Herbal Textual Research on Anemarrhenae Rhizoma in Famous Classical Formulas / 中国实验方剂学杂志

Through reviewing ancient and modern literature， the textual research of Anemarrhenae Rhizoma（AR） has been conducted to verify the name， origin， changes in production areas， quality evaluation， harvesting and processing methods， so as to provide reference for the development and utilization of the famous classical formulas containing AR. Through the herbal textual research， AR was first published in Shennong Bencaojing， and has been used as the proper name for this herb for generations， and the mainstream source of AR used for generations is the rhizome of Anemarrhena asphodeloides. The high-quality production areas that have been revered throughout the ages are Hebei， Shanxi， Shaanxi， Inner Mongolia and Fangshan district of Beijing， etc. In recent times， AR produced in Yixian county of Hebei province（Xiling Zhimu）， is better known and is regarded as a very good source. At present， cultivated AR is mainly produced in Yixian county and Anguo of Hebei province， Bozhou of Anhui province and other places. The medicinal parts of AR in ancient and modern times are all rhizomes， and the quality is better if it has thick flesh， hard wood， yellow outer color and white section color. The harvesting time recorded in ancient medical books is usually in lunar February and August， with exposure to dryness， while modern harvesting is spring and autumn. The processing methods of the past dynasties were mainly to remove the hair when using， avoid iron when cutting， process with wine or salt water， while the two main specifications in modern times are raw and salted products. Based on the systematic research， it is recommended that the dried rhizome of A. asphodeloides in the famous classical formulas be used for AR. If the original formula specifies processing requirements， it should be operated according to the requirements， if the processing requirements are not indicated， the raw products can be used as medicine.

Network pharmacological analysis and experimental validation of Anemarrhenae Rhizoma in treating Alzheimer 's disease / 浙江大学学报·医学版

OBJECTIVES@#To explore the mechanism of action of Anemarrhenae Rhizoma in treatment of Alzheimer's Disease (AD) through network pharmacology analysis and experimental validation.@*METHODS@#The active ingredients and targets of Anemarrhenae Rhizoma for treatment of AD were screened with network pharmacological methods, the protein-protein interaction (PPI) network was constructed and the core targets were analyzed, enriching Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis was performed. The peripheral blood lymphocytes were extracted and lymphoblast like cell lines (LCL) were constructed and an in vitro cell model of LCL-SKNMC was established. MTT/CCK-8 method was used to detect the activity of SKNMC/LCL cells, 2 ´, 7 ´-dichlorodihydrofluorescein diacetate (DCFH-DA) probe was used to detect reactive oxygen species (ROS) generated, and immunofluorescence staining was used to detect the generation of Aβ1-42 in the co-cultured model; Western blotting was used to detect protein expression in the co-culture model. The lifespan of N2 nematodes was observed under oxidative stress, normal state, and heat stress; ROS generated by N2 nematodes was detected by DCFH-DA probes. The paralysis time of CL4176 N2 nematodes was evaluated by paralysis assay, and Aβ deposition in the pharynx was detected by Thioflavin S (Th S) staining.@*RESULTS@#Through network pharmacology, 15 potential active ingredients and 103 drug-disease targets were screened out. PPI analysis showed that the Anemarrhenae Rhizoma might play anti-AD roles through albumin, Akt1, tumor necrosis factor, epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGFA), mammalian target of rapamycin (mTOR), amyloid precursor protein (APP), glycogen synthase kinas (GSK) 3β and other related targets. KEGG analysis showed that the pharmacological effects of Anemarrhenae Rhizoma might involve the biological processes of Alzheimer's disease, endocrine resistance, insulin resistance; and neuroactive ligand-receptor interaction, phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, calcium signaling pathway, AGE-RAGE signaling pathway in diabetes complications, neurotrophic factor signaling pathway and others. The in vitro cell experiments showed that Anemarrhenae Rhizoma was able to reduce the production of ROS and Aβ1-42 (all P<0.01), inhibit the expression of β-secretase 1 (BACE1), APP and Aβ1-42 proteins (all P<0.05), up-regulate the expression of p-PI3K/PI3K, p-AKT/AKT, p-GSK3β/GSK3β in SKNMC cells (all P<0.05). Studies further confirmed that Anemarrhenae Rhizoma prolonged the lifespan of C. elegans under stress and normal conditions, reduced the accumulation of ROS and the toxicity of Aβ deposition.@*CONCLUSIONS@#Anemarrhenae Rhizoma may reduce the production of Aβ in AD and inhibit its induced oxidative stress, which may be achieved by regulating the PI3K/AKT/GSK-3β pathway.

