5TH INTERNATIONAL CONFERENCE AND WORKSHOP ON BASIC AND APPLIED

SCIENCES (ICOWOBAS 2015)

Conference date: 16–17 October 2015

Location: Surabaya, Indonesia

ISBN: 978-0-7354-1364-1

Editors: Moh. Yasin and Professor Dr. Sulaiman W. Harun

Volume number: 1718

Published: 15 March 2016

PRELIMINARY

Preface: 5th International Conference and Workshop on Basic and Applied Sciences (5th

ICOWOBAS) 2015

Source: AIP Conf. Proc. 1718, 010001 (2016); http://dx.doi.org/10.1063/1.4943308

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Committees: 5th International Conference and Workshop on Basic and Applied Sciences (5th

ICOWOBAS) 2015

Source: AIP Conf. Proc. 1718, 010002 (2016); http://dx.doi.org/10.1063/1.4943309

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INVITED SPEAKER

Microstructure and mechanical changes induced by Q-Switched pulse laser on human enamel

with aim of caries prevention

R. Apsari, D. A. Pratomo, D. Hikmawati and N. Bidin

Source: AIP Conf. Proc. 1718, 020001 (2016); http://dx.doi.org/10.1063/1.4943310

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BIODIVERSITY

Sea cucumber species identification of family Caudinidae from Surabaya based on morphological

and mitochondrial DNA evidence

Muhammad Hilman Fu’adil Amin, Ida Bagus Rai Pidada, Sugiharto, Johan Nuari

Widyatmoko and Bambang Irawan

Source: AIP Conf. Proc. 1718, 030001 (2016); http://dx.doi.org/10.1063/1.4943311

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Oil removal from petroleum sludge using bacterial culture with molasses substrate at temperature

variation

Ni’matuzahroh, Alvin Oktaviana Puspitasari, Intan Ayu Pratiwi, Fatimah, Sri

Sumarsih, Tini Surtiningsih and Salamun

Source: AIP Conf. Proc. 1718, 030002 (2016); http://dx.doi.org/10.1063/1.4943312

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MICROBIAL BIOCHEMISTRY AND MOLECULAR BIOLOGY

Immunofluorescence assay method to detect dengue virus in Paniai-Papua

Teguh Hari Sucipto, Nur Laila Fitriati Ahwanah, Siti Churrotin, Norifumi Matake,

Tomohiro Kotaki and Soegeng Soegijanto

Source: AIP Conf. Proc. 1718, 040001 (2016); http://dx.doi.org/10.1063/1.4943313

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Inhibitor candidates’s identification of HCV’s RNA polymerase NS5B using virtual screening

against iPPI-library

Indah Sulistyawati, Sulistyo Dwi K. P. and Mochammad Ichsan

Source: AIP Conf. Proc. 1718, 040002 (2016); http://dx.doi.org/10.1063/1.4943314

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ENVIRONMENTAL AND GREEN CHEMISTRY

Seasonal radon measurements in Darbandikhan Lake water resources at Kurdistan region-

northeastern of Iraq

Adeeb Omer Jafir, Ali Hassan Ahmad and Wan Muhamad Saridan

Source: AIP Conf. Proc. 1718, 050001 (2016); http://dx.doi.org/10.1063/1.4943315

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Effect of digestion time on anaerobic digestion with high ammonia concentration

Nur Indradewi Oktavitri, Hery Purnobasuki, Eko Prasetyo Kuncoro, Indah

Purnamasari and Semma Hadinnata P.

Source: AIP Conf. Proc. 1718, 050002 (2016); http://dx.doi.org/10.1063/1.4943316

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The influence of dicarboxylic acids: Oxalic acid and tartaric acid on the compressive strength of

glass ionomer cements

Ahmadi Jaya Permana, Harsasi Setyawati, Hamami and Irmina Kris Murwani

Source: AIP Conf. Proc. 1718, 050003 (2016); http://dx.doi.org/10.1063/1.4943317

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The effect of glicerol and sorbitol plasticizers toward disintegration time of phyto-capsules

