Analysis on Quality of Sojae Semen Praeparatum Based on Traditional Quality Evaluation / 中国实验方剂学杂志

ObjectiveBased on the quality evaluation experience of "it is better to have a fragrant and strong aroma" summarized by materia medica of past dynasties， the chemical components of Sojae Semen Nigrum（SSN） and Sojae Semen Praeparatum（SSP） were systematically compared and analyzed， and the main fermentation products in different fermentation time were quantitatively analyzed， so as to clarify the transformation law of internal components in the processing process and provide scientific basis for the modern quality control of SSP. MethodUltra performance liquid chromatography-quadrupole tandem time-of-flight mass spectrometry（UPLC-Q-TOF-MS） was used for the structural identification of the chemical constituents of SSN and SSP， and with the aid of Progenesis QI v2.3 software， the negative ion mode was employed for principal component analysis（PCA） pattern recognition， and the data were analyzed with the aid of orthogonal partial least squares-discriminant analysis（OPLS-DA） for two-dimensional data to obtain S-plot， and components with |P|>0.1 were selected as the differential constituents. The contents of isoflavonoids in SSP during fermentation was determined by UPLC， and the samples were taken every 8 h in the pre-fermentation period and every 2 d in the post-fermentation period， and the dynamic changes of isoflavonoid contents in different fermentation stages were analyzed. The contents of amino acids and nucleosides in SSP and SSN from different fermentation stages were quantitatively analyzed by phenyl isothiocyanate（PITC） pre-column derivatization and high performance liquid chromatography（HPLC） gradient elution， and the contribution of flavor substances to the "delicious" taste of SSP was discussed by taste intensity value（TAV）. ResultA total of 19 kinds of differential components were screened out， mainly soybean saponins and isoflavones， and their contents decreased significantly or even disappeared after fermentation. In the pre-fermentation process of SSP， glycoside bond hydrolysis mainly occurred， and isoflavone glycosides in SSN were degraded and converted into the corresponding aglycones， the content of flavor substances such as amino acids increased gradually. In the post-fermentation process， protein degradation mainly occurred， after 8 d of post-fermentation， the content of isoflavones was basically stable， while the total content of amino acids increased by 8-40 times on average. Different amino acids form the special flavor of SSP， such as the TAV of glutamate is always ahead of other flavor substances， and sweet substances such as alanine and valine have made relatively great contributions to SSP. ConclusionBased on the law of constituent transformation， combined with the traditional evaluation index of "fragrant and strong"， it is difficult to control the fermentation degree of SSP by the existing standards in the 2020 edition of Chinese Pharmacopoeia. It is suggested that description of the characteristics of SSP be refined and changed to "fragrant， delicious and slightly sweet"， and at the same time， the post-fermentation index compounds such as glutamic acid， alanine and valine should be added as the quality control indicators of SSP， so as to standardize the production process and improve the quality of SSP.

Comparison of contents variation of six isoflavones in black beans, semifinished and finished Sojae Semen Praeparatum / 药学实践杂志

Objective To compare the contents variation of six flavonoids includingdaidzin，glycitin, genistin, daidzein, glycitein and genisteinin black beans, semifinished and finished Sojae Semen Praeparatum．Methods The contents of flavonoids were determined by HPLC，the condition were Diamonsil C18 column (4.6×250 mm, 5 μm) , column temperature 30 ℃, detection wavelength 260 nm, mobile phase 0.2% acetic acid water (A) - methanol (B), gradient elution, flow rate 1.0 ml/min．Results The linearity of this method to determine 6 isoflavones was good (r≥0.9993) within the determination range, and the recovery rate met the requirements. The RSD of precision, repeatability and stability experiment was less than 4%, 3%and 3%. The results of HPLC showed that the contents of six flavonoidsin Sojae Semen Praeparatum increased significantly compared with black beans. And, the contents of six flavonoids in finished Sojae Semen Praeparatum were slightly more than those in semifinished Sojae Semen Praeparatum. Conclusion The HPLC method established in this study could accurately determine the content of 6 isoflavones in Sojae Semen Praeparatum. The content of six isoflavones in black beans could be increased by the fermentation, and the combined isoflavones were transformed into free isoflavones during the fermentation process.

