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This study focuses on the microbial quality control of the Chinese herbal decoction pieces. In view of the shortcomings of traditional culture methods such as slow detection speed and inability to detect unculturable microorganisms, a new method based on ATP bioluminescence technology combined with statistical analysis methods was established to rapidly predict and quantitatively detect the total aerobic microbial count (TAMC) and total yeast and mold count (TYMC) contaminated Bupleurum chinense DC. decoction pieces. Based on the optimized ATP bioluminesence detection system, accurate detection of pure bacterial solution of Escherichia coli, Bacillus subtilis and Staphylococcus aureus can be achieved, with detection limits of 47.86, 89.13 and 1 862.09 CFU·mL-1, respectively. The detection time was 6.5 h, and the detection cost was as low as 2 yuan/time. The upper and lower warning limits of TAMC were determined by the misjudgment rates of 10% and 20%, respectively. And the warning limit of TYMC was determined by the misjudgment rate of 20%. The proposed crossing method could quickly predict the amount of microbial contamination in Bupleurum chinense DC. decoction pieces. The constructed partial least squares regression (PLSR) model could accurately quantify the quantity of microbial contamination in Bupleurum chinense DC. decoction pieces. The optimal PLSR prediction model for TAMC had a correction coefficient (R2) of 0.826, a root mean square error of correction set (RMSEE) of 0.468 and a root mean square error of cross-validation set (RMSECV) of 0.465. The R2, RMSEE and RMSECV in the prediction model of TYMC were 0.778, 0.543 and 0.541, respectively. The aim of this study is to establish a kind of rapid detection method and prediction models for the microbial limit of traditional Chinese medicine and Chinese herbal decoction pieces, and to provide a more convenient and sensitive detection technology for the microbial quality process control of traditional Chinese medicine products.
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OBJECTIVE To explore the intestinal absorption characteristics of saikosaponins. METHODS Based on everted intestinal sac model， using accumulative absorption amount （Q） and absorption rate constant （Ka） as indexes， UHPLC-MS/MS technique as a method， the absorption of saikosaponin A， B2， C， D and F from total saponins of Bupleurum chinense （8 g/mL， by crude drug） in the duodenum， jejunum and ileum was detected. RESULTS The correlation coefficients （r） of the regression equations for the absorption of saikosaponins A， B2， C and F in the duodenum， jejunum and ileum were all higher than 0.95， while the r of saikosaponin D in the above intestinal segments was lower than 0.95； compared with the absorption of the same composition in the duodenum， the Q and Ka of saikosaponin A and C circulating in jejunum and ileum for 120 min， as well as the Q and Ka of saikosaponin F circulating in the ileum for 120 min were significantly decreased （P＜0.05）. CONCLUSIONS Saikosaponin A and the other 4 saikosaponins are all absorbed in the duodenum， jejunum and ileum； among them， saikosaponin A， B2， C and F are linearly absorbed， which conforms to the zero-order absorption characteristics， but saikosaponin D shows non- linear absorption.
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Bupleuri Radix, serving as the sovereign medicinal in many antidepressant compound preparations, has been proved effective in treating depression in mice, but its effect on the intestinal flora remains unclear. The present study aimed to investigate the effects of Bupleurum chinense(one of the original materials of Bupleuri Radix) on the behaviors and the diversity of intestinal flora of depressed mice. A depression mouse model was induced by repeated social defeat stress. Specifically, C57 BL/6 J male mice were exposed to the attack from the CD-1 mice. Then, C57 BL/6 J male mice were divided into a depression group and a B. chinense group, with normal saline and B. chinense administered(ig) respectively. Sucrose preference test and tail suspension test were conducted during and after the experiment respectively, to analyze the effects of B. chinense on the behaviors of the depressed mice. The feces were collected after the experiment. The V3-V4 16 S rDNA regions of intestinal flora of mice in each group were sequenced by Ion S5 TMXL for the analysis of the number of operational taxonomic units(OTUs), richness, alpha and beta diversity indexes, and differential phyla and genera. The results indicated that B. chinense could decrease depressive-like behaviors of mice, increase sucrose preference, and shorten the time of immobility in tail suspension test. After B. chinense intervention, the relative abundance of Firmicutes was significantly decreased, while that of Bacteroidetes was increased at the phylum level. At the genus level, the relative abundance of Lactobacillus and Lachnoclostridium decreased(P<0.05), while that of Bacteroides, Alistopes, etc. was elevated(P<0.05). The findings demonstrate that B. chinense can regulate the intestinal flora and improve the depressive-like behaviors of mice with depression.