Anti-diabetic Effect of Zishenwan and Its Herbal Components： A Review / 中国实验方剂学杂志

Zishenwan， also known as Tongguanwan， is composed of three herbs：Anemarrhenae Rhizoma， Phellodendri Chinensis Cortex， and Cinnamomi Cortex， which thus is thought to be the representative formula to clear heat， purge fire， nourish Yin， and tonify Qi， and it is often used for treating anuresis and renal arthralgia. In recent years， this formula has become a commonly used combination of herbs and is used to treat diabetes （consumptive thirst disease） diagnosed with "lower consumption" syndrome. This article systematically reviewed the development of traditional Chinese medicine （TCM） theory for Zishenwan. Moreover， based on modern pharmacological research， the previous studies on the components of Zishenwan， the improvement of diabetes-related diseases by Zishenwan， and the relationship between the single herd of Zishenwan and the treatment of diabetes were summarized， and the chemical components and the mechanisms for treating diabetes by the three herbs were discussed. It is found that Zishenwan can alleviate diabetes and diabetic nephropathy by performing anti-inflammation， enhancing insulin sensitivity， and inhibiting pyroptosis of renal tubular epithelial cells. Three herbs in Zishenwan and several components of them， including mangiferin， timosaponins， berberine， jatrorrhizine， cinnamaldehyde， and cinnamic acid can ameliorate diabetes and maintain stable glycometabolism by a variety of mechanisms such as improving insulin resistance in insulin target tissues， suppressing inflammation， anti-oxidation， regulating lipid metabolism， enhancing insulin secretion， and regulating gut microbiota. This review provides a theoretical foundation and reference for subsequent studies on the mechanisms of the anti-diabetic effect of Zishenwan.

Simultaneous determination of five components in Anemarrhenae Rhizoma by HPLC-ELSD / 药学实践杂志

Objective To establish a method for the simultaneous determination of new mangiferin, mangiferin, artemisinin BⅡ, icariin and artemisinin A in Anemarrhenae Rhizoma by high performance liquid chromatography-evaporation light scattering detector (HPLC-ELSD). Methods The column was Agilent Poroshell 120 EC-C18. The mobile phase used acetonitrile-0.2% acetic acid water system with gradient elution. Column temperature was 30 ℃. Flow rate was 0.7 ml/min. Evaporative light scattering detector used nitrogen as atomizing gas. The atomizing gas temperature was 40 ℃ and the drift tube temperature was 90 ℃. The nitrogen volume flow rate was 2.00 L/min and the sample volume was 20 μl. Results The five components were able to achieve baseline separation. Neomangiferin, Mangiferin, Anemaponin BⅡ, Baohuoside I , Anemarrhena saponin AⅢwere determined as 24.1-386 μg/ml (r=0.999 3), 23.2-371 μg/ml (r=0.998 6), 54.2-867.2 μg/ml(r=0.995 6), 5.3-84.8 μg/ml (r=0.996 8), 10-160 μg/ml (r=0.998 9) respectively, which showed a good linear relationship within the concentration range. The average recovery rate of the five components was between 101.8% and 105.0%, and the repeatability RSD was less than 2.4%. The content of the above five components in Zhimu medicinal materials were 1.62%, 0.82%, 7.36%, 0.07%, 0.34%, respectively. Conclusion The method is simple, accurate, and highly sensitive, which could be used as the quantitative determination of multiple index components of Anemarrhenae Rhizoma.

Effect of Compatibility of Anemarrhenae Rhizoma-Phellodendri Chinensis Cortex Couplet Medicines on Glucolipid Metabolism in Type 2 Diabetic Rats Before and After Salt-processing / 中国实验方剂学杂志