Isoflavones from Sojae Semen Praeparatum Regulate Lipid Metabolism in Atherosclerotic Mice Through PPARγ/LXRα/ABCA1 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo study the effect of isoflavones from Sojae Semen Praeparatum （ISSP） on lipid metabolism in atherosclerotic mice， and decipher the underlying mechanism via the peroxisome proliferator-activated receptor gamma/liver X receptor alpha/ATP-binding cassette transporter A1 （PPARγ/LXRα/ABCA1） signaling pathway. MethodFifty ApoE-/- mice were randomly assigned into the model group， western medicine （atorvastatin calcium， 3.03 mg·kg-1） group， and low-， medium-， and high-dose ISSP （2.5， 5， 10 mg·kg-1， respectively） groups， with 10 rats in each group. Atherosclerosis model mice were established by bilateral ovariectomy and feeding high-fat diet. Another 10 ApoE-/- mice receiving ovariectomy and high-fat diet were taken as the sham group. Some mice died of postoperative infection， and finally 6 mice were included in each group. One week after operation， each group was administrated with corresponding drugs or equivalent amount of normal saline. After 12 weeks， the levels of triglyceride （TG）， total cholesterol （TC）， high-density lipoprotein cholesterol （HDL-C）， low-density lipoprotein cholesterol （LDL-C）， and non-esterified fatty acids （NEFAs） in serum and liver tissue were measured. The levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） in serum were detected by enzyme-linked immunosorbent assay （ELISA）. Hematoxylin-eosin （HE） staining and oil red O staining were used for observation of aortic plaque formation and liver lipid deposition. The mRNA and protein levels of PPARγ， LXRα， ABCA1， and ATP-binding cassette transporter G1 （ABCG1） in liver were determined by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot. ResultCompared with the sham group， the modeling of atherosclerosis increased the aortic plaque area （P<0.01）， elevated the serum TC， TG， LDL-C， TNF-α， and IL-6 levels （P<0.01）， decreased the level of HDL-C （P<0.01）， increased the liver index （P<0.05） and the levels of TC， TG， and NEFAs in liver （P<0.01）， and caused obvious hepatic fat vacuoles and lipid deposition. In addition， the modeling down-regulated the mRNA levels of PPARγ， LXRα， ABCA1 in liver （P<0.05， P<0.01），and regulated the mRNA and protein levels of ABCG1（P<0.05， P<0.01）. Compared with the model group， atorvastatin calcium and middle-， high-dose ISSP reduced the serum TC， TG， LDL-C， TNF-α， and IL-6 levels （P<0.01）， decreased the liver index （P<0.01）， alleviated the liver fat vacuoles and lipid deposition， and increased the levels of TC， TG， and NEFAs in the liver （P<0.05， P<0.01）. Furthermore， they up-regulated the mRNA and protein levels of PPARγ， LXRα， ABCA1， and ABCG1 in the liver （P<0.05， P<0.01）. ConclusionISSP may regulate lipid metabolism through PPARγ/LXRα/ABCA1 signaling pathway to down-regulate the expression of inflammatory cytokines in serum and alleviate liver lipid deposition， thereby suppressing the formation of atherosclerotic plaque.

An Overview of Fermentation Technology Evolution and Process Control of Sojae Semen Praeparatum / 中国实验方剂学杂志

The historical evolution， fermentation technology and key links of Sojae Semen Praeparatum （SSP） were sorted out by consulting ancient books and modern literature， and the influencing factors and control methods of quality were analyzed and summarized in order to provide reference for the quality control of SSP. After analysis， it was found that in the fermentation process of SSP， fermentation strains， miscellaneous bacteria， temperature and humidity were all important factors affecting the quality of SSP. The condition control of "post fermentation" process has been paid more attention to in the past dynasties. In addition， the delicious SSP recognized in ancient times should be made from mold fermentation， and the breeding and application of fermented mold may be the key point to solve the quality problem of SSP. Therefore， based on the evaluation indexes of SSP in the past dynasties， it is of great significance to study and optimize the technological conditions such as strain， temperature and humidity in depth to improve the quality of SSP.

