Characterization of CYP82 genes involved in the biosynthesis of structurally diverse benzylisoquinoline alkaloids in Corydalis yanhusuo.

Benzylisoquinoline alkaloids (BIAs) represent a significant class of secondary metabolites with crucial roles in plant physiology and substantial potential for clinical applications. CYP82 genes are involved in the formation and modification of various BIA skeletons, contributing to the structural diversity of compounds. In this study, Corydalis yanhusuo, a traditional Chinese medicine rich in BIAs, was investigated to identify the catalytic function of CYP82s during BIA formation. Specifically, 20 CyCYP82-encoding genes were cloned, and their functions were identified in vitro. Ten of these CyCYP82s were observed to catalyze hydroxylation, leading to the formation of protopine and benzophenanthridine scaffolds. Furthermore, the correlation between BIA accumulation and the expression of CyCYP82s in different tissues of C. yanhusuo was assessed their. The identification and characterization of CyCYP82s provide novel genetic elements that can advance the synthetic biology of BIA compounds such as protopine and benzophenanthridine, and offer insights into the biosynthesis of BIAs with diverse structures in C. yanhusuo.

Functional characterization of CYP81C16 involved in the tanshinone biosynthetic pathway in Salvia miltiorrhiza.

Danshen, the dried roots and rhizomes of Salvia miltiorrhiza Bunge (S. miltiorrhiza), is widely used in the treatment of cardiovascular and cerebrovascular diseases. Tanshinones, the bioactive compounds from Danshen, exhibit a wide spectrum of pharmacological properties, suggesting their potential for future therapeutic applications. Tanshinone biosynthesis is a complex process involving at least six P450 enzymes that have been identified and characterized, most of which belong to the CYP76 and CYP71 families. In this study, CYP81C16, a member of the CYP71 clan, was identified in S. miltiorrhiza. An in vitro assay revealed that it could catalyze the hydroxylation of four para-quinone-type tanshinones, namely neocryptotanshinone, deoxyneocryptotanshinone, and danshenxinkuns A and B. SmCYP81C16 emerged as a potential broad-spectrum oxidase targeting the C-18 position of para-quinone-type tanshinones with an impressive relative conversion rate exceeding 90%. Kinetic evaluations andin vivo assays underscored its highest affinity towards neocryptotanshinone among the tested substrates. The overexpression of SmCYP81C16 promoted the accumulation of (iso)tanshinone in hairy root lines. The characterization of SmCYP81C16 in this study accentuates its potential as a pivotal tool in the biotechnological production of tanshinones, either through microbial or plant metabolic engineering.

Functional liposome loaded curcumin for the treatment of Streptococcus mutans biofilm.

Introduction: Plaque biofilms, mainly formed by Streptococcus mutans (S. mutans), play an important role in the occurrence and development of dental caries. Antibiotic treatment is the traditional way to control plaque. However, problems such as poor drug penetration and antibiotic resistance have encouraged the search for alternative strategies. In this paper, we hope to avoid antibiotic resistance through the antibacterial effect of curcumin, a natural plant extract with photodynamic effects, on S. mutans. However, the clinical application of curcumin is limited due to its low water solubility, poor stability, high metabolic rate, fast clearance rate, and limited bioavailability. In recent years, liposomes have become a widely used drug carrier due to their numerous advantages, such as high drug loading efficiency, high stability in the biological environment, controlled release, biocompatibility, non-toxic, and biodegradability. So, we constructed a curcumin-loaded liposome (Cur@LP) to avoid the defect of curcumin. Methods: Cur@LP functioned with NHS can adhere to the surface of the S. mutans biofilm by condensation reaction. Liposome (LP) and Cur@LP was characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS). The cytotoxicity of Cur@LP was evaluated by CCK-8 assay and LDH assay. The adhesion of Cur@LP to S. mutans biofilm was observed by confocal laser scanning microscope (CLSM). The antibiofilm efficiency of Cur@LP were evaluated by crystal violet staining, CLSM, and scanning electron microscope (SEM). Results: The mean diameter of LP and Cur@LP were 206.67 ± 8.38 nm and 312 ± 18.78 nm respectively. The ζ-potential of LP and Cur@LP were ∼-19.3 mV and ∼-20.8 mV respectively. The encapsulation efficiency of Cur@LP was (42.61 ± 2.19) %, and curcumin was rapidly released up to ±21% at 2 h. Cur@LP has negligible cytotoxicity, and can effectively adhered to the S. mutans biofilm and inhibited its growth. Discussion: Curcumin has been widely studied in many fields such as cancer, which can be attributed to its antioxidant and anti-inflammatory effects. At present, there are few studies on the delivery of curcumin to S. mutans biofilm. In this study, we verified the adhesion and antibiofilm of Cur@LP to S. mutans biofilm. This biofilm removal strategy has the potential to be translated into the clinic.