Pratiwi Pudjiastuti, Esti Hendradi, Siti Wafiroh, Muji Harsini and Handoko

Darmokoesoemo

Source: AIP Conf. Proc. 1718, 050004 (2016); http://dx.doi.org/10.1063/1.4943318

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Speciation and bioavailability of some heavy metals in agricultural soils used for cultivating

various vegetables in Bedugul, Bali

I. Made Siaka, I. Made Supartha Utama, I. B. Putra Manuaba, I. Made Adnyana and

Emmy Sahara

Source: AIP Conf. Proc. 1718, 050005 (2016); http://dx.doi.org/10.1063/1.4943319

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Potential contribution of low cost materials in clean technology

Heman A. Smail, Kafia M. Shareef and Zainab Ramli

Source: AIP Conf. Proc. 1718, 050006 (2016); http://dx.doi.org/10.1063/1.4943320

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Monitoring of coastline change using remote sensing data at South Pamekasan

Thin Soedarti, Onny Z. Rinanda and Agoes Soegianto

Source: AIP Conf. Proc. 1718, 050007 (2016); http://dx.doi.org/10.1063/1.4943321

View Description

The production of sulfonated chitosan-sodium alginate found in brown algae (Sargassum sp.)

composite membrane as proton exchange membrane fuel cell (PEMFC)

Siti Wafiroh, Pratiwi Pudjiastuti and Ilma Indana Sari

Source: AIP Conf. Proc. 1718, 050008 (2016); http://dx.doi.org/10.1063/1.4943322

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NATURAL PRODUCTS AND MEDICINAL CHEMISTRY

Virtual screening using MTiOpenScreen and PyRx 0,8 revealed ZINC95486216 as a human

acetylcholinesterase inhibitor candidate

Sulistyo Dwi K. P., Arindra Trisna W., Vindri Catur P. W., Erna Wijayanti and

Mochammad Ichsan

Source: AIP Conf. Proc. 1718, 060001 (2016); http://dx.doi.org/10.1063/1.4943323

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Three-step crystallization in synthesis of ZSM-5 without organic template

Hartati, Alfa Akustia, Indra Permana and Didik Prasetyoko

Source: AIP Conf. Proc. 1718, 060002 (2016); http://dx.doi.org/10.1063/1.4943324

View Description

Spermatogenic structure and fertility of Mus musculus after exposure of mangosteen (Garcinia

mangostana L) pericarp extract

Alfiah Hayati, Melia Eka Agustin, Farida Ayu Rokhimaningrum, Hasan Adro’i and

Win Darmanto

Source: AIP Conf. Proc. 1718, 060003 (2016); http://dx.doi.org/10.1063/1.4943325

View Description

Double layer structure-based virtual screening reveals 3’-Hydroxy-A-Naphthoflavone as novel

inhibitor candidate of human acetylcholinesterase

Mochammad Ichsan, Ardini Pangastuti, Mohammad Wildan Habibi and Kartika

Juliana

Source: AIP Conf. Proc. 1718, 060004 (2016); http://dx.doi.org/10.1063/1.4943326

View Description

Total flavonoid and phenolic contents of n-butanol extract of Samanea saman leaf and the

antibacterial activity towards Escherichia coli and Staphylococcus aureus

Wiwik Susanah Rita, I. Made Dira Swantara, I. A. Raka Astiti Asih, Ni Ketut

Sinarsih and I. Kadek Pater Suteja

Source: AIP Conf. Proc. 1718, 060005 (2016); http://dx.doi.org/10.1063/1.4943327

Properties of kojic acid and curcumin: Assay on cell B16-F1

Sugiharto, Arbakariya Ariff, Syahida Ahmad and Muhajir Hamid

Source: AIP Conf. Proc. 1718, 060006 (2016); http://dx.doi.org/10.1063/1.4943328

View Description

Phenolic compounds from the stem bark Erythrina Orientalis and detection of antimalaria

activity by ELISA

Tjitjik Srie Tjahjadarie, Ratih Dewi Saputri and Mulyadi Tanjung

Source: AIP Conf. Proc. 1718, 060007 (2016); http://dx.doi.org/10.1063/1.4943329

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Morphology characterization and biocompatibility study of PLLA (Poly-L-Llactid-Acid) coating

chitosan as stent for coronary heart disease

Prihartini Widiyanti, Adanti W. Paramadini, Hajria Jabbar, Inas Fatimah, Fadila N. K.

