Quantification of Luteolin, Apigenin and Chrysoeriol in Tecoma stans by RP-HPLC Method.

Tecoma stans (Fam. Bignoniaceae) is also popularly known as yellow bells and yellow trumpet bush in vernacular terminology. Limited and variable data are available from the literature regarding the quantification of luteolin, apigenin and chrysoeriol, which are considered as the most active pharmacological active constituents. High-performance liquid chromatography-photodiode array detection has been developed for the determination of the bioactive flavonoids, luteolin, apigenin and chrysoeriol, from the methanolic extract of the leaves of T. stans. A column packed with a pentafluorophenyl-based stationary phase was used for the separation of the analytes under gradient elution. The detection wavelength was 345 nm. The validation of the method as per the International Council on Harmonisation (ICH) guidelines (ICH 2005) for linearity, accuracy and precision was investigated and found within limits specified by the ICH guidelines. The method was linear over with a good regression coefficient of more than 0.99. The limit of detection of the method was 0.68, 2.97 and 1.76 µg/mL for luteolin, apigenin and chrysoeriol, respectively. In conclusion, a reliable and reproducible method was devised that can be used for the estimation of the said components from T. stans.

Quality control aspects of herbs and botanicals in developing countries: Coleus forskohlii Briq a case study.

OBJECTIVE: Current trend of commercialization of herbal medicines draw a huge need of maintaining their quality. The declaration of quality, safety and efficacy of medicinal plants as well as poly-herbal formulations has become an important issue. Hence, qualitative and quantitative analysis of herbal drugs and formulations viz., fingerprint profiles and quantification of the various markers become key factors of quality control. MATERIALS AND METHODS: Present investigation is a detailed report for quality control of well-known herb Coleus forskohlii Briq, which includes physicochemical parameter determination, safety evaluation, microscropical evaluation, and chromatographic fingerprinting as well. RESULTS: Physico-chemical characters were evaluated according to Indian Pharmacopoeia, further microscopic evaluation of transverse section of Coleus reveals that periderm, secondary phloem, and wide secondary xylem cylinder, which occupies major portion of the root fragmentary. Chromatographic fingerprint profiles of Coleus have been generated, and a marker based standardization strategy was adopted; using different analytical technique like high-performance thin layer chromatography, high-performance liquid chromatography and gas chromatography-mass spectroscopy to maintain quality and ensure safety as well as efficacy. CONCLUSION: These advancements in modern techniques of analysis can lead to effective quality control of Coleus as well as other herbs. Present report can act as pioneer for quality control of modern herbal medicine.

Characterization of Linum usitatissimum L. oil obtained from different extraction technique and in vitro antioxidant potential of supercritical fluid extract.

AIM: Present investigation was aimed to characterize the fixed oil of Linum usitatissimum L. using five different extraction methods: Supercritical fluid extraction (SFE), ultrasound-assistance, soxhlet extraction, solvent extraction, and three phase partitioning method. MATERIALS AND METHODS: The SFE conditions (temperature, pressure, and volume of CO2) were optimized prior for better yield. The extracted oils were analyzed and compared for their physiochemical parameters, high performance thin layer chromatography (HPTLC), gas chromatography-mass spectrometry (GC-MS), and Fourier-transformed infrared spectroscopy (FT-IR) fingerprinting. Antioxidant activity was also determined using 1,1-diphenyl-2-picrylhydrazyl and superoxide scavenging method. RESULT: The main fatty acids were α-linolenic acid, linoleic acid, palmitic acid, and stearic acid as obtained by GC-MS. HPTLC analysis revealed the presence of similar major components in chromatograms. Similarly, the pattern of peaks, as obtained in FT-IR and GC-MS spectra of same oils by different extraction methods, were superimposable. CONCLUSION: Analysis reported that the fixed oil of L. usitatissimum L. is a good source of n-3 fatty acid with the significant antioxidant activity of oil obtained from SFE extraction method.

Rapid, sensitive, and validated UPLC/Q-TOF-MS method for quantitative determination of vasicine in Adhatoda vasica and its in vitro culture.