Medical Conditions and Preference of Traditional Chinese Medicine: Results from the China Healthcare Improvement Evaluation Survey.

Purpose: Although complementary and alternative medicine (CAM) is used around the world, there has been a lack of comprehensive understanding of major factors affecting patients' decision to use CAM. This study aimed to describe the preferences of Chinese patients regarding what conditions they will use Traditional Chinese Medicine (TCM) for and to determine the factors associated with these preferences. Patients and Methods: This study used data from the China Healthcare Improvement Evaluation Survey in January 2021, a national cross-sectional survey conducted at 163 hospitals across 31 provinces. A convenient sampling method was used to conduct the patient satisfaction survey, and 28,993 patients in an ambulatory setting constituted our study sample on TCM use. A multiple-choice question regarding TCM listed nine medical conditions and asked the patient about what condition he/she and his/her family members would use TCM. In addition to descriptive statistics, we used a binary logistic regression model to investigate factors affecting the likelihood of patients' decision to use TCM for multiple conditions. Results: The majority of the surveyed patients (76.3%) would use TCM for the purpose of disease prevention, and more than half (67.3%) for multiple medical/health conditions, 34.0% for dealing with chronic diseases, 33.0% for common symptoms, 26.9% for rehabilitation, and 26.3% for sleeping disorder. Female and older patients, as well as patients with a higher education level, urban residency, and higher family income, were found to be associated with a higher probability of using TCM for multiple conditions than their counterparts (odd ratios [OR]>1, P<0.05). Conclusion: This study reveals a preference for TCM in a large sample of Chinese patients, especially used for prevention. Generally, patients with a higher socioeconomic status had a more positive attitude toward TCM.

Catalytic promiscuity of O-methyltransferases from Corydalis yanhusuo leading to the structural diversity of benzylisoquinoline alkaloids.

O-methyltransferases play essential roles in producing structural diversity and improving the biological properties of benzylisoquinoline alkaloids (BIAs) in plants. In this study, Corydalis yanhusuo, a plant used in traditional Chinese medicine due to the analgesic effects of its BIA-active compounds, was employed to analyze the catalytic characteristics of O-methyltransferases in the formation of BIA diversity. Seven genes encoding O-methyltransferases were cloned, and functionally characterized using seven potential BIA substrates. Specifically, an O-methyltransferase (CyOMT2) with highly efficient catalytic activity of both 4'- and 6-O-methylations of 1-BIAs was found. CyOMT6 was found to perform two sequential methylations at both 9- and 2-positions of the essential intermediate of tetrahydroprotoberberines, (S)-scoulerine. Two O-methyltransferases (CyOMT5 and CyOMT7) with wide substrate promiscuity were found, with the 2-position of tetrahydroprotoberberines as the preferential catalytic site for CyOMT5 (named scoulerine 2-O-methyltransferase) and the 6-position of 1-BIAs as the preferential site for CyOMT7. In addition, results of integrated phylogenetic molecular docking analysis and site-directed mutation suggested that residues at sites 172, 306, 313, and 314 in CyOMT5 are important for enzyme promiscuity related to O-methylations at the 6- and 7-positions of isoquinoline. Cys at site 253 in CyOMT2 was proved to promote the methylation activity of the 6-position and to expand substrate scopes. This work provides insight into O-methyltransferases in producing BIA diversity in C. yanhusuo and genetic elements for producing BIAs by metabolic engineering and synthetic biology.

[Identification of genes involved in biosynthesis of paeoniflorin in Paeonia lactiflora based on transcriptome analysis].

