Ningxin-Tongyu-Zishen formula alleviates the senescence of granulosa cells on D-galactose-induced premature ovarian insufficiency mice.

Ningxin-Tongyu-Zishen formula (NTZF) is a clinical experience formula for the treatment of premature ovarian insufficiency (POI) in traditional Chinese medicine (TCM), and the potential mechanism is unknown. For in vivo experiments, POI mouse models (C57BL/6 mice), were constructed by subcutaneous injection of D-galactose (D-gal, 200 mg/kg). After treatment of NTZF (10.14, 20.27, 40.54 g/kg;) or estradiol valerate (0.15 mg/kg), ovarian function, oxidative stress (OS) and protein expression of Sirt1/p53 were evaluated. For in vitro experiments, H2O2 (200 µM) was used to treat KGN to construct ovarian granulosa cells (OGCs) cell senescence model. Pretreatment with NTZF (1.06 mg/mL) or p53 inhibitor (Pifithrin-α, 1 µM) was performed before induction of senescence, and further evaluated the cell senescence, OS, mRNA and protein expression of Sirt1/p53. In vivo, NTZF improved ovarian function, alleviated OS and Sirt1/p53 signaling abnormalities in POI mice. In vitro experiments showed that NTZF reduced the level of OS and alleviated the senescence of H2O2-induced KGN. In addition, NTZF activated the protein expression of Sirt1, inhibited the mRNA transcription and protein expression of p53 and p21. Alleviating OGCs senescence and protecting ovarian function through Sirt1/p53 is one of the potential mechanisms of NTZF in the treatment of POI.

Identification of ARF genes in Cucurbita pepo L and analysis of expression patterns, and functional analysis of CpARF22 under drought, salt stress.

BACKGROUND: Auxin transcription factor (ARF) is an important transcription factor that transmits auxin signals and is involved in plant growth and development as well as stress response. However, genome-wide identification and responses to abiotic and pathogen stresses of the ARF gene family in Cucurbita pepo L, especially pathogen stresses, have not been reported. RESULTS: Finally, 33 ARF genes (CpARF01 to CpARF33) were identified in C.pepo from the Cucurbitaceae genome database using bioinformatics methods. The putative protein contains 438 to 1071 amino acids, the isoelectric point is 4.99 to 8.54, and the molecular weight is 47759.36 to 117813.27 Da, the instability index ranged from 40.74 to 68.94, and the liposoluble index ranged from 62.56 to 76.18. The 33 genes were mainly localized in the nucleus and cytoplasm, and distributed on 16 chromosomes unevenly. Phylogenetic analysis showed that 33 CpARF proteins were divided into 6 groups. According to the amino acid sequence of CpARF proteins, 10 motifs were identified, and 1,3,6,8,10 motifs were highly conserved in most of the CpARF proteins. At the same time, it was found that genes in the same subfamily have similar gene structures. Cis-elements and protein interaction networks predicted that CpARF may be involved in abiotic factors related to the stress response. QRT-PCR analysis showed that most of the CpARF genes were upregulated under NaCl, PEG, and pathogen treatment compared to the control. Subcellular localization showed that CpARF22 was localized in the nucleus. The transgenic Arabidopsis thaliana lines with the CpARF22 gene enhanced their tolerance to salt and drought stress. CONCLUSION: In this study, we systematically analyzed the CpARF gene family and its expression patterns under drought, salt, and pathogen stress, which improved our understanding of the ARF protein of zucchini, and laid a solid foundation for functional analysis of the CpARF gene.

[Definitive screening design combined with design space for optimizing purification process of Scutellariae Radix extract].

