Advances in research methods for biosynthetic pathway analysis of active ingredients in traditional Chinese medicine / 中国中药杂志

The active ingredients in traditional Chinese medicine(TCM)are the foundation for the efficiency of TCM and the key to the formation of Dao-di herbs. It is of great significance to study the biosynthesis and regulation mechanisms of these active ingredients for analyzing the formation mechanism of Daodi herbs and providing components for the production of active ingredients in TCM by synthetic biology. With the advancements in omics technology, molecular biology, synthetic biology, artificial intelligence, etc., the analysis of biosynthetic pathways for active ingredients in TCM is rapidly progressing. New methods and technologies have promoted the analysis of the synthetic pathways of active ingredients in TCM and have also made this area a hot topic in molecular pharmacognosy. Many researchers have made significant progress in analyzing the biosynthetic pathways of active ingredients in TCM such as Panax ginseng, Salvia miltiorrhiza, Glycyrrhiza uralensis, and Tripterygium wilfordii. This paper systematically reviewed current research me-thods for analyzing the biosynthetic functional genes of active ingredients in TCM, elaborated the mining of gene elements based on multiomics technology and the verification of gene functions in plants in vitro and in vivo with candidate genes as objects. Additionally, the paper summarized new technologies and methods that have emerged in recent years, such as high-throughput screening, molecular probes, genome-wide association studies, cell-free systems, and computer simulation screening to provide a comprehensive reference for the analysis of the biosynthetic pathways of active ingredients in TCM.

Modification of C20 oxidase in tanshinone biosynthesis pathway / 中国中药杂志

Tanshinones are one of the main effective components of Salvia miltiorrhiza, which play important roles in the treatment of cardiovascular diseases. Microbial heterogony production of tanshinones can provide a large number of raw materials for the production of traditional Chinese medicine(TCM) preparations containing S. miltiorrhiza, reduce the extraction cost, and relieve the pressure of clinical medication. The biosynthetic pathway of tanshinones contains multiple P450 enzymes, and the catalytic element with high efficiency is the basis of microbial production of tanshinones. In this study, the protein modification of CYP76AK1, a key P450-C20 hydroxylase in tanshinone pathway, was researched. The protein modeling methods SWISS-MODEL, Robetta, and AlphaFold2 were used, and the protein model was analyzed to obtain the reliable protein structure. The semi-rational design of mutant protein was carried out by molecular docking and homologous alignment. The key amino acid sites affecting the oxidation activity of CYP76AK1 were identified by molecular docking. The function of the obtained mutations was studied with yeast expression system, and the CYP76AK1 mutations with continuous oxidation function to 11-hydroxysugiol were obtained. Four key amino acid sites that affected the oxidation acti-vity were analyzed, and the reliability of three protein modeling methods was analyzed according to the mutation results. The effective protein modification sites of CYP76AK1 were reported for the first time in this study, which provides a catalytic element for different oxidation activities at C20 site for the study of the synthetic biology of tanshinones and lays a foundation for the analysis of the conti-nuous oxidation mechanism of P450-C20 modification.

Difference in liver injury induced by dictamnine between males and females: based on untargeted metabolomics / 中国中药杂志

In recent years, reports of adverse reactions related to traditional Chinese medicine(TCM) have been on the rise, especially some traditionally considered "non-toxic" TCM(such as Dictamni Cortex). This has aroused the concern of scholars. This study aims to explore the metabolomic mechanism underlying the difference in liver injury induced by dictamnine between males and females through the experiment on 4-week-old mice. The results showed that the serum biochemical indexes of liver function and organ coefficients were significantly increased by dictamnine(P<0.05), and hepatic alveolar steatosis was mainly observed in female mice. However, no histopathological changes were observed in the male mice. Furthermore, a total of 48 differential metabolites(such as tryptophan, corticosterone, and indole) related to the difference in liver injury between males and females were screened out by untargeted metabolomics and multivariate statistical analysis. According to the receiver operating characteristic(ROC) curve, 14 metabolites were highly correlated with the difference. Finally, pathway enrichment analysis indicated that disorders of metabolic pathways, such as tryptophan metabolism, steroid hormone biosynthesis, and ferroptosis(linoleic acid metabolism and arachidonic acid metabolism), may be the potential mechanism of the difference. Liver injury induced by dictamnine is significantly different between males and females, which may be caused by the disorders of tryptophan metabolism, steroid hormone biosynthesis, and ferroptosis pathways.

