Elicitor-mediated enhancement of biomass, polyphenols, mangiferin production and antioxidant activities in callus cultures of Salacia chinensis L.

The present investigation aimed to improve callus biomass, polyphenolic content, biosynthesis of mangiferin and biological potential following application of different elicitor treatments for medicinally important Salacia chinensis L. The leaf-derived callus cultures were established on Murashige and Skoog's (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D: 2.0 mg/l) and 6-benzylaminopurine (BAP: 1.5 mg/l). These cultures were treated with different elicitors viz. jasmonic acid (JA), methyl jasmonate (MeJA) and yeast extracts (YE). The highest calli biomass (five-fold increase within 4 weeks) was achieved when callus was treated with JA (75 µM). The callus obtained on MS medium supplemented with 2,4-D (2.0 mg/l), BAP (1.5 mg/l) and treated with JA (75 µM) displayed augmented values for total phenolics, flavonoids and mangiferin contents. Besides, same treatment elicits the calli for antioxidant properties as evaluated by 2,2-diphenyl-2-picrylhydrazyl (DPPH), ferric-reducing antioxidant power (FRAP) and metal chelating assays. This is the first report on the elicitation study in genus Salacia and, therefore, the discoveries suggested that, S. chinensis calli might be a perfect source for large-scale production of industrially important secondary metabolites. Concurrently data provide accumulated information demonstrating its prominent antioxidant effect revealing its potential without disturbing natural resources.

Molecular Authentication of the Traditional Medicinal Plant "Lakshman Booti" (Smithia conferta Sm.) and Its Adulterants through DNA Barcoding.

BACKGROUND: Smithia conferta Sm. is an annual herb widely used in Indian traditional medical practice and commonly known as "Lakshman booti" in Sanskrit. Morphological resemblance among the species of genus Smithia Aiton. leads to inaccurate identification and adulteration. This causes inconsistent therapeutic effects and also affects the quality of herbal medicine. AIM: This study aimed to generate potential barcode for authentication of S. conferta and its adulterants through DNA barcoding technique. MATERIALS AND METHODS: Genomic DNA extracted from S. conferta and its adulterants was used as templates for polymerase chain reaction amplification of the barcoding regions. The amplicons were directed for sequencing, and species identification was conducted using BLASTn and unweighted pair-group method with arithmetic mean trees. In addition, the secondary structures of internal transcribed spacer (ITS) 2 region were predicted. RESULTS: The nucleotide sequence of ITS provides species-specific single nucleotide polymorphisms and sequence divergence (22%) than psbA-trnH (10.9%) and rbcL (3.1%) sequences. The ITS barcode indicates that S. conferta and Smithia sensitiva are closely related compared to other species. CONCLUSION: ITS is the most applicable barcode for molecular authentication of S. conferta, and further chloroplast barcodes should be tested for phylogenetic analysis of genus Smithia. SUMMARY: The present investigation is the first effort of utilization of DNA barcode for molecular authentication of S. conferta and its adulterants. Also, this study expanded the application of the ITS2 sequence data in the authentication. The ITS has been proved as a potential and reliable candidate barcode for the authentication of S. conferta. Abbreviations used: BLASTn: Basic Local Alignment Search Tool for Nucleotide; MEGA: Molecular Evolutionary Genetic Analysis; EMBL: European Molecular Biology Laboratory; psbA-trnH: Photosystem II protein D1- stuctural RNA: His tRNA gene; rbcL: Ribulose 1,5 bi-phosphate carboxylase/oxygenase large subunit gene.

Extraction efficiency, phytochemical profiles and antioxidative properties of different parts of Saptarangi (Salacia chinensis L.) - An important underutilized plant.