In the pharmacopoeia, many process parameters for the purification process of Scutellariae Radix are unclear. In this study, deterministic screening design combined with design space method was used to optimize the purification process of Scutellariae Radix extract. Nine method parameters such as mass fraction of solution(X_1), first acid precipitation pH(X_2) and first holding time(X_3) in the purification process were firstly studied by definitive screening design. The yield of baicalin was defined as the evaluation index. A stepwise regression method was used then to build quantitative models between evaluation index and method parameters and the three most critical impact parameters were determined. Probability-based design space was calculated and successfully verified with the experimental error simulation method. Finally, the second standing temperature, the first standing temperature and the pH value of the second acid precipitation were determined as the three most critical method parameters. The recommended operating space was as follows: the second standing temperature 5-7 ℃, the first standing temperature 13-15 ℃, and the pH of the second acid precipitation 1.5-1.7. Within this operating space, the baicalin yield in the purification process was over 80%, and the probability of reaching the standard was over 0.96. In this study, we optimized the effect of various parameters for the purification process of the Scutellariae Radix extract in the pharmacopoeia on the yield of baicalin and provided a reference for industrial production of the exact of Scutellariae Radix.

Definitive screening design combined with design space for optimizing purification process of Scutellariae Radix extract / 中国中药杂志

In the pharmacopoeia, many process parameters for the purification process of Scutellariae Radix are unclear. In this study, deterministic screening design combined with design space method was used to optimize the purification process of Scutellariae Radix extract. Nine method parameters such as mass fraction of solution(X_1), first acid precipitation pH(X_2) and first holding time(X_3) in the purification process were firstly studied by definitive screening design. The yield of baicalin was defined as the evaluation index. A stepwise regression method was used then to build quantitative models between evaluation index and method parameters and the three most critical impact parameters were determined. Probability-based design space was calculated and successfully verified with the experimental error simulation method. Finally, the second standing temperature, the first standing temperature and the pH value of the second acid precipitation were determined as the three most critical method parameters. The recommended operating space was as follows: the second standing temperature 5-7 ℃, the first standing temperature 13-15 ℃, and the pH of the second acid precipitation 1.5-1.7. Within this operating space, the baicalin yield in the purification process was over 80%, and the probability of reaching the standard was over 0.96. In this study, we optimized the effect of various parameters for the purification process of the Scutellariae Radix extract in the pharmacopoeia on the yield of baicalin and provided a reference for industrial production of the exact of Scutellariae Radix.

Prevention and control of HIV/AIDS in China: lessons from the past three decades / 中华医学杂志(英文版)

In the past 37 years, human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) has undergone various major transmission routes in China, with the world most complex co-circulating HIV-1 subtypes, even the prevalence is still low. In response to the first epidemic outbreak of HIV in injecting drug users and the second one by illegal commercial blood collection, China issued the Anti-Drug Law and launched the Blood Donation Act and nationwide nucleic acid testing, which has avoided 98,232 to 211,200 estimated infections and almost ended the blood product-related infection. China has been providing free antiretroviral therapy (ART) since 2003, which covered >80% of the identified patients and achieved a viral suppression rate of 91%. To bend the curve of increasing the disease burden of HIV and finally end the epidemic, China should consider constraining HIV spread through sexual transmission, narrowing the gaps in identifying HIV cases, and the long-term effectiveness and safety of ART in the future.

[Research progress on percolation extraction process of traditional Chinese medicines].

Percolation extraction is a conventional extraction method used in the processing of traditional Chinese medicines. After medicinal material powder is placed in a percolation tank, the extraction solvent is continuously added, and percolation extract is collected simultaneously. The percolation equipment is simple. The percolation operation is easy. It is applicable to a wide range of medicinal materials. Components that are unstable under thermal conditions can be effectively extracted. However, there are also disadvantages, such as high solvent consumption, long extraction time, and high energy consumption in subsequent concentration processes. This article mainly reviews the research progress on the common equipment types, affecting factors, parameter optimization methods, and process monitoring. According to analysis on literatures, solvent composition, impregnation time, percolation speed, and solvent consumption are considered as the important factors of percolation processes. At present, near-infrared spectroscopy is widely used in the monitoring of percolation process, and partial least square is a commonly used quantitative modeling method. According to the concept of "Quality by Design", in-depth investigation of the percolation process mechanism and development of process control methods are future development trends. Therefore, process modeling, process optimization and process monitoring shall be improved. The mechanism models and the empirical models of column chromatography can be used as references to construct the percolation process models. The effect of the quality changes of medicinal materials shall be taken into account when optimizing the percolation process parameters. More simple and easy methods shall be developed to monitor the percolation process status and key properties of percolation extracts.