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Animals , Mice , Bupleurum , Feces , Gastrointestinal Microbiome , Lactobacillus , Mice, Inbred C57BLABSTRACT
Objective:To establish a molecular identification method for Bupleurum chinense seeds based on ribosomal DNA internal transcribed spacer (ITS) sequence, ensuring the species authenticity of the cultivated seeds of B. chinense. Method:A total of 59 seeds samples of B. chinense and its main cultivated species, marketed B. chinense were collected. The effect of different sampling amounts and different water bath conditions on DNA extraction quality of the seeds was investigated, a DNA extraction method for seeds of Bupleurum was established. Their ITS sequences were obtained by polymerase chain reaction (PCR) and bidirectional sequencing. In addition, 34 ITS sequences of main cultivated Bupleurum species, such as B. chinense, B. scorzonerifolium, B. falcatum and B. smithii, were downloaded from GenBank to enrich identification database of B. chinense seeds. The neighbor-joining (NJ) dendrogram were constructed by MEGA-X 10.0.5 software to investigate the the species identification ability of ITS sequences for B. chinense seeds. And DNA barcoding identification of marketed B. chinense seeds was conducted based on BLAST method and NJ dendrogram method. Result:In total, 59 ITS sequences were obtained. ITS sequences of B. chinense could be divided into six haplotypes, including seven variable sites. The NJ dendrogram indicated that all the haplotypes of B. chinense could form independent branches, which could be distinguished from other cultivated species of Bupleurum in the collected samples, and possessed the ability to identify species of B. chinense seeds. Based on ITS sequence barcoding identification, 3 of the 19 marketed B. chinense seeds were B. falcatum with a counterfeit rate of 15.8%. Conclusion:DNA barcoding technology based on ITS sequence can accurately and reliably identify B. chinense seeds and its adulterants, providing reference for the standardization construction of Chinese medicinal materials seeds.
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Objective: To study the HPLC fingerprint of Bupleurum chinense and the relationship between the spectrum and the toxicity in vitro, construct the network of the liver toxicity induced by B. chinense, and integrate the spectrum and the network toxicology to predict the material basis of the liver toxicity induced by B. chinense. Methods: The fingerprints of 10 batches of decoction of B. chinense were established by HPLC, the contents of ALT and AST in L02 cells were determined by decoction of B. chinense, and the toxic components of liver were preliminarily determined by the method of grey correlation analysis. Combined with network toxicology, the candidate components of hepatotoxicity were predicted. Integrated analysis on the components of liver toxicity induced by decoction of B. chinense. Results: The established fingerprint of B. chinense was calibrated with 29 common peaks. The results of grey correlation analysis showed that common peaks 13, 12, 8, 26, 10, 14, 27 and 11 had a high correlation degree with the ALT content of L02 cells. The common peaks of 12, 13, 26, 8, 10, 14, 27 and 11 had a high correlation degree with the AST content of L02 cells. According to the network toxicology, 17 components of B. chinense, stigmasterol, baicalin, etc were speculated to be the hepatotoxicity components of B. chinense. The integrated analysis initially determined that the hepatotoxicity components of B. chinense were 13, 12, 8, 26, 10, 14, 27 and 11 peaks in total, and stigmasterol, baicalin, saikosaponin D, etc. Conclusion: The hepatotoxicity of B. chinense is the result of the interaction of various components. This study can provide data support for the further research on the material basis of B. chinense hepatotoxicity.
