Distinct latitudinal patterns and drivers of topsoil nitrogen and phosphorus across urban forests in eastern China.

Nitrogen (N) and phosphorus (P) are the two most important macronutrients supporting forest growth. Unprecedented urbanization has created growing areas of urban forests that provide key ecosystem services for city dwellers. However, the large-scale patterns of soil N and P content remain poorly understood in urban forests. Based on a systematic soil survey in urban forests from nine large cities across eastern China, we examined the spatial patterns and key drivers of topsoil (0-20 cm) total N content, total P content, and N:P ratio. Topsoil total N content was found to change significantly with latitude in the form of an inverted parabolic curve, while total P content showed an opposite latitudinal pattern. Variance partition analysis indicated that regional-scale patterns of topsoil total N and P contents were dominated by climatic drivers and partially regulated by time and pedogenic drivers. Conditional regression analyses showed a significant increase in topsoil total N content with lower mean annual temperature (MAT) and higher mean annual precipitation (MAP), while topsoil total P content decreased significantly with higher MAP. Topsoil total N content also increased significantly with the age of urban park and varied with pre-urban soil type, while no such effects were found for topsoil total P content. Moreover, topsoil N:P ratio showed a latitudinal pattern similar to that of topsoil total N content and also increased significantly with lower MAT and higher MAP. Our findings demonstrate distinct latitudinal trends of topsoil N and P contents and highlight a dominant role of climatic drivers in shaping the large-scale patterns of topsoil nutrients in urban forests.

Single-incision laparoscopic appendectomy versus traditional three-hole laparoscopic appendectomy for acute appendicitis in children by senior pediatric surgeons: a multicenter study from China.

Objective: The aim of this study was to evaluate the clinical efficacy of single-incision laparoscopy appendectomy (SILA) and traditional three-hole laparoscopy appendectomy (THLA) for the treatment of acute appendicitis in children. Methods: The clinical data of children (<14 years old) who underwent laparoscopic appendectomy at Yijishan Hospital of Wannan Medical College, Hubei Provincial Maternal Health Hospital and Qingdao Women and Children's Medical Center from January 2019 to June 2022 were retrospectively analyzed. According to the operation method, the patients were assigned to the SILA group or the THLA group. The clinical data, including the efficacy, and the surgical details, including the complications, of the two surgical methods were compared. The personal information of the children and the time of disease onset were recorded. Results: In this study, the data of 588 patients, including 385 patients in the THLA group and 203 patients in the SILA group were collected. The baseline characteristics between the two groups of patients before surgery were comparable. There was no significant difference in the average operation time between the THLA group and the SILA group (56.31 ± 1.83 min vs. 57.48 ± 1.15 min, P > 0.05). There was also no significant difference in the average length of hospital stay between the THLA group and the SILA group (6.91 ± 0.24 days vs. 7.16 ± 0.36 days, P > 0.05). However, the FLACC scores of the SILA group (3.71 ± 0.78) were significantly lower than those of the THLA group (3.99 ± 0.56) on the second postoperative day, and the difference was significant (P < 0.05). The score of the questionnaire evaluating cosmetic appearance of the postoperative abdomen was significantly higher in the SILA group (15.81 ± 0.36) than in the THLA group (13.10 ± 0.24) (P < 0.05). There was no significant difference in the incidence of postoperative complications between the two groups (P > 0.05). Conclusion: SILA is more advantageous in terms of postoperative FLACC scores and cosmetic appearance in children than THLA. There was no significant difference in the incidence of complications or other aspects between the two surgical methods.

[Gene clone and functional identification of sterol glycosyltransferases from Paris polyphylla var. yunnanensis].

