High-performance Liquid Chromatography Fingerprint Analysis Research Articles

High perfomance liquid chromatography fingerprint analysis for quality control of brotowali (Tinospora crispa)

Brotowali (Tinospora crispa) is widely used in Indonesia as ingredient of herbal medicine formulation. To ensure the quality, safety, and efficacy of herbal medicine products, its chemical constituents should be continuously evaluated. High performance liquid chromatography (HPLC) fingerprint is one of powerful technique for this quality control process. In this study, HPLC fingerprint analysis method was developed for quality control of brotowali. HPLC analysis was performed in C18 column and detection was performed using photodiode array detector. The optimum mobile phase for brotowali fingerprint was acetonitrile (ACN) and 0.1% formic acid in gradient elution mode at a flow rate of 1 mL/min. The number of peaks detected in HPLC fingerprint of brotowali was 32 peaks and 23 peaks for stems and leaves, respectively. Berberine as marker compound was detected at retention time of 20.525 minutes. Evaluation of analytical performance including precision, reproducibility, and stability prove that this HPLC fingerprint analysis was reliable and could be applied for quality control of brotowali.

Inulin-type fructan and infusion of Artemisia vulgaris protect the liver against carbon tetrachloride-induced liver injury

BackgroundInfusions of aerial parts of Artemisia vulgaris L. (Asteraceae) are used in herbal medicine to treat several disorders, including hepatosis. PurposeEvaluation of in vivo hepatoprotective effects of A. vulgaris infusion (VI) and inulin (VPI; i.e., the major polysaccharide of VI). Study designThe hepatoprotective effect of A. vulgaris extracts on carbon tetrachloride (CCl4)-induced hepatotoxicity and the probable mechanism involved in this protection were investigated in mice. MethodsA. vulgaris infusion (VI) was prepared according to folk medicine using the aerial parts of the plant. Carbohydrate, protein, and total phenolic content was determined in VI, and its phenolic profile was determined by high-performance liquid chromatography (HPLC). Male Swiss mice were orally pretreated for 7 days with VI or VPI (once per day). On days 6 and 7 of treatment, the mice were intraperitoneally challenged with CCl4. Liver and blood were collected and markers of hepatic damage in plasma and oxidative stress in the liver were analyzed. Hepatic histology and inflammatory parameters were also studied in the liver. The scavenging activity of VI and VPI were evaluated in vitro using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. ResultsVI contained 40% carbohydrates, 2.9% proteins and 9.8% phenolic compounds. The HPLC fingerprint analysis of VI revealed chlorogenic, caffeic and dicaffeoylquinic acids as major low-molar-mass constituents. Oral pretreatment with VI and VPI significantly attenuated CCl4-induced liver damage, reduced the activity of alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP) in plasma, and prevented reactive oxygen species accumulation and lipid peroxidation in the liver. Comparisons with the CCl4-treated group showed that VI and VPI completely prevented necrosis, increased the levels of reduced glutathione (GSH), and reduced tumor necrosis factor alpha (TNF-α) level in the liver. VI and VPI also exhibited high radical scavenging activity in vitro. ConclusionVI and VPI had remarkable hepatoprotective effects in vivo, which were likely attributable to antioxidant and immunomodulatory properties. The present findings support the traditional use of A. vulgaris infusion for the treatment of hepatic disorders.

Hydroethanolic extract of Baccharis trimera promotes gastroprotection and healing of acute and chronic gastric ulcers induced by ethanol and acetic acid.

Ethanol is a psychoactive substance highly consumed around the world whose health problems include gastric lesions. Baccharis trimera is used in folk medicine for the treatment of gastrointestinal disorders. However, few studies have evaluated its biological and toxic effects. To validate the popular use of B. trimera and elucidate its possible antiulcerogenic and cytotoxic mechanisms, a hydroethanolic extract of B. trimera (HEBT) was evaluated in models of gastric lesions. Rats and mice were used to evaluate the protective and antiulcerogenic effects of HEBT on gastric lesions induced by ethanol, acetic acid, and chronic ethanol consumption. The effects of HEBT were also evaluated in a pylorus ligature model and on gastrointestinal motility. The LD50 of HEBT in mice was additionally estimated. HEBT was analyzed by nuclear magnetic resonance, and a high-performance liquid chromatography fingerprint analysis was performed. Oral HEBT administration significantly reduced the lesion area and the oxidative stress induced by acute and chronic ethanol consumption. However, HEBT did not protect against gastric wall mucus depletion and did not alter gastric secretory volume, pH, or total acidity in the pylorus ligature model. Histologically, HEBT accelerated the healing of chronic gastric ulcers in rats, reflected by contractions of the ulcer base. Flavonoids and caffeoylquinic acids were detected in HEBT, which likely contributed to the therapeutic efficacy of HEBT, preventing or reversing ethanol- and acetic acid-induced ulcers, respectively. HEBT antiulcerogenic activity may be partially attributable to the inhibition of free radical generation and subsequent prevention of lipid peroxidation. Our results indicate that HEBT has both gastroprotective and curative activity in animal models, with no toxicity.