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Objective: To summarize the prescription rules of traditional Chinese medicine (TCM) for treating alcoholic liver disease (ALD) based on TCM inheritance support system (V2.50). Methods: The literatures about TCM prescriptions for treating ALD were collected from CNKI, Wanfang database, and VIP database. The TCM inheritance platform system was used to analyze the prescription rules of TCM in the treatment of alcoholic liver disease. Results: Statistics showed that the majority of prescriptions were used to treat alcoholic fatty liver, alcoholic hepatitis, and alcoholic cirrhosis. Through "frequency statistics" analysis, 107 prescriptions were found involving 149 flavors of TCM, with a cumulative frequency of 1 195 times. Twenty-three Chinese medicines with a frequency of ≥ 15 times were used, and the cumulative frequency was 737 times (62%). The most frequently used medicines were blood-activating and stasis-removing drugs, water-diffusing and damp-permeating drugs, tonics, heat-clearing drugs, antialcoholic poisons and qi-regulating drugs. The commonly used doses of Salvia miltiorrhiza, Poria cocos, Bupleurum chinense, Glycyrrhiza uralensis, Atractylodes macrocephala, Alismatis Rhizoma, and Curcumae Radix in the top 10 medicines ranked in the frequency of medication accorded with the prescribed doses in the Pharmacopoeia of the People's Republic of China (2015 edition), while Crataegi Fructus, Artemisiae Scopariae Herba, and Puerariae Lobatae Radix exceeded the prescribed doses. In the frequency analysis of drug pairs, the combination of S.miltiorrhiza and B. chinense was the most widely used. According to the association rules of drug combination, the correlation between Curcumae Radix and S. miltiorrhiza was the strongest, that was, the probability of S. miltiorrhiza appearing with the emergence of Curcumae Radix was 88%. From the network display chart, it was indicated that S. miltiorrhiza and P. cocos were the main herbs for treatment. Through unsupervised entropy hierarchical clustering algorithm, 14 core combinations for new clustering were extracted, and seven new prescriptions can be obtained by further clustering. Conclusion: The basic principles of TCM treatment of ALD include promoting blood circulation and removing blood stasis, removing dampness, tonifying, detoxifying alcohol, and promoting qi, and with "protecting spleen and stomach function" as its purpose, which accords with the theoretical basis of traditional Chinese medicine in treating alcoholic liver disease. Core combinations and new prescriptions provide references for clinical drug use and new drug research and development, but new prescriptions must be further evaluated with the combination of traditional Chinese medicine theory and clinical practice.
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Objective: Soil is an important factor affecting the formation and accumulation of active ingredients in Chinese medicinal herbs. Taking 29 sample wild and cultivated Bupleurum chinense of 11 areas as study materails, using mathematical statistical analysis methods to explore the relationship between saikosaponin accumulation and soil factors in order to improve the quality of B. chinense by soil ecological regulation. Methods: HPLC analysis of the contents of saikosaponins a, c, d, e, and f from different habitats; pH, organic matter, conductivity, soil water, total nitrogen, alkaline nitrogen, available phosphorus, available potassium, effective calcium, effective magnesium, effective iron, effective copper, and effective manganese were determined by conventional soil physicochemical property assay methods, cluster analysis method was used to analyze the content of saikosaponin from different habitats, analysis of the relationship between soil factors and the content of saikosaponin by Pearson correlation analysis, analysis of soil factors using principal component analysis. Results: Determination of saikosaponin showed that higher content of saikosaponin in Henan province (habitat 5, habitat 6), total content of saikosaponin a and saikosaponin d in habitat10 was 5.16 times the national standard.Cluster analysis of B. chinense from different origins, according to the content of saikosaponin, the B. chinense from 29 habitats were grouped into three types. Pearson correlation analysis of saikosaponin content and soil factors that the organic matter in the soil was significantly positively correlated with saponin a and total saponins (P < 0.05), there was a significant positive correlation between available zinc and available iron and each saikosaponin (P < 0.05) properly improve the organic matter, effective zinc, effective ironcontent in the soil can promote the accumulation of saikosaponin.Analysis of the principal components of soil from different habitats, the higher score was habitat 9, habitat 10, habitat 11, habitat 14, habitat 19, habitat 28. This is basically consistent with the results of cluster analysis of saikosaponin content. Based on the analysis of the main soil indicators of B. chinense from different habitats that in a certain range, the higher organic and effective zinc content, the more favorable were the accumulation of saikosaponins. This was basically consistent with the results of Pearson correlation analysis. Conclusion: These results indicated enhancement of organic matter, effective zinc, in the soil can improve the saikosaponin content in cultivated B. chinense.
