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Objective:To investigate the effect of specnuezhenide on high glucose-induced human retinal microvascular endothelial cells (hRMECs) injury and its mechanism.Methods:The hRMECs were divided into a normal control group cultured in a culture medium containing 5.5 mmol/L glucose, a hypertonic group cultured in a culture medium containing 5.5 mmol/L glucose + 24.5 mmol/L mannitol, a high glucose group cultured in a culture medium containing 30 mmol/L glucose, as well as high glucose+ low-, medium-, and high-dose specnuezhenide groups cultured in culture media containing 30 mmol/L glucose + 25, 50, 100 μmol/L specnuezhenide for 24 hours, respectively.In addition, hRMECs were divided into a high glucose+ small interfering RNA-negative control (si-NC) group cultured in a culture medium containing 30 mmol/L glucose, a high glucose+ si-forkhead box O4 (FOXO4) group cultured in a culture medium containing 30 mmol/L glucose, a high glucose+ specnuezhenide+ pcDNA group cultured in a culture medium containing 100 μmol/L specnuezhenide + 30 mmol/L glucose, and a high glucose+ specnuezhenide+ pcDNA-FOXO4 group cultured in a culture medium containing 100 μmol/L specnuezhenide+ 30 mmol/L glucose for 24 hours after transfection by corresponding reagents.Cell apoptosis was detected by flow cytometry.The malondialdehyde (MDA) concentration and superoxide dismutase (SOD) activity in cells were detected by the thiobarbituric acid method and xanthine oxidase method, respectively.The concentrations of interleukin (IL)-1β and tumor necrosis factor (TNF)-α in the cell culture supernatant were detected by enzyme linked immunosorbent assay.The relative expression level of FOXO4 protein in cells was determined by Western blot.Results:The apoptosis rates of normal control group, hypertonic group, high glucose group, high glucose+ low-, medium- and high-dose specnuezhenide groups were (7.32±0.72)%, (7.44±0.70)%, (23.96±1.32)%, (19.84±1.09)%, (14.13±0.85)% and (9.84±0.70)%, respectively.There were significant differences in cell apoptosis rate, MDA concentration, SOD activity, the concentration of IL-1β, the concentration of TNF-α, and the relative expression level of FOXO4 protein among the six groups ( F=498.545, 1 186.693, 516.629, 654.247, 638.238, 472.655; all at P<0.001). Compared with high glucose group, the apoptosis rate, MDA concentration, IL-1β and TNF-α concentration, FOXO4 protein expression level were significantly decreased in high glucose+ low-, medium- and high-dose specnuezhenide groups, and SOD activity was significantly increased in a dose-dependent manner.Compared with high glucose+ si-NC group, the expression level of FOXO4 protein, cell apoptosis rate, MDA concentration, IL-1β and TNF-α mass concentrations were decreased in high glucose + si-FOXO4 group, while the SOD activity was increased.Compared with high glucose+ specnuezhenide+ pcDNA group, the apoptosis rate, MDA concentration, IL-1β and TNF-α concentrations, FOXO4 protein expression level of hRMECs in high glucose+ specnuezhenide+ pcDNA-FOXO4 group were significantly increased, and SOD activity was significantly decreased (all at P<0.05). Conclusions:Specnuezhenide can protect hRMECs from high glucose-induced apoptosis, oxidative stress and inflammatory response by down-regulating FOXO4.