In this study, the authors cloned a glycosyltransferase gene PpUGT2 from Paris polyphylla var. yunnanensis with the ORF length of 1 773 bp and encoding 590 amino acids. The phylogenetic tree revealed that PpUGT2 belonged to the UGT80A subfamily and was named as UGT80A49 by the UDP-glycosyltransferase(UGT) Nomenclature Committee. The expression vector pET28a-PpUGT2 was constructed, and enzyme catalytic reaction in vitro was conducted via inducing protein expression and extraction. With UDP-glucose as sugar donor and diosgenin and pennogenin as substrates, the protein was found with the ability to catalyze the C-3 hydroxyl ß-glycosylation of diosgenin and pennogenin. To further explore its catalytic characteristic, 15 substrates including steroids and triterpenes were selected and PpUGT2 showed its activity towards the C-17 position of sterol testosterone with UDP-glucose as sugar donor. Homology modelling and molecule docking of PpUGT2 with substrates predicted the key residues interacting with ligands. The re-levant residues of PpUGT2-ligand binding model were scanned to calculate the corresponding mutants, and the optimized mutants were obtained according to the changes in binding affinity of the ligand with protein and the surrounding residues within 5.0 Å of ligands, which had reference value for design of the mutants. This study laid a foundation for further exploring the biosynthetic pathway of polyphyllin as well as the structure of sterol glycosyltransferases.

The most bioactive fraction of stir-fried Radix Paeoniae Alba regulating IL-6/STAT3 signaling pathway in allergic asthma mouse.

ETHNOPHARMACOLOGICAL RELEVANCE: Radix Paeoniae Alba (RPA), a traditional Chinese medicine, has been used frequently in the treatment of asthma. Previous studies demonstrated the dichloromethane fraction of Stir-Frying RPA (FDCM) enhanced the effect of anti-allergic asthma compared with the dichloromethane fraction of RPA (DCM). AIM OF THE STUDY: The significant increasing of Paeoniflorin (PF), ethyl gallate (EG), 1,2,3,4,6-pentagalloylglucose (PGG) had been observed in FDCM. This study aimed to investigate the effects and mechanisms of these compounds from FDCM in ovalbumin (OVA)-induced allergic asthma mouse model. MATERIALS AND METHODS: The significant difference contents compounds fraction (FB-40) and other fractions in FDCM were enriched by Medium Pressure Liquid Chromatography (MPLC). The pharmacodynamics was verified among all fractions in OVA-induced allergic asthma mice. Moreover, the drug dose dependence of FB-40 (0.42 mg/kg, 0.21 mg/kg, and 0.07 mg/kg), which were the most active fraction from FDCM for anti-allergic asthma, was explored. The expression of IL-6, p-STAT3, and STAT3 was analyzed by Western blot analysis. In addition, the main components of FB-40 were identified by UPLC with standards. Finally, the anti-inflammatory effects of the main components from FB-40 were detected by LPS-stimulated BEAS-2B cells using an Elisa assay. RESULTS: The results showed that FB-40 was the most active fraction from FDCM, which could significantly improve the lung tissue pathological condition, and decrease the number of inflammatory cells in bronchoalveolar lavage fluid (BALF). It had greater pharmacological activity than its main component PF. FB-40 also showed dose dependence and regulated the IL-6/STAT3 signaling pathway in allergic asthma mice. Besides, PF, Albiflorin (AF), PGG, EG, and 1,2,3,6-Tetra-O-galloyl-ß-D-glucose (TGG) from FB-40 were identified by UPLC with the standard. At last, in the LPS-induced BEAS-2B cell experiments, EG, PGG, 1,2,3,6-Tetra-O-galloyl-ß-D-glucose (TGG) showed stronger inhibiting activities of cytokine than the monoterpenoid glycosides (PF and AF). CONCLUSION: The research proved that FB-40 was an active fraction in FDCM, which regulates IL-6/STAT3 Signaling Pathway to ameliorate allergic asthma. Gallic acids including TGG and PGG, and EG also play a role in the treatment of allergic asthma in FB-40.

Screening of Bioactive Fraction of Radix Paeoniae Alba and Enhancing Anti-Allergic Asthma by Stir-Frying Through Regulating PI3K/AKT Signaling Pathway.