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Objective To investigate the relationship between the content of flavonoids and the key enzyme genes expression in different tissues of Bupleurum chinense and B. scorzonerifolium. Methods The roots, stems, leaves, and fruits of B. chinense and B. scorzonerifolium were used as test materials, determination of flavonoids (rutin, quercetin, kaempferol, and isorhamnetin) in different tissues by HPLC, determination of total flavonoids by UV spectrophotometry, the tissues expression of key enzyme genes (IFS, F3H, and DFR) in flavonoids synthesis was determined by real-time quantitative PCR, correlation analysis was performed with SPSS. Results The content of flavonoids in the aerial parts (stems, leaves, and fruits) of B. chinense and B. scorzonerifolium was significantly higher than that in roots, the content of flavonoids was mainly rutin, and the content of rutin in the leaves of B. chinense leaves was up to 106.961 mg/g; The distribution of total flavonoids in B. chinense and B. scorzonerifolium was obviously different, the content was from high to low: leaves ≥ fruit > stem > root; The expression of B. chinense IFS, F3H, and DFR gene in the aerial parts was much higher than that in roots, IFS gene was significantly positive correlated with rutin (P < 0.05), F3H gene was significantly positive correlated with DFR gene (P < 0.05), but the expression of IFS, F3H, and DFR gene in each tissues of B. scorzonerifolium was at lower level. Conclusion The content of flavonoids in different parts of B. chinense and B. scorzonerifolium was consistent with the expression of flavonoids synthesis key enzyme genes, the differential expression of key enzyme genes regulates the synthesis and accumulation of flavonoids in different tissues.
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Objective: To select reference genes suitable for the expression analysis of Bupleurum chinense, and analyze the relationship between the content of saikosaponin and the gene expression of key enzymes in different tissues of B. chinense. Methods The roots, stems, leaves and fruits of B. chinense were used as test materials, and five commonly used internal reference genes of Actin, α-tubublin, β-tubulin, Cyclophilin and EF-1α were selected as candidates by real-time quantitative PCR. Based on the selected internal reference gences, tissue expression pattern of ACAT, FPS, HMGR, IPPI, PMD, PMK, SE, SS, β-AS, UGT1, UGT3, UGT6, UGT8, UGT10, P450-7 and P450-12 genes in B. chinense was analyzed. The content of saikosaponin a, saikosaponin c and saikosaponin d were determined by HPLC, and correlation analysis was performed by SPSS. Results:The EF-1α gene with the best stability in the five candidate genes (EF-1α, Cyclophilin, Actin, β-tubulin, α-tubublin) was selected as the internal reference gene. The expression levels of 16 key enzymes in the roots of B. chinense were measured. The results showed that ACAT, PMK, IPPI, SS, SE, UGT1, UGT3, UGT6, and UGT8 were the highest in the aboveground parts, the levels of HMGR, β-AS, P450-7 and P450-12 were higher in the roots than those in the aboveground parts, but PMD, FPS and UGT10 were not significantly different in the tissues. The content of saponins in the root was much higher than that in the aerial parts (stem, leaf and fruit) by HPLC. The results of correlation analysis showed that 16 key enzyme genes in the upstream ACAT, HMGR, PMD, SE and so on were significantly correlated with downstream P450-7, P450-12, UGT3, UGT6 and UGT8 (P < 0.05). It showed that the key enzyme genes were closely related to each other and regulated the synthesis of saikosaponin in common. The correlation analysis between the 16 key enzyme genes and the content of saikosaponin showed: HMGR, P450-7, p450-12 and the total of three saponins were significantly positively correlated (P < 0.01), and β-AS was significantly correlated with the total content of three saponins (P < 0.05), and HMGR, P450-7, P450-12, and β-AS were significantly correlated with the monomer saponins a, c, d (P < 0.05). These four genes jointly regulated the synthesis of saikosaponin and had an important effect on the accumulation of saponin. Conclusion:The HMGR, β-AS, P450-7 and P450-12 genes in the saikosaponin synthesis pathway have a consistent distribution in saikosaponin synthesis and play an important role in the regulation of saponin synthesis.