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ObjectiveTo investigate the quality of Ligustri Lucidi Fructus in the market， and the moisture， extract， determination of Zhuyao and Douchi Ligustri Lucidi Fructus were compared to increase the utilization rate of Ligustri Lucidi Fructus. MethodThe properties， moisture， total ash， alcohol-soluble extract content and thin layer chromatography （TLC） identification were determined by the methods of Ligustri Lucidi Fructus included in the 2020 edition of Chinese Pharmacopoeia， and high performance liquid chromatography （HPLC） fingerprint and determination of specnuezhenide and salidroside were established with the mobile phase of 0.2% phosphoric acid aqueous solution （A）-acetonitrile （B） （0-70 min， 92%-65%A） for gradient elution， and the detection wavelength of 220 nm at 0-14 min and 225 nm at 14-70 min. The two different characters of Ligustri Lucidi Fructus were comprehensively compared by the above indicators. ResultExcept for one batch which did not meet the requirements due to the quality of harvesting， the other 12 batches of samples all met the requirements of the 2020 edition of Chinese Pharmacopoeia， but there were two different characters. Comparing the two different characters of Ligustri Lucidi Fructus， it is found that the moisture， total ash， extract， salidroside and specnuezhenide contents of Zhuyao samples were 2.22%-5.19%， 3.91%-4.49%， 32.56%-40.95%， 0.073%-0.170% and 1.45%-4.14%， and these values of Douchi samples were 3.57%-5.61%， 3.65%-4.44%， 41.31%-46.70%， 0.041%-0.067% and 3.01%-4.20%， respectively. ConclusionThe contents of extract and specnuezhenide of Douchi Ligustri Lucidi Fructus are mostly higher than those of Zhuyao Ligustri Lucidi Fructus， while the content of salidroside is lower than that of Zhuyao samples， and there are no significant differences in moisture， TLC identification and total ash content. Based on the above research， if the main purpose is to extract salidroside， it is recommended to choose Zhuyao Ligustri Lucidi Fructus. If the main purpose is to use Ligustri Lucidi Fructus as medicine， it is recommended to choose Douchi Ligustri Lucidi Fructus.
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To investigate the dynamic change law of the main components in Ligustri Lucidi Fructus during the wine-steaming process and attempt to establish the characteristic quality standard of wine-steamed Ligustri Lucidi Fructus by determining the content of salidroside and specnuezhenide using Ultra Performance Liquid Chromatography (UPLC) technology at different processing time points (12, 15, 18, 21, 24 h). The chromatographic separation was performed on Waters Acquity UPLC®BEH C₁₈ column (2.1 mm×50 mm, 1.7 μm) with acetonitrile and 0.2% formic acid aqueous solution as the mobile phase for gradient elution; and the detection wavelength was set at 280 nm; the flow rate was 0.5 mL·min⁻¹, and the column temperature was set at 40 °C. The results showed that the two components were well separated in the above conditions. The salidroside and specnuezhenide showed a good linear relationship within the range of 10.19-326 ng and 49.53-1 585 ng, respectively. Their average recovery was 103.4% and 101.7% with RSD of 0.81% and 0.79%, respectively. With the extension of processing time, the content of specnuezhenide was decreased, while salidroside was gradually increased. For the 27 batches of Ligustri Lucidi Fructus, the content of salidroside was between 0.042 5% and 0.192 4%, and that of specnuezhenide was between 0.829 7% and 5.218 0%. While for the 25 batches of wine-steamed Ligustri Lucidi Fructus, the content of the first one was between 0.229 2% and 1.045 8%, and the latter one was between 0.743 8% and 3.645 4%. As compared with Ligustri Lucidi Fructus, the ratio of specnuezhenide to salidroside was significantly decreased in the wine-steamed Ligustri Lucidi Fructus. According to the experimental results, the quality standard of Ligustri Lucidi Fructus is tentatively fixed as follows: the content of specnuezhenide shall not be less than 0.80%, and the ratio of specnuezhenide content/salidroside content (Sp/Sa) should not be smaller than 15. As for the wine-steamed ones, the content of salidroside should not be less than 0.20%, and specnuezhenide content should not be less than 0.70%; Sp/Sa should not be greater than 8. The method established in this study is simple and reliable, which could be used for the content detection of salidroside and specnuezhenide in Ligustri Lucidi Fructus samples. The characteristic quality standard established in this study could be used to distinguish the Ligustri Lucidi Fructus and wine-steamed Ligustri Lucidi Fructus.