The study aimed to evaluate extraction efficiency, detection and quantification of phytochemicals, minerals and antioxidative capacity of different parts of Salacia chinensis L. Continuous shaking extraction, steam bath assisted extraction, ultrasonic extraction and microwave assisted extraction with varied time intervals were employed for extraction of phenolics, flavonoids, and antioxidants. Preliminary screening revealed the presence of wide array of metabolites along with carbohydrates and starch. Steam bath assisted extraction for 10 min exposure was found most suitable for extraction phenolics (46.02 ± 2.30 mg of gallic acid equivalent per gram of dry weight and 48.57 ± 2.42 mg of tannic acid equivalent per gram of dry weight) and flavonoids (35.26 ± 1.61 mg of quercetin equivalent per gram of dry weight and 51.60 ± 2.58 mg of ellagic acid equivalent per gram of dry weight). In support, reverse phase-high performance liquid chromatography- diode array detector confirmed the presence of seven pharmaceutically important phenolic acids. Antioxidant capacity was measured by 1, 1- diphenyl-1-picryl hydrazyl (DPPH), ferric reducing antioxidant power (FRAP), 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) scavenging (ABTS) and N, N-dimethyl-p-phenylenediamine (DMPD) assays and represented as trolox equivalent antioxidant capacity (TEAC) and ascorbic acid equivalent antioxidant capacity (AEAC). Antioxidant capacity ranged from 121.02 ± 6.05 to 1567.28 ± 78.36 µM trolox equivalent antioxidant capacity and 56.62 ± 2.83 to 972.48 ± 48.62 µM ascorbic acid equivalent antioxidant capacity. Roots showed higher yields of illustrated biochemical parameters, however fresh fruit pulp was found a chief source of minerals. Gas chromatography-mass spectroscopic analysis revealed the presence of a vast array of phytoconstituents associated with different plant parts. The present study revealed the amounts of minerals and diverse phytoconstituents in various parts of S. chinensis and confirmed its medicinal and nutritional implications.

Storing of Extracts in Polypropylene Microcentrifuge Tubes Yields Contaminant Peak During Ultra-flow Liquid Chromatographic Analysis.

BACKGROUND AND AIM: This study was designed to understand the effect of storage in polypropylene microcentrifuge tubes and glass vials during ultra-flow liquid chromatographic (UFLC) analysis. MATERIALS AND METHODS: One ml of methanol was placed in polypropylene microcentrifuge tubes (PP material, Autoclavable) and glass vials (Borosilicate) separately for 1, 2, 4, 8, 10, 20, 40, and 80 days intervals stored at -4°C. RESULTS: Contaminant peak was detected in methanol stored in polypropylene microcentrifuge tubes using UFLC analysis. The contaminant peak detected was prominent, sharp detectable at 9.176 ± 0.138 min on a Waters 250-4.6 mm, 4 µ, Nova-Pak C18 column with mobile phase consisting of methanol:water (70:30). CONCLUSION: It was evident from the study that long-term storage of biological samples prepared using methanol in polypropylene microcentrifuge tubes produce contaminant peak. Further, this may mislead in future reporting an unnatural compound by researchers. SUMMARY: Long-term storage of biological samples prepared using methanol in polypropylene microcentrifuge tubes produce contaminant peakContamination peak with higher area under the curve (609993) was obtained in ultra-flow liquid chromatographic run for methanol stored in PP microcentrifuge tubesContamination peak was detected at retention time 9.113 min with a lambda max of 220.38 nm and 300 mAU intensity on the given chromatographic conditionsGlass vials serve better option over PP microcentrifuge tubes for storing biological samples. Abbreviations used: UFLC: Ultra Flow Liquid Chromatography; LC: Liquid Chromatography; MS: Mass spectrometry; AUC: Area Under Curve.

RP-HPLC analysis of seco-iridoid glycoside swertiamarin from different Swertia species.

Genus Swertia is valued for its great medicinal potential; mainly Swertia chirayita (Roxb. ex Fleming) H. Karst. is used in traditional medicine for a wide range of diseases. Seco-iridoid glycosides like swertiamarin is referred with enormous pharmacological potentials. The aim of the study was to identify a suitable substitute to S. chirayita by quantifying seco-iridoid swertiamarin from five different Swertia species endemic to the Western Ghats. The reverse-phase high-performance liquid chromatography diode array detector analyses were performed and chromatographic separation was achieved on a Lichrospher 100, C18e (5 µm) column (250-4.6 mm). A mobile phase consisting of acetonitrile and water (25:75) was used for separation. Results indicated that the concentration of the marker compound has been found to vary largely between and within the species from different localities. The content of swertiamarin was the highest in S. chirayita compared to the other species studied herein, advocating the use of Swertia minor as an alternate source to S. chirayita.

Reverse Phase-ultra Flow Liquid Chromatography-diode Array Detector Quantification of Anticancerous and Antidiabetic Drug Mangiferin from 11 Species of Swertia from India.