Objective:To investigate the effect of compatibility of Anemarrhenae Rhizoma-Phellodendri Chinensis Cortex couplet medicines on glucolipid metabolism in type 2 diabetic rats before and after salt-processing. Method:The type 2 diabetic rat model was induced by high-fat and high-glucose diet combined with low dose streptozotocin （STZ）， the model rats were randomly divided into six groups， including the model group， metformin group （200 mg·kg^-1）， and different compatibility groups of raw and salt-processed of Anemarrhenae Rhizoma and Phellodendri Chinensis Cortex （6.48 g·kg^-1）. In addition， The same week old rats fed with normal diet were set as the blank group. After 30 d of continuous intragastric administration， changes of fasting blood glucose （FBG）， fasting serum insulin （FINS）， glycosylated serum protein （GSP）， hepatic glycogen， blood lipid ［total cholesterol （TC）， triglyceride （TG）， low density lipoprotein cholesterol （LDL-C）， high density lipoprotein cholesterol （HDL-C）］， nonesterified fatty acid （NEFA）， adipocytokines ［adiponectin （ADP） and leptin）］， kidney function ［blood urea nitrogen （BUN） and creatinine （CRE）］ and other indicators of rats from different groups were detected， and the insulin sensitivity index （ISI） and insulin resistance index （HOMA-IR） were calculated， hematoxylin-eosin （HE） staining was used to observe the morphological changes of pancreas， liver and kidney of rats from different groups. Result:Compared with the model group， compatibility of Anemarrhenae Rhizoma-Phellodendri Chinensis Cortex couplet medicines before and after salt-processing all could decrease the levels of FBG， GSP， TC， TG， LDL-C， NEFA， leptin， BUN， CRE and HOMA-IR， and increase the contents of FINS， HDL-C， ADP， hepatic glycogen and ISI， among which the compatibility of salt-processed Anemarrhenae Rhizoma and salt-processed Phellodendri Chinensis Cortex had the most significant effect on regulating glucolipid metabolism in type 2 diabetic rats. The compatibility of all couplet medicines could improve the histopathological changes of pancreas， liver and kidney in type 2 diabetic rats， among which the compatibility of salt-processed Anemarrhenae Rhizoma and salt-processed Phellodendri Chinensis Cortex had the most prominent effect on repairing pathological damage. Conclusion:The compatibility of Anemarrhenae Rhizoma and Phellodendri Chinensis Cortex before and after salt-processing can improve glucolipid metabolism in type 2 diabetic rats， while the comprehensive effect of salt-processed Anemarrhenae Rhizoma and salt-processed Phellodendri Chinensis Cortex<italic> </italic>on lowering glucose and regulating lipid is the best.

Analysis on Changes of Chemical Composition Contents and Efficacy of Improving Insulin Resistance in Type 2 Diabetic Mice of Anemarrhenae Rhizoma Before and After Processing with Salt-water / 中国实验方剂学杂志

Objective:By comparing the changing of chemical composition contents and the effects of improving insulin resistance in type 2 diabetic KKAy mice， to explore the processing principle of Anemarrhenae Rhizoma processed with salt-water. Method:Ultra-performance liquid chromatography-tandem mass spectrometry （UPLC-MS） was established for determining the contents of seven saponins and mangiferin in raw and salt-processed products of Anemarrhenae Rhizoma. The mobile phase was 0.1% formic acid aqueous solution （A） and acetonitrile （B） for gradient elution （0-1 min， 90%-80%A； 1-2 min， 80%-78%A； 2-5.5 min， 78%-70%A； 5.5-10.5 min， 70%-40%A； 10.5-12 min， 40%-20%A； 12-12.1 min， 20%-90%A； 12.1-13 min， 90%A）. The flow rate was set at 0.3 mL·min-1. The mass spectrographic analysis employed electrospray ionization （ESI） and negative ion acquisition mode. The acquisition range was m/z 100-1 200. The experimental type 2 diabetic KKAy mice were divided into model group， Anemarrhenae Rhizoma group （7.2 g·kg-1·d-1） and Anemarrhenae Rhizoma processed with salt-water group （7.2 g·kg-1·d-1）. C57BL/6J mice were considered as normal group and were given the same volume of saline. There are nine mice in each group， once a day for 21 consecutive days. The fasting blood glucose （FBG） was measured once a week. Three hours after the last administration， the blood samples of mice were collected by drawing eyeballs and were centrifuged to separate serum for further experiment. The fasting insulin （FINS）， leptin （LEP）， glycated hemoglobin （HbA1c）， glycated albumin （GA）， glucagon-like peptide-1 （GLP-1） levels in serum were detected by enzyme-linked immunosorbent assay （ELISA）. The insulin sensitivity index （ISI） and the homeostasis model assessment insulin-resistance （HOMA-IR） were calculated. The expressions of phosphatidylinositol 3-kinase （PI3K）， phosphoenolpyruvate carboxylkinase （PEPCK） and peroxisome proliferator activated receptor gamma co-activator1 （PGC1） mRNA in hepatic and adipose tissue of mice from each group were detected by real-time fluorescence quantitative polymerase chain reaction method （Real-time PCR）. Result:After being processed with salt-water， the contents of 8 chemical components in Anemarrhenae Rhizoma were increased， among which the contents of timosaponin AⅢ， timosaponin BⅡ， timosaponin BⅢ， anemarrhenasaponin Ⅰ， anemarrhenasaponin Ⅰa， mangiferin were significantly increased， and increased by 43.78%， 38.77%， 25.84%， 28.21%， 22.51%， 24.04%， respectively. Compared with the model group， raw and salt-processed products of Anemarrhenae Rhizoma could significantly decrease the levels of FBG， FINS， HOMA-IR， HbA1c， LEP， GA （P<0.05， P<0.01）， increase the levels of ISI， GLP-1 （P<0.05， P<0.01） in serum of mice with type 2 diabetes， and significantly increase the expression of PI3K and PGC1 mRNA in hepatic and adipose tissue （P<0.05）. It is worth noting that salt-processed products is better than that of raw products. Conclusion:Raw and salt-processed products of Anemarrhenae Rhizoma have obvious hypoglycemic effect. And the hypoglycemic effect of Anemarrhenae Rhizoma can be promoted after being processed with salt-water by promoting insulin secretion and improving insulin resistance. Incremental components are the probably material basis for enhancement of hypoglycemic effect of Anemarrhenae Rhizoma after being processed with salt-water.

