| Objective:Currently,the presence of ligustrazine in traditional Chinese medicine Chuanxiong Rhizoma is a controversial issue.This study aimed to preliminarily explore the origin of ligustrazine in Ligusticum chuanxiong Hort.based on the endopHytic bacteria Bacillus subtilis metabolite pathway in this medicinal plants,and to study the basis of pHarmacodynamic substances on promoting the blood circulation of Chuanxiong Rhizoma.Methods:?1?UPLC-PDA/UPLC-Q-TOF-MS were used to simultaneously analyze the six components of Ferulic acid,Ligustrazine,Senkyunolide H,Senkyunolide I,Senkyunolide A and Z-Ligustilide qualitatively and quantitatively.The mobile pHase is composed of acetonitrile?A?-0.1%formic acid water?B?,and the column is Acquity UPLC BEH C18 column?2.1 mm*100 mm,1.7?m?;?2?Isolation and identification of endopHytic Bacillus subtilis in Chuanxiong Rhizoma.The fresh rhizome of Ligusticum chuanxiong Hort.was collected from the planting base in Pengzhou City of Sichuan Province,and it was used as the source of separation.The endopHytic bacteria were separated by tissue grinding method,suspected of Bacillus subtilis to a single colony,the endopHytic bacteria suspected of Bacillus subtilis were purified to single colonies using a plate scribing method.The traditional identification of bacteria was carried out using pHysiological and biochemical experiments unique to Bacillus subtilis,the strains selected were further identified by 16S rRNA technology;?3?Study on the nutritional fermentation characteristics of endopHytic Bacillus subtilis.Continuous culture for 144 h with YPG medium,the shaking speed was 220 rpm and constant temperature was 37°C.The fermentation liquid was sampled once every 12 h in the ultra-clean workbench,the fermentation products were detected by GC/GC-MS to explore the ability of endopHytic Bacillus subtilis to produce ligustrazine;the residual sugar content was measured by DNS colorimetry method,the viable cells count were measured by plate counting method,the pH of the fermentation liquid was measured by electronic PH meter.Then,the grapHs were made to characterize the nutrient fermentation characteristics of endopHytes.?4?The study on Chuanxiong Rhizoma medicinal substrate medium in solid state and liquid state fermentation experiment.Chuanxiong Rhizoma power was pulverized to pass through 54 mesh sifter for the preparation of solid state and liquid state medium.The Chuanxiong Rhizoma solid medium was prepared according to the ratio of 29%Chuanxiong Rhizoma power and 71%steriled water,the sterilization conditions were 121°C moist heat sterilization for 30 min.Diammonium hydrogen pHospHate is divided into Chuanxiong Rhizoma-Diammonium hydrogen pHospHate?CRD?and non-diammonium pHospHate group?Chuanxiong Rhizoma,CR?for liquid state fermentation experiment.The formula of CRD medium is 60 g Chuanxiong powder,1000 mL sterile water,2 g K2HPO4,2 g KH2PO4,10 g?NH4?2HPO4,pH 7.0-7.2.The CR group was same as the CRD group except for?NH4?2HPO4.The endopHytic Bacillus subtilis was inoculated into the solid state and liquid state medium.The blank group was not inoculated with endopHytic Bacillus subtilis.The solid-state fermentation conditions were cultured in a 37°C incubator for 30 days,and the liquid fermentation conditions were 220 rpm on a shaker at 37°C for 144 h.After two kinds of fermentations were completed,UPLC/UPLC-Q-TOF-MS was used to determine the metabolites of it.?5?Research on the pHarmacodynamics of Chuanxiong Rhizoma for the effect of activating blood circulation to dissipate blood stasis.The acute blood stasis model of SD rats was established by adrenaline combined with ice water bath.We had set up 5groups named according to the drug administered by gavage,the ferulic acid group,the ligustrazine group,the Chuanxiong Rhizoma volatile oil group,the Chuanxiong Rhizoma alcohol extract,positive drug group,model group and the blank group were given for once a day for 7 days.The bioelectrical impedance of each group of rats was measured by ImpediVET BIS1,the indicators include total body water,extracellular fluid,intracellular fluid,fat mass,free fat mass,body mass index.Blood samples were taken 30 min after the last administration,and routine blood test and the blood rheology and coagulation system indexes were detected.Results:?1?Ligustrazine was not detected in detect in Chuanxiong Rhizoma and the fresh Chuanxiong Rhizoma wth the detection limit of ligustrazine in this experiment was 0.1 ng·g-1.The Ferulic