Allergic asthma is a common respiratory inflammation disease. The crude Radix Paeoniae Alba (RPA) and its processed products have been used frequently as antipyretic and anti-inflammatory agents in traditional medicine. To evaluate the effect of honey and bran processing, different fractions of RPA were used for treating anti-allergic asthma in the ovalbumin (OVA)-induced mice model, and then, the most effective fraction of RPA and stir-frying Radix Paeoniae Alba with honey and bran (FRPA) for treating anti-allergic asthma were compared mutually for pharmacological effects. The results showed that the treatment of the dichloromethane fraction of RPA significantly improved the pathological condition of lung tissues, decreased the number of eosinophils and other cells in bronchoalveolar lavage fluid (BALF), and the increased the expression of various inflammatory factors. Furthermore, the study discovered that the lung pathological conditions, compared with the high dose of dichloromethane RPA fraction, could be ameliorated by high dose of dichloromethane FRPA fraction treatment. Moreover, the expression of inflammatory factors and the phosphorylation of the PI3K/AKT signaling pathway could be diminished by FRPA. Finally, the contents of compounds with a significant difference in the FRPA dichloromethane fraction were paeoniflorin, ethyl gallate, pentagalloylglucose, galloylpaeoniflorin, and others by UPLC/Q-TOF-MS analysis. These findings suggest that the dichloromethane fraction of FRPA has an enhancement effect on anti-allergic asthma and provide the experimental basis for exploring the processed mechanism of RPA.

[Protective effect of ginsenoside Rg_1 aganist diabetic retinopathy by inhibiting NLRP3 inflammasome in type 2 diabetic mice].

Ginsenoside Rg_1, one of the main active components of precious traditional Chinese medicine Ginseng Radix et Rhizoma, has the anti-oxidative stress, anti-inflammation, anti-aging, neuroprotection, and other pharmacological effects. Diabetic retinopathy(DR), the most common complication of diabetes, is also the main cause of impaired vision and blindness in the middle-aged and the elderly. The latest research shows that ginsenoside Rg_1 can protect patients against DR, but the protection and the mechanism are rarely studied. This study mainly explored the protective effect of ginsenoside Rg_1 against DR in type 2 diabetic mice and the mechanism. High fat diet(HFD) and streptozotocin(STZ) were used to induce type 2 diabetes in mice, and hematoxylin-eosin(HE) staining was employed to observe pathological changes in the retina of mice. The immunohistochemistry was applied to study the localization and expression of nucleotide-binding oligomerization domain-like receptors 3(NLRP3) and vascular endothelial growth factor(VEGF) in retina, and Western blot was used to detect the expression of nuclear factor-kappa B(NF-κB), p-NF-κB, NLRP3, caspase-1, interleukin-1ß(IL-1ß), transient receptor potential channel protein 6(TRPC6), nuclear factor of activated T-cell 2(NFAT2), and VEGF in retina. The results showed that ginsenoside Rg_1 significantly alleviated the pathological injury of retina in type 2 diabetic mice. Immunohistochemistry results demonstrated that ginsenoside Rg_1 significantly decreased the expression of NLRP3 and VEGF in retinal ganglion cells, middle plexiform layer, and outer plexiform layer in type 2 diabetic mice. According to the Western blot results, ginsenoside Rg_1 significantly lowered the expression of p-NF-κB, NLRP3, caspase-1, IL-1ß, TRPC6, NFAT2, and VEGF in retina of type 2 diabetic mice. These findings suggest that ginsenoside Rg_1 can significantly alleviate DR in type 2 diabetic mice, which may be related to inhibition of NLRP3 inflammasome and VEGF. This study provides experimental evidence for the clinical application of ginsenoside Rg_1 in the treatment of DR.

Protective effect of ginsenoside Rg_1 aganist diabetic retinopathy by inhibiting NLRP3 inflammasome in type 2 diabetic mice / 中国中药杂志

