Phytochemical and Pharmacological Evaluation of Clerodendrum serratum roots with Special Reference to Asthma

Abstract

Clerodendrum serratum (L.) Moon. (Verbenaceae) commonly known as bharangi is an important medicinal plant growing in India has traditional value to treat pain, inflammation, rheumatism, respiratory disorders and fever. The crude roots are employed in many Ayurvedic and herbal formulations as one of the ingredient for the treatment of various ailments. The purposes of the present study were to study phytochemical and pharmacological effects of crude (70% methanolic) extract and fractions (ethyl acetate and n-butanolic) of C. serratum roots (CSR) with reference to asthma in various animal models in comparison with reference drugs to claim the traditional uses of plant. CSR was standardized using pharmacognostical, physicochemical and phytochemical parameters to assess the quality of collected plant material. The pharmacognostical and physicochemical parameters evaluated for the roots were found in compliance with the Pharmacopoeial standards of crude drug. The qualitative and quantitative phytochemical studies of methanolic extract of CSR performed using chemical tests, thin layer chromatography, ultraviolet spectroscopy and quantification of phytoconstituents, showed presence of saponins, phenolics, flavonoids and carbohydrates. The results of pharmacognostical, physicochemical and phytochemical studies would be helpful for the identification and quality control of CSR. The mast cell stabilizing, bronchodilating, anti-histaminic activities along with inherent antioxidant and anti-inflammatory effects of the crude extract and fractions of C. serratum roots were performed using various in-vitro and in-vivo pharmacological experiments. The significant inhibition of mast cell degranulation induced by compound 48/80, increase in preconvulsion time and contraction produced by histamine in guinea pig tracheal chain preparation was observed with extract and fractions of CSR. Furthermore, the dose dependent in vitro free radical (DPPH and ABTS) scavenging and anti-inflammatory (protein denaturation, protease inhibition and lipoxygenase inhibition) activities of crude extract and fractions of CSR were exhibited in studied concentration range. However, the said pharmacological effects produced by an ethyl acetate fraction of CSR (EFCSR) were found to be significantly higher compared to the others though lower than the respective reference standard. To establish the anti-asthmatic mechanism of action, the EFCSR was consequently evaluated using ovalbumin (OVA)-induced airway inflammation in guinea pigs employing dexamethasone as a reference drug. Treatment with the EFCSR displayed significantly lower airway inflammatory cells, cytokine levels, histamine level compared to OVA-sensitized animals, further supported by lung histology. The significant anti-asthmatic actions of EFCSR could be mediated by inhibiting the inflammatory mediators and release/synthesis of Th2 cytokines. The protective effects produced by an ethyl acetate fraction may ascribe to the polyphenolics including flavonoids and terpenoids presence as detected by GC-MS technique. From the effective (ethyl acetate) fraction of CSR, a oleanane-type triterpene acid (oleanolic acid, OA), an ursan-type triterpene acid (ursolic acid, UA) and a flavonoid (isoquercitrin, ISQ) were isolated using chromatographic techniques and structural elucidation was carried out using FT-IR, EIMS, 1H-NMR, 13C-NMR spectroscopy. The anti-asthmatic potential of UA, OA and ISQ isolated from EFCSR might be due to the inhibition of infiltration of inflammatory cells, release/synthesis of T-cell derived cytokines; IL-4, IL-5 and TNF-α. Further, the drug has an ability to inhibit the release of mediators like histamine into the local tissues of the lungs and trachea which was confirmed by histopathogical observations. These findings suggest that the C. serratum roots may be a valuable therapy for asthma; however, a well-designed clinical trial include persistent, mild or moderate asthmatic patients, is warranted.