Literature-based analysis of conversion of components in fermentation process of Sojae Semen Praeparatum / 中国中药杂志

Based on the systematic retrieval and the reported components of Sojae Semen Nigrum and Sojae Semen Praeparatum, this study conducted in-depth analysis of conversion of components in the fermentation process, and discussed types and possible mec-hanisms of conversion of chemical components, so as to provide the basis for studying technology, medicinal ingredients and quality standards. According to the analysis, there is a certain degree of conversion of nutrients(like protein, sugar, lipid), bioactive substances(like isoflavones, saponins, γ-aminobutyric acid) and other substances(like nucleosides, melanoids, biamines, etc) in the process of fermentation.

Screening and identification of fibrinolytic enzyme-producing microbes in fermentation process of Sojae Semen Praeparatum / 中草药

Objective: To screen and identify the dominant strains which produce fibrinolytic enzyme during the processing of Sojae Semen Praeparatum (SSP, Dandouchi in Chinese). Methods: SSP was prepared according to the Chinese Pharmacopoeia (2020 edition), and samples were taken at different time points during the fermenting process of SSP.The casein plate method and fibrin plate method were used to screen the fibrinolytic enzyme-producing microorganisms in samples at different time points. The fibrinolytic enzyme-producing microorganisms were inoculated in the designated liquid medium to obtain single strain fermentation broth, and fibrin plate method was used to measure the fibrinolytic activity of the fermentation broth. The DNA sequences of fibrinolytic enzyme-producing bacteria and fungi were amplified using 16S rDNA and 18S rDNA universal primer by PCR respectively.The amplified products were sequenced, and the sequencing results were identified through NCBI homology comparison. Molecular biological identification was done by phylogenetic tree constructed by MEGA 4.1 software. Results: Three types of fibrinolytic enzyme-producing bacteria were screened out and identified in this study. They were Bacillus subtilis, Stenotrophomonas maltophilia and Micrococcus, respectively. The result of fibrin plate method showed that the fermentation broth of S. maltophilia had the highest fibrinolytic activity, reaching 527.49 IU/mL. Conclusion: There are fibrinolytic enzyme-producing dominant microorganisms existing in the fermenting process of SSP and the thrombolytic effect of SSP is worthy of further study. This study lays the foundation for revealing the formation mechanism of fibrinolytic enzyme in the fermentation process of SSP.

Preliminary analysis of primary and secondary factors of γ-aminobutyric acid enrichment in fermentation process of Sojae Semen Praeparatum / 中草药

Objective: To study the primary and secondary factors of γ-aminobutyric acid (GABA) enrichment in the processing of Sojae Semen Praeparatum (SSP), and lay the foundation for revealing the formation mechanism of high content of GABA in SSP. Methods: The dynamic changes of pH value, temperature, moisture, protease and glutamic acid decarboxylase (GAD) in the processing of SSP were determined by conventional methods. The GABA content of each sample was determined by pre-column on-line derivatization established by our laboratory. The correlation between each indicator and GABA was analyzed by SPSS software. Results: Correlation analysis and multiple regression analysis showed that the correlation coefficient between water, acid protease and GABA was 0.211 and -0.340, respectively, and the P values were 0.324 and 0.228, respectively. The correlation was small and there was no statistical difference. The absolute value of the regression coefficient showed that the primary and secondary status of other indicators in the formation of GABA was pH value (-0.375) > temperature (-0.284) > GAD (0.140) > alkaline protease (0.047) > neu-protease (-0.030), in which pH value, temperature and neutral protease had a negative correlation with GABA, and GAD activity and alkaline protease had a positive correlation with GABA. Conclusion: The temperature, pH value, GAD, neutral and alkaline protease are important factors affecting the formation of GABA in the fermentation process of SSP.