Shugan Jieyu capsule improve sleep and emotional disorder in coronavirus disease 2019 convalescence patients: a randomized, double-blind, placebo-controlled trial.

OBJECTIVE: To evaluate the efficacy of the Shugan Jieyu capsule on improving sleep and emotional disorder during Coronavirus disease 2019 (COVID-19) convalescence. METHODS: We conducted a randomized, double-blind, placebo-controlled trial, and recruit 200 COVID-19 convalescence patients and then divide the subjects into two groups respectively: the experimental group ( 100) and the control group ( 100). Patients in the control group were given doses as a placebo, while those in the experimental group were given Shugan Jieyu capsule. The investigators mainly observed the differences between the two groups before and after treatment in terms of the rate of reduction and the rate of efficiency in Hamilton Depression Scale (HAMD-17) total scores from baseline, and recorded the scores of Hamilton Anxiety Scale (HAMA), Patient Health Questionnaire-15 (PHQ-15), Insomnia Severity Index (ISI) and Treatment Emergent Symptom Scale at 2 week, the 4 week and the 6 week respectively after treatment, and compared the differences between the groups. And the occurrence of adverse events was recorded. RESULTS: After 6-week treatment, there were statistically significant differences in the rate of reduction as well as efficiency in HAMD-17 scores, HAMA Total Scores, PHQ-15 Score, ISI Score from baseline in the experimental group and control group (< 0.05). There were 4 adverse events in the experimental group and 1 in the control group. CONCLUSION: Shugan Jieyu capsule could significantly improve sleep and emotional disorder in patients during COVID-19 convalescence.

Transcriptome analysis to identify genes involved in the biosynthesis of aconitines in Aconitum pendulum / 药学学报

italic>Aconitum pendulum is a Tibetan medicine that is rich in bioactive compounds such as aconitine-type C19-diterpenoid alkaloids. To investigate the key enzymes in the aconitine biosynthesis pathway, roots, leaves and flowers of Aconitum pendulum were subjected to a high-throughput transcriptomic sequencing analysis by Illumina HiSeqTM2000. Trinity de novo assembly yielded 47 264 unigenes with an average length of 1 140 bp and N50 of 1 678 bp, of which 30 231 unigenes (63.96%) were annotated. In the KEGG database, 542 unigenes were implicated in 17 secondary metabolic pathways; the analysis showed that 44 genes encoded 20 key enzymes in the diterpene skeleton of aconitine biosynthesis and 12 BAHD acyltransferase genes were related to the acetylation modification, with differential expression among three organs. For example, ApTPS8 was the only committed enzyme in the upstream aconitine biosynthetic pathway. The high expression level of ApTPS8 in root indicated that it is the main tissue for the production of precursors of diterpene alkaloids. Consistent with the accumulation of aconitine, we propose that ApBAHD1/2/8 is involved in the biosynthesis of 2-hydroxyaconitine, dehydrated 14-benzoylaconitine, 8-O-methyl-14-benzoylaconine, benzoyldeoxyaconitine and benzoylaconitine, and ApBAHD10 is involved in the biosynthesis of acontine, lucidusculine, 14-O-acetylneoline and 14-O-acetylvirescenin. Comparative transcriptome analysis of A. pendulum and A. carmichaeli indicates significant gene loss in the family of diterpene synthases and acyltransferases in A. pendulum, which is in accordance with the significantly fewer type and quantity of aconitine compounds in this species. Therefore, A. pendulum has proved to be an ideal material for the study of the aconitine biosynthesis pathway. This work provides basic scientific data for further study of aconitine biosynthesis, the discussion of molecular mechanisms of toxicity, and the synthesis of genuine medicinal materials.

Biopathway construction of plant natural products / 药学学报

Plant natural products (PNPs) are important sources of innovative drugs. They are mainly obtained by isolation or extraction from plants. Low content and with structural analogues in plants result in high production cost, which restricts the research and application of PNPs. While biopathway construction by synthetic biology provides an alternative for production of PNPs. By biosynthetic pathway analysis of PNPs and reconstructing the biopathway in microorganisms, we can produce PNPs in cell factories efficiently. Recently, several predominantly international reports about biosynthesis of PNPs and its synthetic biology production, triggered the researches of PNPs. Abundant traditional Chinese medicine resources and profound cultural heritage of Chinese medicine make biosynthesis pathway analysis of PNPs to be a research hotspot. And some of the studies have achieved significant progress. Here, recent progress in the biosynthesis of plant natural products and its synthetic biology was reviewed. In particular, the application of new methods and technologies in recent years were summarized and discussed. This will provide reference for the biopathway construction of plant natural products.