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Chromatography, High Pressure Liquid , Drugs, Chinese Herbal , Fruit , Ligustrum , WineABSTRACT
Objective: To optimize the extraction technology of two major iridoid glucosides (specnuezhenide and nuezhenoside G13) from four kinds of Osmanthus fragrans (OF) seeds, and to evaluate the anti-thrombotic activity of OF seeds. Methods: The orthogonal-test experiment was employed to optimize the parameters including ethanol concentration, liquid-material ratio, and extraction time for three extraction methods (ultrasonic extraction, reflux extraction, and microwave extraction). The extraction yield, content, and total peak area of iridoid glucosides were selected for weighted analysis to determine the best extraction method and technology. Additionally, an anti-thrombotic zebra fish model was established for biological evaluation of OF seeds. Results: Microwave extraction was the best method for iridoid glucosides extraction with the optimal conditions of ethanol concentration 55%, material-liquid ratio 1∶10, and microwave time 15 min. HPLC analysis showed that there was no significant difference in chemical composition among the four kinds of OF seeds. In zebra fish biological screening model, OF seeds displayed a weak inhibitory effect on the growth of thrombus and exhibited a pericardial edema effect in high dose-treated group. Conclusion: In this paper, extraction technology of two iridoid glucosides from four different kinds of OF seeds and preliminary anti-thrombotic activity evaluation of OF seeds were investigated. These results can provide the reference for further development and utilization of the agricultural waste of OF seeds.
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AIM To establish an HPLC method for the simultaneous content determination of ginsenoside Re,ginsenoside Rg1,ginsenoide Rb1,specnuezhenide,calycosin-7-O-β-D-glucoside and oleanolic acid in Yitai Cap sules (Ginseng Radix et Rhizoma,Astragali Radix,Ligustri lucidi Fructus,etc.).METHODS The analysis of 70％ ethanol extract of this drug was performed on a 25 ℃ thermostatic Luna C18 column (4.6 mm ×250 mm,5 μm),with the mobile phase comprising of methanol-O.2％ phosphoric acid flowing at 1.0 mL/minin a gradient elution manner,and the detection wavelength was set at 203 nm.RESULTS Six constituents showed good linear relationships within their own ranges (r ＞ 0.999 0),whose average recoveries were 95.58％-102.12％ with the RSDs of 0.82％-1.73％.CONCLUSION This simple and stable method can be used for the rapid quality control of Yitai Capsules.
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Objective: To establish methods of qualitative identification and quantitative determination for Zishen Yangyin Granules (ZYG). Methods: The TLC method was used to identify the herb by mixture of chloroform-methanol (31) as a developing solvent on high performance silica gel precoated plate (HSGF254) and using 5% vanillic aldehyde sulfuric acid as a chromogenic reagent for qualitative identification of Corni Fructus; TLC identification of Eclipta prostrata, Alismatis Rhizoma, and Moutan Cortex was performed on high performance silica gel precoated plate (HSGF254) with petroleum ether (60-90 ℃)-chloroform-ethyl acetate (312) as developing solvent. The same developing method was used to identify E. prostrata, A. Rhizoma, and paneol in M. Cortex of ZYG by different detected method at the same time. The contents of morroniside, loganin, hyperoside, specnuezhenide, and paeonol were analyzed by high performance liquid chromatography on C18 column (250 mm × 4.6 mm, 5 μm) with the mobile phase of acetonitrile-0.1% trifluoroacetic acid by gradient elution. The detection wavelength was set at 254 nm. Results: Morroniside and loganin were used to identify C. Fructus in ZYG, paeonol in M. Cortex can be identified at 254 nm; Some substances in E. prostrata can be identified at 366 nm; Some substances in A. Rhizoma can be detected in sunlight, with 5% phosphomolybdic acid in ethanol as a chromogenic reagent. The TLC separation was desirable with moderate Rf value and clear spot. The methodology validation for the assay of morroniside, loganin, hyperoside, specnuezhenide, and paeonol presented that they were in good linear correlation in the ranges of 4.432-110.8, 4.192-104.8, 4.040-101.0, 4.132-103.3, and 4.076-101.9 μg/mL, The correlation coefficients of indicator were over 0.999 7. The average recoveries were between 96.57% and 98.67%. The RSD value of intra-day precision was less than 2% and the RSD value of inter-day precision was less than 3%. The method has good stability and reproducibility. Conclusion: The methods of quality control are specific, reproducible, accurate, and suitable, which can be successfully applied to the quality control of ZYG.