Paeoniflorin, a representative pinane monoterpene glycoside, is the main active component and quality index of Paeoniae Radix Alba and Paeoniae Radix Rubra.The possible biosynthesis of paeoniflorin is as follows: GPP is derived from mevalonate(MVA) and/or 2-C-methyl-D-erythritol 4-phosphate(MEP) pathway(s) followed by the catalysis with terpene synthase, cytochrome P450(CYP450), UDP-glucuronosyltransferase(UGT), and acyltransferase(AT), respectively.This study aims to explore the genes rela-ted to the biosynthesis of paeoniflorin.To be specific, the cDNA libraries for flowers, leaves, and roots of Paeonia lactiflora were established and sequenced.A total of 30 609 open reading frames(ORFs) were yielded.Through functional annotation and expression analysis of all CYP450 genes in the transcriptome, 11 CYP450 genes belonging to CYP71 A and CYP71 D subfamilies and showing expression trend consistent with monoterpene synthase PlPIN that may be involved in paeoniflorin biosynthesis were screened out.Subsequently, 7 UGT genes and 9 AT genes demonstrating the expression trend consistent with PlPIN which were possibly involved in paeoniflorin biosynthesis were further screened by functional annotation analysis, full-length sequence analysis, expression analysis, and phylogeny analysis.This study provided a systematic screening method with smaller number of candidate genes, thus reducing the workload of functional gene verification.The result laid a foundation for analyzing the biosynthesis pathway of paeoniflorin and the formation mechanism.

Functional Characterization of a 2OGD Involved in Abietane-Type Diterpenoids Biosynthetic Pathway in Salvia miltiorrhiza.

Salvia miltiorrhiza is one of the most commonly used Chinese medicinal herbs. Tanshinones, the most abundant lipid-soluble bioactive constituents of S. miltiorrhiza, are a class of structural highly oxidized abietane-type diterpenoids with multiple pharmacological activities. Although several enzymes, including diterpene synthase, cytochrome P450, and Fe(II)/2-oxoglutarate-dependent dioxygenase (2OGD), have been functionally characterized in biosynthesis of abietane-type diterpenoids, the highly oxidized structure and complex secondary metabolic network of tanshinones imply that more oxidases should be characterized. Here, we identified a new 2OGD (Sm2OGD25) from S. miltiorrhiza. Molecular cloning and functional studies in vitro showed that Sm2OGD25 could catalyze the hydroxylation of sugiol at C-15 and C-16 positions to produce hypargenin B and crossogumerin C, respectively. The phylogenetic analysis of the DOXC family demonstrated that Sm2OGD25 belongs to the DOXC54 clade. Furthermore, structural modeling and site-directed mutagenesis characterization revealed the importance of the hydrogen-bonding residue Y339 and the hydrophobic residues (V122, F129, A144, A208, F303, and L344) in substrate binding and enzyme activity. This study will promote further studies on the catalytic characterization of plant 2OGDs and the secondary metabolic biosynthesis network of diterpenoids.

Recent progress and new perspectives for diterpenoid biosynthesis in medicinal plants.

Diterpenoids, including more than 18,000 compounds, represent an important class of metabolites that encompass both phytohormones and some industrially relevant compounds. These molecules with complex, diverse structures and physiological activities, have high value in the pharmaceutical industry. Most medicinal diterpenoids are extracted from plants. Major advances in understanding the biosynthetic pathways of these active compounds are providing unprecedented opportunities for the industrial production of diterpenoids by metabolic engineering and synthetic biology. Here, we summarize recent developments in the field of diterpenoid biosynthesis from medicinal herbs. An overview of the pathways and known biosynthetic enzymes is presented. In particular, we look at the main findings from the past decade and review recent progress in the biosynthesis of different groups of ringed compounds. We also discuss diterpenoid production using synthetic biology and metabolic engineering strategies, and draw on new technologies and discoveries to bring together many components into a useful framework for diterpenoid production.

Sodium Butyrate Supplementation Inhibits Hepatic Steatosis by Stimulating Liver Kinase B1 and Insulin-Induced Gene.

