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Introduction@#<i>Sophora alopecuroides L</i> has broadly been utilized in traditional medicine and all crude drugs including root, herb, and seed are used to treat numerous diseases. This herb is included in 181 Tibetan-Mongolian medicinal prescriptions and ranks 8<sup>th</sup> among Mongolian medicinal plants in terms of frequency of administration. The <i>S.alopecuroidesL . </i> root standard was developed by the Institute of Traditional Medicine and Technology in 2017 and approved by “ҮФӨ-0307-2017”. Herb and seed are still used in medicine. Therefore, their standard parameters need to be determined and verified.@*Materials and methods@#The quantitative pharmacognosy analysis of herb and seed was carried out in accordance with the methodology specified in the “General requirements for medicinal plant raw materials” of the National Pharmacopoeia of Mongolia. To determine the total alkaloid in standard matrine, a bromcresol green complex was formed, which was measured by spectrophotometer.@*Conclusion@#By developing, standards for the crude drugs of herb and seed of <i>S.alopecuroides L. </i> which are included in numerous medicinal prescriptions, will confirm the rationale for the use of medicinal raw materials and to expand the utilization’s possibilities.
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Objective: To establish the chemical fingerprint of Sophora alopecuroides extracts based on high performance liquid chromatography (HPLC), and determine the LD50 of different extracts of S. alopecuroides to analyze its “spectrum toxicity” relationship. Methods: A series of extracts were prepared by 75% ethanol reflux (ER), water decoction (WD), 75% ethanol ultrasound (EU) and water ultrasound (WU), and their fingerprints were established to determine the acute toxicity LD50 of different extracts. The relationship between chemical composition and acute toxicity LD50 of S. alopecuroides extracts were studied by means of fingerprint similarity evaluation system. Results: The LD50 of ER, WD, EU, and WU extracts were 38.397, 24.994, 18.536, and 19.957 g/kg, respectively. The ocular lesions of mice viscera were mainly manifested in liver and kidney, and the toxicity of ER extracts was the greatest. The 10 common peaks of S. alopecuroides extracts can be divided into two categories; Peaks 4 and 10, oxymatrine and sophocarpidine were negatively correlated with acute toxicity LD50. Conclusion: The spectral toxicity relationship analysis method of S. alopecuroides was constructed. The unidentified peaks 4, 10 and oxymatrine and sophocarpidine were the main chemical components of the toxicity reaction, which laid a good foundation for clinical application and scientific and rational development of S. alopecuroides.
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Objective: In this study, flow cytometry and K-mer analysis were used to estimate genome size and hybridity percentage of Sophora alopecuroides, so as to provide reference for its genome sequencing. Methods: Glycine max served as an internal reference, the multiple relationship between DNA content in Glycine max and S. alopecuroides cells was detected by flow cytometry, and the genome size of S. alopecuroides was calculated. Genome sequencing of S. alopecuroides was carried out using NGS technology, genome size and hybridity percentage of S. alopec uroides were estimated by K-mer analysis. Results: The genome size of S. alopecuroides was about 1 749 Mb measured by flow cytometry. K-mer analysis showed that the genome size of S. alopecuroides was about 1 648 Mb, the hybridity percentage was 1.12%, and the percentage of repetitive sequence was high. Conclusion: The genome size of S. alopecuroides was about 1.7 Gb with high hybridity percentage. The technology of combining Illumina and PacBio is recommended for future genome sequencing.
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Objective To establish a method of detecting the expression of Lysine decarboxylase (LDC) -a key enzyme for the synthesis of alkaloid in the host promoted by the endophytic fungal elicitor of Sophora alopecuroides by using real-time fluorescence quantitative PCR (qRT-PCR). Methods Target gene primers QLDC-F/QLDC-R and reference gene primers Lectin-F/Lectin-R were designed according to LDC and Lectin gene sequences of S. alopecuroids; Five-fold gradient dilution of cDNA was used as the standard sample for the construction of the standard curve of target gene and the reference gene. Reaction system and reaction conditions of qRT-PCR were optimized, and the sensitivity of semi-quantitative PCR and qRT-PCR were analyzed and compared. Under different eliciting time of endophytic fungal elicitors NDZKDF13 of S. alopecuroides, the content of oxymatrine in the host was determined by HPLC, the expression of LDC gene was detected by qRT-PCR, and the relationship between LDC gene expression and the accumulation of OMA was analyzed. Results The results of qRT-PCR were better when the cDNA content in the system was 200 ng/μL and the annealing temperature was 61 ℃. The standard curve of the target gene and the reference gene was constructed, in which the cycle threshold and template concentration showed a good linear relationship, the amplification efficiency was above 99%, and the sensitivity was 25 times that of semi-quantitative PCR. Under the induction effect of endophytic fungal elicitor NDZKDF13, expression of host LDC gene reached the peak on the 6th day, which was 25.58 times that of the control. The increase of OMA content lagged the change of the LDC gene expression and reached the highest amount on the 9th day after the induction. Conclusion The qRT-PCR technique was successfully applied to the functional gene research of S. alopecuroides. Through the optimization of various conditions, a platform for accurate and simple detection of functional gene expression in S. alopecuroides was established.
