ABSTRACT
The many-banded krait, Bungarus multicinctus, has been recorded as the animal resource of JinQianBaiHuaShe in the Chinese Pharmacopoeia. Characterization of its venoms classified chief phyla of modern animal neurotoxins. However, the evolutionary origin and diversification of its neurotoxins as well as biosynthesis of its active compounds remain largely unknown due to the lack of its high-quality genome. Here, we present the 1.58 Gbp genome of B. multicinctus assembled into 18 chromosomes with contig/scaffold N50 of 7.53 Mbp/149.8 Mbp. Major bungarotoxin-coding genes were clustered within genome by family and found to be associated with ancient local duplications. The truncation of glycosylphosphatidylinositol anchor in the 3'-terminal of a LY6E paralog released modern three-finger toxins (3FTxs) from membrane tethering before the Colubroidea divergence. Subsequent expansion and mutations diversified and recruited these 3FTxs. After the cobra/krait divergence, the modern unit-B of β-bungarotoxin emerged with an extra cysteine residue. A subsequent point substitution in unit-A enabled the β-bungarotoxin covalent linkage. The B. multicinctus gene expression, chromatin topological organization, and histone modification characteristics were featured by transcriptome, proteome, chromatin conformation capture sequencing, and ChIP-seq. The results highlighted that venom production was under a sophisticated regulation. Our findings provide new insights into snake neurotoxin research, meanwhile will facilitate antivenom development, toxin-driven drug discovery and the quality control of JinQianBaiHuaShe.
ABSTRACT
OBJECTIVE：To analyze the metabolites of tetrahydroxystilbene glucoside （THSG）and speculate its metabolism pathway in rats. METHODS ：Male SD rats were randomly divided into plasma group （n＝3），urine group （n＝3），bile group （n＝3），and tissue group （n＝9）. Each group was given single dose of THSG 200 mg/kg intragastrically. Plasma samples 10，30 min and 1，1.5，2，4 h after medication ，the unrine 0-6 h after medication ，the bile 0-4 h after medication ，the tissue of heart ， liver，spleen，lung，kidney and stomach 30 min and 1，2 h after medication （3 at each time point ）were collected respectively.After precipitated with methanol ，the metabolites of samples were analyzed and identified by UHPLC-Q-Exactive Orbitrap MS and mass loss filtration （MDF）. Its metabolism pathway was speculated. RESULTS：In the blood ，urine，bile，heart，liver，spleen， lung，kidney，stomach samples ，6，7，11，1，5，1，3，4，4 metabolites were detected ，including two phase Ⅰ（hydrolysis， hydrogenation and hydroxylation ）metabolites，18 phase Ⅱ（glucuronic acid binding and sulfation ）metabolites. There were 12 glucuronic acid binding products. CONCLUSIONS：Most of the metabolites of THSG are found in bile ，mainly glucuronic acid binding products of phase Ⅱ metabolite THSG ； main metabolic pathways involve glucose hydrolysis ， hydrogenation， hydroxylation，glucuronic acid binding and sulfation.