Critical pharmaceutical process identification considering chemical composition, biological activity, and batch-to-batch consistency: A case study of notoginseng total saponins.

Objective: Critical pharmaceutical process identification (CPPI) is an important step in the implementation of quality by design concept to traditional Chinese medicines (TCMs). Risk assessment methods are usually used in CPPI. However, risk evaluation is usually subjective. The purpose of this work is to present a more objective CPPI method. Methods: A CPPI method considering chemical composition, biological activity, and batch-to-batch consistency was presented in this work. The manufacturing process of notoginseng total saponins (NTS) was investigated as an example. The changes of chemical composition, biological activity, and chemical composition consistency after main processes were measured and compared. A significant change of them indicated a critical process. Results: After extraction process and chromatography process, saponin purity and chemical composition similarity remarkably increased, and saponin content variations decreased. Thrombin inhibitory activity was remarkably decreased after chromatography process. Because of the large influences on NTS quality, extraction process and chromatography process were identified to be critical processes of NTS. Conclusion: Based on a comprehensive and objective examination of the role of each process, critical pharmaceutical processes can be identified. A similar method can also be applied to other TCM processes.

Critical process parameter identification of manufacturing processes of Astragali Radix extract with a weighted determination coefficient method.

Objective: Critical process parameters (CPPs) identification is an important step of the implementation of quality by design (QbD) concept. There are many CPP identification methods, such as risk analysis method, sensitivity analysis method, multiple linear regression method, standard partial regression coefficient (SPRC) method, and so on. The SPRC method can consider multiple process critical quality attributes (CQAs) simultaneously, but the determination of CPP number is subjective. Therefore, new CPP identification method is still required. Methods: The manufacturing process of Astragali Radix extract, which contained water reflux extraction, concentration, and ethanol precipitation, was used as an example. First, the multiple process CQAs were determined to be the yield of pigment, dry matter, sugars, and active ingredients. Second, the potential CPPs were determined by a knowledge organization method. Plackett-Burman designed experiments were then performed. A weighted determination coefficient ( R w 2 ) method was presented to identify CPPs. In this method, the importance of different CQAs was considered. Process parameters were removed one-by-one according to their importance index. The decrease in R w 2 was used to characterize the importance of the removed parameter. If the decrease of R w 2 was less than a preset threshold, the removed parameter was not a CPP. Results: During the manufacturing process of Astragali Radix extract, the potential CPPs determined by the knowledge organization method were water consumption, reflux extraction time, extraction frequency, ethanol content, ethanol consumption, and concentration endpoint. Reflux extraction time, the first ethanol consumption, the second ethanol consumption, and the second ethanol precipitation refrigeration temperature were found to be CPPs using the weighted determination coefficient method with the threshold of 10%. Conclusion: Using the weighted determination coefficient method, CPPs can be determined with all the CQAs considered based on their importance. The determination of CPP number is more objective compared with the SPRC method.

Research progress on percolation extraction process of traditional Chinese medicines / 中国中药杂志

Percolation extraction is a conventional extraction method used in the processing of traditional Chinese medicines. After medicinal material powder is placed in a percolation tank, the extraction solvent is continuously added, and percolation extract is collected simultaneously. The percolation equipment is simple. The percolation operation is easy. It is applicable to a wide range of medicinal materials. Components that are unstable under thermal conditions can be effectively extracted. However, there are also disadvantages, such as high solvent consumption, long extraction time, and high energy consumption in subsequent concentration processes. This article mainly reviews the research progress on the common equipment types, affecting factors, parameter optimization methods, and process monitoring. According to analysis on literatures, solvent composition, impregnation time, percolation speed, and solvent consumption are considered as the important factors of percolation processes. At present, near-infrared spectroscopy is widely used in the monitoring of percolation process, and partial least square is a commonly used quantitative modeling method. According to the concept of "Quality by Design", in-depth investigation of the percolation process mechanism and development of process control methods are future development trends. Therefore, process modeling, process optimization and process monitoring shall be improved. The mechanism models and the empirical models of column chromatography can be used as references to construct the percolation process models. The effect of the quality changes of medicinal materials shall be taken into account when optimizing the percolation process parameters. More simple and easy methods shall be developed to monitor the percolation process status and key properties of percolation extracts.