Ginsenoside Rg_1, one of the main active components of precious traditional Chinese medicine Ginseng Radix et Rhizoma, has the anti-oxidative stress, anti-inflammation, anti-aging, neuroprotection, and other pharmacological effects. Diabetic retinopathy(DR), the most common complication of diabetes, is also the main cause of impaired vision and blindness in the middle-aged and the elderly. The latest research shows that ginsenoside Rg_1 can protect patients against DR, but the protection and the mechanism are rarely studied. This study mainly explored the protective effect of ginsenoside Rg_1 against DR in type 2 diabetic mice and the mechanism. High fat diet(HFD) and streptozotocin(STZ) were used to induce type 2 diabetes in mice, and hematoxylin-eosin(HE) staining was employed to observe pathological changes in the retina of mice. The immunohistochemistry was applied to study the localization and expression of nucleotide-binding oligomerization domain-like receptors 3(NLRP3) and vascular endothelial growth factor(VEGF) in retina, and Western blot was used to detect the expression of nuclear factor-kappa B(NF-κB), p-NF-κB, NLRP3, caspase-1, interleukin-1β(IL-1β), transient receptor potential channel protein 6(TRPC6), nuclear factor of activated T-cell 2(NFAT2), and VEGF in retina. The results showed that ginsenoside Rg_1 significantly alleviated the pathological injury of retina in type 2 diabetic mice. Immunohistochemistry results demonstrated that ginsenoside Rg_1 significantly decreased the expression of NLRP3 and VEGF in retinal ganglion cells, middle plexiform layer, and outer plexiform layer in type 2 diabetic mice. According to the Western blot results, ginsenoside Rg_1 significantly lowered the expression of p-NF-κB, NLRP3, caspase-1, IL-1β, TRPC6, NFAT2, and VEGF in retina of type 2 diabetic mice. These findings suggest that ginsenoside Rg_1 can significantly alleviate DR in type 2 diabetic mice, which may be related to inhibition of NLRP3 inflammasome and VEGF. This study provides experimental evidence for the clinical application of ginsenoside Rg_1 in the treatment of DR.

Berberine-loaded M2 macrophage-derived exosomes for spinal cord injury therapy.

Spinal cord injury (SCI) causes immune activation of resident macrophages/microglia. Activated macrophages/microglia have two different phenotypes, the pro-inflammatory classically activated (M1) phenotype and the anti-inflammatory alternatively activated (M2) phenotype. M1 phenotype macrophages/microglia are the key factor in inflammation. The treatment of SCI remains a huge challenge due to the nontargeting and inefficiency of anti-inflammatory drugs through the blood-brain barrier (BBB). The purpose of this experiment was to design M2-type primary peritoneal macrophages exosomes (Exos) as a drug carrier for berberine (Ber), which can be efficiently targeted to deliver drugs to the injured spinal cord due to the natural advantage of Exos across the BBB. The Exos with particle size of 125±12 nm were loaded with by an ultrasonic method and the drug loading reached 17.13 ±1.64%. The Ber release experiment showed that the loaded sample (Exos-Ber) exhibited sustained release effect, and the cumulative release amount reached 71.44±2.86% within 48 h. In vitro and in vivo experiments confirmed that the Exos-Ber could decrease the M1 protein marker iNOS, elevate the M2 protein marker CD206 and reduce inflammatory and apoptotic cytokines (TNF-α, IL-1ß, IL-6, Caspase 9, Caspase 8), which showed that Exos-Ber had a good anti-inflammatory and anti-apoptotic effect by inducing macrophages/microglia from the M1 phenotype to M2 phenotype polarization. Moreover, the motor function of SCI mice was significantly improved after Exos-Ber treatment, indicating that Exos-Ber is a potential agent for SCI therapy. STATEMENT OF SIGNIFICANCE: Efficient targeting strategy for drug delivery. In addition to good biocompatibility and stealth ability, M2 macrophage-derived Exosomes present natural inflammatory targeting ability. The inflammatory microenvironment after spinal cord injury provides motivation for the targeting of exosomes. Natural drug carrier with higher safety. With the rapid development of nanomaterials, drug carriers have become more selective. However, due to the special microenvironment after central nervous system damage, some non-degradable inorganic materials will increase the pressure of self-healing and even secondary damage to neurons, which has been solved by the emergence of exosomes. Some previous studies used tumor cell line exosomes as drug carriers, but the carcinogenic factors carried by themselves have extremely high hidden dangers, and endogenous macrophage exosomes have absolute advantages over their safety.

Joint effects of mitochondrial DNA4977 deletion and serum folate deficiency on coronary artery disease in type 2 diabetes mellitus.