Fingerprint analysis, multi-component quantitation, and antioxidant activity for the quality evaluation of Salvia miltiorrhiza var. alba by high-performance liquid chromatography and chemometrics.

Salvia miltiorrhiza Bge. var. alba C.Y. Wu and H.W. Li has wide prospects in clinical practice. A useful comprehensive method was developed for the quality evaluation of S. miltiorrhiza var. alba by three quantitative parameters: high-performance liquid chromatography fingerprint, ten-component contents, and antioxidant activity. The established method was validated for linearity, precision, repeatability, stability, and recovery. Principal components analysis and hierarchical clustering analysis were both used to evaluate the quality of the samples from different origins. The results showed that there were category discrepancies in quality of S. miltiorrhiza var. alba samples according to the three quantitative parameters. Multivariate linear regression was adopted to explore the relationship between components and antioxidant activity. Three constituents, namely, danshensu, rosmarinic acid, and salvianolic acid B, significantly correlated with antioxidant activity, and were successfully elucidated by the optimized multivariate linear regression model. The combined use of high-performance liquid chromatography fingerprint analysis, simultaneous multicomponent quantitative analysis, and antioxidant activity for the quality evaluation of S. miltiorrhiza var. alba is a reliable, comprehensive, and promising approach, which might provide a valuable reference for other herbal products in general to improve their quality control.

Phytochemical composition, antioxidant activity and HPLC fingerprinting profiles of three Pyrola species from different regions.

The present study was performed to investigate the variation of phytochemical composition, antioxidant activity and High Performance Liquid Chromatography (HPLC) fingerprinting profiles of three Pyrola species. Thirteen samples (eight P. decorata, three P. calliantha and two P. renifolia) were collected from different regions in China. The tannin, hyperoside and quercetin contents of all samples were determined by reverse-phase HPLC and varied within the range 9.77–34.75, 0.34–2.16 and 0.062–0.147 mg/g dry weigh, respectively. Total flavonoid content was evaluated and varied within the range 16.22–37.82 mg/g dry weight. Antioxidant activity was determined by DPPH assay, with IC50 ranging from 7.96 to 50.33 µg/ml, ABTS•+ and FRAP assay, within the range 612.66–1021.05 and 219.64–398.12 µmol equiv. Trolox/g, respectively. These results revealed that there were significant variations in phytochemical profiles and antioxidant activity among all samples. Due to the higher phytochemical content and significant antioxidant activity, P. calliantha was selected as the most valuable species, and the P. calliantha sample from Left banner of Alxa even possessed the strongest antioxidant activity among all the thirteen samples. Futhermore, Emei Mountain was proved to be the most suitable region for producing P. decorata. Moreover, in order to further evaluate the diversities and quality of Pyrola, HPLC fingerprint analysis coupled with hierarchical cluster and discrimination analyses were introduced to establish a simple, rapid and effective method for accurate identification, classification and quality assessment of Pyrola. Thirteen samples were divided into three groups consistent with their morphological classification. Two types of discriminant functions were generated and the ratio of discrimination was 100%. This method can identify different species of Pyrola and the same species from different regions of origin. Also, it can be used to compare and control the quality of Pyrola and other natural products prepared from them.

Piper nigrum: Micropropagation, Antioxidative enzyme activities, and Chromatographic Fingerprint Analysis for Quality Control

A reliable in vitro regeneration system for the economical and medicinally important Piper nigrum L. has been established. Callus and shoot regeneration was encouraged from leaf portions on Murashige and Skoog (MS) medium augmented with varied concentrations of plant growth regulators. A higher callus production (90 %) was observed in explants incubated on MS medium incorporated with 1.0 mg L(-1) 6-benzyladenine (BA) along with 0.5 mg L(-1) gibberellic acid after 4 weeks of culture. Moreover, a callogenic response of 85 % was also recorded for 1.0 mg L(-1) BA in combination with 0.25 mg L(-1) α-naphthalene acetic acid (NAA) and 0.25 mg L(-1) 2,4-dichlorophenoxyacetic acid or 0.5 mg L(-1) indole butyric acid (IBA) along with 0.25 mg L(-1) NAA and indole acetic acid. Subsequent sub-culturing of callus after 4 weeks of culture onto MS medium supplemented with 1.5 mg L(-1) thiodiazoran or 1.5 mg L(-1) IBA induced 100 % shoot response. Rooted plantlets were achieved on medium containing varied concentrations of auxins. The antioxidative enzyme activities [superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX)] revealed that significantly higher SOD was observed in regenerated plantlets than in other tissues. However, POD, CAT, and APX were higher in callus than in other tissues. A high-performance liquid chromatography (HPLC) fingerprint analysis protocol was established for quality control in different in vitro-regenerated tissues of P. nigrum L. During analysis, most of the common peaks represent the active principle "piperine." The chemical contents, especially piperine, showed variation from callus culture to whole plantlet regeneration. Based on the deviation in chromatographic peaks, the in vitro-regenerated plantlets exhibit a nearly similar piperine profile to acclimated plantlets. The in vitro regeneration system and HPLC fingerprint analysis established here brought a novel approach to the quality control of in vitro plantlets, producing metabolites of interest with substantial applications for the conservation of germplasm.

