Study on scrophulariaceae radix reference extract and its application in quality control of Scrophulariae Radix / 中国中药杂志

By establishing the preparation process of Scrophulariaceae Radix reference extract(SRRE) and calibrating it, we discussed its feasibility as a substitute for single reference substance in the quality control of Scrophulariae Radix. The SRREs were prepared by solvent extraction method and chromatographic separation technology, and then calibrated with the reference substances of harpagide, angoroside C and harpagoside. The HPLC content determination method of Scrophulariae Radixl was established with SRREs of the known content and the reference substances of harpagide, angoroside C and harpagoside respectively as the control ones. Then the content of three components in Scrophulariae Radix was determined, and the t-test method was used to compare the results of the two methods. With SRRE as references, harpagide, angoroside C and harpagoside were in a good linear relationship(r≥0.999 8) within each range, and the average recovery rate was 98.55% to 100.6%. The t-test results showed that the P values of two determination methods were 0.493, 0.155 and 0.171 for harpagide, angoroside C and harpagoside respectively, indicating no significant diffe-rence between the two methods of content determination. The SRRE can be used as a substitute for the reference in the quality control of Scrophulariaceae Radix. The SRRE can replace the corresponding reference substance for the quality control of Scrophulariae Radix. The results of this study provide new methods and new ideas for the quality evaluation of Scrophulariae Radix, and provide a scientific basis for the application of reference extracts in the quality research of traditional Chinese medicine.

Determination of harpagide and harpagoside contents in Scrophularia ningpoensis from 15 origins / 药学实践杂志

Objective To establish an HPLC method for the simultaneous determination of harpagide and harpagoside content in Scrophularia ningpoensis (SN). Methods An Eclipse C18 column was used for determination of methanol extract of S. ningpoensis with a HPLC-PDA method and mobile phase of acetonitrile-0.03% phosphate solution in a gradient elution manner. The flow rate of mobile phase was 1.0 ml/min, and the detection wavelengths were 210 nm and 280 nm. Results Harpagide and harpagoside contents in SN showed good linear relationships within 0.1020-0.5100 mg/ml (r=0.9999) and 0.0340-0.1700 mg/ml (r=0.9999). Their average recovery rates were 97.44% and 97.08%.The RSDs were 0.93% and 1.24%.. There were significant differences in the contents of harpagide and harpagoside in SN from 15 origins (P<0.01). The content of harpagoside in Sichuan Long-dong, Zhejiang Lin-an, Zhejiang Pan-an and Henan Nan-feng is higher. Conclusion This method is stable, accurate and reproducible and can be used for the quality control of SN.

Isolation and identification of endophytic fungi producing harpagoside and harpagide from Scrophularia ningpoensis / 中国中药杂志

The endophytic fungi from root,main stem,branch and leaf of Scrophularia ningpoensis were isolated from Zhejiang,whether these strains could yield harpagide or harpagoside were tested by HPLC and LC-MS. According to the morphological characteristic and the similarity of the nucleotide sequence of internal transcribed spacer( ITS) between r DNAs,the strains producing harpagide or harpagoside were identified. The results showed that 210 strains were isolated from the samples,which were classified into 9 orders,13 families and 17 genera by morphological study. Harpagide was detected in endogenous fungi ZJ17 and harpagoside was detected in endogenous fungi ZJ25 by HPLC coupled with LC-MS. ZJ17 was identified as Alternaria alternate and ZJ25 was identified as A.gaisen by its morphology and authenticated by ITS( ITS4 and ITS5 regions and the intervening 5. 8 S rDNA region).

Grade quality of Scrophulariae Radix based on fingerprints and quantitative analysis of multi-index / 中草药

