H. M. Alamgir*
Department of Clinical Pharmacy and Pharmacology, Faculty of Pharmacy, University of Dhaka, Dhaka 1000, Bangladesh.
Sheikh Arman Mahbub
Department of Pharmacy, Jahangirnagar University, Dhaka 1342, Bangladesh.
Muniruddin Ahmed
Department of Clinical Pharmacy and Pharmacology, Faculty of Pharmacy, University of Dhaka, Dhaka 1000, Bangladesh.
Md. Shahidulla Kayser
Department of Clinical Pharmacy and Pharmacology, Faculty of Pharmacy, University of Dhaka, Dhaka 1000, Bangladesh.
Lagenaria siceraria; Cucumis sativus; Cucurbita maxima; Phytoconstituents; Phenols; Flavonoids; Antioxidants; Cytotoxicity; DPPH- 1, 1-diphenyl-2-picrylhydrazyl
Resource Development and Management
Medicinal Plants
2.1 Preparation of the Plant Extracts The whole plants of Lagenaria siceraria, Cucumis sativus & Cucurbita maxima from Cucurbitacae family were collected from Savar, Bangladesh and identified by the taxonomist of the National Herbarium of Bangladesh, Mirpur, Dhaka. The Aerial part (leaves) of the plants were sun-dried separately and then, dried in a hot air oven (Size 1, Gallenkamp) at reduced temperature (not more than 50°C) to make suitable for grinding purpose. After that plant parts were ground into coarse powders using high capacity grinding mill which were then stored in air-tight container with necessary markings for identification and kept in cool, dark and dry place for the investigation. The powdered leaves (200 gm each of aerial part and the remaining part) were used for extraction by Soxhlet apparatus at elevated temperature (65°C) using Petroleum Ether, and Methanol consecutively (500 ml of each solvent). After each extraction the plant material was dried and used again for the next extraction. Extraction was considered to be complete when the plant materials become exhausted of their constituents that were confirmed from cycles of colorless liquid siphoning in the Soxhlet apparatus. Two extracts of each plant were filtered individually through fresh cotton bed. The filtrates obtained were dried at temperature of 40±2°C to have gummy concentrate of the crude extracts. Each extract was kept in suitable container with proper labeling and stored in cold and dry place. 2.2 Phytochemical Screening [6] The crude plant extracts were subjected to different qualitative tests such as Molisch’s test (General test for Carbohydrates), Barfoed’s test (General test for Monosaccharides), Fehling’s test, Test for combined Reducing Sugar, General test for Glycosides, Test for Glucosides, Liebermann-Burchard’s Test, Frothing test, Lead acetate test, General laboratory tests for alkaloids to find out the presence of chemical constituents. 2.3 Pharmacological Investigation 2.3.1 Antioxidant activity evaluation 2.3.1.1 Determination of total phenolics content The content of total phenolic compounds in plant methanolic extracts was determined using the Folin-Ciocalteu Reagent (FCR). The Folin-Ciocalteu reagent (FCR) or Folin's phenol reagent or Folin-Denis reagent is a mixture of phosphomolybdate and phosphotungstate used for the colorimetric assay of phenolic and polyphenolic antioxidants. It works by measuring the amount of the substance being tested needed to inhibit the oxidation of the reagent [16]. 2.3.1.2 Determination of total flavonoids content Total flavonoid was determined using the Aluminum chloride colorimetric method described by Wang and Jiao.The principle of aluminum chloride colorimetric method is that aluminum chloride forms acid stable complexes with the C-4 keto group and either the C-3 or C-5 hydroxyl group of flavones and flavonols. In addition, aluminum chloride forms acid labile complexes with the ortho-dihydroxyl groups in the A- or B-ring of flavonoids, In preliminary experiments, the wavelength scans of the complexes of 15 standards with aluminum chloride showed that the complexes formed by flavonols with C-3 and C-5 hydroxyl groups, such as galangin, morin and kaempferol, as well as those with extra ortho-dihydroxyl groups, such as rutin, quercetin, quercitrin and myricetin, had maximum absorbance at 415-440 nm. However, the λmax of the complexes formed by chrysin and apigenin which have only the C-5 hydroxyl and C-4 keto groups were at 395 and 385 nm, respectively. Another flavone compound investigated, luteolin, which has the C-5 hydroxyl group and the ortho-dihydroxyl groups in B ring formed a complex that showed a strong absorption at 415 nm. In compromise, therefore, the wavelength 415 nm is chosen for absorbance measurement.
2.3.2 Lethality bioassayBrine Shrimp lethality bioassay is a rapid and comprehensive bioassay for the bioactive compounds of natural and synthetic origin. By this method, natural product extracts, fractions as well as the pure compounds can be tested for their bioactivity. The method utilizes in vivo lethality in a simple zoological organism (Brine nauplii) as a convenient monitor for screening and fractionation in the discovery of new bioactive natural products. Brine toxicity is closely correlated with 9 KB (human nasopharyngeal carcinoma) cytotoxicity (p=0.036 and kappa = 0.56). ED50 values for cytotoxicities are generally about one-tenth the LC50 values found in the Brine Shrimp test. Thus, it is possible to detect and then monitor the fractionation of cytotoxic, as well as 3PS (P388) (in vivo murine leukaemia) active extracts using the Brine lethality bioassay. The Brine Shrimp assay has the advantages of being rapid (24 hours), inexpensive, and simple (e.g., no aseptic techniques are required). It easily utilizes a large number of organisms for statistical validation and requires no special equipment and a relatively small amount of sample (2-20 mg or less). Furthermore it does not require animal serum as is needed for cytotoxicities.
European Journal of Medicinal Plants 12(3): 1-13, 2016, Article no.EJMP.22160 ISSN: 2231-0894, NLM ID: 101583475
Journal