[Study on lipid production by reusing herb residues of Songling Xuemaikang Capsules].

Songling Xuemaikang Capsules is a Chinese patent medicine mainly made of the Chineses medicine Puerariae Lobatae Radix and leaves of Pinus massoniana. During its production, a large amount of herb extraction residues would be treated as wastes, resulting in resource wasting and serious environmental pollution. In order to solve this problem, we took the hydrolysates of Puerariae Lobatae Radix, P. massoniana leaves, and whole herb residues of Songling Xuemaikang Capsules as the fermentation substrate to explore the ability of Rhodosporidium toruloides to produce microbial lipid. The results showed that the R. toruloides could produce lipid with use of the residues from Songling Xuemaikang Capsules, and the lipid contents reached 33.6%. The lipid products had similar fatty acid composition profiles to those of vegetable oils. Herb residues were converted into fermentation substrates in this study, and were recycled into the production of high value-added compounds to realize the transformation of the wastes, laying the foundation for the sustainable utilization of herb residues.

Limitation standard of toxic aconitines in proprietary Chinese medicines using on-line extraction electrospray ionization mass spectrometry

Development of rapid analytical methods and establishment of toxic component limitation standards are of great importance in quality control of traditional Chinese medicine. Herein, an on-line extraction electrospray ionization mass spectrometry (oEESI-MS) coupled with a novel whole process integral quantification strategy was developed and applied to direct determination of nine key aconitine-type alkaloids in 20 proprietary Chinese medicines (APCMs). Multi-type dosage forms (, tablets, capsules, pills, granules, and liquid preparation) of APCM could be determined directly with excellent versatility. The strategy has the characteristics of high throughput, good tolerance of matrix interference, small amount of sample (∼0.5 mg) and reagent (∼240 μL) consumption, and short analysis time for single sample (<15 min). The results were proved to be credible by high performance liquid chromatography-mass spectrometry (LC-MS) and electrospray ionization mass spectrometry, respectively. Moreover, the limitation standard for the toxic aconitines in 20 APCMs was established based on the holistic weight toxicity (HWT) evaluation and the severally, and turned out that HWT-based toxicity evaluation results were closer to the real clinical applications. Hence, a more accurate and reliable APCM toxicity limitation was established and expected to play an important guiding role in clinics. The current study extended the power of ambient MS as a method for the direct quantification of molecules in complex samples, which is commonly required in pharmaceutical analysis, food safety control, public security, and many other disciplines.

Research progress of P450 in the biosynthesis of bioactive compound of medicinal plants / 药学学报

Most of the active ingredients of herbs are secondary metabolites of plants. Cytochrome P450s (P450s) are hemoglobin-containing monooxygenases encoded by a super-gene family, which play important roles in the metabolic network of plants. This review focuses on the role of P450s on biosynthesis of secondary metabolites such as terpenoids, alkaloids, flavonoids and phenylpropanoids. This will provide references for biosynthesis and regulation of secondary metabolites in medicinal plants.

Effects of electroacupuncture on skeletal muscle and blood glucose in rats with diabetic amyotrophy / 中国针灸

