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Roxb.) Bosser (Ma-U 162 Maubin University Research Journal 2020, Vol.11 A Study on Physicochemical Properties and Antimicrobial Activities of Phytochemical Constituents Extracted from the Stem Bark of Neolamarckia cadamba (Roxb.) Bosser (Ma-u) Nay Soe Moe1, Aung Kyaw Swar2, Saw Hla Myint3 Abstract This research deals mainly with studies on physicochemical properties and antimicrobial activities of the different crude extracts such as water (H2O), ethanol (EtOH), ethyl acetate (EtOAc) and petroleum ether (PE) of the stem bark of Neolamarckia cadamba (Roxb.) Bosser (Ma-u). The sample was collected from Maubin Township, Ayeyarwady Region. The antimicrobial activities of Ma-u were screened by using agar well diffusion method against Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus pumilus, Candida albicans and Escherichia coli species. In addition, the qualitative and quantitative determination and elemental analysis of the selected sample were determined by phytochemical tests. The soluble matter contents of various organic solvents such as ethanol (5.90 %), methanol (8.07 %), pet-ether (0.31 %), ethyl acetate (1.22 %) and water (9.25 %) were observed. According to the experiments, the moisture (10.68 %), ash (7.82 %), protein (7.35 %), crude fiber (28.80 %), crude fat (1.64 %), carbohydrate (43.71 %) and calories (219 kcal/ 100 g) were found in the bark of Neolamarckia cadamba. The relative abundance of elements in the bark of Neolamarckia cadamba was determined by EDXRF technique which reveals that Ca (5.422 %), K (3.691 %), S (0.412 %) and C (90.289 %) were observed as major constituents. Keywords: Neolamarckia cadamba (Roxb.) Bosser (Ma-u), soluble matter contents, phytochemical tests, physicochemical properties, antimicrobial activities Introduction Research in medicinal plants has gained a renewed focus recently. The main reason is that the other systems of medicine are associated with a number of side effects and can cause serious problems. There has been a number of flora in use for medicinal purposes over past several centuries. Varieties of plants have been used for medicinal treatments, either whole or specific parts (bark, root, leaves, fruit, flowers, seeds) in the dried state. Now they are formulated into suitable preparations such as tablets, pills, extracts, tinctures, lotions, ointments and creams. Neolamarckia cadamba (Roxb.) Bosser (Ma-u) is an evergreen tropical tree widely grown in Australia, China, India, Indonesia, Malaysia, Philippines, Singapore, Vietnam, Bangladesh, Nepal, Myanmar and Sri Lanka. The Cadamba has been known to cure a number of diseases, particularly, the extract prepared from the bark and leaves is crucial. Various researchers across the world have focused their studies on discovering a number of phytochemicals as well as secondary metabolites with pharmacological significance from the Cadamba. (Dwevedi, A., et al., 2015) 1 Lecturer, Department of Chemistry, Maubin University 2 Lecturer, Department of Chemistry, University of Yangon 3Part-time Professor, Department of Chemistry, University of Yangon Maubin University Research Journal 2020, Vol.11 163 Outstanding Features Trees, deciduous, to 30 m tall; trunk with small buttresses; bark thin, grayish brown, fissured and scabrous when old; branches horizontally spreading, flattened becoming subterete, glabrescent. Petiole 20–35 mm, glabrous; leaf blade drying thinly leathery, elliptic or oblong-elliptic, adaxially shiny and glabrous, abaxially glabrous to densely puberulent, base shallowly cordate on juvenile growth, rounded or truncate on adult growth, apex acute; secondary veins 8–12 pairs; stipules lanceolate, acute. Inflorescences flowering heads with peduncle, rather stout. Calyx puberulent to pilosulous; ovary portion ellipsoid to obovoid, partially to deeply lobed; lobes oblong to spatulate, obtuse to rounded. Corolla yellowish white, funnelform, outside glabrous. Fruiting heads yellowish green, 30–40 mm in diam., with peduncles markedly thickened. Fruit cylindrical to ellipsoid or obovoid, glabrous; seeds 3angled. (Kress, W.J., et al., 2003 and Wu, Z.Y., et al., 1994) Botanical Aspects of Selected Sample Kingdom : Planate Family : Rubiaceae Botanical name : Neolamarckia cadamba (Roxb.) Bosser Genus : Neolamarckia Species : cadamba English name : Bur-flower tree Myanmar name : Ma-u, Ma-u-let-tan-she Part used : Stem bark Chemical Constituents and Health Benefits of Neolamarckia cadamba (Roxb.) Bosser Studies on discovering a number of phytochemical constituents of Neolamarckia cadamba showed saponins, indole and quinoline alkaloids, secoindoids, triterpenes, kaempferol and ursolic acid with pharmacological significance. The various extracts have been known to cure a number of diseases; particularly, the extracts