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ObjectiveTo investigate the quality variation of Lonicera japonica flower from different harvesting periods by ultraviolet visible（UV-Vis） fingerprint combined with chemometrics. MethodTwenty-five L. japonica flower samples from five harvesting periods， including young bud stage，green bud stage，white bud stage，silver and golden flower stages， were collected， with five samples for each stage. UV-Vis fingerprints of L. japonica flower from different harvesting periods were established in the context of the optimum extraction method based on the single factor experiment. The results showed that the absorption values at 209，216，226，250，280，303，318， and 350 nm were significantly different. Moreover，after data pretreatment and normalization，multivariate statistical analyses， such as principal component analysis （PCA），partial least squares discriminant analysis （PLS-DA），and orthogonal PLS-DA （OPLS-DA）were performed by SIMCA-P+ to establish the quality variation model of L. japonicas flower from harvesting periods. ResultAs revealed by PCA and PLS-DA， L. japonicas flower samples from five harvesting periods were clustered separately and closely in a harvesting time-dependent manner， suggesting that the content of components contained in samples from different harvesting periods was highly distinct and correlated with harvesting periods. The pairwise comparison of OPLS-DA indicated that triterpenoids or volatile oils were the main components causing the changes from the young bud stage to the green bud stage，and the content of them decreased. The main components from the green bud stage to the white bud stage were triterpenoids （or iridoids），volatile oils，phenolic acids， or flavonoids，and the content of them decreased， which was consistent with the HPLC result of chlorogenic acid. From the white bud stage to the silver flower stage， the main components were iridoids （increasing in content） and triterpenoids （or volatile oils） （decreasing in content）. The main altered components from the silver flower stage to the golden flower stage were triterpenoids （or volatile oils） whose content increased. ConclusionThis method is simple and feasible， which can provide references for the quality control of Chinese medicine.
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Prunella vulgaris(PV) is an edible and traditional medicinal herb which has a wide range application in fighting inflammation and oxidative stress, and protecting liver. Now it has been used to treat various types of liver diseases and has significant clinical efficacy. This study aims to investigate the effects of PV on ethanol-induced oxidative stress injury in rats and its metabolic mechanism. The rats were divided into control group, model group, PV group, and VC group. The liver protection of PV was identified by measuring pharmacological indexes such as antioxidant and anti-inflammatory activity. The metabolic mechanism of long-term ethanol exposure and the metabolic regulation mechanism of PV treatment were studied by LS-MS metabonomics. The pharmacological investigation indicated that ethanol could significantly decrease the contents of SOD, GSH-Px, CAT and other antioxidant enzymes in liver and increase the content of MDA. At the same time, PV could significantly reduce the contents of inflammatory factors(TNF-α, IL-6 and IL-1β) and liver function markers(ALT, AST, ALP) in serum. What's more, long-term ethanol exposure could significantly cause liver injury, while PV could protect liver. Metabolomics based on multiple statistical analyses showed that long-term ethanol exposure could cause significant metabolic disorder, and fatty acids, phospholipids, carnitines and sterols were the main biomarkers. Meanwhile, pathway analysis and enrichment analysis showed that the β oxidation of branched fatty acids was the main influencing pathway. Also, PV could improve metabolic disorder of liver injury induced by ethanol, and amino acids, fatty acids, and phospholi-pids were the main biomarkers in PV treatment. Metabolic pathway analysis showed that PV mainly regulated metabolic disorder of ethanol-induced liver injury through phenylalanine, tyrosine and tryptophan biosynthetic pathways. This study could provide a new perspective on the hepatoprotective effect of natural medicines, such as PV.
Subject(s)
Animals , Rats , Antioxidants/metabolism , Ethanol/toxicity , Liver/metabolism , Metabolomics , Oxidative Stress , PrunellaABSTRACT
Prunellae Spica is a perennial edible and medicinal plant, rich in antioxidant substances. Total flavonoids (TFC), Phenolics (TPC), triterpenoids (TSC), polysaccharides (PC) and their antioxidant capacities (by the FRAP, DPPH and ABTS⁺ methods) of ethyl acetate fraction, n-butanol fraction and other fractions of aqueous extract from Prunellae Spica were investigated in this study. Then the multivariate statistical method was adopted to analyze the relationship between the multiple pharmaceutical ingredients and antioxidant capacities of Prunellae Spica. The results showed that ethyl acetate fraction had relatively high concentration of TFC (0.61±0.10) g·g⁻¹DW, TPC (0.52±0.09) g·g⁻¹DW, and TSC (0.21±0.03) g·g⁻¹DW, with high scavenging capacity of DPPH (3.1±0.38) mmol·L⁻¹·g⁻¹DW and FRAP (2.56±0.35) mmol·L⁻¹·g⁻¹DW. Hierarchical clustering analysis (HCA) and principal component analysis (PCA) results indicated the information from chemical compositions and antioxidant capacity can represent the "differences" of different fractions. Canonical correlation analysis (CCorA) revealed a high positive correlation between the amounts of multiple chemical compositions and the antioxidant capacities (r=0.970 0), and the first canonical variate had been reached. Moreover, ABTS⁺ method showed a low response to the compositions of different fractions, so this method may not be suitable for evaluation of Prunellae Spica antioxidant capacities, while DPPH evaluation method was more suitable for TSC and TPC. The results of this study have important reference significance for the evaluation method on antioxidant activity of Prunellae Spica in the field of food or medicine as well as for the development of related extracts.