Improvement researches on quality standard of Anemarrhenae Rhizoma and its raw processed products / 中国中药杂志

This study is to improve the quality standard and supply the scientific basis for Anemarrhenae Rhizoma and its raw processed products. Steroidal saponin including timosaponin BⅡ, timosaponin AⅢ and flavonoids including neomangiferin and mangiferin were selected as the indicative components. Silica gel G thin layer chromatography(TLC) and polyamide TLC were used to detect the two types of compounds, respectively. The contents of timosaponin BⅡ and timosaponin AⅢ were determined by HPLC-ELSD and the content of neomangiferin, mangiferin and isomangiferin were determined by HPLC-UV. Moisture, total ash and acid insoluble ash were determined according to Chinese Pharmacopoeia(2015 edition). And 80% ethanol was selected as the solvent and the content determination of total extract were determined. The fingerprints of Anemarrhenae Rhizoma and its raw processed products were established by HPLC-UV and HPLC-ELSD. The results showed that the methods of TLC and HPLC have been successfully stablished. There are 2 and 3 peaks which have been identified by HPLC-ELSD and HPLC-UV, respectively. The HPLC fingerprint methods are specific and can be used to identify and quality control for Anemarrhenae Rhizoma and its raw processed products in the mass. Comparing to Chinese Pharmacopoeia(2015 edition), the TLC identification and content determination were revised and the total extract determination and HPLC fingerprints were added in the present study. Our results can be used as the scientific basis of quqlity control for Anemarrhenae Rhizoma and its raw processed products.

Molecular docking combined with network pharmacology to study molecular mechanism of Guizhi Shaoyao Zhimu Decoction in treatment of rheumatoid arthritis / 中草药

Objective: To predict the active constituents and targets of Guizhi Shaoyao Zhimu Decoction (GSZD) in the treatment of rheumatoid arthritis by using molecular docking and network pharmacology, and to analyze the effect of multi component-multi target-multi pathway combined with the theory of compatibility of TCM prescriptions. Methods: The main chemical constituents of nine kinds of Chinese herbal medicines (Cinnamomi Ramulus, Paeoniae Radix Alba, Anemarrhenae Rhizoma, Glycyrrhizae Radix et Rhizoma, Ephedrae Herba, Zingiberis Rhizoma Recens, Atractylodis Macrocephalae Rhizoma, Saposhnikoviae Radix and Aconiti Lateralis Radix Praeparata) were collected from TCMSP, TCM-Datebas@Taiwan and PubChen Compound database. The protein targets to the treatment of rheumatoid arthritis are found through DrugBank and TTD databases and uploaded to the String online database to build the network relationship of protein interaction. Appropriate crystal structures of protein targets were downloaded from PDB database, and molecular docking between compounds and targets was performed by using Discovery studio 4.5.0 software. A drug-compound-target visualization network was constructed by using Cytoscape 3.6.1 software to elucidate the main mechanism of GSZD against rheumatoid arthritis. Results: The results of molecular docking showed that there were 316 potential anti-arthritis active components in GSZD, acting on 26 targets, among which MAPK1, ZADH2, P38, AKR1C2, DHODH, CA2, MMP3, MMP9, RANKL, and other proteins were the main targets. Biological function and pathway analysis indicated that the mechanism of GSZD mainly involved in bone absorption (28%), histone kinase activity (20%), peptide tyrosine phosphorylation (20%), prostaglandin metabolism (12%), and other biological processes. The main pathway was osteoclast differentiation (94.12%). Conclusion: In this study, molecular docking combined with network pharmacology was used to study the pharmacodynamic material basis and molecular mechanism of GSZD in the treatment of rheumatoid arthritis from the perspective of multi-target and multi-approach, providing reference and basis for better clinical use.