prepared from the bark and leaves is crucial. The commercially available compounds from cadamba were cadambagenic acid, quinovic acid, E-sitosterol, cadambine, cadamine, etc., so cadamba plant can produce a lot of biologically active compounds. (Dwevedi, A., et al., 2015) According to traditional medicine in India, the bark and leaves have great significance of biological and pharmacological activities such as analgesic, antipyretic, anti-inflammatory, antifungal, diuretic and laxative, hypolipidemic, antioxidant, antidiabetic, antihepatotoxic, antimalarial, antihelminthic, antivenon and antitumor. (Gautam, R., et al., 2012) Materials and Methods Sample Collection and Botanical Identification of Collected Sample The samples were randomly collected in Maubin Township, Ayeyarwady Region in August, 2017. The collected samples were identified as Neolamarckia cadamba (Roxb.) Bosser according to the authorized botanist at Department of Botany, Maubin University. 164 Maubin University Research Journal 2020, Vol.11 Figure 1 Stem bark, leaf and flowers of Neolamarckia cadamba (Roxb.) Bosser Sample Preparation The collected cadamba samples were washed with water and dried at room temperature. The dried samples were ground into purely fine powder by using an electric grinder and then stored in air-tight container to prevent moisture changes and other contaminations. Extraction of Matter Contents in the Stem Bark of Neolamarckia cadamba (Roxb.) Bosser The extractable matter contents (1:20 w/v) will be determined with various organic solvents such as ethanol, methanol, pet-ether, ethyl acetate and water. 2 g of dry powder sample were taken into each of five conical flasks of 150 mL. 40 mL of each EtOH, MeOH, PE, EtOAc and H2O were added to each flask. Then, the flasks were shaken continuously for 24 hours and allowed to stand for 24 hours. The supernatant solution was filtered by the filter paper (< 2 μm pore size). The filtrate was dried, weighed and the percent yield was calculated. Phytochemical Investigation for Classification of Compounds The phytochemical constituents were qualitatively determined by using Dragendorff’s/Mayer’s reagent, ninhydrin, Benedic’s solution, 10% lead acetate, 5% FeCl3, 70% HCl, concentrated H2SO4, 10% NaOH, 1% CuSO4 and acetic anhydride. Quantitatively, total alkaloid content was determined by Harborne’s method (1984), total glucose-related carbohydrate content was determined by Phenol- sulphuric acid method and total saponin content was determined by Obadoni and Ochukos’ method (2001). Determination of Physicochemical Parameters of Stem Bark Sample The moisture content of dried powdered samples was determined by oven dry method. The crude protein content was determined by micro-Kjeldahl method. The crude fat content was determined by Soxhlet extraction method. The fiber content was determined by digestion method. The total carbohydrate content was determined by subtraction method (the difference between 100 and the sum of the percentages of ash, fat, fibre, moisture and protein).The percentage of ash was obtained by ashing method. The energy value of samples was calculated by usual equation. Energy value (kcal/100g) = (4 × protein %) + (4 × carbohydrate %) + (9 × fat %) Determination of Elemental Analysis of Stem Bark Sample The elemental contents (Relative abundance) in the stem bark of Neolamarckia cadamba (Roxb.) Bosser were determined by EDXRF technique. Screening of Antimicrobial Activity of Cadamba Stem Bark For the examination of vitro antimicrobial activity, solvent extracts such as PE, EtOAc, EtOH, MeOH and H2O of the cadamba stem bark were investigated by using agar well diffusion method. Then, the extracts were tested against six pathogenic microorganisms (Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus pumilus, Candida albicans and Escherichia coli) by using agar well diffusion method. Maubin University Research Journal 2020, Vol.11 165 Determination of Glucose-related Carbohydrate Content of Selected Sample Glucose-related carbohydrate contents of two different extracts (H2O and EtOH) were determined from absorbance at 490 nm by using PD-303UV spectrophotometer. The concentrations were measured by two-fold dilution. Results and Discussion The phytochemical tests revealed that alkaloids, α-amino acids, carbohydrates, flavonoids, glycosides, phenolic compounds, proteins, reducing sugars, steroids, saponins, tannins and terpenoids were present in Neolamarckia cadamba (Roxb.) Bosser (Ma-u). Their positive and negative quantities are observed in Table 1. The presence of secondary metabolites such as alkaloids, flavonoids, glycosides, steroids, saponins, tannins and terpenoids in the plant sample can contribute to their medicinal values. The percentages of extractable
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