BASIC PHYTOCHEMICAL SCREENING AND ANTIBACTERIAL, ANTIFUNGAL AND ANTIOXIDANT PROPERTIES OF SYZYGIUM CUMINI, A TREE FROM GUYANA

CRIBADO FITOQUÍMICO BÁSICO Y PROPIEDADES ANTIBACTERIANAS, ANTIFÚNGICAS Y ANTIOXIDANTES DE SYZYGIUM CUMINI, UN ÁRBOL DE GUYANA

Syzygium cumini or Jamun is an evergreen tropical tree belonging to the flowering plant family myrtaceae. The scientific classification of Syzygium cumini is given as follows:

Kingdom: Plantae

Angiosperms

Endicots

Rosids

Order: Myrtales

Family: Myrtaceae

Genus: Syzygium

Species: Syzygium cumini

Syzygium cumini is also known as black plum, java plum, duhat plum and malabar plum. More than one thousand species were classified as belonging to the genus Syzygium. It is a slow growing species. It can reach up to 30 meters and live more than 100 years. Syzygium cumini is original from India, Nepal, Pakistan, Bangladesh, Sri Lanka, Philippines and Indonesia. It was is man-introduced to Florida, USA, Suriname, Brazil, Trinidad &Tobago and Guyana. Syzygium cumini leaves are used as food for live stocks. Wine (and vinegar) are also made from the fruits. It is rich in vitamins A and C. Leaves and bark of S. cumini are used for controlling blood pressure. Syzygium cumini seeds are used in various alternative healing system like Unani, Chinese and Ayurveda medicine. Syzygium cumini wood is water resistance, so it is used in railway sleepers and to installing motors in wells. Kaur et al. [1] investigated drying (50, 70, 90 and - 55o C) characteristics and antioxidant properties of java plum seeds and skin waste. A review on ethnobotanical uses, antimicrobial potential, pharmacological properties and phytoconstituents of Syzygium cumini was reported by Singh and Navneet [2]. Mitra et al. [3] examined effects of summer, winter, rainy and autumn seasons on UV absorbing properties of the acetone extract of S. cumini leaves pointing out the rainy season as responsible of maximum absorbing properties and it can be regarded as anti-solar agent for preparation of sun screen lotions. The UV absorption properties of the methanolic, ethanolic, benzenic, acetone and ethyl acetate leave extract of S. cumini were examined by Mitra et al. [4]. The acetone leave extract of S. cumini was found to have maximum UV absorption potential. Arunpandian et al. [5] presented a review focused on the information on traditional and medicinal use of S. cumini. Phytochemical contents and antioxidant capacity of S. cumini from Indonesia were investigated by Sukmasari et al. [6]. They have also compared the properties of the leave extract of S. cumini and S. polyanthum. Prema et al. [7] studied the phytochemical constituents and antidiabetic properties of S. cumini seeds extract. S. cumini seeds powder was found to possess antidiabetic properties in type - 2 diabetic model rats. The potential use of S. cumini methanolic leave extract for dengue by increasing platelet and leukocyte levels were described by Bandiola and Corpuz [8]. A review on pharmacological properties and therapeutic potential of S. cumini was discussed by Kumawat et al. [9]. Antimicrobial activity of S. cumini juice extract on enteric pathogenic bacteria (viz. Salmonella typhimurium, Shigella flexneri, Staphylococcus aureus and Escherichia coli) was described by Haque et al. [11]. Khan et al. [12] studied the antifungal potential of the methanolic extract of bark and leaves of S. cumini against Rhizoctonia solani. Kuhn. Hasanuzzaman et al. [13] performed phytochemical screening of methanolic, acetone, chloroform and n-hexane extracts of S. cumini seeds, root, stem, bark, leaves. The antimicrobial potential of methanolic, aqueous, and petroleum ether extracts of S. cumini leaves was tested by Elfadil et al. [14] against bacteria (viz. Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subitlis) and fungi (Aspergillus niger and Candida albicans). Ramos and Bandiola [10] has examined phytochemicals in extracts from methanol, ethanol, water, ethyl acetate and hexane macerations out of the leaves of S. cumini. Praphakaran et al. [15] reported that ethanolic extract of leaves and aqueous extracts of seeds S. cumini were found to have very high antimicrobial property for wide range of gram positive and gram negative bacterial strains. The natural antimicrobial potential of extract of all parts of Syzygium cumini (L) over the chemical synthetic antibiotic was studied by Mohit et al. [16]. Jabeen and Javaid [17] evaluated the antifungal potential of the ethanolic, aqueous and n-hexane extracts from leaves, fruit, root bark and stem bark of Syzygium cumini (L) against Ascochyt arabiei, the cause of blight disease of chick pea (Cicer arietinum). The antioxidant activity of S. cumini leaf extracts was investigated using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenging and ferric-reducing antioxidant power (FRAP) arrays was described by Ruan et al. [18]. A significant linear relationship between antioxidant potency, free radical-scavenging ability and the content of phenolic compounds of leaves extracts were observed. The plant of Syzygium cumini with fruits is shown in Figure 1.