Protective Effect and Mechanism of Extracts from Ginseng Radix et Rhizoma,Anemarrhenae Rhizoma and Paeoniae Radix Rubra on Hippocampal Neurons in Rats with Vascular Dementia / 中国实验方剂学杂志

Objective: To observe the effect of extracts from Ginseng Radix et Rhizoma,Anemarrhenae Rhizoma and Paeoniae Radix Rubra on N-methyl-D-aspartate receptors(NMDAR1) in hippocampal neurons in rats with vascular dementia and investigate its possible mechanism. Method: The 60 SPF male rats were randomly divided into normal group, sham-operated group,model group, traditional Chinese medicine group(0.20 g·kg-1)and memantine group(2.1 mg·kg-1),with 12 rats in each group. The model was established by repeated ischemia-reperfusion combined with intraperitoneal injection of sodium nitroprusside. After modelling, normal group, sham-operated group and model group were dosed the similar volume of normal saline once a day for 14 days. The learning and memory capacity was assessed by Morris water maze; pathologic change in the CA1 district of hippocampus was assessed by hematoxylin-eosin (HE) staining, and the expression level of NMDAR1 in hippocampal neuron membrane protein was detected by Western blot and immunohistochemistry(IHC),the NMDAR1 mRNA in hippocampal tissue was detected by Real-time PCR. Result:Compared with normal and sham-operated group, the latency period was prolonged in model group(PPPPPPPPConclusion:The extracts from Ginseng Radix et Rhizoma,Anemarrhenae Rhizoma and Paeoniae Radix Rubra can improve the learning and memory capacity of rats with vascular dementia, and alleviate the injury in CA1 district of hippocampus. The mechanism may be related to the down-regulation of NMDAR1 expression in hippocampal neurons.

Effects of different Chinese meteria medica pair on regulation of adrenal function in mice with hydrocortisone-induced syndrome / 中草药

Objective: To study the effects of different Chinese materia medica combinations on the regulation of adrenal function. Methods Mice with 1.65 mg/(kg•d) hydrocortisone for 14 d to induce drug-induced syndrome was treated synchronously with assissting yang and dissipating cold (Aconm Lateralis Radix Praeparaia-Cinnamomi Cortex), nourishing yin and reducing fire (Anemarrhenae Rhizoma-Phellodendri Chinensis Cortex), tonifying qi and promoting fluid production (Ginseng Radix et Rhizoma-Astragali Radix), and tonifying blood and benefiting spirit (Rehmanniae Radix Praeparata-Corni Fructus). The body mass of mice was dynamically observed every day, and the characteristic information such as body mass, holding power, axillary temperature, activity degrees of open field, and infrared temperature were detected by the experimental methodology of mice syndrome differentiation; The mice were sacrificed whose spleen, thymus was weighed and the organ index was calculated. Serum corticosterone, ACTH, adrenaline, and noradrenaline were measured by ELISA. The gene expressions of Star, Cyp11a1, Cyp21a1, Cyp11b1, Cyp11b2, Dbh, Ddc, Pnmt, and Th in the adrenal were detected by real-time fluorescent quantitative PCR. The protein’s expressions of LDLR, SRB1, and StAR were detected by Western blotting. Results:The effects of four different treatments on the reduction of body weight and body temperature caused by hydrocortisone were not significant. The holding power of mice in Aconm Lateralis Radix Praeparaia-Cinnamomi Cortex group was significantly higher than that of hydrocortisone group (P < 0.05). Compared with the hydrocortisone group, the serum corticosterone content of mice in Anemarrhenae Rhizoma-Phellodendri Chinensis Cortex group increased significantly (P < 0.01); The genes expression of Star, Cyp11b1, and Cyp11b2 was significantly up-regulated (P < 0.05, 0.01); The proteins expression of SRBI and StAR were significantly up-regulated. Conclusion: The nourishing yin and purging fire treatment group (Anemarrhenae Rhizoma-Phellodendri Chinensis Cortex) is the best to correct and protect the adrenal cortex function in mice with hydrocortisone induced syndrome at low dose.