Nisak and Rahma A. Puspitasari

Source: AIP Conf. Proc. 1718, 060008 (2016); http://dx.doi.org/10.1063/1.4943330

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ANALYTIC AND FORENSIC CHEMISTRY

Preparation and characterization Al

3+-bentonite Turen Malang for esterification fatty acid

(palmitic acid, oleic acid and linoleic acid)

Abdulloh Abdulloh, Nanik Siti Aminah, Triyono, Mudasir and Wega Trisunaryanti

Source: AIP Conf. Proc. 1718, 070001 (2016); http://dx.doi.org/10.1063/1.4943331

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Electrochemical degradation of malachite green using nanoporous carbon paste electrode

Muji Harsini, Faizatul Fitria and Pratiwi Pudjiastuti

Source: AIP Conf. Proc. 1718, 070002 (2016); http://dx.doi.org/10.1063/1.4943332

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Imprinted zeolite modified carbon paste electrode as a potentiometric sensor for uric acid

Miratul Khasanah, Alfa Akustia Widati and Sarita Aulia Fitri

Source: AIP Conf. Proc. 1718, 070003 (2016); http://dx.doi.org/10.1063/1.4943333

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Potential complex of rhodamine B and copper (II) for dye sensitizer on solar cell

Harsasi Setyawati, Aning Purwaningsih, Handoko Darmokoesoemo, Hamami, Faidur

Rochman and Ahmadi Jaya Permana

Source: AIP Conf. Proc. 1718, 070004 (2016); http://dx.doi.org/10.1063/1.4943334

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Gas chromatography-mass spectrometry of ethyl palmitate calibration and resolution with ethyl

oleate as biomarker ethanol sub acute in urine application study

Ni Made Suaniti and Manuntun Manurung

Source: AIP Conf. Proc. 1718, 070005 (2016); http://dx.doi.org/10.1063/1.4943335

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ENVIRONMENTAL BIOCHEMISTRY AND BIOTECHNOLOGY

Tailoring folic acid and methotrexate-attributed quantum dots for integrated cancer cell imaging

and therapy

Mochamad Zakki Fahmi and Jia-Yaw Chang

Source: AIP Conf. Proc. 1718, 080001 (2016); http://dx.doi.org/10.1063/1.4943336

View Description

The effect of aqueous extract of Kalanchoe Folium on methylprednisolone pharmacokinetic

profile

Niken Indriyanti, Afrillia Nuryanti Garmana, Finna Setiawan, Elin Yulinah

Sukandar and I. Ketut Adnyana

Source: AIP Conf. Proc. 1718, 080002 (2016); http://dx.doi.org/10.1063/1.4943337

View Description

Microbial consortium role in processing liquid waste of vegetables in Keputran Market Surabaya

as organic liquid fertilizer ferti-plus

Fauziah Rizqi, Agus Supriyanto, Intan Lestari, Lita Indri D. L., Elmi Irmayanti A. and

Fadilatur Rahmaniyah

Source: AIP Conf. Proc. 1718, 080003 (2016); http://dx.doi.org/10.1063/1.4943338

View Description

Isolation, transformation, anticancer, and apoptosis activity of lupeyl acetate from Artocarpus

integra

Hery Suwito, Wan Lelly Heffen, Herry Cahyana and Wahyudi Priyono Suwarso

Source: AIP Conf. Proc. 1718, 080004 (2016); http://dx.doi.org/10.1063/1.4943339

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COMPUTATIONAL PHYSICS, CHEMISTRY & MATHEMATICS

Contrastive studies of potential energy functions of some diatomic molecules

Hassan H. Abdallah and Hewa Y. Abdullah

Source: AIP Conf. Proc. 1718, 090001 (2016); http://dx.doi.org/10.1063/1.4943340

View Description

Determination the total neutron yields of several semiconductor compounds using various alpha

emitters

Ramadhan Hayder Abdullah and Barzan Nehmat Sabr

Source: AIP Conf. Proc. 1718, 090002 (2016); http://dx.doi.org/10.1063/1.4943341

View Description

Forward problem solution as operator of filter and back projection matrix to reconstruct the

various of data collection in electrical impedance tomography

Khusnul Ain, Deddy Kurniadi, Suprijanto, Oerip Santoso and R. Arif Wibowo

Source: AIP Conf. Proc. 1718, 090003 (2016); http://dx.doi.org/10.1063/1.4943342

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Influence of geometrical factor on binding energy of Cooper pairs in YBa 2 Cu 3 O 7-δ compound

