical software recommended by the State Food and Drug Administration （SFDA）. The results indicate that Nan Chaihu raw materials and Chaihu decoction pieces vary markedly in chemical quality, while Bei Chaihu raw materials are relatively more stable. Furthermore, it is obvious that Nan Chaihu is chemically very different from Bei Chaihu, suggesting that Nan Chaihu may not be suitable for medicinal use. In addition, the obvious differences between the chromatograms of decoction pieces and raw materials, especially the peaks between 30 and 40 rain and after 45 rain, indicate possible effects of the processing procedures on the chemicals. By analyzing the fingerprints of all samples, 12 main saponin-like fingerprint peaks, of which at least three are characteristic peaks of saikosaponins a, c, and d, are proposed to be considered for further characterization and quality evaluation of Chaihu.

柴胡皂苷抗肿瘤的生物医学功能研究

柴胡是伞形科柴胡属植物,药用部位柴胡的干燥根。柴胡皂苷是从柴胡中提取出来的,具有多种药理特性。为了探讨柴胡皂苷的生物医学功能和机理,本研究进行了在体外和小鼠体内抑制肾肿瘤癌细胞分化研究。研究表明柴胡皂苷联合干扰素能够呈浓度依赖方式显著抑制肾癌细胞生长活力(P<0.05),使得肾癌细胞阻滞在G0/G1期,能加快细胞凋亡。细胞凋亡基因p53的表达量呈上升趋势;能够显著降低肾癌小鼠移植瘤体积和重量;凋亡蛋白p53阳性表达率显著上调。本研究初步显示出柴胡皂苷联合干扰素对肾肿瘤小鼠有明显抑制作用,其作用机制可能是柴胡皂苷联合干扰素将肾癌细胞阻滞在G0/G1期,诱导细胞凋亡,使p53蛋白上调表达。

Polyclonal antibody preparation of BcbZIP134 for transcriptional regulation analysis on saikosaponin biosynthesis

Objective: We are trying to verify how a transcription factor BcbZIP134 regulates the synthesis of saikosaponin using specific antibody. However, it is hard to obtain this soluble protein expressed in vitro, a prerequisite for antibody preparation. So we explored a condition in which the soluble protein can efficiently express followed by preparation of the specific polyclonal antibody.Methods: Firstly, the cDNA of BcbZIP134 from Bupleurum chinense was expressed in Transetta(DE3) E.coli. Different concentrations of IPTG(0.05 and 0.5 mmol/L) and different culture temperatures(16 and37 °C) were explored for efficient expression of target protein. Then, the expressed protein with His Tag was purified using Ni Sepharose 6 Fast Flow. Different concentrations of imidazole elution(15, 60, and300 mmol/L) were used to elute the target protein. The purified protein of BcbZIP134 was used to immunize rabbits. Using the purified polyclonal antibody, the expression of BcbZIP134 in transgenic B. chinense hairy root lines was analyzed by Western blot assays.Results: Under the conditions, IPTG 0.5 mmol/L, 16 °C, and overnight, recombinant protein of BcbZIP134 was obtained in high content using pEASY?-Blunt E1 vector and Transetta(DE3) E. coli. Anti-serum against BcbZIP134 was obtained with a high titer of 1: 51 200 after immunization in rabbits. The polyclonal antibody could react with BcbZIP134 overexpressed in transgenic B. chinense hairy root lines.Conclusion: An efficient protein expression system in E. coli was constructed and the soluble recombinant protein for BcbZIP134 was obtained. By using this protein, the specific and high-performance polyclonal antibody was prepared. Furthermore, by using BcbZIP134 overexpressed hairy roots, the availability of the polyclonal antibody was verified by Western Blotting.