Objective In order to provide a scientific basis for the grade quality standard criterion of Scrophulariae Radix, a HPLC-DAD method was established to acquire fingerprint and detect the content of multiple components. Methods The chromatographic separate was achieved on Elipse XDB-C18 (250 mm × 4.6 mm, 5 μm) column, with the temperature of 25 ℃, using acetonitrile (A)-0.03% phosphoric acid water (B) as the mobile phase gradient elution, a flow rate of 1.0 mL/min, and the detection wavelength was set at 207 nm for fingerprint, 210 nm for Harpagide, 280 nm for harpagoside, and 264 nm for cinnamic acid. The injection volume was 10 μL. The fingerprints of different grades of Scrophulariae Radix from 30 batches were evaluated with a chromatographic fingerprint similarity evaluation system (version 2012), and the content of harpagide, harpagoside, and cinnamic acid was also determined at the same time. Results There were seven common peaks in the fingerprints, the RSD values of relative retention time were lower than 2.0% and those of relative peak area were quite different, which indicated that the main chemical compounds can exist stably in Scrophulariae Radix with different content. Compared with control, the results of fingerprintsimilarity were presented as follows: 5% under 0.7, 12.5% arranged 0.7 to 0.8, 40% between 0.8 and 0.9, and another 42.5% exceed 0.9, demonstrated that there was qualitative difference in various batches of Scrophulariae Radix. Also, the quantitative analysis of multi-index showed the differences in three main compounds, the content respectively was 0.18%—2.89% in harpagide, 0.01%—0.35% in harpagoside, and 0.01%—0.24% in cinnamic acid. Conclusion Suggestions were provided in the formulation of new grade quality standard, such as adding the fingerprints and multi-index detection of main chemical components based on the original criterion of classification.

Optimization of steaming technology for decoction pieces of Scrophularia ningpoensis by D-optimal response surface methodology based on AHP- entropy weight method / 中草药

Objective :To determine the weight coefficient and optimize the steaming technology of decoction pieces of Scrophularia ningpoensis (SN). Methods The contents of harpagide, harpagoside, aucubin, acteoside, angoroside C, and cinnamic acid were simultaneously determined by HPLC. The weight coefficient of each component was evaluated by AHP-entropy (analyitc hierarchy process) method. With composite score as index, D-optimal response surface methodology was adopted to investigate the effects of soaking time, steaming time, and drying temperature on the quality of processed products and optimize the processing technology of decoction pieces of SN. Results :Optimal processing parameters were as follows: soaking time was 15.63 min, steaming time was 85 min, drying temperature was 60 ℃, and the synthetical mark was 97.20. Considering the actual situation, the optimum processing technology of SN was obtained by fine-turning the soaking time. The soaking time was 15 min, the steaming time was 85 min, and the drying temperature was 60 ℃. Also, the synthetical mark were 98.53, 99.39, 98.86, and its RSD was 0.47% through the obtained conditions in parallel with three batches of samples. Conclusion: The optimized steaming technology is simple and feasible, which can provide a reference for the steaming of decoction pieces of SN. The method established to simultaneously determine the contents of six components in decoction pieces of SN is rapid and reliable for controlling the quality of decoction pieces of SN.

Simultaneous determination of six constituents in Xuanmai Ganju Granules by HPLC / 中成药

AIM To establish an HPLC method for the simultaneous content determination of six constituents in Xuanmai Ganju Granules (Scrophulariae Radix,Ophiopogonis Radix,Glycyrrhizae Radix et Rhizoma,Platycodonis Radix).METHODS The analysis of 80％ methanol extract of this drug was performed on a 35 ℃ thermostatic ZORBAX SB-C18 column (4.6 mm × 250 mm,5 μm),with the mobile phase comprising of acetonitrile0.1％ phosphoric acid flowing at 1.0 mL/min in a gradient elution manner,and the detection wavelengths were set at 210,250,278 nm.RESULTS Harpagide,liquiritin apioside,liquiritin,harpagoside,cinnamic acid and glycyrrhizic acid showed good linear relationships within the ranges of 2.177-43.539 μg/mL(r =0.999 6),1.713-34.261 μg/mL (r =0.999 5),1.946-38.916 μg/mL(r =0.999 6),2.070-41.395 μg/mL(r =0.999 7),2.06-41.2 pg/mL (r =0.999 6) and 3.623-72.454 μg/mL (r =0.999 6),whose average recoveries (RS-Ds) were96.08％ (2.1％),95.55％ (2.5％),95.04％ (2.6％),94.86％ (2.7％),95.70％ (1.9％) and 95.47％ (1.9％),respectively.CONCLUSION This simple and accurate method can be used for the quality control of Xuanmai Ganju Granules.