Technology of combining field processing and crude drug processing of Anemarrhenae Rhizoma / 中草药

Objective To optimize the method for integration of field processing and crude drug preparation of Anemarrhenae Rhizoma (AR) and to provide scientific evidence for the procedure of AR. Methods Four main factors including drying temperature of the fresh herbal medicine, the water content of the medicine, thickness of the products, and the drying temperature of decoction pieces were studied by using single factor experiment and orthogonal test. The content of timosaponin B-Ⅱ and mangiferin and the appearance of the products were selected as evaluation indexes to optimize the integral processing of AR by using comprehensive evaluating method. The changing of the body temperature of yeast-induced rat model was used to compare the antipyretic activity between the two processing technologies (the integration technology and traditional technology). The content of the blood glucose, insulin, and glycosylated serum proteins (GSP) in the blood of the STZ-induced diabetic rat model were selected as the evaluation indexes to compare the hypoglycemic activity between the two processing technologies. Results AR was dried at 50 ℃ for 11 h (water content of medicinal materials was 45%—50%), and then the flosses were knocked off in the drum for 30 min. The results showed that 40—50 ℃ was the best drying temperature, 45%—50% was the best water content, and then unhairing process were conducted for AR for 30 min. The thickness of the slice was 4 mm and the best drying temperature was 50 ℃. There were no significant differences in chemical composition and hypoglycemic activity between the integration technology and traditional technology, while the antipyretic effect of the integrated processing was better than that of the traditional technology. Conclusion The technology of integration of field processing and crude drug processing is feasible and it can be used in industrial production.

Analysis of chemical markers in sulfur-fumigated Anemarrhenae Rhizoma / 中国中药杂志

Sulfur-containing Anemarrhenae Rhizoma decoction pieces were prepared by using sulfur-fumigating procedure. The difference components before and after sulfur fumigation in Anemarrhenae Rhizoma were analyzed and on-line identified by UPLC-Q-TOF-MSE combined with UNIFI informatics platform, principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) respectively. As a result, 16 major differences components were identified, and among them, 9 components were mainly from sulfur-fumigated samples. The main chemical markers in sulfur-fumigated Anemarrhenae Rhizoma were identified as the sulfite derivatives newly produced after sulfur-fumigating. Meanwhile, UPLC-Q-TOF-MSE was used to find the main chemical marker anemarrhena saponin BⅡ sulfite (m/z 983). By using this method, a rapid screening method for sulfur-fumigated Anemarrhenae Rhizoma was established. This was a convenient and accurate detection method for sulfur dioxide residue, and it can be used as an effective assistant method to control the quality of Anemarrhenae Rhizoma. At the same time, it was the first time to identify sulfited derivatives of steroidal saponins, and screen the sulfur-fumigated Anemarrhenae Rhizoma.

Determination of eight constituents in Anemarrhenae Rhizoma before and after salt processing by HPLC-MS / 中草药

Objective: To establish an HPLC-MS method for the simultaneous quantitation of neomangiferin, mangiferin, timosaponin E1, timosaponin BII, timosaponin BIII, anemarrhenasaponin I, timosaponin AIII, and timosaponin AII in Anemarrhenae Rhizoma, and to analyze the content change before and after salt processing. Methods: Chromatography was performed on Agilent SB-C18 (50 mm × 2.1 mm, 1.8 μm) column by gradient elution using 0.05% formic acid in water (A)-acetonitrile (B) at the flow rate of 0.4 mL/min. The column temperature was kept at 30 ℃ and the inject volume was 10 μL. HPLC-MS was used for quantification in negative ion mode under ESI. Results: The eight components had good linearity (r ≥ 0.9997) within the linear range. The average recovery rate was 97.55%-103.01% with RSD < 2.80%. The contents of the eight components in Anemarrhenae Rhizoma varied according to the different habits. The contents of mangiferin, timosaponin BII, and timosaponin AIII were high. After salt processing, contents of timosaponin E1, timosaponin BII, and anemarrhenasaponin I were reduced, content of timosaponin BIII was increased and contents of neomangiferin, mangiferin, timosaponin AIII, and timosaponin AII had no obvious changes. Conclusion: The changes of components in Anemarrhenae Rhizoma before and after salt processing are proved by the method of HPLC-MS which is simple, sensitive, and accurate.

Effects and mechanism of Anemarrhenae Rhizoma-Phellodendri Chinensis Cortex medicine effective parts to improve cognitive impairment in type 2 diabetic rats / 中草药