Disparity of human immunodeficiency virus incidence and drug resistance in college student, non-student youth and older men who have sex with men: a cross-sectional study from seven major cities of China / 中华医学杂志(英文版)

BACKGROUND@#Human immunodeficiency virus (HIV) prevalence among student men who have sex with men (MSM) in college is more than 5.0% and keeps on increasing in China. This study aims to clarify the proportion of HIV recent infection, its propeller and the source among college student MSM.@*METHODS@#We conducted a multicenter cross-sectional study in seven major Chinese cities during 2012-2013. HIV recent infections (≤ 168 days) and incidence was measured and estimated by BED HIV-1 capture enzyme immunoassay (BED-CEIA) testing strategy. HIV-related behaviors and transmitted drug resistance (TDR) were investigated and compared between the college student MSM, <25-year-old non-student youth MSM (NSYM), and ≥25-year-old non-student non-youth MSM (NSNYM), using structured survey, and analyses of drug resistance.@*RESULTS@#Overall, 4,496 (4496/4526, 99.3%) were eligible for enrollment, comprising 565 college student MSM, 1,094 NSYM, and 2,837 NSNYM. The proportion of HIV recent infection were 70.3% (26/37), 50.8% (65/128) and 35.1% (95/271), the HIV incidence rate were 10.0 (95% CI: 6.2-13.9)/100PY, 12.9 (95% CI: 9.8-16.1)/100PY, 6.8 (95% CI: 5.4-8.2)/100PY, and TDR prevalence were 7.4% (2/27), 2.0%, (2/98) and 4.9% (11/226), among student MSM, NSYM, and NSNYM, respectively. Among HIV positive student MSM with age< 21-year-old, the proportion of HIV recent infection is 90.9% (10/11). Factors independently associated with HIV recent infection in student MSM was usage of recreational drug in the past 6 months (AOR: 2.5; 95% CI: 1.0-5.8).@*CONCLUSIONS@#College student MSM had higher proportion of HIV recent infection and TDR than the youth and older MSM in China during 2012-2013. The HIV infections were more likely to happen during the early year of college life among student MSM.

[Quantitative chromatographic fingerprint analysis of Sanye Tangzhiqing Decoction based on quality by design concept].

In this work,a high performance liquid chromatography-ultraviolet( HPLC-UV) detection technology was used to establish fingerprint analysis method for Sanye Tangzhiqing Decoction following an analytical quality by design( AQb D) approach. Firstly,column temperature,flow rate,and gradient elution conditions were determined as the method parameters needing to be optimized. Then according to the results of definitive screening design,three critical method attributes( CMAs) were identified,including peak number,the percentage of common peak area to total peak area,and retention time of the last peak. A stepwise regression method was used then to build quantitative models between CMAs and method parameters. Probability-based design space was calculated and successfully verified using the experimental error simulation method. After the analysis conditions were optimized,the contents of six components,namely chlorogenic acid,paeoniflorin,rutin,hyperoside,quercetin-3-O-ß-D-glucuronide,and salvianolic acid B were simultaneously determined. There were 19 common peaks in the fingerprint and their common peak area accounted for 96% of the total peak area. Both fingerprint and quantitative analysis methods were validated applicable in methodology study,and they can be applied to determine new samples.

[Research on quality standards of standard decoction of Fructus Corni piece].