Application of QAMS in quality control of Scrophularia Radix / 中草药

Objective To establish a measurement method for the content of five compounds (acteoside, harpagide, harpagoside, angoroside-C, and cinnamic acid) in Scrophularia Radix quantitative analysis multi-components by single-marker (QAMS), and verify the accuracy and feasibility of QAMS in the quality control. Methods Taking harpagide as internal standard substance, the relative correlation factor (RCF) of acteoside, cinnamic acid, harpagoside, and angoroside C was established. And the content of each component in Scrophularia Radix was determined by the above-mentioned RCF. In order to prove the scientificity and feasibility of this method, the results were compared with the external standard method. Results The relative correction factors of acteoside, cinnamic acid, harpagoside, and angoroside-C were 0.068 (RSD = 0.53%), 0.060 (RSD = 0.81%), 0.142 (RSD = 1.17%), 0.197 (RSD = 1.82%). No significant differences were found among the quantitative results of four components in 25 batches of Scrophularia Radix determined by the two methods. Conclusion It is feasible and accurate to evaluate the quality of Scrophularia Radix by QAMS.

Simultaneous Determination of 5 Components in Xiaoer Qingyan Granules by HPLC / 中国药房

OBJECTIVE:To develop a method for simultaneous determination of harpagide,harpagoside,chlorogenic acid, caffeic acid and phillyrin in Xiaoer qingyan granules. METHODS:HPLC method was adopted. The determination was performed on Zorbax SB-C18 column with mobile phase consisted of acetonitrile-0.1% phosphoric acid solution (gradient elution) at a flow rate of 1.0 mL/min. The detection wavelength was set at 210 nm (0-13 min,harpagide),327 nm (13-25 min,chlorogenic acid and caffeic acid),277 nm(25-29 min,phillyrin),210 nm(29-40 min,harpagosid);the column temperature was 30℃,and sam-ple size was 10 μL. RESULTS:The linear ranges of harpagide,harpagosid,chlorogenic acid,caffeic acid and phillyrin were 8.400-168.0 ng(r=0.9996),11.30-226.0 ng(r=0.9998),128.8-257.6 ng(r=0.9993),8.110-162.2 ng(r=0.9996),29.69-593.8 ng(r=0.9994),respectively. LOQs of the 5 components were 33.39,451.2,515.2,324.5,1188 ng/mL;LODs were 8.348, 112.8,128.8,81.12,297.0 ng/mL,respectively;RSDs of precision,stability and reproducibility tests were all lower than 2.0%. The recoveries were 96.39%-98.64%(RSD=0.83%,n=6),96.60%-98.89%(RSD=0.89%,n=6),96.28%-99.22%(RSD=1.25%,n=6),96.49%-99.54%(RSD=1.16%,n=6),96.26%-99.70%(RSD=1.30%,n=6),respectively. CONCLUSIONS:The method is simple,accurate and suitable for simultaneous determination of 5 components in Xiaoer qingyan granules

Simultaneous Determination of Aygdalin, Aucubin, Harpagide, Peimisine, Peimine and Peiminine in Keling Capsules by HPLC-ELSD / 中国药师

Objective: To develop an HPLC-ELSD method for the determination of amygdalin, aucubin, harpagide, peimisine, peimine and peiminine in Keling capsules simultaneously.Methods: An Ultimate XB C 18 (250 mm× 4.6 mm , 5 μm) chromatographic column was adopted with an ELSD (the drift tube temperature was 105℃, the flow rate of nitrogen was 2.0 L·min-1).The mobile phase was acetonitrile-methanol (1∶1) and 0.4% acetic acid solution with gradient elution at a flow rate of 0.7 ml·min-1 , and the column temperature was set at 35 ℃.Results: Amygdalin, aucubin, harpagide, peimisine, peimine and peiminine was linear within the range of 13.56-271.20 μg·ml-1 (r=0.999 2), 8.48-169.60 μg·ml-1 (r=0.999 9), 4.89-97.80 μg·ml-1 (r=0.999 7), 2.66-53.20 μg·ml-1 (r=0.999 4), 1.82-36.40 μg·ml-1 (r=0.999 8) and 2.04-40.80 μg·ml-1 (r=0.999 6), respectively.The average recovery and the corresponding RSD were 97.90% (1.20%), 99.21% (1.62%), 97.68% (0.75%), 98.36% (1.38%), 99.70% (0.79%) and 97.95% (1.56%)(n =6), respectively.Conclusion: The method is simple and specific, and the results are accurate and repeatable.The method is helpful to the quality control of Keling capsules.