BACKGROUND & AIMS: The 4977-bp mitochondrial deletion (mtDNA4977 deletion), as a hallmark of mitochondrial oxidative damage, may play an important role in coronary artery disease (CAD), but its interaction with folate deficiency among diabetic patients is largely unknown. We aimed to explore the joint association of leukocyte mtDNA4977 deletion and serum folate status with obstructive CAD in Chinese adults with type 2 diabetes. METHODS: We cross-sectionally analyzed the angiographic data of 2017 diabetic patients without B-vitamin supplementation. Of the 2017 participants, 756 who received percutaneous coronary intervention (PCI) completed prospective follow-up of one year. In vitro, we explored the mediation effects of mitochondrial reactive oxygen species (mtROS) in folic acid (FA)-deficient human aortic smooth muscle cells (HASMCs) under hyperglycemic conditions. RESULTS: Cross-sectionally, the multivariate odds ratios (ORs) for obstructive CAD were 1.41 (95% CI: 1.29-1.55) for greater mtDNA4977 deletion, and 1.15 (95% CI: 1.05-1.25) for lower folate levels. Particularly, the combination of high mtDNA4977 deletion (top tertile) and folate deficiency (serum folate < 6 ng/mL) was associated with more than 2-fold increased odds of having obstructive CAD and higher degrees of coronary stenosis. Prospectively, the hazard ratio for all-cause death at 1-year after PCI was up to 2.37 (95% CI: 1.21-4.63) for folate-deficient participants in the top tertile of mtDNA4977 deletion. In HASMCs, the adverse effects of FA deficiency were aggravated by induction of mtROS, and attenuated by scavenging of mtROS. CONCLUSIONS: The risk of obstructive CAD may be greatly increased by the interaction between greater mtDNA4977 deletion and folate deficiency among diabetic patients.

Clinical features and outcome of Wilson's disease with generalized epilepsy in Chinese patients.

OBJECTIVE: Generalized epilepsy is rarely reported in patients with Wilson disease (WD) and lacks experience in clinical practice. We aim to provide better experience for the diagnosis and treatment for WD patients with epilepsy in the future. METHODS: A retrospective study was performed in 13 Chinese WD patients with generalized epilepsy. Each patient was diagnosed with WD by clinical evaluation and genetic screening. Patients were given small doses of antiepileptic drugs (AEDs), followed by copper-chelation therapy when the seizures stabilized. Clinical manifestations, brain imaging changes, and treatment and outcome after a long-term follow-up were analyzed. RESULTS: Four out of 13 (30.8%) patients stopped taking copper-chelation drugs for more than 1 year before they were admitted for epilepsy. The incidence of epilepsy of WD patients in our cohort is 1.43% (13/910), lower than those (4.5%-5.9%) in other populations. After the attack of epilepsy, frontal lobes were the most common abnormalities (13/13, 100%) in patients, followed by brain stem (8/13, 61.5%) and thalamus (7/13, 53.8%). After a long-term follow-up, brain imaging and clinical manifestations of 8 (8/9, 88.9%) WD patients were significantly improved. CONCLUSIONS: We firstly described WD patients with generalized epilepsy in the Chinese population. WD patients with aggravation of neuropsychiatric symptoms are prone to occur epilepsy; thus, brain MRI should be performed regularly in those patients. Cortical abnormality in brain MRI is a warning sign of epilepsy. Irregular use of copper-chelation drugs and excessive copper deposition in the brain may be the cause of seizures. Long-term standardized treatment for WD can effectively prevent the extensive brain damage and reduce the incidence of epilepsy in WD patients.

Processing and Polyherbal Formulation of Tetradium ruticarpum (A. Juss.) Hartley: Phytochemistry, Pharmacokinetics, and Toxicity.