Figure 1

The Syzygium cumini plant with fruits

(i) Syzygium cumini leave extracts were found to have antimicrobial, antifungal and antioxidant properties.

(ii) The antioxidant power of Syzygium cumini’s leaves in extracts in various solvents follows the order:

methanol> ethyl acetate > ethanol> chloroform

(iii) The antioxidant power of Syzygium cumini’s leaves in extracts in various solvents was found to increase with increase of its concentrations.

(iv) The chloroform extract was found to have more antibacterial property in comparison to methanol, ethylacetate, and ethanol extracts.

(v) In disc diffusion method inhibitory zone for antibacterial activity follows the order:

Chloroform > ethanol > methanol > ethyl acetate

(vi) In poison plate method inhibitory zone for antibacterial activity follows the order:

Ethanol > ethyl acetate > chloroform > methanol

(vii) In poison plate method all extracts have showed nearly same inhibitory zone or antibacterial activity.

Collecting of plant material

The plant material (leaves) of Syzygium cumini was collected at the Institute of Applied Science and Technology (IAST), University of Guyana, Turkeyen Campus, Georgetown. Guyana.

Preparation of plant materials:

The collected leaves sample of Syzygium cumini was weighted on Citizen CTG 3000E electronic balance. The leaves dried in oven (Gallenhamp Incubator Model IH-150) at 50-60ºC. The dried leaves were cooled at room temperature and weighted again on Citizen electronic balance. Weight of green leaves, dried leaves and value of percentage moisture content in various samples of Syzygium cumini is given in Table 1. The weight of ground leaves of Syzygium cumini was found to be 560 grams.

Collection of test organism

Three micro-organisms (Escherichia coli, Staphylococcus aureus and Candidus albicans) were used for the study. All the tested strains are reference strains and were collected from the microbiology laboratory of Georgetown Public Hospital Corporation, Georgetown (GHPC). All cultures are maintained in nutrient broth (Himedia, M002) at 37ºC and maintained on nutrient agar (Himedia MM012) slants at 4ºC.

Extraction and preparation of test solutions

The grounded leaves of Syzygium cumini were extracted in each solvent, ethanol, ethyl acetate, chloroform and methanol. 20 g of dried pulverized leaves were soaked with 200 mL of each solvent for 48 h separately. Solvent is decanted each time and residue again soaked with same solvent for 24 h. The total extract is combined and filtered. The evaporation of solvent was done on rotavapour (Buchi). The respective solvent was added to viscous semi solid liquid extract to make up the desired volume of extract solution.