BACKGROUND: Genus Swertia is valued for its great medicinal potential, mainly Swertia chirayita (Roxb. ex Fleming) H. Karst. is used in traditional medicine for a wide range of diseases. Mangiferin one of xanthoids is referred with enormous pharmacological potentials. OBJECTIVE: The aim of the study was to quantify and compare the anticancerous and antidiabetic drug mangiferin from 11 Swertia species from India. The study also evaluates hierarchical relationships between the species based on mangiferin content using multivariate analysis. MATERIALS AND METHODS: The reverse phase-ultra flow liquid chromatography-diode array detector analyses was performed and chromatographic separation was achieved on a Lichrospher 100, C18e (5 µm) column (250-4.6 mm). Mobile phase consisting of 0.2% triethylamine (pH-4 with O-phosphoric acid) and acetonitrile (85:15) was used for separation with injection volume 20 µL and detection wave length at 257 nm. RESULTS: Results indicated that concentration of mangiferin has been found to vary largely between Swertia species collected from different regions. Content of mangiferin was found to be highest in Swertia minor compared to other Swertia species studied herein from the Western Ghats and Himalayan region also. The same was also evident in the multivariate analysis, wherein S. chirayita, S. minor and Swertia paniculata made a separate clade. CONCLUSION: Conclusively, the work herein provides insights of mangiferin content from 11 Swertia species of India and also presents their hierarchical relationships. To best of the knowledge this is the first report of higher content of mangiferin from any Swertia species. SUMMARY: The present study quantifies and compares mangiferin in 11 species of Swertia from India. The study also evaluates hierarchical relationships between the species based on mangiferin content using multivariate analysis. The mangiferin content was highest in S. minor compared to the studied Swertia species. To the best of our knowledge this is the first report of higher content of mangiferin from Swertia species. Abbreviations used: LOD: Limit of detection, LOQ: Limit of quantification, RP-UFLC-DAD: Reverse phase-ultra flow liquid chromatography-diode array detector, RSD: Relative standard deviation, SAN: Swertia angustifolia, SAP: Swertia angustifolia var. pulchella, SBI: S. bimaculata, SCH: S. chirayita, SCO: S. corymbosa, SDE: S. densifolia, SDI: S. dialatata, SLA: S. lawii, SMI: S. minor; SNE: S. nervosa, and SPA: S. paniculata.

Quantitative determination of three pentacyclic triterpenes from five Swertia L. species endemic to Western Ghats, India, using RP-HPLC analysis.

Aim of this study was to identify pentacyclic triterpenoids betulinic acid (BA), oleanolic acid (OA) and ursolic acid (UA) from five Swertia species endemic to Western Ghats, which are used as substitutes as well as adulterants to Swertia chirayita. Our results indicate that the concentration of active compound vary largely among and within the species from different localities. OA was found in a higher amount in all species compared to BA and UA. From the results, it was clear that BA, OA and UA are present in the endemic species collected from Western Ghats, thus advocating the use of these species as alternate sources to S. chirayita. This in due course may release pressure of exploitation from natural resources of S. chirayita and help to bring it out from an endangered category from conservation point of view.

Highly efficient in vitro regeneration, establishment of callus and cell suspension cultures and RAPD analysis of regenerants of Swertia lawii Burkill.

Highly efficient in vitro regeneration system has been developed for Swertia lawii Burkill, an important herb used as substitute for Swertia chirayita. Shoot tips explants were cultured on MS medium with various phytohormones for multiple shoot production. The best shoot production frequency (100%) and maximum shoots (10.4 ± 0.8) were obtained on MS media containing TDZ (3.0 mg l-1) in combination with IBA (0.3 mg l-1). Maximum callus induction (95 ± 4.8%) and callus growth (1.7 ± 0.4 gm) was achieved on MS medium with 2, 4-D (3.0 mg l-1). Cell suspension cultures were established and studied for their growth kinetics. Shoots were rooted best (22.1 ± 2.5) in 1/2 MS medium with IAA (3.0 mg l-1). The genetic uniformity of the micropropagated clones was assessed using RAPD markers. Out of 405 bands, 400 (98.76%) were monomorphic and rest 5 (1.24%) were polymorphic. High multiplication frequency and low risk of genetic instability ensures the efficacy of this protocol.

Seasonal variation in content of camptothecin from the bark of Nothapodytes nimmoniana (Grah.) Mabb., using HPLC analysis.

OBJECTIVE: To study and compare seasonal variation in camptothecin (CPT) content from bark samples of Nothapodytes nimmoniana obtained from geographically and climatologically isolated populations. METHODS: A standard High Performance Liquid Chromatography methodology was used to analyze and quantify CPT from bark samples of N. nimmoniana. RESULTS: Sample collected from Amboli yielded highest CPT content 1.337 g/100 g dry bark powder during the monsoon compared to other localities in study. Monsoon (August) showed to accumulate higher levels of CPT in barks of N. nimmoniana as compared to summer (May). Amboli averaged highest accumulation of CPT compared to other localities under study. CONCLUSION: These findings indicate season to have control over accumulation of CPT. Locality Amboli has highest CPT content in all seasons and were the elite population during the study. The study also suggests the need for further investigation in lights of biosynthesis in the plant.