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Phytochemical and antibacterial properties of the ethanolic leaf extract of Merremia peltata (L.) Merr. and Rubus spp.

INTRODUCTION

Today, plant materials remain an important resource for combating illnesses, including infectious diseases and many of these plants have been investigated for novel drugs or new therapeutic agents, food additives, agrochemicals, and industrial chemicals [2]. Despite the increasing use of synthetic drugs, it persisted as the "treatment of choice" since the therapy with synthetic antibiotics is not always possible because of their high cost as well as toxicity due to their extended use [14]. The medicinal value of plants lies in some chemical substances that produce a definite physiological action on the human body. The most important of these bioactive compounds of plants are alkaloids, flavonoids, tannins and phenolic compounds [7].

Phytochemical analysis of medicinal plants has shown that numerous secondary compounds in plants traditionally used for medicinal purposes have chemical properties effective at treating illness. This was observed in the work done by Wadood et.al [20], on the phytochemical analysis of medicinal plants occurring on local area of Mardan, where all extracts used have displayed anti-inflammatory, antianalgesic, anti-cancer, anti-viral, anti-malarial, anti-bacterial and anti-fungal. Studies like this are important and have commercial interest in both research institutes and pharmaceutical companies for the manufacturing of new drugs for treatment of various diseases.

Moreover, there is a continuous and urgent need to discover new antimicrobial compounds with diverse chemical structures and novel mechanisms of action because there has been an alarming increased in the incidence of new and reemerging infectious diseases [16, 18]. There is therefore a need to search for more herbal medicines with the aim of validating the ethno-medicinal use and subsequently the isolation and characterization of compounds which will be added to the potential list of drugs [15].

This study used two plants specifically Merremia peltata (L.) Merr. and Rubus spp. which has been used ethno-medicinally among the Higa-onon tribe of Rogongon Iligan City as--remedy to various illnesses. The genus Rubus is comprised of more than 250 species of shrubs, often with prickles on stems and leaves within this genus from the rose (Rosaceae) family. They are found throughout the world, some are cultivated for their ornamental value, as a useful food source, while others are regarded as weeds [1]. Rubus spp. is a spreading, low, spiny shrub, rarely exceeding a meter in height. Leaves are pinnate with 5 to 7 leaflets. Leaflets are smooth or hairy, with lobed margins, oblong-ovate or broadly lanceolate, and 2 to 7 centimeters in lenght. Flowers are borne upon slender, prickly, hairy, 3 to 5 centimeter-long stalks and arise from the uppermost leaf axils. Calyx is hairy. Petals are white, broadly oblong, and constricted toward the base. Berries are red, conically elongated, and 1.5 to 2 centimeters across.

Merremia peltata (L.) Merr. is under the family Convolvulaceae known as bindweed or morning glory family. This plant is a coarse and widely spreading woody vine. Stems are 5 or more centimeters thick, and porous. Leaves are alternate, smooth, somewhat rounded, about 20 centimeters wide, those toward the ends of the branchlets being much smaller, heart-shaped at the base, pointed at the tip. Peduncle grows solitary from each of the upper leaf axils, erect and longer than the leaves. Flowers are large, golden-yellow, few to many, or clustered. Sepals are smooth, thick, oblong, 2 centimeters long. Corolla has a wide limb.

Thus, this study was conducted to determine their phytochemical property and investigate the bioactivity of ethanolic and water leaf extracts of Merremia peltata (L.) Merr. and Rubus spp. through Kirby-Bauer disc diffusion method against Gram-positive and Gram-negative bacteria inorder to assess their ethnomedicinal viability.

MATERIALS AND METHODS

Collection of the Plant Samples:

The Merremia peltata (L.) Merr. and Rubus spp. plant samples (Fig. 1) were collected from Rogongon, Iligan City during early morning. Disease free-looking and fresh plants were selected and its leaves were carefully washed with tap water thrice. A total of 700 g plant samples were zip locked inside polyethylene bags. Pressed samples were identified and authenticated at the Herbarium of the Department of Biological Sciences Mindanao State University, Iligan Institute of Technology where voucher specimen was prepared and deposited. The collected samples were hanged for days until sufficiently dried samples were obtained. The dried samples were placed in polyethylene bags ready for further processing in the laboratory.

Preparation of the Plant Extracts:

Five hundred (500) grams of the two air-dried plant samples were pounded till its powder state was achieved. Powdered samples were mixed in 100 ml 95% ethanol and enclosed in a sterile, dark container for 72 hours.

Phytochemical Screening:

The screening was done to determine the presence of bioactive chemical components in the two plant samples such as alkaloids, antraquinones, cyanogenic glycosides, unsaturated sterols, flavonoids and saphonins according to the standard protocols described previously [2, 6, 15].