Identification and Analysis of Harpagide Metabolites in Rats in vivo / 中国药房

OBJECTIVE:To study the metabolites,distribution,metabolic type and the possible activity of harpagide which is the active component from Scrophularia ningpoensis in rats in vivo. METHODS:4 SD rats were divided into blank group (ul-tra-pure water) and administration group (harpagide reference solution),2 in each group,ig,160 mg/kg,twice a day,for 3 d. Urine and feces were collected every 12 h before administration and the first administration;sample blood 8 mL was taken after 0.5,1 h of last administration;heart,liver,spleen,lung,kidney,stomach and small intestine were taken. The blood,urine,fe-ces and other tissue solutions were prepared,HPLC-MS was conducted to detect and identify the harpagide metabolites in rats in vi-vo and presume metabolic pathways,and PharmMapper software was used to predict metabolites activity. RESULTS:12 harpagide metabolites were identified in rats in vivo,the form of prototypes and metabolites were distributed in heart,liver,spleen,lung, kidney,stomach and small intestine. The metabolic type mainly included hydrolysis,dehydration,reduction,methylation,sul-fation,glucuronic acid binding,grade A coumaric acid binding,etc. The 12 compounds may have activities in the treatment of epi-lepsy,amyotrophic lateral sclerosis,diabetes,stroke,etc. CONCLUSIONS:Harpagide may be effective in the form of prototypes and metabolites. The study has provided basis for attributing the origins of metabolite,studying the effective form of S. ningpoensis clarifying its pharmacological mechanism and processing mechanism.

Effects of harpagide on cerebral ischemia and mitochondria mediatedCaspase dependent apoptotic signaling pathway in mice / 中国药理学通报

Aim To investigate the effects of harpagide on cerebral ischemia and the mitochondria mediated Caspase dependent apoptotic signaling pathway in mice.Methods The MCAO was employed to establish MCAO model.When the models were established, the mice were given harpagide (4, 8, 12 mg·kg-1) and edaravone (3.2 mg·kg-1) [0.1 ml·(10 g)-1] by tail vein injection after MCAO immediately.And the model and control mice were given equivalent normal saline by the same way.After MCAO for 6 h, the behavior, volume of cerebral ischemia and pathological changes in the brain were observed.Westernblot was employed to determine the contents of Cyt C in mitochondrion and pro-caspase-3 in endochylema.Results Compared with the model group, harpagide (4, 8, 12 mg·kg-1) could significantly decrease the increased nerve functional score, brain index, brain water content and volume of cerebral ischemia induced by cerebral ischemia.Harpagide (4, 8, 12 mg·kg-1) could reduce the contents of Cyt C in mitochondrion and pro-caspase-3 in endochylema.Conclusion Harpagide may have protective effect on the cerebral ischemia injury in mice, which might be related to the inhibition of the cerebral mitochondria mediated Caspase dependent apoptotic signaling pathway.

Effects of harpagide on nerve cell and caspase-independent apoptotic signaling pathway in MCAO mice / 中国药理学通报