Saeed O. Ibrahim and Bassam M. Mustafa

Source: AIP Conf. Proc. 1718, 090004 (2016); http://dx.doi.org/10.1063/1.4943343

View Description

Size dependence lattice thermal conductivity for Si nanofilm

Hawkar T. Taha and Abdulrahman Kh. Alassafee

Source: AIP Conf. Proc. 1718, 090005 (2016); http://dx.doi.org/10.1063/1.4943344

View Description

PHYSICS AND RENEWABLE ENERGY

The effect of nitrogen on biogas flame propagation characteristic in premix combustion

Willyanto Anggono, Fandi D. Suprianto, Tan Ivan Hartanto, Kenny Purnomo and

Tubagus P. Wijaya

Source: AIP Conf. Proc. 1718, 100001 (2016); http://dx.doi.org/10.1063/1.4943345

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Porous carbon materials synthesized using IRMOF-3 and furfuryl alcohol as precursor

Pemta Tia Deka and Ratna Ediati

Source: AIP Conf. Proc. 1718, 100002 (2016); http://dx.doi.org/10.1063/1.4943346

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Fiber optic displacement sensor for medal detection using fiber bundled probe

M. Yasin, Samian, Supadi, Pujiyanto and Y. G. Yhun Yhuwana

Source: AIP Conf. Proc. 1718, 100003 (2016); http://dx.doi.org/10.1063/1.4943347

STATISTICS, PURE AND APPLIED MATHEMATICS

Estimation of median growth curves for children up two years old based on biresponse local

linear estimator

Nur Chamidah and Marisa Rifada

Source: AIP Conf. Proc. 1718, 110001 (2016); http://dx.doi.org/10.1063/1.4943348

View Description

oa

Segmentation of breast cancer cells positive 1+ and 3+ immunohistochemistry

Ause Labellapansa, Izzati Muhimmah and Indrayanti

Source: AIP Conf. Proc. 1718, 110002 (2016); http://dx.doi.org/10.1063/1.4943349

View Description

Search and selection hotel system in Surabaya based on geographic information system (GIS)

with fuzzy logic

Purbandini and Taufik

Source: AIP Conf. Proc. 1718, 110003 (2016); http://dx.doi.org/10.1063/1.4943350

View Description

Fuzzy multinomial control chart and its application

Wibawati, Muhammad Mashuri, Purhadi and Irhamah

Source: AIP Conf. Proc. 1718, 110004 (2016); http://dx.doi.org/10.1063/1.4943351

View Description

An implementation of continuous genetic algorithm in parameter estimation of predator-prey

model

Windarto

Source: AIP Conf. Proc. 1718, 110005 (2016); http://dx.doi.org/10.1063/1.4943352

View Description

BIOMEDICAL ENGINEERING

Chlorophyll mediated photodynamic inactivation of blue laser on Streptococcus mutans

Suryani Dyah Astuti, A. Zaidan, Ernie Maduratna Setiawati and Suhariningsih

Source: AIP Conf. Proc. 1718, 120001 (2016); http://dx.doi.org/10.1063/1.4943353

View Description

Nearest patch matching for color image segmentation supporting neural network classification in