Preparation of Microorganism:

In this study, pure cultures of four different pathogens (Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa) were obtained from the Philippine National Collection of Microorganisms, National Institute of Molecular Biology and Biotechnology, University of the Philippines Los Banos. Prior to the antimicrobial activity study, the test microorganisms were subcultured on Nutrient Agar (NA) media, incubated at 37oC for 24 hrs then inoculums of the test microorganisms were grown into Nutrient Broth (NB) and adjusted according to Mac Farlands Standard to achieve approximately 1 x [10.sup.8] CFU/ml before inoculating to the test media, Mueller-Hinton agar (MHA).

Preparation of Controls:

Streptomycin and Chloramphenicol were used as the positive control for the Gram-positive (S. aureus and B. subtilis) and Gram-negative (E. coli and P. aeruginosa) respectively. Standard dosage used in the study for Streptomycin was 10 [micro]g/ml and for Chloramphenicol was 30pg/ml based on the Kirby Bauer Chart for antibacterial susceptibility testing [10]. Ninety-five percent (95%) ethanol was used as the negative control.

Preparation of the Two Leaf Extracts as treatments:

The treatments used were 5 [micro]g/ml, 10 [micro]g/ml, 15 [micro]g/ml, 20 [micro]g/ml and 25 [micro]g/ml for both Merremia peltata (L.) Merr. and Rubus spp. extracts with three replications. The solvent used for the preparation of the extract was 95% ethanol.

Antimicrobial Activity:

The Kirby-Bauer Disk Diffusion Method [12] which is commonly used method for antimicrobial activity test was employed. Using the sterile micropippetor tips, 100 [micro]L or 0.1 mL of bacterial culture in broth were transferred to the MHA plates and was spread using a sterile L-rod asceptically to minimize contamination and erroneous result. Sterile Whatmann's No. 4 filter paper with a 6mm diameter was used as paper discs. The specific filter paper discs with 10 [micro]L each of the test extracts of various concentrations and the controls were aseptically placed into the centre of divisions on each MHA plate using a forcep to test the activity of each extract as an antimicrobial agent. The plates were incubated at 37[degrees]C for 24 hours. After incubation, zones of inhibition were measured using a standard ruler by measuring the diameter of the clear zone in the nearest millimeter. This was then subtracted with the diameter of the disc and the result was the measurement considered in this study.

Data Analysis:

In order to determine whether different concentrations of the extracts were significantly different or not with the controls, the average measurement of zone of inhibition for each concentrations were analysed using Analysis of Variance (ANOVA).with 95% level of significance. Post-hoc test was also applied to assess differences within treatments.

Results:

Phytochemical Sreening of the Ethanolic Leaf Extracts of Merremia peltata (L.) Merr. and Rubus spp:

Results of the phytochemical screening (Table 1) showed that only Merremia peltata (L.) Merr. leaf extract had alkaloid while Rubus spp. extract had tannins. Both plants had more steroids and flavonoids except for the two phytochemicals, anthraquinones and cyanogenic glycosides which were absent.

Antimicrobial Activity of the Ethanolic and Water Leaf Extract of Merremia peltata (L.) Merr. and Rubus spp:

A. Against Gram-positive ([G.sup.+]) Bacteria:

The Merremia peltata (L.) Merr. and Rubus spp. ethanolic and water leaf extract concentrations: 5 [micro]g/ml, 10 [micro]g/ml, 15 [micro]g/ml, 20 [micro]g/ml, and 25 [micro]g/ml had antimicrobial effect on the Gram-positive bacteria S.aureus and B. subtilis as compared to the negative controls: ethanol and water. A mean zone of inhibition of 5.7 mm was observed to be highly significant (p < 0.05) for the ethanolic leaf extract of Merremia peltata (L.) Merr against S. aureus and B. subtilis at concentrations 20 ug/ml and 10 ug/ml respectively (Fig.1A). For Fig.1B, the mean zones of inhibition of 4.8 mm against S. aureus and 5.2 mm against B. subtilis were observed to be highly significant (p < 0.05) for the ethanolic leaf extract of Rubus spp. The observed results showed higher inhibitory activity than that of the standard antibiotic Streptomycin which has a mean zone of inhibition of 2 mm.