Subject(s)
Antioxidants , Flavonoids , Phenols , Plant ExtractsABSTRACT
Flavonoids have attracted much attention due to their good anti-inflammatory, anti-oxidation and anti-tumor effects. At present, the extraction of flavonoids is mainly based on organic solvent, while the researches on the use of green and safe solvents are quite limited. Therefore, in the present study, different types of deep eutectic solvents (DESs) were applied to investigate their effect on extraction of flavonoids and optimize the process, also investigate the recovery efficiency of DESs and evaluate the recovery method for total flavonoids. The extraction yield of the total flavonoids acted as the comprehensive evaluation indexes, and a central composite design (CCD) of response surface methodology (RSM) was employed to further optimize the alcohol-based DES extraction conditions. The results showed that the optimized extraction conditions were as follows: water-DES ratio of 27%, solid-liquid ratio of 15 mL·g⁻¹, extraction temperature of 83 °C and extraction time of 42 min in ChCl-glycerol at 1:4 ratio. Under these conditions, the mean experimental value of the extraction yield (75.05 mg·g⁻¹) corresponded well with the predicted value (77.86 mg·g⁻¹). Moreover, these experimental results showed more advantages such as in higher efficiency, economy and environmental protection as compared with previously reported conventional extraction methods. In addition，the recovery yield of the total flavonoids from the DESs extraction solution achieved 97.88% by using AB-8 macroporous resin, and 88.12% desorption ratio can be achieved by 100% ethanol with 5 times resin content. After the above treated DESs were collected, the extraction yield with the same method reached 95.23%, indicating that the method of macroporous resin can be used for efficient and simple recovery and reuse. This study suggests that DESs can be used as a kind of sustainable and efficient natural extraction solvents for extraction of flavonoids from Prunella vulgaris.
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To establish a random forest algorithm for identifying and classifying different brands of Xiasangju granules, and provide effective reference for identifying multi-index complex fingerprint. HPLC method was used to collect the fingerprint of 83 batches of Xiasangju granules from different manufacturers. The classification of Xiasangju granules samples based on chromatographic fingerprints was identified by chemometric methods including principal component analysis (PCA), partial least squares discriminate analysis (PLS-DA) and random forest analysis (RF). The superiority of the above three chemometric methods was compared. The results showed that the fingerprints of 83 batches of Xiasangju granules were established in this study. PCA could only explicate 56.52% variance contribution rate and could not completely classify the samples; PLS-DA analysis was superior to PCA, explicating 63.43% variance contribution rate and could obtain certain separation; RF could well classify the samples into 3 types, and the predication accuracy of the proposed method was 96.5%. Therefore, The results indicate that RF combined with HPLC fingerprint could effectively construct traditional Chinese medicine quality control and analysis system.
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To establish a content determination method for 2,3,5,4'-tetrahydroxy stilbene-2-O-β-D-glucoside (TSG) of the crude/processed root of Polygonum multiflorum from different habitats in China and set up the fingerprint by using UPLC. Various samples were pretreated by macro-porous resin. Then UPLC analysis was performed on Waters ACQUITY UPLC@BEH C18 chromatographic column (2.1 mm×50 mm, 1.7 μm) at (25±5) ℃. A binary gradient elution system was composed of acetonitrile (phase A) and 0.5% acetic acid solution (phase B). Detection was performed at the wavelength of 254 nm, and the mobile flow rate was set at 0.3 mL•min⁻¹. Results showed that the yield of extraction of the 2,3,5,4'-tetrahydroxy stilbene-2-O-β-D-glucoside from root of P. multiflorum was all over 25.0% after macro-porous resin separation; an exclusive UPLC fingerprint method of the crude/processed root of P. multiflorum from different habitats was successfully set up and 17 chromatographic peaks were calibrated. Cluster analysis can not entirely distinguish the crude one from the processed one, while principal component analysis absolutely can. 2,3,5,4'-tetrahydroxy stilbene-2-O-β-D-glucoside is the composition that has largest differences in variable importance in projection (VIP) between crude and processed root of P. multiflorum. The separating method can gain high-purity 2,3,5,4'-tetrahydroxy stilbene-2-O-β-D-glucoside, and the determination method is simple, sensitive, reliable and can be used in fast identifying the crude/processed root of P. multiflorum or as a method for overall quality control of root of P. multiflorum.