OBJECTIVE@#To explore the effects of electroacupuncture (EA) on skeletal muscle and blood glucose in rats with diabetic amyotrophy.@*METHODS@#Among 40 SD rats, 10 rats were randomly selected into the control group and received no treatment. The remaining 30 rats were treated with intraperitoneal injection of streptozotocin (STZ, 60 mg/kg) to establish diabetes mellitus (DM) model, and then the rats were treated with vascular ligation at right posterior limb to establish amyotrophy model. The rats with diabetic amyotrophy were randomly divided into a model group and an EA group, 10 rats in each group (10 rats were excluded due to unsuccessful model establishment and death). The rats in the EA group was treated with EA at right-side "Yishu (EX-B 3)" "Shenshu (BL 23)" "Zusanli (ST 36)" and "Sanyinjiao (SP 6)", disperse-dense wave, 2 Hz/ 15 Hz, 20 minutes each time, once a day for 3 weeks. Before and after EA treatment, the blood sample was collected from inner canthus and the "glucose oxidase-peroxidase" method was used to detect fasting blood glucose level; ELISA method was used to detect insulin content. At the end of the treatment, HE staining method was used to observe the morphology of ischemic skeletal muscle in the right hindlimb; the real-time PCR method was used to detect the mRNA expression of muscle atrophy F-box (MAFbx), muscle ring finger-1 (MuRF1) and forkhead box O3a (FOXO3a) in the ischemic skeletal muscle tissue of right hindlimb.@*RESULTS@#Before the treatment, the body mass in the model group and EA group was lower than that in the control group (<0.01); after the treatment, the body mass in the control group was increased, while the body mass in the model group and EA group was decreased (<0.01). Compared with the control group, the fasting blood glucose was significantly increased and insulin content was significantly decreased in the model group (<0.01); compared with the model group, the fasting blood glucose was significantly decreased and the insulin content was significantly increased in the EA group after treatment (<0.01). The muscle fibers of the model group were obviously broken, the number of the nuclei decreased, and the nuclei shrinked or even dissolved; the morphology of the muscle tissue of the EA group after intervention was improved compared with the model group. Compared with the control group, the cross-sectional area of ischemic skeletal muscle cells in the right hindlimb in the model group was decreased (<0.01); compared with the model group, the cross-sectional area of ischemic skeletal muscle cells in the right hindlimb was increased in EA group (<0.05). Compared with the control group, the levels of MAFbx, MuRF1 and FOXO3a mRNA in the right hindlimb ischemic skeletal muscle in the model group were increased significantly (<0.01, <0.05); compared with the model group, the levels of MAFbx, MuRF1 and FOXO3a mRNA in the EA group were decreased significantly (<0.05, <0.01).@*CONCLUSION@#EA may play a role in the treatment of diabetic amyotrophy by inducing FOXO3a to reduce the transcription of MAFbx and MuRF1.

Expert consensus on Injection of Breviscapine in clinical practice / 中国中药杂志

With the advancement of the aging process, cerebrovascular disease has become China's first cause of death. Injection of Breviscapine is a type of traditional Chinese medicine injections published in the Chinese Pharmacopoeia of 2015 Edition and the National Basic Medical Insurance, Industrial Injury Insurance and Maternity Insurance Drug Catalogue, and used to treat ischemic cerebrovascular disease in clinic. In order to further improve clinicians' understanding of the drug and guidance of its rational clinical use, we gave full consideration of clinical research evidences and expert experience, followed the procedures developed based on expert consensus of Chinese Academy of Traditional Chinese Medicine, and then offered recommendations for clinical problems summarized by clinical first-line investigations and evidence-based clinical problems according to internationally accepted evidence grading and recommendation standards, i.e. Grade. As for clinical problems without evidence, we reached through nominal group method, and formed consensus recommendations. Safety issues of Injection of Breviscapine, such as indication, syndrome, dosage, course of treatment, precautions, suggestions and contraindications, were defined to improve clinical efficacy, promote rational drug use and reduce drug risks. This consensus needs to be revised in the future based on emerging clinical issues and evidence-based updates in practical applications.

Accumulation and biosynthetic of curcuminoids and terpenoids in turmeric rhizome in different development periods / 中国中药杂志

The dynamic accumulation rule of active substances in medicinal plants is of great value not only for medicinal material production and application,but also for the genetic mechanism study on the formation of medicinal ingredients,especially vital to guide medicinal material collection as well as experiment material selection and candidate gene screening in the analysis of biosynthesis pathway. This study investigated the accumulation of curcumins and terpenoids,and the biosynthesis of these metabolites,which are the active metabolites in Curcuma longa,a commonly used traditional Chinese medicine. Rhizoma of C. longa from leaf growing period,rhizome swelling period and dry matter accumulating period were used as experimental materials,to analyze the changes of metabolites and biosynthesis in the three periods by comparative transcriptome and metabolomes analysis.The results indicated that terpenoids accumulation and biosynthesis mainly occurred in leaf growing period,while curcumin accumulation and biosynthesis mainly occurred in dry matter accumulating period. Therefore,we suggested that turmeric rhizomes in leaf growth period were suitable for terpenoids biosynthetic pathway characterization,and rhizome in accumulation of dry matter period was suitable for curcuminoid biosynthesis pathway characterization. This study provides references for medicinal materialproduction and application,as well as biopathway analysis of active compounds for C. longa.