BACKGROUND: Adhatoda vasica a perennial herb has been used in Ayurvedic and Unani system of medicines since last 2000 years and has been employed for the treatment of respiratory tract ailments. OBJECTIVE: To develop and validate new, rapid, and highly sensitive high throughput ultra-performance liquid chromatography/quadrupole-time-of-flight mass-spectrometry (UPLC/Q-TOF-MS) method for the quantitative estimation of vasicine in the leaves and to establish in vitro cultures of Adhatoda vasica for production of vasicine. MATERIALS AND METHODS: The chromatographic separation was achieved on a Waters ACQUITY UPLC™ BEH C8 (100.0 × 2.1 mm; 1.7 µm) column packing using isocratic mobile phase consisting of acetonitrile: 20 mM ammonium acetate (90:10; v/v) in a multiple reactions monitoring mode using the transitions m/z 189.09 → 171.08 for vasicine. RESULTS: The vasicine was eluted at 2.58 ± 0.05 min and established a dynamic range of linearity over the concentration range of 1-1000 ng/ml (r (2) = 0.999 ± 0.0005). The lower limit of detection and quantification was 0.68 and 1.0 ng/ml, respectively. There was no significant difference observed in the content of vasicine (0.92-1.04%w/w) among the eleven samples collected from different locations of India. The in vitro cultures developed showed that addition of extra 28 mM KNO3 and 100 mM NaCl in MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) + benzyladenine (BA) + indole acetic acid (IAA) (1 ppm each) produces faster biomass and higher amount of quinazoline alkaloids. CONCLUSION: Rapid, efficient, and sensitive UPLC/Q-TOF-MS method was developed for the estimation of vasicine and an efficient protocol for development of in vitro cultures was proposed, which can be used at large scale for industrial production of vasicine using bioreactors.

In vitro production of alkaloids: Factors, approaches, challenges and prospects.

The wide diversity of plant secondary metabolites is largely used for the production of various pharmaceutical compounds. In vitro cell tissue or organ culture has been employed as a possible alternative to produce such industrial compounds. Tissue culture techniques provide continuous, reliable, and renewable source of valuable plant pharmaceuticals and might be used for the large-scale culture of the plant cells from which these secondary metabolites can be extracted. Alkaloids are one of the most important secondary metabolites known to play a vital role in various pharmaceutical applications leading to an increased commercial importance in recent years. The tissue culture techniques may be utilized to improve their production of alkaloids via somaclonal variations and genetic transformations. The focus of this review is toward the application of different tissue culture methods/techniques employed for the in vitro production of alkaloids with a systematic approach to improve their production.

Genetic and metabolic diversity in Stevia rebaudiana using RAPD and HPTLC analysis.

CONTEXT: Stevia rebaudiana Bertoni (Asteraceae) is an important medicinal plant and is much used due to its zero calories sweetening property. Stevia leaves as well as its extracts and pure compounds are currently used in the preparation of several medicines, food products and neutraceuticals. OBJECTIVE: To study the genetic and metabolic variability in S. rebaudiana among accessions of different geographical regions of India using random amplified polymorphic DNA (RAPD) markers and high-performance thin layer chromatography (HPTLC) analysis. MATERIALS AND METHODS: The RAPD analysis of Stevia rebaudiana (11 accessions) was carried out using 20 random operon primers. Dendrogram was constructed for cluster analysis based on the unweighted pair group method with arithmetic means (UPGMA) using Winboot. The HPTLC analysis of all samples was carried out on silica using acetone:ethyl acetate:water (5:4:1, v/v/v) for fingerprinting and quantification of stevioside and rebaudioside A at 360 nm after spraying with anisaldehyde sulphuric acid. RESULTS: Ten out of 20 primers screened were found most informative; amplification products of the genotypes yielded a total of 87 scorable bands (67 polymorphic), whereas genetic similarity (GS) coefficient (0.01-0.08) and polymorphism (67.24-92.40%) showed huge variability. Similarly, HPTLC analysis showed large variation among different samples with respect to their presence or absence of metabolite and their concentration. CONCLUSION: Out of the 11 Stevia accessions, Delhi and Mohali varieties showed much relatedness with each other and were concluded to be the superior genotype in context to RAPD and HPTLC analysis. The information obtained here could be valuable for devising strategies for cultivating this medicinal plant.

Metabolic diversity in Coleus forskohlii Briq. of Indian subcontinent.

New economic, easy, specific, accurate, robust, validated high performance thin layer chromatography (HPTLC) and high performance liquid chromatography methods with good range of linearity and sensitivity were developed for quantification of forskolin in ten samples collected from different regions of Indian subcontinent, which showed a large variation among samples (0.074-0.282%, w/w). Metabolic diversity of all the samples using HPTLC fingerprint method showed a total of 16 well separated spots. There is no significant metabolic diversity among the samples collected from different locations of Indian subcontinent, which was obtained from HPTLC fingerprinting. The results of locational variation showed highest content of forskolin in Bengaluru sample by both analytical methods. The validated quantification methods and fingerprint profile together can act as a good authentication tool for coleus as well as for other medicinal plants.