Herbal medicine is a major part of traditional Chinese medicine (TCM), which is evolved as a system of medical practice from ancient China. The use of herbal medicine is mainly based on practice and theories and concepts rooted in ancient philosophy. In the era of evidence-based medicine, it is essential to accurately evaluate herbal remedy with standard/modern medical practice approaches. Tetradium ruticarpum (A. Juss.) Hartley (TR), a medicinal plant with diversify bioactive components, has been broadly used to treat pain and gastrointestinal disorders in TCM. However, TR has also been reported to have potential toxicity by long-term use or excessive doses, though the associated compounds are yet to be identified. TR is usually processed, and/or combined with other herbs in TCM formulas in order to achieve a synergistic effect or reduce its toxicity. Since processing or polyherbal formulation of TR may lead to changes in its chemical composition and contents, quality, efficacy and toxicity, comparison of TR samples before and after processing, as well as its combination with other medicines, would provide useful knowledge of bioactive compounds, efficacy and toxicity of this valuable medicinal plant. Here we reviewed the recent studies about the phytochemistry, pharmacokinetic behaviors and toxicity of TR under various processing or polyherbal formulation conditions, which would expand our understanding of mechanisms of TR's efficacy and toxicity and be valuable for quality control in industrial manufacturing, future medicinal research, and safety and rational use of TR in TCM.

Folate functionalized pH-sensitive photothermal therapy traceable hollow mesoporous silica nanoparticles as a targeted drug carrier to improve the antitumor effect of doxorubicin in the hepatoma cell line SMMC-7721.

In this paper, we prepared doxorubicin-loaded folic acid-functionalized pH-sensitive photothermal therapy (PTT) traceable hollow mesoporous silica nanoparticles (DOX-HPCF) as a drug carrier for liver cancer treatment. According to TEM characterization, hollow mesoporous silica nanoparticles (HMSN) are monodispersed spherical particles with hollow structure. In vitro drug release experiments showed that HPCF exhibited pH-sensitive release. Cell uptake experiments showed that HPCF was successfully absorbed by SMMC-7721 cells. In addition, DOX-HPCF significantly inhibited the proliferation of SMMC-7721 cells, and the near-infrared (NIR) light group showed a more obvious inhibitory effect. In vivo anti-tumor experiments showed that DOX-HPCF-assisted PTT inhibited tumor growth significantly. Therefore, HPCF is a promising photothermotherapy carrier for the treatment of liver cancer.

[Cloning and characterization of a monoterpene synthase gene from Tripterygium wilfordii].

Tripterygium wilfordii is a medicinal plant commonly used in the treatment of rheumatoid arthritis,and with pharmacological activities in anti-tumor and obesity treatment. The known active ingredients in T. wilfordii are mainly terpenoids,but with very low content. Therefore,the analysis of the biosynthesis pathway of terpenoids in T. wilfordii has become a research hotspot to solve the problem of its resources. Terpenoid synthase( TPS) is a key enzyme that catalyzes the formation of a wide variety of terpenoid skeletons. In this study,a gene fragment with an ORF of 1 785 bp was cloned from T. wilfordii. Bioinformatics analysis was performed using NCBI's BLASTP,ProtParam and Interpro online tools and MEGA 6.0 software. The response of this gene to methyl jasmonate was also detected by real-time fluorescent quantitative PCR,and its catalytic function was verified by prokaryotic expression and in vitro enzymatic assay. Bioinformatics analysis indicated that the amino acid sequence encoded by this gene had both N-terminal domain and C-terminal domain of TPS,as well as the DDxx D conserved domain of the class I of TPS family. And Tw MTS gathered together with TPS-b subfamily in the Neighbor-Joining Tree constructed with known homologous TPSs. The results of RT-PCR showed that 50 µmol·L-1 MeJA 12 h could increase the expression of Tw MTS to 735 times in the control group at 12 h,and 1 644 times at 24 h. In addition,in vitro enzymatic reaction results showed that Tw MTS can catalyze the production of ß-citronellol with GPP as substrate,indicating that Tw MTS was a monoterpene synthase. The above results provided a new element for the synthetic biology database of T. wilfordii terpenoids,and laid the foundation for future biosynthesis research.