Screening of long non-coding RNA related to CYP450s involved in biosynthesis of tanshinones / 中国中药杂志

Tanshinones are abietane-type norditerpenoid quinones that make up the main bioactive ingredients of traditional Chinese medicine Salvia miltiorrhiza. Cytochrome CYP450 plays an important role in the post-structural modification of tanshinone biosynthesis pathway. Long non-coding RNA( lncRNA) have been defined as transcripts longer than 200 nucleotides,which have been functionally characterized in regulating the growth and development,secondary metabolism and stress of medicinal plants. In this study,we perform a comprehensive identification of lncRNAs in response to tanshinone metabolism induced by yeast extract( YE) and Ag~+ S. miltiorrhiza hairy roots. Deep RNA sequencing was used to identify a set of different 8 942 lncRNAs,of which 6 755 were intergenic lncRNAs. We predicted a total of 1 115 814 lncRNA-coding gene pairs,including 122 lncRNA-coding gene as cis pairs. The correlation analysis between lncRNA and CYP450 related to tanshinone biosynthesis was carried out and a total of 16 249 lncRNA-CYP450 target gene pairs were identified. Further analysis with functional known CYP76 AH1,CYP76 AH3 and CYP76 AK1 involved in tanshinone biosynthesis,we also identified a set of 216 target genes. These candidate genes will be the important target in the downstream regulation mechanism analysis of the tanshinone biosynthesis pathway.

Type I and Ⅱ Cardiorenal Syndrome-related Biomarkers / 中国实验方剂学杂志

The physiology and pathology of the heart and kidney are interdependent and interact with each other, and dysfunction of any one of them causes dysfunction of the other, namely cardiorenal syndrome, in which type I and type Ⅱ have the highest incidence rate and are the commonest in clinic. Traditional Chinese medicine has a long history of treating the cardiorenal syndrome. It believes that the disease is located in the heart and kidney, and Wenyang Yiqi, Huoxue Lishui and other methods shall be adopted to effectively improve the heart and kidney function of patients. However,the pathogenesis of cardiorenal syndrome is complicated, and the clinical manifestations are diverse, which makes it difficult to diagnose and treat in the early stage, and causes missing of the best intervention timing and a poor prognosis. Biomarkers play a vital role in predicting the occurrence and development of cardiorenal syndrome. Therefore, efforts shall be made to look for biomarkers with better specificity and sensitivity, accurately evaluate physiological and pathological changes in heart and kidney, so as to achieve early diagnosis and early intervention of cardiorenal syndrome, and improve the effect of disease diagnosis and treatment. At present, domestic and foreign scholars have studied and applied more markers mainly in renal tubular injury, including neutrophil gelatinase-associated lipocalin, kidney injury molecule-1 and urinary interleukin-18. In addition, other studies have found cell cycle arrest inducing factors, such as insulin-like growth factor binding protein 7, tissue inhibitor metallo proteinase-2, and fibroblast growth factor 23 associated with mineral metabolism. The increase of the content of these biomarkers in the body is earlier than the rise of serum creatinine, which can better predict the occurrence of early cardiorenal syndrome, and has a high application value and research value. After summarization of the biomarkers relating to type I and Ⅱ cardiorenal syndrome in domestic and foreign literatures, the research progress of several representative markers were reviewed to provide reference for related research.

Multivariate data analysis of volatile metabolites in rhizomes and radixes of four medicinal plants from Curcuma L / 药学学报

The commonly used traditional Chinese medicines Curcumae Rhizome (Ezhu) and Curcumae Radix (Yujin), are representatives of multi-plant sources. The relationship among the original source plant species is intricate. In this study, by using multivariate data analysis, volatile metabolites in rhizomes and radixes of source plants of Curcumae Longae rhizome (Jianghuang), Yujin and Ezhu in traditional Chinese medicine were compared and analyzed. The source plants included Curcuma longa, Curcuma kwangsuensis, Curcuma wenyujing and Curcuma phaeocaulis. The results indicated that:① volatile metabolites were similar in quality but variation in quantity for rhizomes and radixes origin from the same plant species; ② volatile metabolites of C. longa rhizomes showed bigger difference compared with others; ③ although common volatile metabolites were observed in rhizome of C. kwangsuensis, C. wenyujing and C. phaeocaulis, the difference among them were significant; and ④ significant differences were observed for the four kinds of radixes. Results in this study revealed the differences of the four source plants species, and similar metabolites in source plants of Curcumae Rhizome (Ezhu) and Curcumae Radix (Yujin) from the level of volatile metabolites. These results provided a reference for the clinical use of the three kinds of traditional Chinese medicine.