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Objective To evaluate the effect of total alkaloids of Sophora alopecuroides L on the nervous behavior and the expression of neurotransmitters in rats. Methods 48 male SD rats were randomly divided into 4 groups:blank group,total alkaloids of Sophora alopecuroides L treatment with 4 mg·kg-1 ·d-1 ( low dose group) ,8 mg·kg-1 ·d-1( medium dose group) and 16 mg·kg-1 ·d-1( high dose group) groups. After successive intragastric administration for 30 days,the locomotor activity was applied to test the nervous behavior and emotional state of rats in each group. After behavioral tests were finished,the contents of trypto-phan (Trp),5-hydroxytryptophan (5-HTP),5-serotonin (5-HT),5-hydroxyindoleacetic acid (5-HIAA), norepinephrine (NE),epinephrine (E) and dopamine (DA) were detected by ELISA in serum and brain. Results In the experiment of locomotor activity,compared with blank group ((95.33±12.75) times),the numbers of horizontal movement of Sophora alopecuroides L in medium and high dose group ( ( 61. 64 ± 5.91),(64.62±5.79)times both P0.05). Correlation analysis indicated that the degree of au-tonomic activity in rats with the content of 5-HT,5-HIAA and DA in serum was negatively correlated (P<0.05, P<0.01) ,the degree of emotional stress and the content of 5-HT,5-HIAA in brain was negatively cor-related (P<0.05, P<0.01) . Conclusion The total alkaloids of Sophora alopecuroides L can reduce the ac-tivity of rats and increase the degree of emotional stress. And the mechanism may be correlated with the in-creasing level of 5-HT and 5-HIAA in serum and brain.
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Objective: To study the structure of Sophora alopecuroides polysaccarides (SAP) and its antitumor activity on CT26 transplanted tumor in mice. Methods: The structure was characterized by high-performance gel permeation chromatography (HPGPC), gas chromatography-mass spectrometer (GC-MS), infrared spectroscopy (IR), and nuclear magnetic resonance spectroscopy (NMR). And then the anticancer activity of CT26-bearing mice was investigated in vivo. Results: The results showed that SAP was consisted of D-mannopyranose and D-galactopyranose residues. The main chain of the galactomannan comprises β-(1,4)-linked D-mannopyranose residues, with branches of galactose, linked to the carbohydrate core through α-D-Gal (1,6) linkage. SAP could inhibit the tumor proliferation of CT26-bearing mice in a dose-dependent manner with inhibitory rate of 44.03% at high dose. Conclusion: Structure determination of SAP provides the theoretical basis for structure-activity relationship study. And it is expected to be further developed as a new antitumor drug with high efficiency and low toxicity.
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Objective:To study the quality standard for Fujie lotion. Methods:The qualitative identification of Sophora alopecu-roides L, Cnidium monnieri ( L. ) Cuss and borneol was detected by TLC. The quantitative determination of matrine was detected by HPLC. Results:The identification by TLC was highly specific, and the content determination method was accurate and repeatable. The linear range of matrine was 0. 013 0-1. 30 mg·ml-1, and the average recovery was 97. 1% with RSD of 1. 7%(n=6). Conclu-sion:The standard can effectively control the quality of Fujie lotion.
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OBJECTIVE: To study the effect of lysine decarboxylase (LDC) gene on the accumulation of matrine (MA) and oxymatrine (OMA) in cotyledon of Sophora alopecuroides L germinating seeds. METHODS: The S. alopecuroides germinating seeds were stressed by different mass fractions of PEG 6000, and the contents of MA and OMA were determined by high performance liquid chromatography (HPLC) and the expression level of LDC was analyzed by real-time fluorescence quantitative PCR (qPCR) after 72 h treatment. RESULTS: The contents of MA and OMA decreased in the cotyledon under light stress (PEG mass fraction 20%). The analysis of qPCR revealed that the LDC expression level was decreased first, and then increased with the stress rising. The changes of the contents of MA and OMA were parallel with the expression level of LDC especially under light and severe stress. CONCLUSION: There is certain association between the accumulation of MA and OMA and the gene expression quantity of LDC. The results is of significance for illustrating the role of LDC in the biosynthetic pathways of MA and OMA.