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Objective: The fingerprint of Bupleurum chinense roots was developed with ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry(UPLC/Q-TOF-MS),the main chromatographic peaks were preliminary identified,and combined with principal component analysis(PCA) pattern recognition method to evaluate the quality of this herb from different origins. Method: The chromatographic separation was performed on a ZORBAX Eclipse Plus C18 column(2.1 mm×100 mm,1.8 μm) with a gradient elution of acetonitrile-0.1%formic acid aqueous solution.The mass spectrometer equipped with electrospray ionization(ESI) was used as detector and operated under the negative ion mode.Taking mass spectrometry data processing software of PeakView 1.2 and metabolomics analysis software of MarkerView 1.2.1,the different origins of B. chinense roots were analyzed by PCA. Result: The fingerprint of B. chinense roots was established within 35 min by UPLC/Q-TOF-MS.The samples from different origins were apparently classified by PCA.Eight compounds with significant differences were screened out,and the structures of three of them were identified as 3″-O-acetyl saikosaponin A,3″-O-acetyl saikosaponin D,6″-O-acetyl saikosaponin D. Conclusion: UPLC/Q-TOF-MS can be used for the rapid identification of fingerprint of B. chinense roots from different origins.IBM SPSS Statistics 21.0 and PCA can comprehensively distinguish the differences of chemical components in B. chinense roots from different origins and can be used to evaluate the quality of this herb.
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OBJECTIVE： To establish the method for simultaneous determination of saikosaponin a and saikosaponin d in Bupleurum chinense water extract， and to optimize its water extraction technology for electromagnetic cracking. METHODS： HPLC method was used. The determination was performed on SB-C18 column with mobile phase consisted of acetonitrile-water （gradient elution） at the flow rate of 1.0 mL/min. The column temperature was 40 ℃. The detection wavelength was set at 210 nm， and the sample size was 10 μL. Based on single factor experiment， using extraction time， particle size， solide-liquid ratio as factors， total extraction rate of saikosaponin a to saikosaponin d as indexes， the extraction technology was optimized by using Box-Behnken response surface methdology， and compared with the results of ultrasound method and decoction method. RESULTS： The linear range of saikosaponin a and saikosaponin d were 50.70-202.80 μg/mL （r＝0.999 9） and 50.50-202.00 μg/mL （r＝0.999 9）， respectively. The quantitation limits were 0.16 and 0.13 μg/mL， respectively. The detection limits were 0.05 and 0.04 μg/mL，respectively. RSDs of precision， stability and reproducibility tests were all lower than 2％. The average recoveries were 98.23-102.47％ （RSD＝1.80％， n＝6） and 98.84％-102.06％ （RSD＝1.60％， n＝6）. The optimal extraction technology was as follows： the extraction time of 2.50 min； the particle size of 80 mesh， solid-liquid ratio of 1 ∶ 28 （g/mL）. Results of 3 times of validation tests showed that the optimal technology included the average total extraction rates of saikosaponin a and saikosaponin d were 8.42 mg/g， which was higher than that of ultrasonic method （8.34 mg/g） and decoction method （8.06 mg/g）， and the extration time was shorter. CONCLUSIONS： Established method is simple and accurate， and can be used for simultaneous determination of saikosaponin a and saikosaponin d in B. chinense water extract. The optimized water extraction technology for electromagnetic cracking is stable and feasible.
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An HPLC method was established for the simultaneous determination of saikosaponin a, b2, c, d, e, f of Bupleurum chinense DC. in order to study the content difference of saikosaponins in different producing areas, different harvest time and different processed products of Bupleurum chinense DC. The Agela Venusil MP C18 (4.6 mm×250 mm, 5 μm) column was used with a gradient elution of acetonitrile-water at the wavelength of 210 and 254 nm with the flow rate of 1.0 mL·min-1 and the column temperature at 30℃. Based on the content of six kinds of saikosaponins, the differences of saikosaponins in four producing areas, eight harvest periods and 11 processing methods of Bupleurum chinense DC. were systematically studied. The results showed that the content of saikosaponins in Bupleurum chinense DC. was higher in May and August of Liaoning, Shaanxi and Gansu, but only in August from Shanxi in the four producing areas. The content of saikosaponins in 11 processed products was as follows:raw product > bran-stir-fried product > stir-fried product > wine-moistened product > turtle blood-stir-fried product > bran-wine-stir-fried product > wine-stir-fried product > vinegar-moistened product > turtle blood-wine-stir-fried product > vinegar-stir-fried product > honey-stir-fried product > honeymoistened product.