pulmonary tuberculosis identification

Riries Rulaningtyas, Andriyan B. Suksmono, Tati L. R. Mengko and Putri Saptawati

Source: AIP Conf. Proc. 1718, 120002 (2016); http://dx.doi.org/10.1063/1.4943354

View Description

Infant breathing rate counter based on variable resistor for pneumonia

Novi Angga Sakti, Ardy Dwi Hardiyanto, La Febry Andira R. C., Kesa Camelya and

Prihartini Widiyanti

Source: AIP Conf. Proc. 1718, 120003 (2016); http://dx.doi.org/10.1063/1.4943355

View Description

Total flavonoid and phenolic contents of n-butanol extract of Samanea saman leaf andthe antibacterial activity towards Escherichia coli and Staphylococcus aureusWiwik Susanah Rita, I. Made Dira Swantara, I. A. Raka Astiti Asih, Ni Ketut Sinarsih, and I. Kadek Pater Suteja Citation: AIP Conference Proceedings 1718, 060005 (2016); doi: 10.1063/1.4943327 View online: http://dx.doi.org/10.1063/1.4943327 View Table of Contents: http://scitation.aip.org/content/aip/proceeding/aipcp/1718?ver=pdfcov Published by the AIP Publishing Articles you may be interested in Antimicrobial activity of honey of stingless bees, tiúba (Melipona fasciculata) and jandaira (Melipona subnitida)compared to the strains of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa AIP Conf. Proc. 1702, 120006 (2015); 10.1063/1.4938905 Isolation, identification, and antibacterial activity of chemical compounds from ethanolic extract of suji leaf(Pleomele angusifolia NE Brown) AIP Conf. Proc. 1589, 431 (2014); 10.1063/1.4868836 Antibacterial efficacy of silver nanoparticles against Escherichia coli AIP Conf. Proc. 1512, 372 (2013); 10.1063/1.4791066 Interaction Of ZnO Nanoparticles With Food Borne Pathogens Escherichia coli DH5α and Staphylococcusaureus 5021 & Their Bactericidal Efficacy AIP Conf. Proc. 1393, 153 (2011); 10.1063/1.3653655 Fine Encapsulated ZnO Nanophosphors And Their Potential Antibacterial Evaluation On The Gram NegativeBacillus Escherichia coli AIP Conf. Proc. 1147, 528 (2009); 10.1063/1.3183485

Reuse of AIP Publishing content is subject to the terms at: https://publishing.aip.org/authors/rights-and-permissions IP: 114.4.97.218 On: Tue, 29 Mar 2016 03:40:25

Total Flavonoid and Phenolic Contents of n-Butanol Extract of Samanea saman leaf and The Antibacterial Activity towards Escherichia coli and Staphylococcus

aureus

Wiwik Susanah Rita1,2, a), I Made Dira Swantara1,2, b), I A Raka Astiti Asih2, Ni Ketut Sinarsih1, and I Kadek Pater Suteja2

11 Department of Applied Chemistry, Postgraduate Program Udayana University, Denpasar, Bali, Indonesia2Department of Chemistry Udayana University, Jimbaran, Bali, Indonesia

a) Email: wiwiksr@yahoo.comb)m_dira_swantara@yahoo.co.id

Abstract. Total flavonoid and phenolic contents in some natural products was suspected of having apositive correlation to its activity in inhibiting the growth of bacteria. The aim of this study was to determine the total flavonoid and phenolic contents of n-butanol extract of Samanea saman leaf, and to evaluate the antibacterial activity towards Escherechia coli and Staphylococcus aureus. Extraction of compounds was done by ethanol 96%, followed by fractionation into n-hexane, ethyl acetate, and n-butanol. Determination of total flavonoid and phenolic contents was done by UV-Vis Spectrophotometer using standard of quersetin and galic acid respectively. In addition, antibacterial activity was evaluated by agar disc diffusion method. Extraction of 1000 g of Samanea saman leaf was obtained 80 g of ethanol extracts, fractionation of the extract was obtained 8.02 g of n-hexane extracts, 7.11 g of ethyl acetate extracts, 13.5 g of n-butanol extracts, and 14.16 g of aqueous extracts. Phytochemical screening of the n-butanol extracts revealed the presence of flavonoid and phenolic compounds. Total flavonoid and phenolic contents were successively 43.5798 mg QE/100g and 34.0180 mg GAE/100g. The butanol extracts inhibited the growth of S.aureus higher than the growth of E.coli. At the concentration of 2, 4, 6, 8 % (b/v), and positive control (meropenem 10 g/disc), inhibition zone towards S. aureus was successively 5.67, 9.33, 10.33, 12.00, and 32.33 mm, while the inhibition zone towards E. coli was1.33, 3.33, 4.33, 5.43, and 34.00 mm.

Keywords: Flavonoid contents; Phenolic content; Samanea saman; Escherechia coli; Staphylococcus aureus

1. INTRODUCTION

Diarrhea is a clinical symptoms and gastrointestinal (gut) that are characterized by increased frequency of defecation more than usual (repeatedly). Jawetz et al. [1] reported that one of the bacteria that can cause diarrhea was Escherichia coli. Other diseases caused by E. coli were urinary tract infections, sepsis, and meningitis. According to Melliawati [2], the spread of E. coli can occur by means of direct contact (touching, shaking hands, and so on) is then passed through the mouth.