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OBJECTIVE: To establish a method for simultaneous determination of six alkaloid components of Sophora alopecuroides L, including cytisine, matrine, oxymatrine, sophocarpine, oxysophocarpine, and sophoridine by HPLC and determine the contents of the six alkaloids in different parts of Sophora alopecuroides L from four different areas.
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Objective: To analyze the genetic polymorphism and relationship of Sophora alopecuroides from Ningxia, Gansu, Qinghai, Xinjiang, and Inner Mongolian in China, and to offer some information for the domestication and protection of wild S. alopecuroides germplasms. Methods: ISSR primers were used to analyze the genetic diversity of 22 populations of S. alopecuroides. Cluster analysis of the unweighted pair-group method with arithmetic average (UPGMA) and principal component analysis (PCA) were carried out based on the molecular data obtained, and a dendrogram was constructed. Results: Totally 433 alleles were amplified using the 51 ISSR primers. The number of allels in per primer range was 5-12, with an average of 8.49. The average percentage of polymorphic loci (PPB) was 93.30%. The Nei's gene diversity (H) and Shannon's information index (I) were 0.3351 and 0.4998, respectively. The genetic distances range was 0.1736-0.6502. Conclusion: The genetic polymorphism among 22 populations of S. alopecuroide is relatively abundant and there are no direct relationship between genetic distance and geographical distribution in them.
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OBJECTIVE: To assess the allozymic variation and genetic diversity of Sophora alopecuroides populations at biochemical level. METHODS: Vertical slab polyacrylamide gel electrophoresis was adopted to study the genetic difference of 720 samples from 24 populations in Ningxia, Gansu, Qinghai, Xinjiang and Inner Mongolia. Data was analyzed by POPGEN 32, and a cluster diagram was presented by UPGMA. RESULTS: A total of 26 loci in 5 enzyme systems (esterase, EST; malic enzyme, ME; malate dehydrogenase, MDH; peroxidase, PER; shikimate dehydrogenase, SKD) were detected and a high level of genetic diversity was revealed at population level. The percentage of polymorphic loci (P) was 73.08%; the mean expected heterozygosity (He) was 0.337; Shannon's index (I) was 0.517; the coefficient of gene differentiation (FST) among 24 populations was 0.427, and the mean gene flow (Nm) was 0.687, respectively. The genetic identity among the populations ranged from 0.5815 to 0.9261 with the average of 0.7690. The genetic differentiation was found among the populations based on the clustering analysis. CONCLUSION: There is a high level of genetic diversity among different populations, which mainly comes from genetic differentiation. No obvious relationship between the genetic distance and geographical distribution of the 24 populations is identified.
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Objective: To study the influence of drought stress on imbibition germination of Sophora alopecuroides and the drought resistance of its seedling. Methods: To measure the imbibition rate, germination rate, germination index, seedling growth, and relative tissue moisture content under the drought stress by PEP-6000. Results: With the increased level of stress, the imbibition rate, germination rate, germination index, vigor index, seedling hight, root length, and the relative tissue moisture content tended to decrease, while the seedling dry weight, root dry weight, and root-shoot ratio presented rising tendency then followed by the decrease. Conclusion: The appropriate drought stress could improve the drought resistance of seedling, which is beneficial to cultivate strong seedling.
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To study the alkaloid constituents in the seed of Sophora alopecuroides L. and isolation of lehmannine by transhydrogenation of sophocarbine to matrine present in the mixture. Methods Various processes of exchange, ion exchange and column chromatograph with very similar physical and chemical properties was successfully separated by transhydrogenation. Results 5 alkaloids were obtained and identified by elementary analysis, IR, MS, 1HNMR and 13CNMR as oxymatrine, oxysophoridine, matrine, sophoridine and lehmannine (12, 13-dehydromatrine). Conclusion Lehmannine was isolated from the seed of S. alopecuroides for the first time.
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Object To study the alkaloid constituents in the seed of Sophora alopecuroides L and isolation of lehmannine by transhydrogenation of sophocarbine to matrine present in the mixture Methods Various processes of exchange, ion exchange and column chromatograph with very similar physical and chemical properties was successfully separated by transhydrogenation Results 5 alkaloids were obtained and identified by elementary analysis, IR, MS, 1HNMR and 13 CNMR as oxymatrine, oxysophoridine, matrine, sophoridine and lehmannine (12, 13 dehydromatrine) Conclusion Lehmannine was isolated from the seed of S alopecuroides for the first time