Objective To study the effects of Anemarrhenae Rhizoma-Phellodendri Chinensis Cortex medicine effective parts on phosphatidyl inositol 3 kinase (PI3K)/protein kinase B (Akt) signaling pathway and explore its mechanism of action to improve brain cognitive of type 2 diabetes rats. Methods A total of 50 male SPF SD rats, divided into five groups with 10 for each group, named as control group, model group, high/low dosage of Anemarrhenae Rhizoma-Phellodendri Chinensis Cortex medicine effective parts (ZBH and ZBL) group, and Ebixa (MHT) treatment group; The rats were immunized with ip of streptozotocin (STZ) jointly fed high-fat diet for three weeks. Then the rats were continuously gavaged with ZBH, ZBL, and MHT for 20 weeks, the serum Aβ1-42 levels were determinated in 8th and 16th week respectively, the changes of the cognitive impairment were analyzed, combined with Barnes maze and Morris water maze to detects the cognitive ability of each group rats; At the end of 20 weeks of administration, dissecting and preservatting the rat pancreatic tissue, the hippocampus to made into routine pathological sections and brain tissue pathology morphology inspection and PI3K, Akt, and Bcl-2 mRNA expression by fluorescence quantitative PCR method detection. Results After treatment for 8 weeks and 16 weeks, compared with control group, Aβ1-42 levels of model group were significantly increased (P < 0.05, 0.01); Administration 20 weeks, compared with control group, pathological section results showed normal hippocampus cells arranged in neat rows, morphological rules, and color is very even. DM models of hippocampal tissue cells arranged scattered through the administration significantly after repair. Compared with model group, ZBH and ZBL could effectively improve the form of neurons and cell arrangement of rat's hippocampus, repairing nerve injury; Compared with control group, PI3K and Akt mRNA expression of hippocampal tissue of model group rats decreased, Bcl-2 mRNA expression was abate (P < 0.05). After delivery, MHT and ZBH group could significantly improve the level of PI3K, Akt, and Bcl-2 mRNA expression (P < 0.05, 0.01). Conclusion Anemarrhenae Rhizoma-Phellodendri Chinensis Cortex medicine effective parts can significantly reduce the accumulation of Aβ1-42 protein, and decreased the expression of PI3K and Akt, indicating that the effects of improvement of cognitive impairment in type 2 diabetic rats may be achieved through regulation of PI3K/Akt pathway.

HPLC fingerprint of Yinqiao Qingre Tablet / 中草药

Objective: To establish HPLC fingerprint for Yinqiao Qingre Tablet (YQT), and analyze the chemical composition of YQT by ESI-Q-TOF MS. Methods: The Kromasil C18 column (250 mm × 4.6 mm, 5 μm) was used with a mobile phase of acetonitrile-0.1% phosphoric acid in gradient elution, the flow rate was 1.0 mL/min, the column temperature was 25℃, and the detection wavelength was 230 nm. Ultra performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF/MS) was used for qualitative analysis in the positive and negative ion modes. Results: The HPLC fingerprint for 11 batches of YQT and 28 common peaks were obtained, belonging to six medicinal herbs. Among them, seven common peaks came from Forsythiae Fructus, nine common peaks came from Lonicerae Japonicae Flos, six common peaks came from Puerariae Lobatae Radix, three common peaks came from Arctii Fructus, one common peak (peak 2) came from Forsythiae Fructus and Cimicifuga Rhizoma, another two common peaks came from Anemarrhenae Rhizoma and Cimicifuga Rhizoma, respectively. The similarity of 11 batches of YQT was over 0.95.Totally 42 chemical components were identified by UPLC-Q-TOF/MS, 16 of which were identified by references, such as puerarin, daidzin, daidzein, neochlorogenic acid, chlorogenic acid, cryptochlorogenin acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, secoxyloganin, rutin, phillyrin, forsythoside A, arctiin, mangiferin, and timosaponin B II, respectively. Conclusion: This study perfects the system of quality standard and provides the reference for the study of substance basis and quality control of the same type of compound preparations.

Research on HPLC-ELSD fingerprint of Anemarrhenae Rhizoma / 中草药

Objective: To provide the HPLC-ELSD fingerprint analysis method of Anemarrhenae Rhizoma, and to reflect and comprehensively control the quality of Anemarrhenae Rhizoma. Methods: The HPLC-ELSD separation was performed on a C8 analytical column (4.6 mm × 250 mm, 5 μm) gradient eluted with a mixture consisting of acetonitrile (A) and 0.2% acetic acid-water (B) at a flow rate of 1 mL/min with ELSD detector. Using a linear gradient of 0 min, 5% A; 5 min, 7% A; 5-15 min, 15% A; 15-36 min, 22% A; 36-43 min, 35% A; 43 min, 40% A; 51 min, 50% A; 51-60 min, 100% A. The temperature of column was 25 ℃. The flow rate of ELSD detector atomizer (air) was 2.6 mL/min. The temperature of the drift tube was 100 ℃ and the injection volume was 20 μL. Results: The fingerprint with better separation effect by using gradient elution method within 60 min was established, and also the 12 peak which are common peak were determined. The most of chemical components of Anemarrhenae Rhizoma were chromatographic separated in this HPLC-ELSD fingerprint. Conclusion: The method is simple and reliable, and in this way for the steroidal saponins and flavonoids of Anemarrhenae Rhizoma can be identified at the same time. Moreover, it can fully reflect the characteristics of the chemical composition of Anemarrhenae Rhizoma, therefore it can be used as an effective quality control method for Anemarrhenae Rhizoma.