Cloning and characterization of a monoterpene synthase gene from Tripterygium wilfordii / 中国中药杂志

Tripterygium wilfordii is a medicinal plant commonly used in the treatment of rheumatoid arthritis,and with pharmacological activities in anti-tumor and obesity treatment. The known active ingredients in T. wilfordii are mainly terpenoids,but with very low content. Therefore,the analysis of the biosynthesis pathway of terpenoids in T. wilfordii has become a research hotspot to solve the problem of its resources. Terpenoid synthase( TPS) is a key enzyme that catalyzes the formation of a wide variety of terpenoid skeletons. In this study,a gene fragment with an ORF of 1 785 bp was cloned from T. wilfordii. Bioinformatics analysis was performed using NCBI's BLASTP,ProtParam and Interpro online tools and MEGA 6.0 software. The response of this gene to methyl jasmonate was also detected by real-time fluorescent quantitative PCR,and its catalytic function was verified by prokaryotic expression and in vitro enzymatic assay. Bioinformatics analysis indicated that the amino acid sequence encoded by this gene had both N-terminal domain and C-terminal domain of TPS,as well as the DDxx D conserved domain of the class I of TPS family. And Tw MTS gathered together with TPS-b subfamily in the Neighbor-Joining Tree constructed with known homologous TPSs. The results of RT-PCR showed that 50 μmol·L-1 MeJA 12 h could increase the expression of Tw MTS to 735 times in the control group at 12 h,and 1 644 times at 24 h. In addition,in vitro enzymatic reaction results showed that Tw MTS can catalyze the production of β-citronellol with GPP as substrate,indicating that Tw MTS was a monoterpene synthase. The above results provided a new element for the synthetic biology database of T. wilfordii terpenoids,and laid the foundation for future biosynthesis research.

Multilayer emulsions as a strategy for linseed oil and α-lipoic acid micro-encapsulation: study on preparation and in vitro characterization.

BACKGROUND: Linseed oil and α-lipoic acid are bioactive ingredients, which play an important role in human nutrition and health. However, their application in functional foods is limited because of their instabilities and poor solubilities in hydrophilic matrices. Multilayer emulsions are particularly useful to protect encapsulated bioactive ingredients. The aim of this study was to fabricate multilayer emulsions by a high-pressure homogenization method to encapsulate linseed oil and α-lipoic acid simultaneously. Tween 20 and lecithin were used as surfactants to stabilize the oil droplets of primary emulsions. Multilayer emulsions were produced by using an electrostatic layer-by-layer deposition process of lecithin-chitosan membranes. RESULTS: Thermal treatment exhibited that chitosan encapsulation could improve the thermal stability of primary emulsions. During in vitro digestion, it was found that chitosan encapsulation had little effect on the lipolysis of linseed oil and bioaccessibility of α-lipoic acid. The oxidation stability of linseed oil in multilayer emulsions was improved effectively by chitosan encapsulation and α-lipoic acid. Chitosan encapsulation could inhibit the degradation of α-lipoic acid. A physical stability study indicated that multilayer emulsions had good centrifugal, dilution and storage stabilities. CONCLUSION: Multilayer emulsion is an effective delivery system to incorporate linseed oil and α-lipoic acid into functional foods and beverages. © 2018 Society of Chemical Industry.

Cloning and functional analysis of two sterol-C24-methyltransferase 1 (SMT1) genes from Paris polyphylla.

The biosynthetic pathways of phytosterols and steroidal saponins are located in two adjacent branches which share cycloartenol as substrate. The rate-limiting enzyme S-adenosyl-L-methionine-sterol-C24-methyltransferase 1 (SMT1) facilitates the metabolic flux toward phytosterols. It catalyzes the methylation of the cycloartenol in the side chain of the C24-alkyl group, to generate 24(28)-methylene cycloartenol. In this study, we obtained two full-length sequences of SMT1 genes from Pari polyphylla, designated PpSMT1-1 and PpSMT1-2. The full-length cDNA of PpSMT1-1 was 1369 bp long with an open reading frame (ORF) of 1038 bp, while the PpSMT1-2 had a length of 1222 bp, with a 1005 bp ORF. Bioinformatics analysis confirmed that the two cloned SMTs belong to the SMT1 family. The predicted function was further validated by performing in vitro enzymatic reactions, and the results showed that PpSMT1-1 encodes a cycloartenol-C24-methyltransferase, which catalyzes the conversion of cycloartenol to 24-methylene cycloartenol, whereas PpSMT1-2 lacked this catalytic activity. The tissue expression patterns of the two SMTs revealed differential expression in different organs of Paris polyphylla plants of different developmental stage and age. These results lay the foundation for detailed genetic studies of the biosynthetic pathways of steroid compounds, which constitute the main class of active substances found in P. polyphylla.