BACKGROUND AND AIMS: Butyric acid is an intestinal microbiota-produced short-chain fatty acid, which exerts salutary effects on alleviating nonalcoholic fatty liver disease (NAFLD). However, the underlying mechanism of butyrate on regulating hepatic lipid metabolism is largely unexplored. METHODS: A mouse model of NAFLD was induced with high-fat diet feeding, and sodium butyrate (NaB) intervention was initiated at the eighth week and lasted for 8 weeks. Hepatic steatosis was evaluated and metabolic pathways concerning lipid homeostasis were analyzed. RESULTS: Here, we report that administration of NaB by gavage once daily for 8 weeks causes an augmentation of insulin-induced gene (Insig) activity and inhibition of lipogenic gene in mice fed with high-fat diet. Mechanistically, NaB is sufficient to enhance the interaction between Insig and its upstream kinase AMP-activated protein kinase (AMPK). The stimulatory effects of NaB on Insig-1 activity are abolished in AMPKα1/α2 double knockout (AMPK-/-) mouse primary hepatocytes. Moreover, AMPK activation by NaB is mediated by LKB1, as evidenced by the observations showing NaB-mediated induction of phosphorylation of AMPK, and its downstream target acetyl-CoA carboxylase is diminished in LKB1-/- mouse embryonic fibroblasts. CONCLUSIONS: These studies indicate that NaB serves as a negative regulator of hepatic lipogenesis in NAFLD and that NaB attenuates hepatic steatosis and improves lipid profile and liver function largely through the activation of LKB1-AMPK-Insig signaling pathway. Therefore, NaB has therapeutic potential for treating NAFLD and related metabolic diseases.

Diversity of O-Glycosyltransferases Contributes to the Biosynthesis of Flavonoid and Triterpenoid Glycosides in Glycyrrhiza uralensis.

Licorice (Glycyrrhiza uralensis) is a popular medicinal plant containing more than 70 flavonoid and triterpenoid glycosides. Thus far, only a few reports are available on the glycosylation enzymes involved in their biosynthesis. In this work, we mined the transcriptome data of G. uralensis and discovered 43 candidate genes for O-glycosyltransferase (O-GT). Among them, 17 genes could be expressed in E. coli, and functions of the enzymes were analyzed by catalyzing eight native substrates. As a result, we characterized 11 O-GTs, including isoflavone 7-O-GTs, flavonol 3-O-GTs, and promiscuous O-GTs catalyzing flavones, chalcones, and triterpenoids. They could efficiently synthesize key licorice compounds such as liquiritin, isoliquiritin, ononin, and 3-O-ß-d-glucuronosyl glycyrrhetinic acid. The diversity of O-GTs contributes to the biosynthesis of various glycosides in licorice. These enzymes could also be used as biocatalytic tools to synthesize other bioactive O-glycosides.

Molecular and Structural Characterization of a Promiscuous C-Glycosyltransferase from Trollius chinensis.

Herein, the catalytic promiscuity of TcCGT1, a new C-glycosyltransferase (CGT) from the medicinal plant Trollius chinensis is explored. TcCGT1 could efficiently and regio-specifically catalyze the 8-C-glycosylation of 36 flavones and other flavonoids and could also catalyze the O-glycosylation of diverse phenolics. The crystal structure of TcCGT1 in complex with uridine diphosphate was determined at 1.85 Šresolution. Molecular docking revealed a new model for the catalytic mechanism of TcCGT1, which is initiated by the spontaneous deprotonation of the substrate. The spacious binding pocket explains the substrate promiscuity, and the binding pose of the substrate determines C- or O-glycosylation activity. Site-directed mutagenesis at two residues (I94E and G284K) switched C- to O-glycosylation. TcCGT1 is the first plant CGT with a crystal structure and the first flavone 8-C-glycosyltransferase described. This provides a basis for designing efficient glycosylation biocatalysts.

A comprehensive review on phytochemistry, pharmacology, and flavonoid biosynthesis of Scutellaria baicalensis.