Rapid Determination of Timosaponin B Ⅱ in Anemarrhenae Rhizomaby NIRS with HPLC / 世界科学技术-中医药现代化

This study was aimed to establish a rapid detection method for timosaponin BⅡ in Anemarrhenae Rhizoma in order to determine its concentration quickly,conveniently and efficiently.The concentration of timosaponin BⅡ in A.Rhizomadetected by HPLC in the Chinese Pharmacopeia was used as the actual measured value.The near-infrared spectroscopy (NIRS) was used to collect the spectrogram of A.Rhizomasamples.The partial least squares (PLS) of TQ Analyst 8.0 were used in the data analysis.Through the pretreatment,wavelength range and principal component number selection,the actual measured value and NIRS information were associated for the establishment of the optimal quantitative analysis model of timosaponin BⅡ.The results showed that the correlation coefficients (R2),root-mean-square error of calibration (RMSEC),root-mean-square error of prediction (RMSEP),root-mean-square error of cross-validation (RMSECV) and the performance index (PI) of the established model were 0.975 15,0.094 2,0.080 0,0.369 20,and 91.0,respectively.It was concluded that the established quantitative analysis model by NIRS with HPLC was able to determine the concentration of timosaponin BⅡ in A.Rhizomaquickly and accurately.

Separation and Content Comparison of Vitexin from Anemarrhenae Rhizoma Before and After Salt Processing / 中国中医药信息杂志

Objective To study the chemical components of Anemarrhenae Rhizoma;To establish a method to determine the content of vitexin;To compare the content difference of vitexin from Anemarrhenae Rhizoma before and after salt processing. Methods 70% ethanol extract of Anemarrhenae Rhizoma was separated and purified by silica gel column chromatography technique, and compound was identified by physicochemical properties and spectral data. HPLC was used with Ecosil column (4.6 mm×150 mm, 5μm), mobile phase of acetonitrile-0.2%acetic acid solution (14.5∶85.5), velocity of 1.0 mL/min, and determine wavelength of 340 nm. Results The compound was vitexin, a good linearity (r=0.999 8) in the range of 0.039 8-1.99μg. The average recovery rate was 98.02%, RSD=0.21%. Vitexin content was 0.008 5% in crude Anemarrhenae Rhizoma, 0.008 1% in processed products. Conclusion This is the first time separation of vitexin from Anemarrhenae Rhizoma. The method for content determination of vitexin is accurate, specific, and highly sensitive in the present experiment. Content of vitexin decreases slightly in processed products of Anemarrhenae Rhizoma compared with crude Anemarrhenae Rhizoma.

UPLC determination of six active components including mangiferin in Anemarrhenae Rhizoma / 中国药学杂志

OBJECTIVE: To establish an analytical method to determine 7-O-glucopyranosylmangiferin, mangiferin, timosapon-inB II, 2, 6, 4'-trihydroxy-4-methoxybenzophenone, broussonin B and cis-hinokiresinol in Anemarrhenae Rhizoma quickly and accurately. METHODS: A UPLC method was established on an HSS T3 column (2.1 mm × 100 mm, 1.8 μm). The mobile phase consisted of 0.03% phosphoric acid solution and acetonitrile eluted in gradient mode, and the detection wavelength was set at 192 and 210 nm. RESULTS: The standard curves of 7-O-glucopyranosylmangiferin, mangiferin, timosaponin B II, 2, 6, 4'-trihydroxy-4-methoxy-benzophenone, broussonin B and cis-hinokiresinol showed good linearity in 15.00-299.6 μg · mL-1, 8.80- 175.6 μg · mL-1, 40.00-800.0 μg · mL-1, 2.300- 45.60 μg · mL-1, 4.200-8.50 μg · mL-1 and 0.400-9.00 μg · mL-1, respectively. The mean recoveries (n=9) of the 6 components were 97.7%-100.6% with RSD of 1.2%. CONCLUSION: The method is quick and accurate and can be used to determine 7-O-glucopyranosylmangiferin, mangiferin, timosaponin B II, 2, 6, 4'-trihydroxy-4-methoxy-benzophenone, broussonin B and cis-hinokiresinol in Anemarrhenae Rhizoma.