Given the Escherichia coli bacteria harmful to human health, it is necessary to mitigation or prevention of the development of E. coli, one of which is to utilize the active ingredients from plants that can be used as an antibacterial or suppress the growth of E. coli. One of the plants is rain tree (Samanea saman). Prasad et al. [3] has studied that rain tree leaf extract can inhibit the growth of bacteria (Escherichia coli, and Staphylococcus aureus). Based on phytochemical screening indicated the presence of tannin, flavonoids, saponins, steroids, cardiac glycosides, and terpenoids in tamarind leaf extract. Rita [4] reported that rain tree leaf collectedt from Denpasar contained triterpenoids, steroids, flavonoids, tannins, alkaloids, and saponins.

According to Staples et al. [5], rain tree leaves can be used as a traditional medicine like drug fever, diarrhea, headache, and abdominal pain. Tamarind leaf usefulness as diarrhea medicine, closely related to the growth of Escherichia coli in the gut. Orhan et al. [6] revealed that some flavonoids were formed as antimicrobial barriers in plants response to microbial infection. Parubak [7] reported that flavonoids in the leaves of Akway (Drimys beccariana Gibbs) has antibacterial

5th International Conference and Workshop on Basic and Applied Sciences (ICOWOBAS 2015)AIP Conf. Proc. 1718, 060005-1–060005-6; doi: 10.1063/1.4943327

© 2016 AIP Publishing LLC 978-0-7354-1364-1/$30.00

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activity against Escherichia coli and Bacilus subtilis. Hendra et al. [8] also reported that the presence of flavonoids in the fruit of the gods crown contribute to its activity as an antibacterial.

Considering the antimicrobial activity of rain tree leaf, antibacterial activity of flavonoids extracted from some plants, as well as the presence of flavonoids in leaf of rain tree, there is a possibility that antibacterial activity of rain tree leaf has correlation with flavonoid or phenolic contents. The objectives of this study were to investigate the total flavonoid and phenolic contents of n-butanol extract of Samanea saman leaf and to study the antibacterial activity. Antibacterial activity was assessed in vitro against two bacterial: Escherichia coli and Staphylococcus aureus.

2. MATERIAL AND METHODS

Plant Material

Materials used in this study was rain tree leaf collected around Denpasar Bali. Fresh leaf was washed with tap water, then dried at room temperature for 15 days, powdered, and used for extraction.

Bacterial Agents

Pure cultures of two species of bacterial namely Escherichia coli and Staphylococcus aureus were obtained from the Laboratory of Microbiology of Faculty of Medicine, University of Udayana. The isolates were purified and maintained at 4 °C until use.

Extraction

Around 1 kg of rain tree leaf powder was extracted with 10 L of ethanol 70% for 24 h at room temperature (25 °C). Ethanol extract was evaporated in vacuum and fractionated with solvents of increasing polarity, hexane, ethyl acetate, and n-butanol. Phytochemical test for flavonoids and phenolic compounds were applied to all extracts. The extracts were kept for 24 h at 4 °C, filtered through Whatman No. 4 filter paper, evaporated to dryness under vacuum and stored at 4 °C until analysis.

Total Flavonoid content

Total flavonoids were measured by an aluminum chloride method according to Khatiwora et al. [10] with modification. Aliquots of extract solutions were taken and made up the volume 2 ml with ethanol. Then 0.1ml AlCl3 (10%), 0.1ml Na-K tartarate and 2.8 ml distilled water were added sequentially. The test solution was vigorously shaken. Absorbance at 415 nm was recorded after 30 minutes of incubation. A standard calibration plot was generated at 415 nm using known concentrations of quercetin. The concentrations of flavonoid in the test samples were calculated from the calibration plot and expressed as mg quercetin equivalent /100g of sample. Total phenols can be calculated by the following formula: 1 = . . . 100% (1)where: F1 = total flavonoids , C = Equality of quercetin (g/mL), V = total volume of extract (mL), F= the dilution factor (10), m = weight of sample (g)