Aim To study the effects of harpagide on hippocampal neurons, mitochondrial function and caspase-independent apoptosis pathway after cerebral ischemia in mice. Methods The middle cerebral ar-tery occlusion (MCAO ) was employed to establish MCAO model. After that,the mice were given harp-agide (4,8,12 mg·kg - 1 )and edaravone (3. 2 mg ·kg - 1 )by tail vein injection after MCAO immediate-ly,and the model and control mice were given equal a-mounts of saline by the same way. After MCAO for 6 h,the apoptosis rate of hippocampal neuron and the mitochondrial membrane potential (MMP)of MCAO mice were detected by flow cytometry. We observed the clarity of inner and outer membrane of the hipp-ocampal neuronal mitochondrial,the integrity of the mitochondrial cristae,the changes of matrix electron density of mitochondria,and mitochondria swelling by transmission electron microscopy. Western blot was employed to determine the expression of apoptosis in-duced factor (AIF)and endonuclease G (Endo G)in mitochondrion and pro-caspase-3 in endochylema. qPCR was employed to determine the expression of AIF and Endo G. Results Compared with control group, the apoptosis rate of hippocampal neuron of MCAO mice significantly increased(P < 0. 01),the MMP of hippocampal neuron significantly decreased and the mi-tochondrial ultrastructure of cerebral ischemic area was severely damaged, loosely arranged and obviously swollen;the expression of AIF and Endo G in mito-chondria of cerebral tissue of MCAO mice significantly decreased,and the releases of AIF and Endo G signifi-cantly increased(P < 0. 01);the expressions of AIF, Endo G mRNA were evidently up-regulated (P <0. 01). Compared with model group,each dose of harpagide could significantly decrease the apoptosis rate of hippocampal neurons of mice brain (P < 0 . 05 ,P < 0. 01);MMP markedly increased in hippocampal nerve cells(P < 0. 01);the ultrastructure of neuronal mitochondria was obviously improved. Compared with model group,harpagide (8,12 mg·kg - 1 )could sig-nificantly increase the expression of AIF and Endo G protein in mitochondria of mouse brain,and the release of mitochondrial AIF and Endo G protein decreased(P< 0. 05,P < 0. 01);harpagide (4 mg·kg - 1 )could significantly increase the expression of Endo G protein in mitochondria of mouse brain,and the release of En-do G protein markedly decreased (P < 0. 05);harp-agide (8,12 mg·kg - 1 )could significantly decrease the expression of AIF and Endo G mRNA in hippocam-pus of mice(P < 0. 05,P < 0. 01). Conclusion The protective effect of harpagide on MCAO may be related to the protective effects on cerebral nerve cells,the ac-tivity of the mitochondria and the inhibition of caspase-independent apoptotic signaling pathways.

Chemical constituents from the whole plants of Lagopsis supina / 中国药学杂志

OBJECTIVE: To investigate the chemical constituents from the whole plants of Lagopsis supina. METHODS: The compounds were isolated and purified by various column chromatography, and their structures were identified based on their physiochemical properties and spectroscopic data. RESULTS: Thirteen compounds were isolated from the n-hexane, dichloromethane, and water extracts of the whole plants of Lagopsis supina by using various chromatographic methods. Their structures were identified as phytol(1), daucosterol(2), 8-O-acetylharpagide(3), antirrinoside(4), ajugoside(5), ajugol(6), harpagide(7), 1-O-caffeoyl-β-D-glucopyranose(8), 1-O-coumaroyl-β-D-glucopyranose(9), 2-hydroxy-5-(2-hydroxyethyl)phenyl-1-O-β-D-glucopyranoside(10), methyl 2-O-β-D-glucopyranosylbenzoate(11), adenosine(12), and sucrose(13), respectively. CONCLUSION: Compounds 1 and 3-13 are isolated from the plants of Lagopsis genus for the first time.

Simultaneous determination of eight constituents in Compound Rhodiola Capsule by HPLC-UV-ELSD / 中草药

Objective: To develop an HPLC-UV-ELSD method for the simultaneous determination of crenelatin, gallic acid, salidroside, tyrosol, harpagide, harpagoside, angoroside C, and cinnamic acid in Compound Rhodiola Capsule. Methods: Kromasil C18 column (250 mm×4.6 mm, 5 μm) was adopted. The mobile phase was composed of acetonitrile (A) and 0.3% HAC (B) with gradient elution. The flow rate was 1.0 mL/min and the detection wavelength was 275 nm. the column temperature was 30℃, and the evaporative light-scattering detector (ELSD) drift tube temperature was 40℃, and gas pressure of 1.5 bar (150 kPa). Results: Crenelatin, gallic acid, salidroside, tyrosol, harpagide, harpagoside, angoroside C, and cinnamic acid were separated well. The linear calibration curves were obtained in 67.084-670.84 μg/mL for crenelatin, R2=0.9992; 11.410-114.100 μg/mL for gallic acid, R2=0.9994; 78.995-789.95 μg/mL for salidroside, R2=0.9996; 19.625-196.25 μg/mL for tyrosol, R2=0.9997; 59.368-593.68 μg/mL for harpagide, R2=0.9998; 62.585-625.85 μg/mL for harpagoside, R2=0.9995; 55.045-550.45 μg/mL for angoroside C, R2=0.9996; and 6.895-68.95 μg/mL for cinnamic acid, R2=0.9998. The average recoveries of the eight constituents were 100.8%, 98.9%, 100.1%, 100.8%, 98.9%, 99.6%, 100.7%, and 99.2% with RSD of 0.64%, 0.56%, 0.35%, 0.65%, 0.26%, 0.58%, 1.00%, and 0.64%. Conclusion: The method is convenient, accurate, and can be used for the simultaneous determination of the preparation.