Effects of Shenqi Xingnao Prescription on Learning and Memory Ability, Contents of Choline Acetyltransferase and Acetylcholine Esterase in Brain Tissue of Mice Models with Alzheimer Disease / 中国中医药信息杂志

Objective To observe the effects of Shenqi Xingnao Prescription on learning and memory ability, contents of choline acetyltransferase (ChAT) and acetylcholine esterase (AChE) in brain tissue in mice models with scopolamine-induced Alzheimer disease (AD); To investigate its mechanism for prevention and treatment for AD. Methods Totally 110 ICR mice were randomly divided into control group, control+Shenqi Xingnao Prescription high-dose group,model group,donepezil group,model+Shenqi Xingnao Prescription high-,medium-,and low-dose groups. The control and model group were given distilled water for gavage, and the other groups were given the corresponding medicine for gavage, once a day, for 14 days. On the 15th day, Morris water maze test and object recognition test were used to evaluate the learning and memory ability of each group. The model mice of memory impairment induced by intraperitoneal injection of scopolamine was established 20 minutes before the behavioral test. The expressions of ChAT and AChE in cortex and hippocampus were detected by Western blot. Results The results of Morris water maze test showed that compared with the control group, the model group had significant longer escape latency(P<0.05);Compared with the model group,Shenqi Xingnao Prescription medium-and high-dose groups could shorten the escape latency (P<0.05). The results of the object recognition test showed that compared with the control group, the ability of the model group to explore new things decreased and the discrimination index (DI) decreased (P<0.001);Compared with the model group,Shenqi Xingnao Prescription groups could increase the DI of model mice (P<0.05, P<0.01, P<0.001). The results of Western blot showed that the expression of AChE protein in the cortex and hippocampus of the model group was significantly higher than the control group (P<0.01); Compared with the model group, Shenqi Xingnao Prescription low- and medium-dose groups could decrease the expression of AChE in the cortex in different degrees(P<0.01);Shenqi Xingnao Prescription groups could decreaed the expression of AChE in the hippocampus (P<0.001); There was no significant statistical significance in the expression of ChAT in the cortex and hippocampus in each group.Conclusion Shenqi Xingnao Prescription can significantly improve the learning and memory ability of AD model mice induced by scopolamine, which may be related to the descent expression of AChE protein in the cortex and hippocampus of the model mice.

Cloning and expression analysis of squalene epoxidase genes from Siraitia grosvenorii / 中国中药杂志

Siraitia grosvenorii, vine plant of Cucurbitaceae family, has been used as natural sweetener and folk medicine. The major components and sweet substances are both known as mogrosides which are cucurbitane-type tetra-triterpenoids. Squalene epoxidase (SQE) has been generally recognized as the common rate-limiting enzyme in triterpenes and phytosterols, catalyzing into their common precursor 2,3-oxidosqualene (OS); however, in the biosynthesis of mogrosides, the precursor was 2,3,22,23-dioxidosqualene (DOS) instead of OS. To explore the specific SQE in S. grosvenorii, we cloned two full-length SQEs (SgSQE1, SgSQE2), performed bioinformatic analysis, analyzed the expression patterns in different periods of fruits by Real-time PCR, and induced the prokaryotic expressions. Finally, the interactive sites between SQE and substrate were predicted by docking, which would provide evidence for SQE gene function study of mogrosides and also lay foundation for triterpene biosynthesis in other plants. SgSQE1 and SgSQE2 both encoded predicted proteins of 524 amino acids, and shared 84% identity to each other at residues level, but had high specificity at N-terminal region. They both accumulated in fruits, but with different patterns, SgSQE1 increased rapidly and reached the highest level at 15 d, which had identical co-expression pattern with cucurbitadienol synthase (CS). SgSQE2 had a relatively constant level. The docking results showed that predicted proteins of SgSQE1 and SgSQE2 can interact with OS, with different contact sites (R348 for SgSQE1, H349 for SgSQE2). The recombinant proteins had no activities by prokaryotic expression, which were caused by transmembrane regions. However, all the results strongly suggested that SgSQEs were both involved in secondary metabolites biosynthesis in S. grosvenorii. SgSQE1 might be involved in mogrosides biosynthesis and SgSQE2 might participate in other cucurbitane-type triterpenes or phytosterols biosynthesis.