Development of an HPLC fingerprint for quality control and species differentiation ofUncaria rhynchophylla(Miq.) ex Havil

Summary The stems of Uncaria rhynchophylla (Miq.) ex Havil have a long history of use in traditional Chinese medicine to treat diseases and improve health. There is evident evidence that alkaloids constituents are mainly responsible for the beneficial effects of this plant medicine. The amounts of the major bioactive alkaloids in this plant vary widely with species, habitat, and as such, and establishment of a high-performance liquid chromatography (HPLC) fingerprint for quality control of this herbal medicine is of particular importance. The most alkaloids are used for medicine treatment and research. On the basis of the chromatographic data, a consistent HPLC fingerprint pattern containing 15 common peaks was obtained. Among these common peaks, four were identified as rhynchophylline, isorhynchophylline, corynoxeine, and isocorynoxeine. On the basis of this HPLC fingerprint and principal-components analysis, the quality of fifteen samples from different producing areas of China was objectively assessed....

Development of a novel method combining HPLC fingerprint and multi-ingredients quantitative analysis for quality evaluation of traditional chinese medicine preparation

A novel method combining high performance liquid chromatography (HPLC) fingerprint and simultanous quantitative analysis of multiple acitve components was developed and validated for quality evaluation of one type of traditional Chinese medicine preparations: Shuang-huang-lian (SHL) oral liquid formulation. For fingerprint analysis, 45 peaks were selected as the common peaks to evaluate the similarities among several different SHL oral liquid preparations collected from manufacturers. Additionally, simultanous quantification of eleven markers, including chlorogenic acid, caffeic acid, rutin, forsythiaside, scutellarin, baicalin, forsythin, luteoloside, apigenin, baicalein and wogonin, was performed. Statistical analysis of the obtained data demonstrated that our method has achieved desired linearity, precision and accuracy. Finally, concentrations of these eleven markers in SHL oral liquid prepared by different manufacturers in China were determined. These results demonstrated that the combination of HPLC chromatographic fingerprint and simultaneous quantification of multi-ingredients offers an efficient and reliable approach for quality evaluation of SHL oral liquid preparations.

Plant regeneration of Erigeron breviscapus (vant.) Hand. Mazz. and its chromatographic fingerprint analysis for quality control

An efficient micropropagation system for Erigeron breviscapus (vant.) Hand. Mazz., an important medicinal plant for heart disease, has been developed. Shoot organogenesis occurred from E. breviscapus leaf explants inoculated on a medium supplemented with a combination of plant growth regulators. On average, 17 shoots per leaf explant were produced after 30 days when they were cultured on MS basal salts and vitamin medium containing 5 microM 6-benzylaminopurine (BAP) and 5 microM 1-naphthaleneacetic acid (NAA). All the regenerated shoots formed complete plantlets on a medium containing 2.5-10 microM indole-3-butyric acid (IBA) within 30 days, and 80.2% of the regenerated plantlets survived and grew vigorously in field conditions. Based on the variation in common peaks and the produced amount of the most important bioactive component, scutellarin, a high performance liquid chromatography (HPLC) fingerprinting system was developed for quality control of these micropropagated plants. Chemical constituents in E. breviscapus micropropagated plants varied during plant development from regeneration to maturation, the latter of which showed the most similar phytochemical profile in comparison with mother plants. The regeneration protocol and HPLC fingerprint analysis developed here provided a new approach to quality control of micropropagated plants producing secondary metabolites with significant implications for germplasm conservation.

Fingerprint analysis of Psoralea corylifolia L. by HPLC and LC–MS

High-performance liquid chromatography (HPLC) was developed for fingerprint analysis of Psoralea corylifolia. Liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI-MS n ) technique was first employed to identify the components of the fingerprint. The samples were separated with an Alltima C 18 column (250 mm × 4.6 mm, 5 μm) by linear gradient elution using water–acetic acid (A; 100:0.1, v/v) and acetonitrile (B; 0 min, 40%; 15 min, 50%; 35 min, 60%; 45 min, 70%; 55 min, 80%; and maintained for 5 min) as mobile phase at a flow rate of 1.0 ml/min and detector wavelength at 245 nm. A standard procedure was developed for HPLC fingerprint analysis. Average chromatogram of 10 batches of P. corylifolia L. from Sichuan and Henan Provinces, PR China, which has been considered as the original and genuine herbal medicine for a long time, was first established as the characteristic fingerprint. There are 12 common peaks in this fingerprint. Ten of these common peaks were identified by MS data. This profile was then used to identify and assess the differences among the herb grown in various areas of China. The HPLC fingerprint analysis is specific and may serve for quality identification and comprehensive evaluation of P. corylifolia.