Simultaneous Determination of the Content of Catalpol,Harpagide and Hapagoside in AIDS Oral Gargle by Double-wavelength UPLC / 中国药房

OBJECTIVE:To establish a method for simultaneous determination of the content of catalpol,harpagide and hapa-goside in AIDS oral gargle. METHODS:Dual-wavelength UPLC was performed on the column of Eclipse Plus C18 with mobile phase of acetonitrile-0.05% phosphoric acid (gradient elution) at the flow rate of 0.2 ml/min. Wavelength was switching,it was set at 210 nm to determine the contents of catalpol and harpagide in the first 8 min and then changed to 280 nm to determine the con-tent of hapagosi- de in 8-15 min. The column temperature was 30 ℃,and injection volume was 2 μl. RESULTS:The linear range was 0.018 5-0.129 2 μg (r=0.999 6) for catalpol,0.014 0-0.098 3 μg (r=0.999 7) for harpagide and 0.015 7-0.110 0 μg(r=0.999 7)for hapagoside;RSDs of precision,stability and reproducibility tests were no more than 0.77%;average recoveries were respectively 99.44%(RSD=0.75%,n=6),99.90%(RSD=0.82%,n=6) and 100.20%(RSD=0.73%,n=6). CONCLUSIONS:The method is simple can reproducible,and can be used for effectively quality control.

Simultaneous determination of harpagide and harpagoside in Mailuoning injection by HPLC / 中国生化药物杂志

Objective To establish a method for content determination of harpagide and harpagoside in Mailuoning injection by HPLC. Methods The experimental condition of HPLC method was as follows: SunfireTM C18 column (4.6 mm ×150 mm, 5 μm), with gradient elution using acetonitrile and 0.03% phosphoric acid; the detected wavelength was 210 nm, and the flow rate was 1.0mL/min.ResuIts Harpagide and harpagoside demonstrated good linear relationship in the range 0.1424~0.8544 μg/mL(r=0.9998) and 0.0732~0.4392μg/mL (r=0.9997) respectively.The average recovery rate were 98.22% and 99.27% with RSD of 1.46% and 1.42%(n=6)respectively.ConcIusion The method is simple, reliable, accurate, reproducible and stable, and it could be used in the determination of harpagide and harpagoside in Mailuoning injection.

Neuro-protective effect of harpagide on acute cerebral ischemic injury in mice and its mechanism involving mitochondria / 中国药学杂志

OBJECTIVE: To observe the neuro-protective effects of harpagide on acute cerebral ischemic injury in mice and its mechanism involving mitochondria. METHODS: Acute cerebral ischemia were achieved by operation of MCAO in the left brain, random allocation was taken to divide ICR mice into sham group, model group, nimodipine group and harpagide (5,10,15 mg·kg-1) groups. Mice were intraperitoneal injected harpagide immediately after surgeiy. Nerve function score, content of brain water, brain index and the common changes of brain pathological structure in HE staining were measured: Ability of mitochondria Ca2+-Mg2+-AT-Pase and protein expression level of caspase-3 in the MCAO mice' brains was determined: Ultrastructure change of mitochondria under the TEM was observed. RESULTS: Compared with model group, the harpagide groups could decreased the nerve function score, the content of brain water, brain index and the volume of ischemia in mice with different degrees in MCAO mice (P-1 of harpagide could increased the activity of Ca2+-Mg2+-ATPase obviously (P<0.01): And significantly decreased the protein expression level of caspase-3 (P<0.01): harpagide groups could protect the pathogeny structure and the ultrastructure of mitochondria with different degrees in MCAO mice, decrease edema of mitochondria obviously. CONCLUSION: Harpagide could obviously protect acute cerebral ischemia in mice, its therapeutical effects are approached to protecting the activity of brain mitochondria and decreasing protein expression level of caspase-3.