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OBJECTIVE：To establ ish the fingerprint of Sojae Semen Nigrum and content determination method of 5 kinds of isoflavones，so as to provide reference for controlling its quality better. METHODS ：HPLC method was adopted to establish the fingerprint and detect the contents of 5 kinds of isoflavones. The determination was performed on Phenomenex C 18 column with mobile phase consisted of acetonitrile- 0.12％ formic acid solution （gradient elution ）at the flow rate of 1 mL/min. The detection wavelength was set at 260 nm；the column temperature was 30 ℃ and sample size was 10 μL. Using daidzin as reference，HPLC fingerprints of 12 batches of samples were determined. The similarity of 12 batches of samples was evaluated by TCM Chromatographic Fingerprint Similarity Evaluation System （2012A） to confirm common peak. Cluster analysis and principal component analysis were performed by using SPSS 20.0 software and SIMCA 13.0 software. RESULTS ：There were 19 common peaks in HPLC fingerprints of 12 batches of samples ，the similarity of which was higher than 0.94. Totally 5 components were identified，such as daidzin ，glycitin，genistin，daidzein，genistein. Cluster analysis showed that 12 batches of Sojae Semen Nigrum were clustered into 2 categories，i.e. S 1-S3 clustered into one category ，and S 4-S12 clustered into the other category. By principal component analysis ，the contribution rates of two principle components were 53.261％ and 40.715％；accumulative contribution rate was 93.976％. The linear range of above 5 components were 5.97-191.00 µg/mL（r＝0.999 9），1.05-33.46 µg/mL（r＝0.999 9）， 8.93-285.61 µg/mL（r＝0.999 5），0.82-26.33 µg/mL（r＝0.999 9），0.93-29.64 µg/mL（r＝0.999 7），respectively. The limits of quantitation were 0.881 1，0.611 6，0.078 6，0.243 3，0.511 6 μg/mL，respectively. The limits of detection were 0.264 3，0.244 7， 0.021 4，0.124 8，0.106 7 μg/mL，respectively. RSDs of precision ，stability，reproducibility and durability tests were all lower than 5％. Recoveries were 95.15％-96.56％（RSD＝0.51％，n＝6），98.52％-103.45％（RSD＝1.88％，n＝6），95.37％-97.91％ （RSD＝0.95％，n＝6），99.75％-102.00％（RSD＝0.78％，n＝6），100.26％-103.65％（RSD＝1.21％，n＝6）. Among 12 batches of Sojae Semen Nigrum ，the contents of above 5 components were 0.178 3-0.265 9，0.021 7-0.096 2，0.288 5-0.597 2，0.014 1- 0.058 8，0.012 9-0.082 9 mg/g. CONCLUSIONS ：Established HPLC fingerprint and content determination method of 5 kinds of isoflavones can be used for quality control of Sojea Semen Nigrum. The Isoflavone components are similar ，but the contents are different among Sojae Semen Nigrum from different producing areas.
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In this study,DNA molecular identification technology and chemical fingerprint method were adopted to evaluate the quality system of precise powder decoction pieces (PPDP) of S.suberectus dunn (SSD).ITS2 sequence was taken as DNA barcode to indentify SSD.Different specifications of PPDP were prepared,their dry extract contents were quantified in contrast with that of original slices.Three batches of SSD original slices were gleaned and the content uniformity,fingerprint and similarity evaluation before and after the mixing and pulverization were valued by HPLC-DAD.As a result,ITS2 successfully and accurately identified the SSD in this study.The extract rate of PPDP was 15.5％,1.11 times as much as the original slices.RSD of inter-assay dissolution of cepicatechin from the original slices was 11.0％,which was reduced to 1.0％ after mixing and preparing into PPDP.The relative peak area of the 14 common peaks identified by fingerpringts were larger,while the RSD values significantly decreased.It was concluded that the PPDP of SSD improved the extraction efficiency and uniformity of the original slices,featuring quite prospective in more reasonable and scientific clinical use.
ABSTRACT
This study aimed at investigating the drug preparation of precise powder decoction pieces (PPDP) system,Fallopia multiflora radix preparata (FMRP) was employed in this study.Different specifications of PPDP were prepared,their extract rates were in contrast with the original pieces.Compared the quality uniformity of three batches between FMRP original slices and its PPDP extraction,the similarity of the chemical fingerprints was evaluated,and the contents of common peaks and quality uniformity were compared by relative peak areas.ITS2 sequence was taken as a DNA barcode to identify F.multiflora radix (FMR).As a result,the extract rate of PPDP was 2.5 times as much as the original slices.The average content of stilbene glucoside from the three original slices and the PPDP extraction were 3.56 ± 2.61 and 13.23 ± 0.37 mg·g-1,respectively;while the RSD were 73.28％ and 2.82％.The similarity of the fingerprints of the PPDP extraction was almost the same as that of the original slices,but the content and the uniformity of the common peaks of the PPDP extraction were significantly improved.Thus,FMR was accurately identified using ITS2 sequences.It was concluded that the PPDP considerably improve the decocting rate and quality uniformity,indicating that PPDP could save resources and improve the clinical efficacy.