This study aims to develop the quality standards of Fructus Corni piece standard decoction. Morroniside and loganin were considered as index components. The content determination method of morroniside and loganin were developed. The fingerprint analysis method was also established. The standard decoctions of 15 batches of Fructus Corni pieces from Henan, Zhejiang, and Shaanxi were analyzed. The similarity values of fingerprint were all above 0.99. The transfer rates of morroniside were all higher than 100%. The quality evaluation indices of standard decoction were discussed. The transfer rate of an index component was not easy to be measured accurately and its concept was not rigorous. Therefore, index component yield was suggested as an evaluation index of standard decoction. Two methods for setting quality standards of standard decoctions, which were the ■ method and the ■ method, were compared. It was found that the standard range of ■ method was wider and more suitable for smaller sample size of standard decoction. The quality standards of Fructus Corni standard decoction were as follows, dry matter extraction ratio 37.48%-69.60%; morroniside yield 8.719-16.19 mg·g~(-1) piece; loganin yield 4.342-8.064 mg·g~(-1) piece.

Research on quality standards of standard decoction of Fructus Corni piece / 中国中药杂志

This study aims to develop the quality standards of Fructus Corni piece standard decoction. Morroniside and loganin were considered as index components. The content determination method of morroniside and loganin were developed. The fingerprint analysis method was also established. The standard decoctions of 15 batches of Fructus Corni pieces from Henan, Zhejiang, and Shaanxi were analyzed. The similarity values of fingerprint were all above 0.99. The transfer rates of morroniside were all higher than 100%. The quality evaluation indices of standard decoction were discussed. The transfer rate of an index component was not easy to be measured accurately and its concept was not rigorous. Therefore, index component yield was suggested as an evaluation index of standard decoction. Two methods for setting quality standards of standard decoctions, which were the ■ method and the ■ method, were compared. It was found that the standard range of ■ method was wider and more suitable for smaller sample size of standard decoction. The quality standards of Fructus Corni standard decoction were as follows, dry matter extraction ratio 37.48%-69.60%; morroniside yield 8.719-16.19 mg·g~(-1) piece; loganin yield 4.342-8.064 mg·g~(-1) piece.

Quantitative chromatographic fingerprint analysis of Sanye Tangzhiqing Decoction based on quality by design concept / 中国中药杂志

In this work,a high performance liquid chromatography-ultraviolet( HPLC-UV) detection technology was used to establish fingerprint analysis method for Sanye Tangzhiqing Decoction following an analytical quality by design( AQb D) approach. Firstly,column temperature,flow rate,and gradient elution conditions were determined as the method parameters needing to be optimized. Then according to the results of definitive screening design,three critical method attributes( CMAs) were identified,including peak number,the percentage of common peak area to total peak area,and retention time of the last peak. A stepwise regression method was used then to build quantitative models between CMAs and method parameters. Probability-based design space was calculated and successfully verified using the experimental error simulation method. After the analysis conditions were optimized,the contents of six components,namely chlorogenic acid,paeoniflorin,rutin,hyperoside,quercetin-3-O-β-D-glucuronide,and salvianolic acid B were simultaneously determined. There were 19 common peaks in the fingerprint and their common peak area accounted for 96% of the total peak area. Both fingerprint and quantitative analysis methods were validated applicable in methodology study,and they can be applied to determine new samples.

[Quantitative analysis method of Shengxuebao Mixture by HPLC-UV-MS based on quality by design concept].

In this study, the HPLC-UV-MS method for the simultaneous determination of eight active ingredients of Shengxuebao Mixture were developed based on the concept of quality by design（QbD）with a stepwise optimization approach. After the analytical target profile（ATP）had been defined, albiflorin, paeoniflorin, 2, 3, 5, 4'-tetra-hydroxy-stilbene-2-O-ß-D-glucopyranoside, specnuezhenide, ecliptasaponin D, emodin, calycosin-7-glucoside, and astragaloside Ⅳ were identified as the indicator components. The resolution and the signal-to-noise ratio of indicator components were then selected as critical method attributes (CMA) for the first step optimization. According to the results collected from fractional factorial design, critical method parameters (CMP) were determined with a multiple linear regression method, which included the amount of acid addition in the mobile phase, temperature, gradient, and wavelength. After that, the amount of acid addition and the wavelength were optimized to improve the resolution and the signal-to-noise ratio of the indicator components. The peak symmetry factors of specnuezhenide and emodin were then set as CMA for the second step optimization. The Box-Behnken designed experiments were conducted. The temperature and gradient were optimized after modelling. The design space were calculated and verified. The optimized analytical method was validated, and the results showed a good precision, accuracy and stability, which means that it can be used for the quantification of the indicator components in Shengxuebao Mixture.