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To establish and analyze the HPLC specific chromatograms of Xingnaojing injection manufactured by different factories. The separation was performed on a Thermo BDS Hypersil C₁₈ column (4.6 mm×250 mm, 5 μm), with the mobile phase consisting of acetonitrile-0.02% formic acid aqueous solution for gradient elution. The flow rate was 1.0 mL•min⁻¹, and the column temperature was 35 ℃. The detection wavelength was set at 254 nm, and the sample size was 20 μL. Eleven chromatographic peaks were identified as characteristic peaks of HPLC specific chromatograms of Xingnaojing injection, after analyzing 29 batches of Xingnaojing injection samples. Compared with the reference substances, seven of them were identified as eucarvone, camphor, curcumenone, curcumenol, curdione, curzerenone and germacrone, respectively. HPLC specific chromatograms of Xingnaojing injection manufactured by three factories could be easily classified into three categories after investigation with computer-aided similarity evaluation system combined with principal component analysis. The established HPLC specific chromatograms provide a basis for scientific evaluation and effective control of the quality of Xingnaojing injection.
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To establish the fingerprints of Xiasangju granules (with sugar and non-sugar forms) by HPLC, and provide reference for their identification and effective quality control. High performance liquid chromatography (HPLC) method was used to collect the fingerprints of 20 batches of non-sugar Xiasangju granules and 34 batches of sugar type Xiasangju granules. Their main different components were classified and screened by mode identification methods (principal component analysis, PCA, and orthogonal partial least squares discriminate analysis, OPLS-DA). The principal components were identified by comparing with reference standards. The fingerprints of Xiasangju granules (sugar type and non-sugar type) were established. PCA could not fully classify the two types of granules, while OPLS-DA could obviously classify these two different types of Xiasangju granules. Six components showed greatest difference between two types of granules, including salviaflaside, luteoloside and linarin. The developed mode identification method is helpful to control the overall quality of Xiasangju granules, and it provides an effective approach to quality evaluation.
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This paper aims to investigate the correlation between the antioxidant activity of Prunella vulgaris and its total phenolic acids content by measuring the antioxidant activity of different sources and different organs of P. vulgaris and the total contents of protocatechuic acid, protocatechuic aldehyde, caffeic acid, salviaflaside and rosmarinic acid in these samples. Using the 50% methanol extract of P. vulgaris samples as the research object, DPPH method and HPLC method were used respectively to determine the antioxidant activities and the total contents of the above-mentioned five analytes in P. vulgaris samples. 0.5 mL of 50% methanol extract of P. vulgaris reacts with 0.1 mmol•L⁻¹ DPPH ethanol solution for 60 min, then the absorbance of the reaction solution was measured at 517 nm, scavenging rate and IC₅₀ values were calculated by the absorbance and the sample concentration for evaluating the antioxidant activity. HPLC analysis was made on a C₁₈ Epic column, with acetonitrile-0.1% formic acid aqueous solution as mobile phase (gradient elution), and the detection wavelength was set at 280 nm. The correlation between the antioxidant capacity of different habitats and different organs of P. vulgaris and the total contents of five kinds of phenolic acids was analyzed by partial least squares method. The reaction dose-response range of 50% methanol extract of P. vulgaris with 0.1 mmol•L⁻¹ DPPH ethanol solution was 0.300-1.65 g•L⁻¹. When the quantities of potocatechuic acid, protocatechuic aldehyde, caffeic acid, salviaflaside and rosmarinic acid were respectively in 0.007 84-0.980, 0.011 5-1.44, 0.008 64-1.08, 0.080 0-1.00 and 0.079 8-0.998 μg range, their quantities were in good linear relationship with the corresponding peak areas. The average recovery of 5 components were 97.76%, 96.88%, 100.3%, 102.1%, 104.5%, with RSD of 1.8%, 1.6%, 1.7%, 1.6% and 1.7%, respectively. In a certain range of crude drug quantity, the antioxidant activity of each organ of P. vulgaris and total phenolic acids content inside has a good linear correlation. Therefore, in certain quality range of crude drug, DPPH bioassay combined with HPLC content determination can be used for the quality control of P. vulgaris, as is a new method for the quality control of P. vulgaris.
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This paper was aim to screen microorganisms with attenualed efficiency for Chinese medicine containing aristolochic acid A by liquid-state fermentation. Twelve Chinese medicine were detected by UPLC and aristolochic acid A was only founded in four species of Aristolochia, those were Caulis Aristolochiae Manshuriensis, Aristolochiae Radix, Aistolochia Contorta Bunge and Herba Aristolochiae Mollissima,but not in the others. With the four Chinese medicine containing aristolochic acid A as raw material, ten microorganisms were tested, and the content of aristolochic acid A was detected by UPLC. The results showed that one microorganism can decrease content of aristolochic acid A in all those four Chinese medicine.