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This study aimed at evaluating the quality of precise powder decoction pieces (PPDP) of E.Folium (EF) compared with the traditional commercial slices by chemical fingerprint methods and DNA molecular identification technology.Different specifications of PPDP were prepared,and their dry extract contents were in contrast with that of commercial slices.The slices of EF were identified using ITS2 and psbA-trnH sequences.Three batches of commercial slices were collected,and the content uniformity,fingerprint and similarity evaluation before and after the mixing and pulverization were detected by HPLC-DAD and DNA sequence alignment.It was found that the paste rate of PPDP was slightly higher than that of the traditional commercial slices.The dissolution of chlorogenic acid of PPDP was higher than that of the traditional commercial slices.RSD of inter-assay dissolutions of chlorogenic acid of commercial slices was 15.56％,which was reduced to 6.82％ after mixing and preparing into PPDP.The fingerprints showed that the similarity of the PPDP of EF was elevated with the inceases of 10 marketed common peaks.The PPDP of EF was accurately identified by ITS2 and psbA-trnH sequences.In conclusion,compared with traditional commercial slices of EF,the PPDP apparently improved the dissolution rate and the quality uniformity,demonstrated that the boiled powder of CRP achieved obvious clinic advantages.
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This study aimed at evaluating the quality of the precise powder decoction pieces (PPDP) of L.japonicae Flos (LJF) compared with the traditional commercial slices with chemical fingerprint methods and DNA molecular identification technology.Different specifications of PPDP were prepared,their dry extract contents were in contrast with that of commercial slices.The three batches of commercial slices were collected,and the content uniformity,fingerprint and similarity evaluation before and after the mixing and pulverization were studied by HPLC-DAD and DNA sequence alignment.As a result,the paste rate of PPDP was slightly higher than that of the traditional commercial slices.The dissolution of chlorogenic acid of PPDP was higher than that of the traditional commercial slices.RSD of inter-assay dissolutions of chlorogenic acid of commercial slices was 11.93％,which was reduced to 8.29％ after mixing and preparing into PPDP.The fingerprint showed that the slimilarity of the fringerprint of the mixed and powdered LJF was elevated with 7 common peaks.All the common peaks were increased at different levels.In conclusion,compared with traditional commercial slices of LJF,PPDP apparently improved the dissolution rate and the quality uniformity,indicating that the boiled powder of CRP obviously presented vantages in clinic.
ABSTRACT
This study aimed at comparing the precise powder decoction pieces and market raw TCM slices of P.cacumen over the decocting quality.ITS2 sequence was adopted as a DNA barcode to identify P.cacumen.The chemical composition of the medicinal materials was characterized by HPLC fingerprints for the evaluation of the similarity of precise powder decoction pieces and market TCM slices.The concentrations of quercitrin were determined using UPLC,and the characteristic common peaks were identified.In addition,the extraction efficiency between the market TCM slices and the precise powder decoction pieces was also compared by standard decoction method.It was found that P.cacumen was accurately identified by ITS2 sequences.HPLC fingerprints showed that the extraction efficiency and similarity of the precise powder decoction pieces increased compared with the market TCM slices.However,the extraction yield rate of the precise powder decoction pieces was improved by 20％ increased in accordance with the standard decoction method,while the contents of the index component,quercitrin,presented rare increase and the decocting rates of the other chemical components little change in the study.In conclusion,it was indicated that precise powder decoction pieces improved the extraction efficiency and uniformity in comparison with TCM slices.
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Objective To establish an HPLC method to determine the contents of dauricine in Menispermi Rhizoma from different producing areas. Methods C18 was set as chromatographic column filler, with acetonitrile-water-triethylamine (45:55:0.1) as the mobile phase, 284 nm as the ultraviolet wavelength detection, 1 mL/min as the flow rate, 30 ℃ as the column temperature. HPLC chromatograms of eight different batches of Menispermi Rhizoma were established. Results HPLC testing conditions of Menispermi Rhizoma was established. Within 20-100 μg/mL, there was a good linear relationship between the injection volume of the reference substance and the peak area (r=0.9995). The average recovery of dauricine was 100.30%, RSD=1.000%. The contents of dauricine in Menispermi Rhizoma from different producing areas were different. Conclusion The HPLC method is with sensitivity, accuracy, precision, good reproducibility and simple operation, which can be used as detection method to determine the content of dauricine in Menispermi Rhizoma.