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Bupleuri Radix, originally appeared in Chinese materia medica (CMM) dating from the Han Dynasty, is a commonly used herb in clinical practice. According to ancient Chinese medical literatures, the morphological description of Bupleuri Radix was vague based on the species-rich and similar patterns, thus it exhibited significantly problems in species identification of Bupleuri Radix which applied in ancient Chinese medicine clinical practice. This paper summarizes important descriptions of morphology and efficacy of Bupleuri Radix from CMM in past dynasties. Research results from modern scholars about identification Bupleuri Radix are summarized to find out which species of Bupleuri Radix in clinic by traditional Chinese physicians. A kind of Bupleurum plants, having consistent efficacy, were used by traditional Chinese physicians before the Tang Dynasty, then Bupleurum scorzonerifolium., B. yinchowense, and Stellariae dichotoma var. lanceolata were used as Bupleuri Radix simultaneously. Stellariae Radix, its efficacy is different from the efficacy of Bupleuri Radix, was distinguished from Bupleuri Radix in the Ming and Qing Dynasties. With the deepening of research of taxonomy, “Bei Chai Hu” (corresponded to B. chinese DC.) and “Nan Chai Hu” (corresponded to B. scorzonerifolium Willd.) first appeared in the Ming Dynasty and the Republic of China era respectively.
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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 and 37 °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, and 300 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.
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Objective To research the effects of drought stress on activity of key enzyme in saikosaponin biosynthesis and saponins content in Bupleurum chinense; To reveal response of Bupleurum chinense to drought stress from protein level. Methods Bupleurum chinense was cultivated in potted water control experiment with 70%–80%, 60%–70%, 40%–50% and 20%–30% saturated soil water contents. The enzyme activities of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR), isoprenyl-based coke (IPPI), farnesyl pyrophosphate synthase (FPS) and β-carotenoid synthase (β-AS) of six months and one year old Bupleurum chinense were measured. The contents of saikosaponins a and d in samples of different soil water contents were determined by HPLC. Results When the saturated moisture was 40%–50%, the enzymatic activities of HMGR, IPPI, FPS and β-AS were the highest, followed by saturated moisture 60%–70%、70%–80% and 20%–30%. The trend of saikosaponin was similar to that of the activity of key enzyme. Conclusion 40%–50% soil saturated water content can significantly increase the activity of the key enzymes in the saikosaponin synthesis pathway. The saponin content and enzyme activity show a significant positive correlation, indicating that under drought stress, Bupleurum chinense regulates the synthesis of saikosaponin by regulating the key enzyme activity of saikosaponin synthesis pathway to respond drought stress.
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Objective:To research the best processing method for Bupleurum chinense DC.by orthogonal tests.Methods:With the contents of saikosaponin a and saikosaponin d as the indices,the L9(34) orthogonal table was used to study three factors including the amount of wheat bran,pot temperature before heating and processing time.The orthogonal design was applied to study the processing technology of Bupleurum chinense DC.fried with wheat bran.Results:The best processing method was as follows:100 g Bupleurum chinense DC.was mixed with 10 g wheat bran and fried at 290 ℃ for 80 seconds.Conclusion:The optimized processing technology is reasonable,reliable and highly reproducible,which provide reference for the processing of Bupleurum chinense DC.with wheat bran.