Total Phenolic Content

Total phenols were assayed according to Medini et al. [10] with modification. As many as 1 g extract (1 g) was dissolved into 5 ml ethanol 85%, homogenized, centrifuged at 3000 rpm for 15 minutes, and filtered obtain a filtrate. The filtrate was diluted to a volume of 5 mL. 0.4 mL of Filtrate was placed in a test tube, added 0.4 mL of Folin-Ciocalteu reagent, homogenized and added 4.2 mL of sodium carbonate 5%. The mixture was allowed to stand 90 minutes at room temperature. Absorbance at 760 nm was read versus a prepared blank. The total phenol content of n-

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butanol extract was expressed as milligrams of gallic acid equivalents per 100 gram of sample calibration curve with gallic acid with various concentrations of 10-100 mg/L. Total phenols can be calculated by the following formula: 2 = . . . 100% (2)

where: F2 = total phenol, C = Equality of galic acid (g/mL), V = total volume of extract (mL), F= the dilution factor (10), m = weight of sample (g).

Antimicrobial activity

Preparation of agar medium

Medium was made from 20.4 grams of powder Mueller Hinton Agar (MHA), which was dissolved in600 mL of distilled water in erlenmeyer, then heated to dissolve, plugged with cotton and, covered withaluminum foil. Furthermore agar medium sterilized by autoclaving at 121 °C for 15 minutes.

Preparation of the bacterial suspension test

One to three loops of Escherichia coli colonies were taken and suspended in a tube containing 5 mL ofphysiological saline solution. The turbidity of suspension was compared with a standard of Mc Farland 0.5%.

Disc diffusion assay

Antibacterial activity test of the rain tree leaf extract was performed at concentrations of 0, 2.0, 4.0, and 8.0 % (w/v). Each blank discs soaked with 20 mL extract, negative control, and antibiotics (meropenem 10) for 2 hours. MHA then placed on the media that have been implanted bacterial suspension and incubated at 37°C for 24 hours in an inverted position. Diameter of inhibition was observed after incubation period. The diameter of the inhibition was evaluated in milli metres. Each assay was repeated in triplicate. Statistical analysis was then performed using ANOVA test Duncan's Multiple Range Test.

RESULT AND DISCUSSION

Extraction of 1000 g of Samanea saman leaf was obtained 80 g of ethanol extracts, fractionation of the extract was obtained 8.02 g of n-hexane extracts, 7.11 g of ethyl acetate extracts, 13.5 g of n-butanol extracts, and 14.16 g of aqueous extracts. Phytochemical screening of the extracts showed that n-butanol extracts contained flavonoid and phenolic compounds.

The calibration plot for the determination of phenols and flavonoids were presented at Figure 1 and Figure 2, respectively.

Figure 1. Calibration plot for flavonoid determination (quercetin)

y = 0.0522x - 0.0035R² = 0.9959

-0.5

0

0.5

1

1.5

0 5 10 15 20 25

Abso

rban

ce

Concentration mg/L

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Figure 2. Calibration plot for phenolic determination (gallic acid)

Based on the calibration plot equation of quercetin (Figure1), y = 0.052x - 0.003 and the calibrationplot equation of gallic acid (Figure2), Y= 0.181x – 0.190, total flavonoids and phenols in n-butanol extract of rain tree leaves respectively could be determine (Table 1).

Table 1. Total flavonoid and phenolic compounds of rain tree leaf butanol extract

Comp. Sample Weight Abs

(Y)Cons Volume

DilutionContents

(g) (x) (mL) % mg/100g

Flavonoids 0.1277 0.287 5.5651 1.00 10 0.0436 43.5798

Phenols 0.1026 0.317 2.7922 1.25 10 0.0340 34.0180

Flavonoids as one of the most diverse and widespread group of natural compounds are probably the most important natural phenols. From Table 1, it can be shown that total flavonoid was hinger than total phenol, this is likely due to not all flavonoids in rain tree leaves contain phenol groups. total phenol content in the butanol extract relatively low, because according Puspita et al [11], the best solvent to extract phenols was acetone.

Antibacterial activity test against E. coli and S. aureus of rain tree leaf butanol extract was conducted at various concentrations of 0, 2, 4, 6, and 8% with a positive control of meropenem. Antibacterial test results are shown in Table 2 and Figure 3.