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Objective To establish the HPLC fingerpint method for simultaneous determination of four representative components (salidroside, echinacoside, specnuezhenide and oleuropein) of Ligustri Lucidi Fructus, so as to provide a reference for the quality control. Methods The method was performed on an Diamonsil C18 anlytical column (250 mm × 4.6 mm, 5 μm) at the column temperature of 30 ℃. The gradient mobile phase consisted of acetonitrile (A)-0.1% formic acid (B) with a flow rate of 1.0 mL/min. The detection wavelengths were 224 nm. HPLC-Q/TOF-MS were used for identifing the common peaks. Results By studying comparatively the fingerprints of 11 samples, 14 common peaks have been confirmed. There were 12 common peaks were identified by HPLC-Q/TOF-MSE. The similarity of 10 batches are greater than 0.990. Salidroside, echinacoside, specnuezhenide and oleuropein were baseline separated with good linearity relationships (r > 0.999) between concentration and peak areas over the linear ranges. The average recoveries of the investigated compounds were 97.40%, 98.99%, 97.03%, and 100.55%, respectively. Conclusion The method is accurate, reliable and with good reproducibility. It could be used for quality control and evaluation of Ligustri Lucidi Fructus.
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AIM To analyze the dynamic changes of five constituents in Ligustri lucidi Fructus at five picking time (August,September,October,November,December).METHODS The HPLC analysis of Ligustri lucidi Fructus ethanol extract was performed on a 25 ℃ thermostatic Aglient Zorbax SB-C1s column (4.6 mm ×250 mm,5 μm),with the mobile phase comprising of acetonitrile-0.1％ phosphoric acid flowing at 1 mL/min in a gradient elution manner,and the detection wavelength was set at 224 nm.RESULTS Salidroside,tyrosol,luteolin-7-O-glucoside,ligustroflavone and specnuezhenide showed good linear relationships within their own ranges (r ＞0.999 0),whose average recoveries were 99.56％-100.30％ with the RSDs of 0.89％-1.23％.The contents of various constituents (except for tyrosol) were the highest in samples picked up in September,followed by those picked up in October.CONCLUSION The suitable picking time of Ligustri lucidi Fructus is September and October.
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OBJECTIVE: To study the change rule of specnuezhenide content and antoxidant activity of Ligustri Lucidi Fructus with temperature and time, and determine its validity period. METHODS: Dynamic parameters were established under constant temperature, and specnuezhenide content of Ligustri Lucidi Fructus was determined by HPLC after accelerated test by classical constant temperature method. The shelf life of Ligustri Lucidi Fructus at 25℃ were determined by Arrhenius theory. The antioxidant activities of different samples of Ligustri Lucidi Fructus were evaluated by ABTS assay. RESULTS: The shelf life of Ligustri Lucidi Fructus was forecasted to be 1.83 years at 25℃. The antioxidant activitiy of Ligustri Lucidi Fructus showed a trend of decline after rising first. CONCLUSION: High temperature and storage time would affect the chemical composition of Ligustri Lucidi Fructus, so Ligustri Lucidi Fructus should be kept in the shade for no more than 1.83 years for a good therapeutic effect.
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This study aimed to trace sources and quantitatively analyze the specnuezhenide content of circular Fructus Ligustri Lucidi for clinical use. Different specifications of Fructus Ligustri Lucidi were identified using DNA barcoding technology and the specnuezhenide content was analyzed by High Performance Liquid Chromatography (HPLC). The ITS sequence of circular Fructus Ligustri Lucidi was identical to that of standard privet, which was determined through botanical identification. ITS sequence similarity between circular Fructus Ligustri Lucidi and Fructus Ligustri Lucidi which was registered in NCBI ranged from 99.5% to 100%. The sequences of circular and other Fructus Ligustri Lucidi were clustered in a Neighbor-Joining tree with bootstrap value of 95, and these sequences could be distinguished from adulterants. Conforming to pharmacopoeia standard, the average specnuezhenide content of circular Fructus Ligustri Lucidi was higher than that of chicken waist Fructus Ligustri Lucidi. Circular Fructus Ligustri Lucidi derived from Ligustrum lucidum Ait. and the specnuezhenide content was higher in circular Fructus Ligustri Lucidi than that in chicken waist Fructus Ligustri Lucidi.