Determination of eight active components in Xuanmai Ganjie Granules by UPLC-MS/MS / 中草药

Objective: To develop a UPLC-MS/MS method for simultaneously determining harpagide, liquiritin, harpagoside, platycodin D, ammonium glycyrrhetate, ophiopogonin D, methylophiopogonanone A, and methylophiopogonanone B in Xuanmai Ganjie Granules (composed with Scrophulariae Radix, Ophiopogonis Radix, Glycyrrhizae Radix et Rhizoma, and Platycodonis Radix) from different pharmaceutical companies. Methods: The chromatographic separation was achieved on Phenomenex Kenetix C18 column (50 mm × 2.1 mm, 5 μm) by using a mobile phase consisted of acetonitrile and 0.1% formic acid water at the flow rate of 0.3 mL/min for gradient elution. Simultaneous monitoring of positive and negative ions and multiple reaction monitoring (MRM) scan mode were applied to the quantification of the components in Xuanmai Ganjie Granules; Sample volume was 5 μL. Results: There was good linearity between the absorption peak area and the concentration for harpagide, liquiritin, harpagoside, platycodin D, ammonium glycyrrhetate, ophiopogonin D, methylophiopogonanone A, and methylophiopogonanone B in the ranges of 9-2250, 8-2000, 3.4-850, 96-24000, 12.4-3100, 3.6-1900, 1.7-425, and 1.5-375 ng/mL, respectively. The average recoveries were ranged from 97.2% to 102.8% (RSD ≤ 2.7%). The contents of harpagide, liquiritin, harpagoside, platycodin D, ammonium glycyrrhetate, ophiopogonin D, methylophiopogonanone A, and methylophiopogonanone B in eight batches of samples were in the ranges of 32.8-107.6, 54.8-178.0, 14.6-70.7, 31.2-280.0, 106.4-287.9, 0.1-0.6, 0.01-0.07, and 0.03-0.17 μg/g, respectively. Conclusion: The developed method is simple, effective, and credible for determining the eight components in Xuanmai Ganjie Granules. It provides more helpful information for the comprehensive quality evaluation of Xuanmai Ganjie Granules.

Protection of harpagide on cytotoxicity in PC12 cells induced by Aβ25-35 peptides / 中草药

Objective: The main purpose of the present study is to investigate the influence of harpagide on amyloid-β25-35 (Aβ25-35)- induced cytotoxicity. Methods: Oxidative stress was assessed by measuring MDA, glutathione (GSH), and superoxide dismutase (SOD) levels. Cell viability was assessed by MTT assay. Cell apoptosis detection was performed using an Annexin-V-FITC Apoptosis Detection Kit. The production of ROS was determined using a ROS Assay Kit. Western blotting detection was carried out to detect the protein expression. Results: Our studies showed that pretreatment with harpagide could reduce Aβ25-35-induced oxidative stress. Harpagide markedly inhibited cell apoptosis in a dose dependent manner. More importantly, harpagide increased the PI3K/AKt and Bcl-2 family protein ratios on pre-incubation with cells for 3 h. Conclusion: One possible mechanism of harpagide to improve Aβ25-35-induced PC12 cytotoxicity may be through the up-regulation of SOD and activation of the PI3K/AKt pathway.

Simultaneous Determination of 8-O-acetylharpagide and Harpagide inAjuga decumbens Thunb. by HPLC-ELSD / 中国中医药信息杂志

Objective To build an HPLC-ELSD method for simultaneous determination of 8-O-acetylharpagide and harpagide inAjuga decumbens Thunb.;To determineAjuga decumbens Thunb. samples from different area and of different batches.Methods Chromatographic separation was achieved on a Dikma C18 column (4.6 mm×150 mm, 5μm). The mobile phase consisted of water (A) and acetonitrile (B), using a gradient elution of 5% B at 0-6 min, 5%-15% B at 6-11 min, 15% B at 11-17 min, and the flow rate was 1.0 mL/min. An evaporative light scattering-detector (ELSD) was used with the temperature of drift tube at 103℃ and the gas flow rate of air was at 1.6 L/min.Results Good linearity was shown at the concentration range of 0.017-17.32μg for 8-O-acetylharpagide, and 0.003-1.62μg for harpagide. The validated method was of great precision and accuracy. The contents of 8-O-acetylharpagide and harpagide inAjuga decumbens Thunb. from Fujian province were higher than the contents in other localities.Conclusion The content of harpagide can be used as index component to monitor quality control ofAjuga decumbens Thunb..