CONTEXT: Scutellaria baicalensis Georgi (Lamiaceae) is a popular medicinal plant. Its roots are used as the famous traditional Chinese medicine Huang-Qin, which is recorded in Chinese Pharmacopoeia, European Pharmacopoeia, and British Pharmacopoeia. OBJECTIVE: This review comprehensively summarizes research progress in phytochemistry, pharmacology, and flavonoid biosynthesis of S. baicalensis. METHODS: English and Chinese literature from 1973 to March 2018 was collected from databases including Web of Science, SciFinder, PubMed, Elsevier, Baidu Scholar (Chinese), and CNKI (Chinese). Scutellaria baicalensis, chemical constituents, phytochemistry, biological activities, and biosynthesis were used as the key words. RESULTS: A total of 126 small molecules (1-126) and 6 polysaccharides have been isolated from S. baicalensis. The small molecules can be classified into four structural types, namely, free flavonoids, flavonoid glycosides, phenylethanoid glycosides, and other small molecules. Extracts of S. baicalensis and its major chemical constituents have been reported to possess anti-viral, anti-tumor, anti-bacterial, antioxidant, anti-inflammatory, hepatoprotective, and neuroprotective activities. Key steps in the biosynthetic pathways of Scutellaria flavonoids have also been summarized. CONCLUSIONS: This article could be helpful for researchers who are interested in the chemical constituents, bioactivities, biosynthesis, and clinical applications of S. baicalensis.

Berberine attenuates hepatic steatosis and enhances energy expenditure in mice by inducing autophagy and fibroblast growth factor 21.

BACKGROUND AND PURPOSE: Berberine, a compound from rhizome coptidis, is traditionally used to treat gastrointestinal infections, such as bacterial diarrhoea. Recently, berberine was shown to have hypoglycaemic and hypolipidaemic effects. We investigated the mechanisms by which berberine regulates hepatic lipid metabolism and energy expenditure in mice. EXPERIMENTAL APPROACH: Liver-specific SIRT1 knockout mice and their wild-type littermates were fed a high-fat, high-sucrose (HFHS) diet and treated with berberine by i.p. injection for five weeks. Mouse primary hepatocytes and human HepG2 cells were treated with berberine and then subjected to immunoblotting analysis and Oil Red O staining. KEY RESULTS: Berberine attenuated hepatic steatosis and controlled energy balance in mice by inducing autophagy and FGF21. These beneficial effects of berberine on autophagy and hepatic steatosis were abolished by a deficiency of the nutrient sensor SIRT1 in the liver of HFHS diet-fed obese mice and in mouse primary hepatocytes. SIRT1 is essential for berberine to potentiate autophagy and inhibit lipid storage in mouse livers in response to fasting. Mechanistically, the berberine stimulates SIRT1 deacetylation activity and induces autophagy in an autophagy protein 5-dependent manner. Moreover, the administration of berberine was shown to promote hepatic gene expression and circulating levels of FGF21 and ketone bodies in mice in a SIRT1-dependent manner. CONCLUSIONS AND IMPLICATIONS: Berberine acts in the liver to regulate lipid utilization and maintain whole-body energy metabolism by mediating autophagy and FGF21 activation. Hence, it has therapeutic potential for treating metabolic defects under nutritional overload, such as fatty liver diseases, type 2 diabetes and obesity.

Cyclic bisbibenzyls induce growth arrest and apoptosis of human prostate cancer PC3 cells.

AIM: To investigate the cytotoxic effects of four cyclic bisbibenzyls, Riccardin C (Ric), Pakyonol (Pak), Marchantin M (Mar), and Plagiochin E (Pla) against chemoresistant prostate cancer PC3 cells. METHODS: Cell growth was assayed by MTT method, and apoptotic related protein Bcl-2 and Bax, poly(ADP-ribose) polymerase (PARP) were examined by Western blotting. Cell cycle and apoptosis of PC3 cells were evaluated with flow cytometry and morphologic examinations. RESULTS: The four compounds inhibited proliferation and elicited cell death in a dose- and time-dependent manner with IC(50) values of 3.22 micromol/L for Ric, 7.98 micromol/L for Pak, 5.45 micromol/L for Mar, and 5.99 micromol/L for Pla, respectively. Furthermore, exposed to these chemicals caused a decrease in the antiapoptotic protein Bcl-2 and an increase in proapoptotic Bax expression. PARP cleavage and caspase-3 activity were also observed. CONCLUSION: The results suggest that cyclic bisbibenzyls could be used for the development of novel therapeutic chemicals against prostate cancer.