Origin of lifting and lowering theory and its herb pair study / 中国中药杂志

Lifting and lowering theory is one of the important basis for guiding clinical medication. Through the study of ancient books and literature, we learned that lifting and lowering theory was originated in Huangdi Neijing, practiced more in the Shanghan Zabing Lun, established in Yixue Qiyuan, and developed in Compendium of Materia Medica and now. However, lifting and lowering theory is now mostly stagnated in the theoretical stage, with few experimental research. In the clinical study, the guiding role of lifting and lowering theory to prescriptions?mainly includes opposite?role?of lift and lower medicine property, mutual promotion of lift and lower medicine property, main role of lift medicine property and main role of lower medicine property. Under the guidance of lifting and lowering theory, the herb pair compatibility include herb combination of lift medicine property, herb combination of lift and lower medicine property and herb combination of lower medicine property. Modern biological technology was used in this study to carry out experimental research on the lifting and lowering theory, revealing the scientific connotation of it, which will help to promote clinical rational drug use.

Identification and classification of traditional Chinese medicine syndrome types among senior patients with vascular mild cognitive impairment using latent tree analysis / 中西医结合学报

<p><b>OBJECTIVE</b>To treat patients with vascular mild cognitive impairment (VMCI) using traditional Chinese medicine (TCM), it is necessary to classify the patients into TCM syndrome types and to apply different treatments to different types. In this paper, we investigate how to properly carry out the classification for patients with VMCI aged 50 or above using a novel data-driven method known as latent tree analysis (LTA).</p><p><b>METHOD</b>A cross-sectional survey on VMCI was carried out in several regions in Northern China between February 2008 and February 2012 which resulted in a data set that involves 803 patients and 93 symptoms. LTA was performed on the data to reveal symptom co-occurrence patterns, and the patients were partitioned into clusters in multiple ways based on the patterns. The patient clusters were matched up with syndrome types, and population statistics of the clusters are used to quantify the syndrome types and to establish classification rules.</p><p><b>RESULTS</b>Eight syndrome types are identified: Qi deficiency, Qi stagnation, Blood deficiency, Blood stasis, Phlegm-dampness, Fire-heat, Yang deficiency, and Yin deficiency. The prevalence and symptom occurrence characteristics of each syndrome type are determined. Quantitative classification rules are established for determining whether a patient belongs to each of the syndrome types.</p><p><b>CONCLUSION</b>A solution for the TCM syndrome classification problem for patients with VMCI and aged 50 or above is established based on the LTA of unlabeled symptom survey data. The results can be used as a reference in clinic practice to improve the quality of syndrome differentiation and to reduce diagnosis variances across physicians. They can also be used for patient selection in research projects aimed at finding biomarkers for the syndrome types and in randomized control trials aimed at determining the efficacy of TCM treatments of VMCI.</p>

Cloning, subcellular localization, and heterologous expression of ApNAC1 gene from Andrographis paniculata / 中国中药杂志

Andrographis paniculata is widely used as medicinal herb in China for a long time and andrographolide is its main medicinal constituent. To investigate the underlying andrographolide biosynthesis mechanisms, RNA-seq for A. paniculata leaves with MeJA treatment was performed. In A. paniculata transcriptomic data, the expression pattern of one member of NAC transcription factor family (ApNAC1) matched with andrographolide accumulation. The coding sequence of ApNAC1 was cloned by RT-PCR, and GenBank accession number was KY196416. The analysis of bioinformatics showed that the gene encodes a peptide of 323 amino acids, with a predicted relative molecular weight of 35.9 kDa and isoelectric point of 6.14. To confirm the subcellular localization, ApNAC1-GFP was transiently expressed in A. paniculata protoplast. The results indicated that ApNAC1 is a nucleus-localized protein. The analysis of real-time quantitative PCR revealed that ApNAC1 gene predominantly expresses in leaves. Compared with control sample, its expression abundance sharply increased with methyl jasmonate treatment. Based on its expression pattern, ApNAC1 gene might involve in andrographolide biosynthesis. ApNAC1 was heterologously expressed in Escherichia coli and recombinant protein was purified by Ni-NTA agarose. Further study will help us to understand the function of ApNAC1 in andrographolide biosynthesis.