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Objective: To investigate the influencing factors of sulfated modification of Bupleurum chinense polysaccharides (BCP),and to elucidate the possible mechanism of improving the antioxidant ability of sulfated BCP (S-BCP).Methods:BCP was sulfated by chlorosulfonic acid-pyridine method.The degree of substitution (DS)of S-BCP was observed by adjusting the volume ratios of chlorosulfonic acid to pyridine (1:2,1:4,and 1:8).The structures of BCP and S-BCP were analyzed by infrared (IR)spectroscopy,the morphology of BCP and S-BCP were observed under scanning electron microscopy (SEM).The antioxidant model was established by using 1, 1-diphenyl-2-picrylhydrazyl (DPPH)free radical scavenging.The experiment was divided into positive control group,BCP group and S-BCP group,and the scavenging rates of DPPH free radical in various groups were compared. Results:When the volume ratio of chlorosulfonic acid to pyridine was 1 : 4,the reaction time was 2 h and the reaction temperature was 60 ℃,the maximum sulfur content percentage of S-BCP was 18.62% and the DS was the highest (DS = 2.32 ).Compared with BCP group, the scavenging rate of DPPH free radical of S-BCP was significantly increased (P <0.05).Conclusion:The volume ratio of chlorosulfonic acid to pyridine can affect the DS of S-BCP.The sulfated modification can increase the anti-oxidant capacity of BCP by changing its physic-chemical characters and spatial conformation.
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Objective To identify stable reference genes in different growth stages and different organs of Bupleurum chinese Methods All Ct values of 18 candidate internal reference genes were obtained by real-time quantitative PCR. Three software (Bestkeeper, NormFinder, and GeNorm) based on different algorithms were used to analyze the stability of the internal reference gene. The Pearson correlation coefficient was also analyzed. Results The Ct values of all candidate genes were relatively broad. ADF1b, ADF5, ADF7, eIF2b, and ACT2 were the most stable reference genes, whereas the gene of eIF6 was the least stable of the reference gene. The results of three softwares showed significant correlation. Conclusion Real-time fluorescence quantitative PCR combined with three different algorithms for the screening and validation of the reference gene of B. chinese is feasible. The homogenization of the target gene by the reference genes of the present study is helpful to improve the accuracy and reliability of gene expression analysis in molecular genetic research of B. chinese.
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Objective To identificate the development process and impact factors of lateral root in Bupleurum chinense. Methods The dynamic growth of lateral root primordia was observed by applying paraffin sectioning, and by light microscopy. In addition, the influence of growth regulators treatment on lateral root growth was also investigated. Results The lateral root primordium formation of B. chinense started near the pericycle cells, part of the endodermis cells also participated in this formation; 10 μmol/L IAA and Ca2+ greatly promoted the development of lateral root, but its high concentration inhibited lateral root emergence; 0.1 μmol/L TIBA and 10 μmol/L EDTA promoted the development of lateral root, but its high concentration inhibited lateral root emergence. Conclusion This research method can also be applied in other pharmaceutical plants, such as Angelica sinensis and Panax ginseng. So the study is very important to medicinal plant cultivation and breeding.
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Objective: To study the effects of different pretreatment methods and culture temperatures on Seed Germination and seedling growth of Bupleurum chinense, in order to provide scientific basis for artificial cultivation of B. chinense. Methods: Several physiological indexes such as the weight, pure rate, content of moisture, the rate of water absorption per thousand seeds were measured. Adopting a double-layer filter paper culture method, the pretreated seeds were cultured in the incubator at 20 ℃ in the 40% light. In different culture temperature treatment groups, the seeds were soaked in distilled water 24 h and then cultured in the corresponding temperature incubator. The germination energy, germination percentage, germination index, vigor index of seeds, and the root length and the shoot height of the seedling were recorded. Results: The pure rate of seeds was 91.51%, the weight of one thousand seeds was (2.83 ± 0.03) g and the weight of seed after absorbing water was about 2.24 times more than the weight of naturally dried seed. The content of moisture of seeds was 7.3%. Different hormones had different effects on seeds, among them the effect of 0.6 mg/L 6-BA treatment was the best. Water bath could increase the seedling vigor index, and 40 ℃ of its temperature significantly promoted the seed germination and seedling growth. The germination rate of seeds was the highest under the condition of 15 ℃, but the root length and seedling height were lower. Conclusion: The suitable condition for seed pretreatment and culture temperature of B. chinense established in this research can effectively improve the germination rate and seedling rate, which is of great significance for seed breeding and artificial cultivation of B. chinense.