Antibacterial Activity and Chemical Constituents of Sojae Semen Praeparatum / 中国实验方剂学杂志

Objective:To study the antibacterial activity and chemical constituents of Sojae Semen Praeparatum as a traditional Chinese medicine. Method:Sojae Semen Praeparatum was extracted with 80% ethanol,and the extracts were suspended in an appropriate amount of distilled water and extracted with petroleum ether,ethyl acetate and water saturated n-butanol in turn. The antibacterial activity in vitro was used for screening the active fractions. Using activity-guided-isolation,the active fractions were isolated and purified by silica gel column chromatography and preparative HPLC and other methods. Their structures were identified by physicochemical properties and spectroscopic analysis, such as 1H-NMR,13C-NMR and MS. Result:The ethyl acetate fraction of 80% ethanol extract from Sojae Semen Praeparatum showed a stronger activity and was studied further. Eighteen compounds were isolated, and eleven compounds were identified as daidzein (1),daidzin (2),genistein (3),genistin (4),glycitein (5),glycitin (6),daucosterol (7),thymine (8),adenine (9),uracil (10),uridine (11). Conclusion:The ethyl acetate fraction of 80% ethanol extract from Sojae Semen Praeparatum shows an antibacterial activity. Compounds 1-6 are isoflavones. Compounds 7-11 are isolated from Sojae Semen Praeparatum for the first time.

Screening and identification of GABA-producing microbes in fermentation process of Sojae Semen Praeparatum / 中国中药杂志

A high-content GABA was found in Sojae Semen Praeparatum(SSP), which is a famous traditional Chinese medicine and officially listed in Chinese Pharmacopoeia. To screen out and identify GABA-producing microbes from samples at different time points during the fermenting process of SSP, traditional microbiological methods combined with molecular biological methods were used to study the predominant GABA-producing microorganisms existing in the fermenting process of SSP. This study would lay a foundation for further studying the processing mechanism of SSP. The fermenting process of SSP was based on Chinese Pharmacopoeia(2010 edition), and samples were taken at different time points during the fermenting process of SSP. The bacteria and fungi from samples at different time points in the fermenting process of SSP were cultured, isolated and purified by selective medium, and dominant strains were selected. The dominant bacteria were cultured in the designated liquid medium to prepare the fermentation broths, and GABA in the fermentation broth was qualitatively screened out by thin-layer chromatography. The microbial fermentation broth with GABA spots in the primary screening was quantitatively detected by online pre-column derivatization and high performance liquid chromatography established in our laboratory. GABA-producing microorganisms were screened out from predominant strains, and their GABA contents in fermentation broth were determined. The DNA sequences of GABA-producing bacteria and fungi were amplified using 16S rDNA and 18S rDNA sequences by PCR respectively. The amplified products were sequenced, and the sequencing results were identified through NCBI homology comparison. Molecular biological identification was made by phylogenetic tree constructed by MEGA 7.0 software. Through the homology comparison of NCBI and the construction of phylogenetic tree by MEGA 7.0 software, nine GABA-producing microorganisms were screened out and identified in this study. They were Bacillus subtilis, Enterococcus faecium, E. avium, Aspergillus tamarii, A. flavus, A. niger, Cladosporium tenuissimum, Penicillium citrinum and Phanerochaete sordida respectively. For the first time, nine GABA-producing microorganisms were screened out and identified in the samples at different time points during the fermenting process of SSP in this study. The results indicated that multiple predominant GABA-producing microorganisms exist in the fermenting process of SSP and may play an important role in the formation of GABA.

History of processing of Sojae Semen Praeparatum / 中国中药杂志

Sojae Semen Praeparatum (SSP) is commonly used as a type of dietetic Chinese herb. By collecting and analyzing ancient and recent literatures, a textual criticism was conducted on the historical evolution of the processing of SSP. Fermented soybean was recorded in Shijing, and relevant rational processing was described in Qimin Yaoshu. In the early time, fermented soybean included the type of "salty" and "light". After the Ming Dynasty, "light" fermented soybean or SSP was recognized as a better medicinal matter than salty fermented soybean, and the fermentation processing was recorded more clearly. In modern time, many characteristic methods for processing SSP have been developed. Today, the processing of SSP is mainly based on the Chinese Pharmacopoeia, which records soybean as a main ingredient and Artemisiae Annuae Herba, Mori Folium as excipients.