B. Against Gram-negative ([G.sup.-]) Bacteria:

The measured zones of inhibition around the treated discs placed on the surface of the MHA plates showed that the ethanol and water leaf extracts of Merremia peltata (L.) Merr. and Rubus spp. also inhibited the growth of all Gram-negative bacteria. However, their antibacterial effectiveness varied and ethanol extract exhibited more pronounced inhibition than water extract in the present study. Figure 2 showed that the various Merremia peltata (L.) Merr. and Rubus spp. ethanolic and water leaf extract concentrations: 5 [micro]g/ml, 10 [micro]g/ml, 15 [micro]g/ml , 20 [micro]g/ml, and 25 [micro]g/ml had antibacterial effect also on the Gram-negative bacteria E. coli and P. aeruginosa as compared to the negative controls: ethanol and water.

Merremia peltata (L.) Merr. showed to be more effective in inhibiting Gram-negative bacteria than Rubus spp. and had the most significant concentrations at 5 [micro]g/ml and 15 [micro]g/ml for E. coli and P.aeruginosa respectively with mean zones of inhibition of 2.66 mm and 4.66 mm which was observed to be highly significant (p < 0.05) for the ethanolic leaf extract (Fig 2A). For Fig. 2B, the mean zone of inhibitions of 4.8 mm against E. coli and 4.7 mm against P. aeeruginosa were observed to be highly significant (p < 0.05) for the ethanolic leaf extract of Rubus spp. at the same concentration of 25 [micro]g/ml. The observed results showed higher inhibitory activity than that of the standard antibiotic Chloramphenicol which has a mean zone of inhibition of 1 mm at 30 [micro]g/ml.

Also, based on the results shown in Fig. 2 and 3 in comparing the inhibitory activity between ethanol and water as solvent used for extraction, the solvent ethanol for both M. peltata and Rubus spp. plants showed higher inhibitory activity than water extracts on all Gram-negative and Gram-positive microorganisms used. The inhibitory activity of the ethanolic leaf extracts of the two plants was even higher than that of the positive controls (Streptomycin in [G.sup.+] bacteria and Chloramphenicol in [G.sup.-] bacteria) used.

Discussion:

Phytochemical analysis of medicinal plants has shown that numerous secondary compounds in plants traditionally used for medicinal purposes have chemical properties effective at treating illness. Alkaloids are nitrogenous compound that function in the defense of plants against herbivores and pathogens, and are widely exploited as pharmaceuticals, stimulants, narcotics and poisons due to their potent biological activities. In nature, alkaloids exists in large portions in the seeds and roots of plants and often in combination with vegetable acids. Alkaloids have pharmacological applications as anesthetics and CNS stimulants [13]. In this study, Merremia peltata (L.) Merr. leaves had an abundant amount of alkaloids but none was found in Rubus spp. The presence of alkaloids in Merremia peltata (L.) Merr means that the plant could be used as anesthetics, CNS stimulants, as antioxidant or free radical scavenger [11, 13]. However, in Rubus spp. there are more tannins present but none in Merremia peltata (L.) Merr. Accordingly, these tannins have significant antibacterial property which was observed in the work of Clark [3] and Ekpo and Etim [8]. Flavonoids and steroids were found in both plants extracts. The former substance, flavonoids are chemical compounds active against microorganisms. They have been found in-vitro to be effective antimicrobial substance against a wide array of microorganisms [4, 9, 19]. This suggests that both M. peltata and Rubus. spp. may have an active antimicrobial property.

The high presence of the latter component in the two plants, the steroids, is of importance and interest in pharmacy due to sex hormones, corticosteroids and contraceptives [5, 17].

Merremia peltata (L.) Merr. and Rubus spp. leaf extracts inhibited the growth of all Gram-positive and Gram-negative bacteria, but their effectiveness varied and ethanol extract exhibited higher inhibition than water extract in the present study. This could be because ethanol being an organic solvent could dissolve organic compounds better, hence liberate the active component required for antimicrobial activity since ethanol was found easier to penetrate the cellular membrane to extract the intracellular ingredients from the plant material [9, 21]. Also, the presence of higher amounts of polyphenols in ethanolic extracts as compared to aqueous extracts means that the former are more efficient in cell walls and seeds degradation which have nonpolar character and cause polyphenols to be released from cells.

The ethanolic leaf extracts of both plants have higher inhibitory activity compared with the positive controls, Streptomycin and Chloramphenicol against Gram+ (B. subtilis and S. aureus) and Gram- (E. coli and P. aeruginosa) bacteria respectively. This suggests that both ethnomedicinal plants have pharmaceutical potentials against wide arrays of microbes.