acid,Senkyunolide H,Senkyunolide I,Senkyunolide A,and Z-ligustilide can be detected both in fresh Chuanxiong Rhizoma and Chuanxiong Rhizoma.The content of each component in Chuanxiong Rhizoma is ferulic acid.0.8770mg·g-1,Senkyunolide H 0.3144 mg·g-1,Senkyunolide I 2.028 mg·g-1,Senkyunolide A7.662 mg·g-1,Z-ligustilide 12.88 mg·g-1;the content of each component in the fresh Chuanxiong Rhizoma is ferulic acid 0.2785 mg·g-1,chuanchuan azide A 0.4512 mg·g-1,ligustilide 30.89 mg·g-1.?2?Five stranis of endopHytic Bacillus subtilis were isolated from fresh Chuanxiong Rhizoma,which were named as LB3,LB3-2-1,LB4,LB5,LB6-2,respectively.The endopHytic bacteria of five strains were positive for Gram staining and had spores.The methyl red reaction test was negative and the V-P reaction was positive.After 16S rRNA alignment,99.99%homology was found in Bacillus subtilis.The strains are preserved in the Guangdong Provincial Institute of Microbiology and the China Industrial Microbial Culture Collection Management Center.?3?GC/GC-MS results of nutrient fermentation showed that all the five strains of endopHytic Bacillus subtilis had the ability to produce ligustrazine.The production of each strains was LB3 2.091 g·L-1,LB3-2-1 1.092 g·L-1,LB4 0.8034 g·L-1,LB5 0.7446g·L-1,LB6-2 1.773 g·L-1.The highest yield was produced by LB5 with a production of10.6945 g·L-1.?4?UPLC/UPLC-Q-TOF-MS was used to detect the metabolites of the solid-state fermentation medium of Chuanxiong Rhizoma matrix.In addition to the blank group,five kinds of endopHytic Bacillus subtilis groups were produced with ligustrazine(LB3235.2?g·g-1,LB3-2-1 188.2?g·g-1,LB4 259.0?g·g-1,LB5 909.3?g·g-1,LB6-2 215.8?g·g-1).Except for the blank group,acetoin,the biosynthesis precursor,was generated in each group.(LB3 70.18?g·g-1;LB3-2-1 61.29?g·g-1;LB4 60.70?g·g-1;LB5 262.8?g·g-1;LB6-2 67.21?g·g-1);UPLC/UPLC-Q-TOF-MS was used to detect the metabolites of the liquid fermentation medium of Chuanxiong Rhizoma matrix.The acetoin was detected in the CR medium,but no tetramethylpyrazine was detected.In the CRD medium,except for the blank group,ligustrazine and acetoin were both generated in each endopHyte group.The yield of acetoin in the CR group was LB3 9.690 mg·mL-1,LB3-2-1 14.81 mg·mL-1,LB4 14.88 mg·mL-1,LB5 15.36 mg·mL-1,LB6-2 14.63mg·mL-1,respectively;the yield of acetoin in the CRD group was LB3 151.18 mg·mL-1,LB3-2-1 127.8 mg·mL-1,LB4 168.73 mg·mL-1,LB5 142.64 mg·mL-1,LB6-2 123.4mg·mL-1,respectively.The yield of ligustrazine in the CRD group was LB3 0.2054mg·mL-1,LB3-2-1 0.2869 mg·mL-1,LB4 0.4814 mg·mL-1,LB5 1.027 mg·mL-1,LB6-20.3427 mg·mL-1,respectively.?5?Compared with the normal group,the whole blood viscosity and plasma viscosity of the model group increased significantly,and the erythrocyte sedimentation index and erythrocyte deformation index increased significantly?p<0.01?.Fibrinogen increased significantly?p<0.01?,prothrombin time and activated partial thromboplastin time were significantly shortened?p<0.01?.Compared with the model group,the positive group,the Chuanxiong Rhizoma volatile oil group,the Chuanxiong Rhizoma group,and the ligustrazine group can significantly reduce the blood rheology indexes?p<0.05?,significantly prolong the prothrombin time and activated partial thromboplastin time?p<0.05?,and significantly reduce the fibrinogen content.The improvement effect of the ferulic acid group on each index was not obvious.To a certain extent,this suggests that the volatile oil of Chuanxiong Rhizoma and ligustrazine can significantly improve the hemorheology and coagulation abnormality of blood stasis rats;the ferulic acid group has no obvious effect,which may be due to the intrahepatic administration.ConclusionsThe content of ligustrazine in Chuanxiong Rhizoma is low,and it is not suitable as a quality control component of Chuanxiong Rhizoma.Under nutrient fermentation conditions,Bacillus subtilis can produce a large amount of ligustrazine,which confirm the endopHyte of Chuanxiong Rhizoma has high ability to produce ligustrazine.The endopHytic Bacillus subtilis could metabolize the Chuanxiong Rhizoma in vitro as the sole carbon source and nitrogen source,this is an indication that ligustrazine may derived from the interaction between endopHytic Bacillus subtilis and Ligusticum chuanxiong Hort.The volatile oil?lactones?in Chuanxiong are the pHarmacodynamic basis of Chuanxiong to promote blood circulation and remove blood stasis. |
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