. Inhibition zone of the growth of E. coli and S. aureus of rain tree leaf butanol extract in various concentrations

Bacterial Treatment (%) Diameter Zone (mm)

Inhibition (mm)

E. coli

0 7.33 0.00a*2 8.33 1.00a

4 10.33 3.00b

6 11.33 4.00bc

8 12.67 5.34c

Meropenem (10) 34.00 26.67d

S. aureus

0 6.00 0.00a

2 11.60 5.67b

4 15.33 9.33c

6 16.33 10.33c

8 18.00 12.00c

Meropenem (10) 32.33 26.33d

y = 0.1819x - 0.1909R² = 0.9976

-0.2

0

0.2

0.4

0.6

0.8

1

0.0 2.0 4.0 6.0 8.0

Abso

rban

ce

Concentration mg/L

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* values followed by the same letters are not significantly different (P>0.05) according to the Duncan’s Multiple Range Test at 5% level.

(a) (b). Antibacterial activity of rain tree leaf butanol extract against a) E.coli

b) S. aureus

Table 2 and Figure 3 showed that treatment with 2% of extract was not significantly different (P>0.05) with the control towards E-coli, but signicantly inhibited the growth of S.aureus. The inhibition of E-coliincreased with increasing concentrations of the extract. The inhibition of S.aureus rose significantly until 4% extract treatment, but the treatment over 4% was not significantly increase. It can be said that the concentration of 4% was the optimum concentration to inhibit the growth of S.aureus.

Inhibition of butanol extract of rain tree leaf against E. coli and S. aureus at concentrations of 8%were respectively 5.34 and 12.00 mm. According to Ardiansyah [12], if zone of inhibition 20 mm: the inhibition was very strong; 10-20 mm: strong; 5-10 mm: moderate; and <5 mm: the inhibition is low. Base on the result, it can be concluded that butanol extract of rain tree leaf led to a low inhibition on the growth of E.coli and strong inhibition on the growth of S. aureus.

Cushnie and Lamb [13] reported that there was a different mechanism for a variety of flavonoids, such mechanisms include inhibiting the synthesis of nucleic acids, cytoplasmic membrane function, and energy metabolism. The mechanism involves the interaction between the enzyme in bacterial with flavonoids. Enzymes are active proteins consisting of various kinds of amino acids, resulting in the formation of hydrogen bonds between flavonoids with amino acids causes the enzyme to be disturbed.

According to Kumar and Pandey [14], Antibacterial flavonoids might be having multiple cellular targets, rather than one specific site of action. One of their molecular actions is to form complex with proteins through nonspecific forces such as hydrogen bonding and hydrophobic effects, as well as by covalent bond formation. Thus, their mode of antimicrobial action may be related to their ability to inactivate microbial adhesins, enzymes, cell envelope transport proteins, and so forth. Lipophilic flavonoids may also disrupt microbialmembranes.

According to Singh [15], phenolic compounds have the mechanism of action in inhibiting the growth of bacteria by inactivation of the protein (enzyme) on the cell membrane. According Susanti [16], phenols bind to the proteinthrough hydrogen bonding resulting protein structure become damaged. Where most of the structure of the cell wall and bacterial cytoplasmic membrane protein and fat. Instability in the cell wall and the cytoplasmic membrane of bacteria causing the function of selective permeability, active transport function, controlling the composition of proteins from bacterial cells to be disrupted, which will result in escape macromolecules, and ions from the cell. So the bacterial cells becomes lost its shape, and there is a lysis.

Mailoa et al. [17] reported the antimicrobial activity of phenolic compounds (tannin) extract of guava leaves against microbial pathogens. The results showed that the inhibitory activity of compounds on five microbial pathogens was different. This was because the composition of the microbial cell wall five microbes was different. The results showed that the tannin extract can inhibit the growth of Escherichia coli, Pseudomonas aureginosa, Staphilococcus aureus, Aspergillus niger and Candida albicans.

CONCLUSION

Based on the results of research and discussion, it can be concluded as follows:

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Total flavonoid and phenolic contents of rain tree leaf butanol extract were successively 43.5798 mg QE/100gand 34.0180 mg GAE/100g.The butanol extract of rain tree leaf led to a low inhibition on the growth of E. coli and strong inhibition on thegrowth of S. aureus.

ACKNOWLEDGEMENTS

This work was supported by a grant from DP2M Ditjen Dikti Indonesia (Competitive research Grand). We wish to express our gratitude to head of Research and Community Institutions of Udayana University facilitating all the needs in the disbursement of research funds.

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