Microscopic, molecular authentications, and flavonoid contents in selected bauhinia species and pharmacognostic specifications of bauhinia malabarica leaves

Abstract

There are more than 40 Bauhinia species throughout Thailand. Many pharmacologically active compounds have been reported, particularly flavonoids such as quercetin and its glycoside (quercitrin). This study aimed to characterize 20 Bauhinia species throughout Thailand using microscopic, molecular analysis as well as quercetin and quercitrin quantification. The pharmacognostic specification of B. malabarica leaf which has been used in Thai’s remedies was established. Twenty Bauhinia species leaves were collected from 3 places throughout Thailand. The lamina of fresh mature leaves were microscopically evaluated for the stomatal number, stomatal index, palisade ratio, trichome number, trichome index, and epidermal cell area. The anatomy of midrib transverse sections were illustrated. ISSR-PCR was performed to classify these Bauhinia species. Preliminary quantification of quercetin and quercitrin in 20 Bauhinia species were done using RP-HPLC. Dried mature leaves were exhaustively extracted with 95% ethanol using Soxhlet apparatus. The extracts were injected to Inersil® ODS-3 C18 column and eluted by 0.5% phosphoric acid and methanol (1 : 1) at 35 ºC. Photo-diode array detector was set at 255 nm. Pharmacognostic specification of B. malabarica dried leaves collected from 15 places throughout Thailand was determined for the contents of loss on drying, water, total ash, acid insoluble ash, extractive matter as well as active compounds (quercetin and quercitrin). Chemical fingerprint of the ethanolic extracts was performed by TLC. All twenty Bauhinia species showed paracytic stomata type. B. aureifolia, B. bracteata, B. integrifolia, B, lakhonensis, B. purpurea, B. scandens, B. strychnifolia, B. variegata, and B. winitii were amphistomatic. Unicellular and multicellular nonglandular trichomes were found except B. integrifolia, B. pulla, B. scandens, B. sirindhorniae, B. strychnifolia, and B. winitii contained no trichomes. B. winitii showed two layers of palisade cells at upper epidermis. B. purpurea displayed the highest numbers of stomata (1120 – 1208 per mm2). B. saccocalyx had trichome number upto 200 per mm2 of lower epidermis. The anatomical characteristics of the midribs were illustrated. Their uniqueness could be used for species identification. Six ISSR primers produced 100% polymorphic DNA bands. A dendrogram generated by UPGMA could classify Bauhinia species in this study especially B. malabarica. Phytochemical analysis revealed quercetin and/or quercitrin contents in dried leaves of these 20 Bauhinia species. The highest contents of quercetin and quercitrin were found in B. malabarica. Pharmacognostic specification of B. malabarica dried leaves revealed the loss on drying, total ash, acid insoluble ash, and water contents should be not more than 8.00, 7.08, 1.79, and 8.28 g/100 g while ethanol and water soluble extractive matters should be not less than 13.78 and 16.47 g/100 g of dried leaves respectively. TLC-fingerprint was demonstrated. The contents of quercetin and quercitrin were 0.1796 ± 0.0678 and 0.3833 ± 0.2138 g/100 g of dried leaves, respectively.