[Cloning and protein expression analysis of geranyl diphosphate synthase genes in Tripterygium wilfordii].

Based on the transcriptome data, the study cloned full-length cDNA of TwGPPS1 and TwGPPS2 genes from Tripterygium wilfordii suspension cells and then analyzed the bioinformation of the sequence and protein expression. The cloned TwGPPS1 has a 1 278 bp open reading frame (ORF) encoding a polypeptide of 425 amino acids. The deduced isoelectric point (pI) was 6.68, a calculated molecular weight was about 47.189 kDa. The full-length cDNA of the TwGPPS2 contains a 1 269 bp open reading frame (ORF) encoding a polypeptide of 422 amino acids. The deduced isoelectric point (pI) was 6.71, a calculated molecular weight was about 46.774 kDa.The entire reading frame of TwGPPS1,2 was cloned into the pET-32a(+) vector and expressed in E. coli BL21 (DE3) cells to obtain the TwGPPS protein, which laid a basis for further study on the regulation of terpenoid secondary metabolism and biological synthesis.

[DNA molecular identification of Manis pentadactyla].

The study was aimed to establish a stable, accurate site specific PCR identification system to identify Manis pentadactyla and its adulterants using DNA molecular identification. The genomic DNA was extracted from experimental samples using the DNA extraction kit. The Cytb and CO Ⅰ genes were amplified using PCR and sequenced bi-directionally. Obtained sequences were assembled using the BioEdit software. The neighbor-joining tree was constructed by MEGA 6.0. Specific identification primers were designed according to the specific allelets, and PCR reaction system was optimized. The results indicated that the Cytb and CO Ⅰ sequence both were able to be used to identify M. pentadactyla and its adulterants. With the specific primers CO Ⅰ-S10/A5, the M. pentadactyla could be amplified a 400 bp DNA band when the annealing temperature ranged from 55 to 60 ℃ and the amount of DNA template ranged from 3 to 100 ng within 35 PCR cycles. However, other adulterants displayed no relevant bands. So that primers CO Ⅰ- S10 / A5 can be used to identify the M. pentadactyla with the adulterants.

Yinzhihuang attenuates ANIT-induced intrahepatic cholestasis in rats through upregulation of Mrp2 and Bsep expressions.

BACKGROUND: The treatment of intrahepatic cholestasis has been limited, and development of an effective drug is needed. Clinical studies have shown that Yinzhihuang (YZH), a traditional Chinese decoction, enhances bilirubin clearance. The goal of this study was to determine the protective effect of YZH on experimental intrahepatic cholestasis in young rats and to explore its underlying molecular mechanisms. METHODS: Intrahepatic cholestasis in rats was induced by α-naphthylisothiocyanate (ANIT) on days 1 and 8. The rats received YZH, ursodeoxycholic acid (UDCA), or vehicle for 9 d and were killed on either day 3 or day 10. Serum biomarkers, liver histology, and the distribution of protein and mRNA expression of Mrp2 and Bsep were analyzed. RESULTS: YZH treatment resulted in decreased levels of serum biomarkers except γ-glutamyl transpeptidase, attenuated liver histological injuries, increased protein expressions of Mrp2 and Bsep, and upregulated expressions of Mrp2 and Bsep mRNAs. The effects of YZH on serum biomarkers (aminotransferase, alanine aminotransferase, and direct bilirubin), liver histology, and Mrp2 mRNA expressions were significantly greater and earlier than those of UDCA. CONCLUSION: Our results suggest that YZH has protective effect against ANIT-induced intrahepatic cholestasis in rats, through upregulation of Mrp2 and Bsep expressions.