Subject(s)
Chromatography, High Pressure Liquid , DNA Barcoding, Taxonomic , DNA, Plant , Fruit , Chemistry , Glucosides , Ligustrum , Chemistry , Classification , Genetics , Medicine, Chinese Traditional , Polymerase Chain Reaction , Pyrans , Quality ControlABSTRACT
Aim To research the protective effects of specnuezhenide, an active compound of traditional Chinese medicine, against CCl4-induced hepatic dam-age in mice. Methods The model of acute hepatic damage caused by CCl4 intraperitoneal injection on mice was obtained. The levels of ALT, AST, TNF-α, IL-1β, and IL-6 in serum, the activities of MDA, SOD, and GSH-Px in liver homogenates, and the his-topathological organization of liver sections were also examined to observe the protective effects of specnu-ezhenide. Results Specnuezhenide markedly de-creased the CCl4-induced elevation of serum ALT and AST activities, and improved hepatic histopathology changes. Specnuezhenide also significantly decreased the content of MDA in liver tissues, meanwhile in-creased the activities of antioxidant enzymes SOD and GSH-Px. In addition, specnuezhenide reduced the lev-els of TNF-α, IL-1β and IL-6 . Conclusions Specnu-ezhenide has significant protective effects against CCl4-induced hepatic damage in mice, and the possible un-derlying mechanisms of the activity could be due to its antioxidant and anti-inflammatory effects.
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Objective: To optimize the processing technology of Ligustri Lucidi Fructus (LLF) stewed with wine. Methods: The contents of salidroside, specnuezhenide, and oleanolic acid were simultaneously determined using HPLC. The weight coefficient of each component was evaluated by the analytic hierarchy process. With composite score as index, an orthogonal test was adopted to investigate the effects of wine amount, soaking time, and steaming time on the quality of the processed products and to optimize the technical parameters for the processing of LLF stewed with wine. Results: Wine amount and steaming time had significant influence on the quality of products, but soaking time had no significant effect. Optimal processing parameters were as follows: 30 g of wine was added to 100 g of herbs, soaking time was 0.5 h, and steaming time was 8 h. Conclusion: The optimized processing technology is reasonable and reliable, and it can provide a reference for the processing of LLF. The method established to simultaneously determine the contents of three components in LLF is simple, rapid, and reproducible for controlling the quality of LLF.
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Objective:To establish a method for the determination of specnuezhenide in traditional decoction and granules of Zish-enwufa formula, and compare the content between them. Methods:HPLC was used for the determination. The samples were analyzed on a Waters Symmetry C18 column (150 mm × 3. 9 mm,5 μm) with acetonitrile-water (16 ∶84) as the mobile phase. The flow rate was 1. 0 ml·min-1 and the detection wavelength was 224nm. Results: A good linearity of specnuezhenide was within the range of 0.179 2-3.316 μg(r=0.999 9),and the average recovery of berberine hydrochloride was 101.65% with RSD of 1.49%(n=6). Conclusion:The determination method is simple, accurate and sensitive with good reproducibility,which can be used for the determi-nation of specnuezhenide in the preparation. The content of specnuezhenide in the traditional decoction and granules of Zishenwufa for-mula is similar.
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Objective Former standards for Yishen Pills only identify angelica root , astragalus root and ligustrum lucidum by thin layer chromatography ( TLC) and there is no test for the content of effective components .The study was to improve the quality of Yishen Pills by perfecting the quality standards . Methods We determined the content of specnuezhenide in Yishen Pills by high-per-formance liquid chromatography(HPLC), with a mobile phase of MeOH-Water, a chromatography column of Agilent TC-C18(2) (4.6 × 250 mm, 5μm), a flow rate of 1.0 mL/min, a column temperature of 25℃, a detection wavelength of 224 nm, and a sample loop volume of 10μL. Results The linear relationship of specnuezhenide content was good in the range of 15.375μg/mL~246.000μg/mL.The relative standard deviation of precision experiment , stablity experiment and repeatablity experiment was 0.44%, 0.95%and 2.65%re-spectively.The average recovery was 99.60%.The qualified standard for specnuezhenide in Yishen Pills was ≥0.3 mg/g. Conclusion The method is simple , accurate and reliable , with good reproducibility , and it is applicable for the quality control of Yishen Pills .
