Isolation, characterisation and in vitro biological activity of bioactive principles in Hermannia geniculata Eckl. & Zeyh. leaf extracts
Mojau, Pheello Jeremia
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Hermannia geniculata has been used widely as traditional medicine for treatment against infectious human pathogens. The aim of the present study was to determine the phytochemical constituents, antioxidant, antidiabetic and antimycotic activities of Hermannia geniculata leaf extracts; and to isolate and test the activity of bioactive compounds from the extract with better activity. The antidiabetic potential of the acetone, hexane, ethanol and ethyl acetate leaf extracts of H. geniculata was investigated against activities of α-amylase, and α-glucosidase enzymes; while the antioxidant activity of the extracts was determined using metal chelation, 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl radical scavenging and 2,2'-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid (ABTS) assays. Fresh leaves of Hermannia geniculata were collected from vegetation along Wetsi café at Monontsha village, Qwaqwa, Eastern Free State Province, South Africa. The roots were thereafter authenticated and a Voucher Specimen (Mojamed/1/2016/Qhb) was prepared and deposited at the Herbarium of Plant Sciences Department, University of the Free State, Qwaqwa Campus, South Africa. The fresh leaves were cut into smaller pieces and washed under running water to remove all debris, afterwards dried in an Ecotherm oven at 40°C. Dried plant materials were then powdered usingWaring laboratory blender (Labcon, Durban, South Africa). Powdered plant materials (150 g each) were extracted separately in ethanol (1500 ml), ethyl acetate (1500 ml), acetone (1500 ml), hexane (1500 ml), the plant in different solvents were put on a Labcon platform shaker for 24 h at the speed of 100 rpm Extracts were filtered using Whatman no. 1 filter paper and each filtrate was concentrated to dryness under reduced pressure at 40°C using rotary evaporator (Cole-Parmer) as depicted in. Finally, extracts were dried to yield ethanol extract (33 g), acetone (10 g), ethyl acetate (44.2 g), hexane (4.5 g). Each extract was re-suspended in its respective solvent to make a 50 mg/ml stock solution. Phytochemical constituents of Hermannia geniculata leaf was determined in the all the extracts adopting standard methods. Antioxidant activity of Hermannia geniculata extracts as per ABTS•+ decolorization assay was measured. The α-glucosidase and α-amylase inhibitory activities were assayed. For the fractions and the isolated compound, only α-amylase and α- glucosidase assays were used. Fractionation of the ethyl extract was done by thin layer chromatography (TLC) profiles, and further purification of semi-pure compounds was achieved using preparative thin layer chromatography (pTLC) to obtain pure compounds. Isolated compound was characterised using nuclear magnetic resonance (NMR), mass spectroscopy (MS) and Fourier Transform Infrared (FTIR). Phytochemical analysis of the extracts revealed the presence of alkaloids, saponins, flavonoids phenols and triterpenes for all the extracts. The isolated compound exhibited significant α-amylase activity with the lowest IC50 value at 0.172 compared to other extracts and acarbose (standard) with 2.344 mg/mL. α-glucosidase activity of the isolated compound gave a lower IC50 of 4.760 compared to acarbose at 10.450 value. For DPPH activity, the isolated compound had lower value of IC50 of 0.474 compared to silymarin (standard) at 16.647. For hydroxyl radical, the isolated compound was more active than the ascorbic acid (standard). However, for metal chelating assay, the compound showed a significantly lower IC50 value of 5.242 compared to silymarin at 2.734. For antimycotic activity, the isolated compound showed activity against candida albicans with MIC values at high concentrations of 6.5 mg/ml which was higher than that of a positive control (Fluconazole). The ethyl acetate extract and the isolated 1,3-dibutyl1-2,8-dihydroxy-9H-xanthen-9-one exhibited best inhibitory activity on the assays studied. Overall the presence of phytochemicals in the leaves of H. geniculata may be suggested to have contributed greatly to the biological activities of the plant.