• AKINLEYE STEPHEN AKINRINDE^1*,
• et al.: Phytochemical Composition¹,
• Antioxidant Activities of Dianthus Thunbergii¹,
• Hypoxis Argentea¹

1. ¹  Pharmacognosy Magazine, Volume 14, Issue 54, April-June 2018 that possess medicinal properties against several diseases that affect humans. The pathogenesis of most diseases involves oxidative and inflammatory components, usually arising from excessive production of reactive oxygen species (ROS).[4] ROS include activated oxygen metabolites called free radicals, such as superoxide anion radicals (O2 −) and hydroxyl radicals (OH), as well as nonradicals including hydrogen peroxide (H2O2) and singlet oxygen (1O2). Nonradicals have the potential to participate in reactions that eventually give rise to free radicals. Free radicals and nonradicals alike are capable of reacting in vivo with tissue macromolecules, forming products such as guanine adducts with DNA, or lysyl adducts with proteins.[5] The body of aerobic organisms is equipped with antioxidant defense mechanisms (enzymic and nonenzymic) that protect against tissue damage by ROS.[6] These are, however, not always sufficient to counteract the severity of oxidative challenge that characterizes many disease or toxic states. The imbalance that results between production of oxidants (ROS) and antioxidants, in favor of the oxidants, is described as oxidative stress and is a major underlying factor in the pathogenesis of many chronic diseases such as diabetes, cancer, and atherosclerosis.[3] In recent times, considerable research efforts have been directed at finding naturally occurring compounds, especially of botanical origin, which offer protection against diseases through their antioxidative and/or anti‑inflammatory properties. Dietary supplementation with phytochemicals represents an effective protective strategy to complement the activities of natural antioxidant defense systems, as well as a viable alternative to synthetic drugs which generally have limitations of associated adverse effects.[7,8] The South African population is one of those plagued with a rising incidence of chronic noncommunicable diseases such as diabetes mellitus.[9] A considerable proportion of the population resorts to the use of herbs for the treatment of various diseases such as diabetes. Ethnobotanical surveys have revealed a large number of these plants reported to be useful in the treatment of diabetes mellitus.[10,11] Dianthus thunbergii, of the family Caryophyllaceae, belongs to a genus of over 300 species, which are traditionally used for medicinal purposes as infusions for the treatment of chest complaints, severe colic, soothing of wounds, urolithiasis, boils and carbuncles, eczema, and itching.[12,13] Extracts from the fresh crushed roots are reportedly used against diabetes and other conditions and are taken as 2 teaspoonfuls orally three times daily.[11] Hypoxis argentea is one of the several species of the genus Hypoxis which are popular in African herbal medicine for the treatment of septic sores, headaches, dizziness, testicular tumors, tuberculosis, asthma, and diabetes.[14‑16] The corm, the underground part, is usually sought for medicinal use. For diabetes treatment, it is reportedly boiled in water and the infusion is administered orally until a patient is healed. Despite traditional claims for the effectiveness of D. thunbergii and H. argentea against diabetes mellitus, they have not been investigated scientifically for their phytochemical composition and biological activities. As part of ongoing comprehensive analysis of the medicinal potentials of these plants, the present study aimed to quantify the content of major phytochemicals and also to evaluate the antioxidant activities of the aqueous and ethanol extracts of the roots and corms of D. thunbergii and H. argentea. These properties were compared between the two plants and correlations were made between the phytochemical contents and antioxidant activities of the plants. MATERIALS AND METHODS Chemicals Folin–Ciocalteu reagent, 1,1 diphenyl‑2‑picrylhydrazyl (DPPH), 2,2’‑azino‑bis (3‑ethylbenthiazoline‑6‑sulfonic acid) (ABTS), vanillin, ferric chloride, butylated hydroxytoluene (BHT), rutin, Vitamin C potassium ferricyanide, trichloroacetic acid (TCA), thiobarbituric acid, glacial acetic acid, sodium nitroprusside, tannic acid, gallic acid, quercetin, sodium carbonate, aluminum chloride, and potassium acetate were purchased from Merck (Pty) Ltd., Gauteng, South Africa. All the other chemicals and solvents were of analytical grade. Collection of plant materials and preparation of extracts The roots of D. thunbergii and corms of H. argentea were collected in May, 2015, at Alice, Eastern Cape, South Africa. They were authenticated at the Giffen Herbarium, University of Fort Hare, South Africa. Both plants were identified with voucher specimen numbers CRY‑2502 for D. thunbergii and HYP‑1230 for H. argentea. The roots and corms were initially washed free of soil attachments and then oven‑dried to constant weight at 30°C. The dried plant materials were then milled into fine powder using an electric blender (Commercial Blender type GB27, Hamilton Beach Brands, Inc., China). A composition of 200 g of each of the powdered plant materials was extracted separately in distilled water and 99.99% ethanol using an orbital shaker (Labcon laboratory service [Pty], South Africa) for 24 h. The suspensions in ethanol were thereafter filtered using Whatman No. 1 filter papers in a Buchner funnel. The aqueous suspensions were initially filtered through a thick layer of sterile cotton wool before their filtration with the filter paper. The ethanol extracts were concentrated to dryness using a rotary evaporator (Heidolph Laborota 4000, Heidolph Instruments, GmbH and Co, Germany) while the aqueous extracts were initially frozen at −40°C and then dried using a freeze dryer. The different extracts were reconstituted in their respective solvents for use in the assays conducted in this study. Phytochemical screening Total phenolic content The content of phenols in the different extracts was determined spectrophotometrically by the Folin–Ciocalteu reagent according to the method of Ozkok et al.[17] A calibration curve was prepared with gallic acid as standard (0.025–0.125 mg/ml in 70% methanol v/v). To 0.5 ml of each of the gallic acid concentrations or extracts (mg/ml), 2.5 ml Folin–Ciocalteu reagent (previously prepared as 10% v/v dilution in distilled water) was added. Thereafter, 2 ml anhydrous sodium carbonate (7.5%) was added, producing a blue‑colored solution. The mixtures were vortexed thoroughly and placed in a water bath for 30 min at 45°C. The absorbance was then read at 765 nm. The equation of the calibration curve obtained (Y = 14.885x; R2 = 0.9961) was used to establish the gallic acid equivalence (mg/ml). The total phenolic content was calculated using the formula: T = C × V/m, where T is the total phenolic content, V is the volume of the extract (ml) used in the assay, C is the gallic acid equivalent (GAE) (mg/ml), and m is the weight of the pure plant extract used in the assay. Values were expressed as GAE per gram of dry plant extract (mg GAE/g). All assays were performed in triplicate. Total flavonoid content Flavonoid contents in the extracts were determined using the aluminum chloride method as described by Ozkok et al.[17] A calibration curve was prepared with quercetin (0.025–0.125 mg/ml in 80% methanol v/v). Briefly, 0.5 ml of the extracts (prepared at a concentration of 1 mg/ml) or the standard at the different concentrations was mixed with 3 ml of 95% ethanol, 0.2 ml of aluminum chloride (prepared as a 10% aqueous dilution), and 0.2 ml of 1 M potassium acetate, and the whole mixture was made up to 10 ml with distilled water. The resulting solutions, prepared in triplicate, were yellow and were thoroughly vortexed and allowed to stand.

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