Reducing antioxidant power

Ammonium thiocyanite, ferric chloride, linoleic acid (99.5 %), thiobarbituric acid, trichloroacetic acid, potassium ferrocyanide, butylated hydroxyl anisol, sodium dihydrophosphate, sodium monhydrophosphate, potassium dihydrophosphate obtained from Aldrich. The reducing antioxidant power of the plants methanolic, ethanolic, ethyl acetate and chloroform extract was determined by the method reported in the literature [23,24]. Different concentration of plant extracts (100 - 1000 µL) in 1 mL of distilled water were mixed with phosphate buffer (2.5 mL, 0.2 M ,pH 6.6) and potassium ferricynaide K₃[ Fe(CN)₆] , (2.5 mL,1 %) . The mixture was incubated at 50o C for 20 min. Then 2.5 mL of trichloroacetic acid 10 % was added to mixture, which was then centrifuged for 10 min at 3000 rpm. The upper layer (2.5 mL) was mixed with distilled water 92.5 mL and FeCl3, 0.5 mL, 1 %. The absorbance was measured at 700 nm against a blank using UV-Vis spectrophotometer (Phillips X500). Increased absorbance of the reaction mixture indicates increase in the reducing power.

Anti-Microbial Assay

Materials

Mueller Hinton Agar, Agar plates and microbial discs were purchased from the International Pharmaceutical Association (IPA) in Guyana. Solvents like chloroform, ethanol, ethyl acetate and methanol obtained from Aldrich. Scitilation vials 20 mL were obtained from Meditron Scientific Sales, Georgetown, Guyana.

Aseptic chamber

Aseptic chamber consists of a wooden box of L = 1 meter, B = 1 meter and D = 0.5 meter area. Chamber is cleaned with 70 % ethanol twice and irradiated with short wave UV light for 1 h.

Potato dextrose agar (PDA) medium

Potato Dextrose Agar (PDA) medium prepared according to method reported by Talaro [25]. This is the medium on which cultured bacteria Escherichia coli and Staphylococcus aureus were grown. The 200 g potato was peeled finely chopped and boiled to a mash in distilled water. Each 12.5 g dextrose and 12.5 g agar was placed in a 1 L measuring cylinder. The potato mash was stirred and poured in to the same measuring cylinder. Distilled water was added to make the solution up to 500 mL. The content was stirred until the consistency of solution mixture. The stirred mixture poured into conical flasks, plugged with cotton wool and tightly wrapped by aluminum foil. The flasks were autoclaved at 121º C, 15 psi, for 15 minutes.

Mother plates

Mother plates were prepared by pouring PDA mixture into Petri dishes and to cool at room temperature, in the aseptic chamber.

Antimicrobial assay was done by disc diffusion and poisons plate method [26] using plants Syzygium cumini extracts (ethanol, methanol, ethyl acetate and chloroform) and commonly used antibiotics. The test quantities of specific extracts were dissolved in depending upon the solubility of the extracts. The dissolution of the organic extracts (chloroform, methanol, ethanol and ethyl acetate) was aided by water, which did not affect the growth of microorganism, in accordance with our control experiments. The surfaces of media were inoculated with bacterial from a broth culture. After 18 h of incubation at a specific temperature 28o C - 30o C for Escherichia coli, Staphylococcus aureus and Candidus albicans the plates were examined and the diameters of the inhibition zones were measured to the nearest millimeter.

Phytochemical analysis of the plant extracts

Materials

Glacial acetic acid, thyonil chloride, dichloromethane, copper sulfate, lead acetate, diethyl ether, ferric chloride, acetic anhydride, antimony chloride, all obtained from Aldrich.

Method

Phytochemical analysis of all the aqueous plant extracts was carried out by suitable methodologies in search of active ingredient responsible for antimicrobial toxicity. The phytochemicals include under study were saponins, terpenoids, alkaloid, glycoside, carbohydrates, protein and amino acids, tannins, and flavonoids the analysis was carried out according to the methodologies of Edeoga et al. [27].