Conclusion:

The phytochemical screening of M. peltata (L.) Merr. and Rubus spp. ethanolic leaf extracts showed the presence of flavonoids, steroids, alkaloids and tannins except tannins and alkaloids for the former and latter plants respectively. Both ethanolic leaf extracts of the two plants have higher inhibitory activity compared with the positive controls: Streptomycin and Chloramphenicol and the negative control. The study showed that the two leaf extracts for both plants had inhibitory activity on both Gram-positive (S. aureus and B. subtilis) and Gram-negative (E.coli and P. aeruginosa) bacteria however, the effect were varied as seen in the different concentrations employed. Moreover, the ethanolic and not the water extract did show a significant inhibitory activity against the different bacterial strains used. This is maybe attributed to the property of the ethanol as organic solvent itself which could extract better the active intracellular ingredients from the plant material. Overall, the results demonstrate and indicate that the Merremia peltata (L.) Merr. and Rubus spp. ethanolic and water leaf extracts can be a source of bioactive substances that have a broad spectrum antimicrobial activity. This confirms the traditional use of the leaves of Merremia peltata (L.) Merr. and Rubus spp. by the Higao-onon tribes in Rogongon, Iligan City, Philippines for the treatment of diseases.

REFERENCES

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[8] Ekpo, M.A. and P.C. Etim, 2009. Antimicrobial activity of ethanolic and aqueous extracts of Sida acuta on microorganisms from skin infections. Journal of Medicinal Plants Research, 3(9): 621-624.

[9] Ekwenye, U.N. and N.N. Elegalam, 2005. Antibacterial Activity of Ginger (Zingiber officinale) Roscoe and Garlic (Allium sativum L.) Extracts on Escherichia coli and Salmonella typhi. International Journal of Molecular Medicine and Advance Sciences, 1: 411-417.

[10] HUGO, 2013. Hardydisk Antimicrobial Sensitivity Test (AST). Retrieved from http://hardydiagnostics.com

[11] Kar, A., 2007. "Pharmacognosy and pharmacobiotechnology (Revised-Expanded Second Edition)", New Age Internaional Limited publishers New Delhi, pp: 332-600.

[12] Lalitha, M., 2005. Manual on antimicrobial susceptibility testing. Indian Association of Medical Microbiologists. Retrieved from http://www.ijmm.org/documents/Antimicrobial.doc

[13] Madziga, HA., S. Sanni and U.K. Sandabe, 2010. "Phytochemical and Elemental Analysis of Acalypha wilkesiana Leaf', Journal of American Science, 6(11): 510-514.

[14] Matu, E.N. and J. Van Staden, 2003. "Antibacterial and Anti-inflammatory activities of some plants used for medicinal purposes in Kenya", Journal of Ethnopharmacology, 87(1): 35-41.

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[17] Okwu, D.E., 2001. Evaluation of the Chemical Composition of indigenous spices and Flavouring Agent. Global J. Pure and Applied Sci., 7(3): 455-459.

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[20] Wadood, A., G.M. Jamal, S.B. Naeem, M. Khan, A. Ghaffar and R. Asnad, 2013. "Phytochemical analysis of Medicinal Plants Occuring in Local Area of Mardan", Journal of Biochemistry and Analytical Chemistry, 2(4): 1000144.

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Kristine Jay B. Perez, Mark Anthony I. Jose, Edgardo Aranico and Ma. Reina Suzette B. Madamba

Department of Biological Sciences, College of Science and Mathematics, Mindanao State University-Iligan Institute of Technology, A. Bonifacio Ave., Iligan City, 9200 Philippines

ARTICLE INFO

Article history:

Received 23 June 2015

Accepted 25 July 2015

Available online 30 August 2015

Corresponding Author: Ma. Reina Suzette B. Madamba, Department of Biological Sciences, Mindanao State University-Iligan Institute of Technology. A. Bonifacio Ave. Tibanga, Iligan City. 9200. Philippines.

Tel: +6309177181101 E-mail: mrsmadamba_ps31@yahoo.com

Table 1: Phytochemical Screening Results of Merremia peltata
(L.) Merr. and Rubus spp ethanolic leaf extracts.

Phytochemicals          Plant Samples

                        Merremia peltata   Rubus spp.
                           (L.) Merr.

Alkaloids                     (+)             (-)
Anthraquinones                (-)             (-)
Cyanogenic glycosides         (-)             (-)
Flavonoids                    (++)           (+++)
Steroids                     (+++)           (+++)
Tannins                       (-)             (++)

Legend: (+) presence, (++) abundant, (+++) very abundant
and (-) absence
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Author:Perez, Kristine Jay B.; Jose, Mark Anthony I.; Aranico, Edgardo; Madamba, Ma. Reina Suzette B.
Publication:Advances in Environmental Biology
Article Type:Report
Geographic Code:9PHIL
Date:Aug 1, 2015
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