Comparative Morpho-micrometric Investigations in Six Indigenous Ocimum Species of India with DOE Based HPTLC Method for Multi-class Component Analysis.

The Ocimum genus is one of India's prominent botanical classes of traditional medicinal culture comprising medicinally and agronomically important plants. Morphological resemblances, overlapping geographical distribution, and history of traditional nomenclature have necessitated a comprehensive qualitative report for effective quality control and removing the species ambiguity pertaining to this genus. This paper provides detailed morpho-micrometric characteristics used to differentiate between six indigenous Ocimum species of India. Among them, O. gratissimum was distinguished as the only shrub with a fleshy petiole. In green and purple forms, O. tenuiflorum leaves had serrate margins and showed no particular anatomical differences except for the anthocyanins containing epidermal cells of the latter. O. basilicum had glabrous leaves except for the veins, which were puberulous. O. filamentosum had tenuous anther filaments and was the least aromatic while O. africanum had a citrusy odour, which along with the number of xylary rows, size of mesophyll cells, and epidermal cell wall architecture, distinguished it from O. americanum. An HPTLC method was developed using experimental design and validated for quantification of multi-class compounds from terpenoic, phenolic acids, and flavonoids in Ocimum leaves. It was found linear (r 2 > 0.99) with recoveries between 95 - 100% for all compounds. The eluted bands of marker compounds were subjected to HPTLC-MS analysis as a confirmative tool. This is the first anatomical and analytical report of O. filamentosum Forssk. The obtained results could be effectively used for species identification using vegetative characters alone with the anatomical-HPTLC data backing up the former as a rapid and economical tool.

Development and Validation of RP-HPLC Method for Vicenin-2, Orientin, Cynaroside, Betulinic Acid, Genistein, and Major Eight Bioactive Constituents with LC-ESI-MS/MS Profiling in Ocimum Genus.

BACKGROUND: Ocimum genus, known as Tulsi or Basil, is a prominent botanical class in Asian culture, especially in India. The leaves have immunomodulatory, antioxidant, stress-relieving, and adaptogenic roles in traditional and modern medicine, with prominent usage in herbal teas and nutraceuticals. OBJECTIVE: An high-performance liquid chromatography-photodiode array (HPLC-PDA) method was developed and validated for quantification of vicenin-2, orientin, cynaroside, betulinic acid, genistein with syringic acid, rosmarinic acid, eugenol, carnosic acid, oleanolic acid, ursolic acid, luteolin, and apigenin and was confirmed using a novel electrospray ionisation-mass spectrometry (ESI-MS/MS) method in the Ocimum genus samples. METHOD: The methodology parameters were developed on an reverse phase (RP) C18 column with a gradient elution of 1 mL/min flow rate for 0.1% o-phosphoric acid and acetonitrile at 210 and 340 nm wavelengths. RESULTS: The validation data for 13 bioactive compounds showed good linearity (r2 > 0.99) with sensitive LOD (0.034-0.684 µg/mL) and LOQ (0.100-2.068 µg/mL) with recoveries (83.66-101.53%). The results of the quantification were found to be precise (RSD, <5.0%) and accurate (relative error (RE), -0.60-1.06). The method performance was verified by analyzing 10 samples of O. tenuiflorum from the 10 geographical states of India (RSD, <5.0%) and were found to be robust. This HPLC-PDA method with ESI-MS/MS confirmation was applicable to the 13 cultivars from O. thyrsiflorum, O. citriodorum, O. americanum, O. africanum, O. basilicum, O. gratissimum, and O. tenuiflorum species. CONCLUSIONS: The validated HPLC-PDA and LC-ESI-MS/MS method was found to be selective and suitable for analyzing 13 compounds in O. tenuiflorum and 12 cultivars from the Ocimum genus as a quality control tool. This method can be used in routine analysis as an inexpensive alternative to advanced techniques. HIGHLIGHTS: This work is the first to report for vicenin-2, orientin, cynaroside, betulinic acid, and genistein, with simultaneous analysis of eight bioactive compounds in the Ocimum genus.

Quantitative Determination and Characterization of a Kashmir Saffron (Crocus sativus L.)-Based Botanical Supplement Using Single-Laboratory Validation Study by HPLC-PDA with LC-MS/MS and HPTLC Investigations.

Food ingredients hold a higher nutritional value as a botanical supplement playing a vital role in modifying and maintaining the physiological conditions that improve human health benefits. The Kashmir saffron (Crocus sativus L; KCS) obtained from dried stigmas is known for its aroma precursors and apocarotenoid derivatives, imparting a wide range of medicinal values and therapeutic benefits. In the present study, a simultaneous determination of apocarotenoids and flavonoids in stigma-based botanical supplements was carried out using analytical investigations. The high-performance thin-layer chromatography-based qualitative analysis of the raw material (stigmas, stamens, and tepals) and stigma extract has been carried out to identify apocarotenoids and flavonoids. The rapid HPLC-PDA method for the simultaneous quantification of KCS apocarotenoids was robust, precise (<5.0%), linear (R 2 > 0.99), and accurate (80-110%) as per the single-laboratory validation data. Furthermore, the combined-expanded uncertainty (95%; K = 2) was calculated and found as 0.0035-0.007% (<5.0%) as per the EURACHEM guide for this HPLC analysis. Additionally, an untargeted identification of 36 compounds in the botanical supplement was based on the elution order, UV-vis spectra, mass fragmentation pattern, and standards by ESI-MS/MS analysis with comprehensive chromatographic fingerprinting. Thus, these analytical approaches enable a composite profile of the stigma-based extract as a potential supplement for human health benefits.

Simultaneous Identification and Quantification of Three Xanthones and Two Polyisoprenylated Benzophenones in Eight Indian Garcinia Species Using a Validated UHPLC-PDA Method.

Background: Xanthones and polyisoprenylated benzophenones (PIBs) are two important classes of plant secondary metabolites with a wide range of bioactivities. Garcinia species synthesize numerous xanthones and PIBs. As per the literature, no data claiming simultaneous identification and quantification of three xanthones, α-mangostin, ß-mangostin, γ-mangostin, and two PIBs, xanthochymol, isoxanthochymol, were found. Methods: A validated ultra-HPLC (UHPLC)-photodiode array (PDA) method for the simultaneous identification and quantification of five compounds in different extracts of eight Indian Garcinia species was developed. The compounds were separated on a Waters ACQUITY™ UPLC H-Class column using a mobile phase consisting of solvents 0.1% formic acid in water (A) and methanol (B) in gradient elution mode. The total run time was 9 min. Results: From fruit rinds of eight Indian Garcinia species, namely Garcinia cambogia, G. cowa, G. indica, G. loniceroides, G. mangostana, G. morella, G. pedunculata, and G. xanthochymus, extracts were prepared using solvents of varying polarity. These extracts were analyzed for five biologically important compounds, namely α-mangostin, ß-mangostin, γ-mangostin, xanthochymol, and isoxanthochymol. The results revealed that there is a wide variation in concentration of these compounds in extracts of Garcinia species. Conclusions: The developed and validated UHPLC-PDA method could be used for simultaneous identification and quantification of these five compounds for bioprospection of other Garcinia species.