[Comparison of two algorithms for development of design space-overlapping method and probability-based method].

In this work, two algorithms (overlapping method and the probability-based method) for design space calculation were compared by using the data collected from extraction process of Codonopsis Radix as an example. In the probability-based method, experimental error was simulated to calculate the probability of reaching the standard. The effects of several parameters on the calculated design space were studied, including simulation number, step length, and the acceptable probability threshold. For the extraction process of Codonopsis Radix, 10 000 times of simulation and 0.02 for the calculation step length can lead to a satisfactory design space. In general, the overlapping method is easy to understand, and can be realized by several kinds of commercial software without coding programs, but the reliability of the process evaluation indexes when operating in the design space is not indicated. Probability-based method is complex in calculation, but can provide the reliability to ensure that the process indexes can reach the standard within the acceptable probability threshold. In addition, there is no probability mutation in the edge of design space by probability-based method. Therefore, probability-based method is recommended for design space calculation.

[Detection of antioxidant activity of Danhong injection and its intermediate with a paper-based analytical device].

The paper-based analytical device (PAD) was applied in this study to analyze the antioxidant activity of Danhong injection and its intermediates. First polycaprolactone was printed on the surface of a filter paper with a 3D printing device. The modified filter paper was then prepared using polycaprolactone and solid paraffin as the modifiers. The PAD was prepared after adding DPPH ethanol solution to the modified filter paper. Ascorbic acid solutions with different concentrations were used as the positive drug on PAD. After the occurrence of color reactions, the PAD was dried, and the data of color were collected by a cell phone. The color component G and grayscale were selected as the potential indices for measurement according to the values of determination coefficients, detection limits, and effective number of digits. Qualitative and quantitative analysis of Danhong injection and the concentrate of aqueous extract were realized with the PAD. Because no significant differences were observed between the results obtained using the two potential indices, the average values of these two were used for analysis, and the antioxidant activity of Danhong injection and the concentrate of aqueous extract was equivalent to ascorbic acid solutions of 3.7, 46 g·L⁻¹, respectively. The PAD method presented in this work can be a simple method to determine biological activities of Chinese medicines and their intermediates.

Efficient production of trans-4-hydroxy-l-proline from glucose using a new trans-proline 4-hydroxylase in Escherichia coli.

trans-4-Hydroxy-l-proline (trans-4Hyp) is widely used as a valuable building block for the organic synthesis of many pharmaceuticals such as carbapenem antibiotics. The major limitation for industrial bioproduction of trans-4Hyp is the low titer and productivity by using the existing trans-proline 4-hydroxylases (trans-P4Hs). Herein, three new trans-P4Hs from Alteromonas mediterranea (AlP4H), Micromonospora sp. CNB394 (MiP4H) and Sorangium cellulosum (ScP4H) were discovered through genome mining and enzymatic determination. These trans-P4Hs were introduced into an l-proline-producing chassis cell, and the recombinant strain overexpressing AlP4H produced the highest concentration of trans-4Hyp (3.57 g/L) from glucose in a shake flask. In a fed-batch fermentation with a 5 L bioreactor, the best strain SEcH (pTc-B74A-alp4h) accumulated 45.83 g/L of trans-4Hyp within 36 h, with the highest productivity (1.27 g/L/h) in trans-4Hyp fermentation from glucose, to the best of our knowledge. This study provides a promising hydroxylase candidate for efficient industrial production of trans-4Hyp.