Study on dynamic changing regularity of microflora in fermentation process of Sojae Semen Praeparatum by PCR-DGGE / 中草药

Objective: To reveal the dynamic changing regularity of microflora in the fermentation process of Sojae Semen Praeparatum (SSP) and lay the foundation for revealing the mechanism of SSP processing by denaturing gradient gel electrophoresis (DGGE). Methods: The dynamic changes of microflora, both bacteria and fungi in fermentation process were monitored by PCR-denaturing gradient gel electrophoresis. According to the unweighted pair group method using arithmetic average clustering, the samples of SSP in various stages were analyzed. Results: Bacterial flora had diversity, and Aspergillus was the major fungus in the first stage called "yellow cladding". The major bacteria was Lactobacillus, while the major fungus was Cryptococcus at the "secondary fermentation" stage. The major microorganism was Bacillus subtillis and Pseudomonas putida on day 1, and Stenotrophomonas maltophilia, Sphingobacterium sp, and A. oryzae on day 3. Then on day 6, B. amyloliquefaciens, Aspergillus, and Trichosporon ovoides became the primary microorganisms. B. subtillis, T. ovoides, and A. niger were the major microorganism on day 3 of "secondary fermentation". On day 9 of this stage, the major strains were B. subtilis, L. concavus, L. nasuensis, and Cryptococcus randhawi. On day 15 of "secondary fermentation", they were B. subtilis, L. concavus, C. randhawi, Trichosporon, and two fungi cannot be cultured. Klebsiella oxytoca, B. subtilis, and L. concavus were dominant strains in the whole fermentation process. The composition of microflora in "yellow cladding" stage was different to that of the "secondary fermentation". The microbial community on day 3 and 6 was similar to 76.4%. While the lowest similarity between the samples on day 3 and 9, it was similar to 24.5% during samples on day 6 and 9 in "secondary fermentation" stage. The highest similarity of fungal composition was between day 3 and 6 samples, and the lowest one was between day 3 and 15 of "secondary fermentation", which was similar to 11.2% only. Conclusion: The results show that the unique flavor and function of SSP may be determined by the dynamic microbial communities and microbial flora in the fermentation process, and the secondary fermentation is proved to be irreplaceable from the microbiological point of view.

Screening and Enzymatic Activity Analysis of Dominant Fermentive Bacteria of Sojae Semen Praeparatum from Different Production Places / 中国药房

OBJECTIVE:To provide reference for the standardized production of Sojae semen praeparatum (SSP). METH-ODS:SSP samples from Heilongjiang,Hebei,Gansu,Shandong,Anhui and Yunnan were respectively collected. The fermentive bacteria were cultured with the selective medium contained artemisiae annuae herba and mori folium. Foline-phenol method,fibrous protein plate method and p-nitrophenol-β-D-glucoside colorimetric method were respectively conducted to determine the activities of protease,plasmin and β-glucosidase of the strains to screen dominant fermentive bacteria. RESULTS:Totally 14 wild strains were separated from SSP samples from 6 production places, including 3 strains of bacteria and 11 strains of molds. 1 strain of rod-shaped bacteria and 1 strain of Mucor sp. were separated from SSP from Heilongjiang;2 strains of Mucor sp. and 1 strain of rod-shaped bacteria were separated from SSP from Hebei;1 strain of Mucor sp.,1 strain of Penicillium sp.,1 strain of Streptococ-cus sp. and 1 strain of Aspergillus sp. were separated from SSP from Gansu;2 strains of Mucor sp. were separated from SSP from Shandong;1 strain of Mucor sp. and 1 strain of Aspergillus sp. were separated from SSP from Anhui;and only 1 strain of Mucor sp. was separated from SSP from Yunnan. According to the strains category and enzyme activities,No.1 bacillus,No.9 Aspergillus sp.,No.11 and No.14 Mucor sp. were preliminary authenticated as dominant fermentation microorganism,total enzyme activities of the 4 strains were 22.77,25.49,41.32,39.13 U/g respectively. CONCLUSIONS:The fermentive bacteria of SSP from different pro-duction places were different,and the dominant one can be screened preliminary through enzyme activity analysis.