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OBJECTIVE:To establish a method for simultaneous determination of the contents of ligustroflavone,specnuezhe-nide,demethylwedelolactone and wedelolactone in Zibu ganshen pill. METHODS:Dual-wavelength HPLC was performed on the column of Elite C18 with mobile phase of acetonitrile-0.5%acetic acid(gradient elution)at flow rate of 0.9 ml/min,column temper-ature was 25 ℃,detection wavelengths were 224 nm(0-30 min) and 351 nm (30-50 min),and the injection volume was 20 μl. RESULTS:The linear range was 6.75-135.00μg/ml(r=0.999 5)for ligustroflavone,6.54-130.80μg/ml(r=0.999 8)for specnuezhe-nide,4.90-98.00μg/ml(r=0.999 4)for demethylwedelolactone and 6.42-128.40μg/ml(r=0.999 6)for wedelolactone;RSDs of pre-cision,stability and reproducibility tests were no more than 1.25%;average recoveries were 96.15%-99.96%(RSD<2%,n=6). CONCLUSIONS:The method is rapid,sensitive and accurate,and can be used for the contents determination of ligustroflavone, specnuezhenide,demethylwedelolactone and wedelolactone in Zibu ganshen pill.
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Objective To determine the dissolution rate of Erzhi pill which was prepared by using different crushing technologies with specnuezhenide as index - and study the influence of superfine pulverization technology on the dissolution of Erzhi pill. Methods Crude drugs were crushed into the fine powder by employing general crusher and ultra-micro pulverizer-respectively. The water-honeyed pills were prepared by using these fine powders. The method of rotating basket and HPLC were used for the determination of dissolution of specnuezhenide in vitro. The data were analyzed by applying the law of Weibull. Results Compared with Erzhi pill B- the dissolution parameters of specnuezhenide in Erzhi pill A - including T50, T60, T70, T80, T90, F1h and F2h- showed significant differentce(P<0.05). Conclusion Applying the superfine pulverization technology can improve the dissolution of Erzhi pills in vitro.
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Objective To determine the dissolution rate of Erzhi pill which was prepared by using different crushing technologies with specnuezhenide as index , and study the influence of superfine pulverization technology on the dissolution of Erzhi pill. Methods Crude drugs were crushed into the fine powder by employing general crusher and ultra-micro pulverizer,respectively. The water-honeyed pills were prepared by using these fine powders. The method of rotating basket and HPLC were used for the determination of dissolution of specnuezhenide in vitro. The data were analyzed by applying the law of Weibull. Results Compared with Erzhi pill B,the dissolution parameters of specnuezhenide in Erzhi pill A ,including T50、T60、T70、T80、T90、F1h and F2h, showed significant differentce(P<0.05). Conclusion Applying the superfine pulverization technology can improve the dissolution of Erzhi pills in vitro.
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OBJECTIVE: To establish a micellar electrokinetic chromatography(MEKC) method for simultaneous determination of salidroside, tyrosol, ligustroflavone, specnuezhenide, oleanolic acid, and ursolic acid in Ligustri Lucidi Fructus. METHODS: 60 mmol·L-1 borax-10 mmol·L-1 SDS-30 mmol·L-1 hydroxypropyl-beta-cyclodextrin-10% methyl alcohol was used as the buffer solution (pH 9.03). Uncoated fused silica capillary (75 μm×64.5 cm, 56 cm of effective length) was used with separation voltage of 20 kV. The detection wavelength was set at 210 nm. The column temperature was maintained at 25°C, and the sample was injected at 5 kPa×6 s. RESULTS: The calibration curves of the six index components showed good linearity (r>0.95) in the range of the tested concentrations, and the average recoveries of the method were between 94.57% and 102.07% (RSD<5%). CONCLUSION: The method is simple, accurate and reproducible, and can be used for the quality control of Ligustri Lucidi Fructus.
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Objective To set up a method for determining Specnuezhenide in Shengxinfa Capsules. Methods With HPLC method, Agilent TC-C18 column (4.6 mm×250 mm, 5 μm) was used. Mobile phase was methanol-water (35∶65), the flow rate was 1.0 mL/min, and the detecting wavelength was at 224 nm. Results The linearity was in the range of 0.15-0.90 μg. The average recovery was 100.35%, and RSD was 1.43%. Conclusion The method is accurate, reliable, and can be used for the determination of Specnuezhenide in Shengxinfa Capsules for its quality control.