Research Journal of

Chemistry and Environment

Vol. 22 (Special Issue II), August 2018

E-ISSN: 2278 - 4527 ! PRINT-ISSN No. 0972 - 0626

Journal is indexed in SCOPUS andChemical Abstracts

ii

GOD IS.

E-ISSN: 2278 – 4527 PRINT-ISSN No. 0972-0626

RESEARCH JOURNAL OF CHEMISTRY AND ENVIRONMENT

An International Research Journal of Chemical Sciences and Environmental Sciences

Res. J. Chem. Environ., Volume 22(Special Issue II), Pages 1-350, August (2018)

Editor- in- Chief (Hon.)

Dr. D.K. Vardhan Mumbai, INDIA

Correspondence Address:

Research Journal of Chemistry and Environment

Sector AG/80, Scheme No. 54, Indore 452 010 (M.P.) INDIA

Phone and Fax: +91-731-4004000

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E-mail: info@worldresearchersassociations.com

CONTENTS

Research Papers:

1. Biosynthesis of Silver Nanoparticles using Jicama Extract and Its Application for Colorimetric

Sensing of Mercury Ions

- Firdaus M. Lutfi, Megarani Juwita, Purawiardi R. Ibrahim, Eddy Diana Rakhmawaty and Rahayu Iman

1-3

2. Synthesis and Structural Analysis of Aurivillius Phase, Ca1-xBi3+xNdTi4-xMnxO15

- Zulhadjri, Bilian Sahiga J.I., Wendari Tio Putra and Emriadi

4-9

3. Digital-Based Image Detection System in Simple Silver Nanoparticles-based Cyanide Assays

- Tambaru David, Nomi Anastasia Grandivoriana and Nitti Fidelis

10-14

4. Preparation of Mixed Matrix Polymeric Membrane for removing of contaminants in Crude Biodiesel

- Saiful, Ramli Muliadi, Maulana Ilham, Fadli and Yusuf M.

15-21

5. A new zinc (II) responsive MRI contrast agent

- Adhitiyawarman and Lowe Mark P.

22-30

6. Synthesis of Benzyl-Protected Robtein (2,3,4,4,5-Pentahydroxychalcone) as an Intermediate to (–)-

Robidanol

- Suharti, Achmadi Suminar S., Irfana Luthfan, Sugita Purwantiningsih, Wukirsari Tuti, Ilmiawati Auliya

and Arifin Budi

31-38

7. Antibacterial and antioxidant activities of pyrogallol and synthetic pyrogallol dimer

- Cynthia, Ignatia Florence, Sutanto Hery and Darmawan Akhmad

39-47

8. Preparation and Characterization of Titania/Bentonite Composite Application on the Degradation of

Naphthol Blue Black Dye

- Lubis Surya, Sheilatina, Putra Vicky Praja and Nika Syahrinta Sepia

48-53

9. Optimization of Light Exposure on Superoxide Scavenger Test of Manganese (III)-Salen Acetate

Complex - Nurrahmajanti, Asiyah Onggo, Djulia Deawati and Yusi

54-57

10. Application of Molecularly Imprinted Polymer Technique on Chitosan Membranes for Increasing

Creatinine Transport Effectiveness

- Sulistyaningsih Eka

58-64

iii

11. Total Phenolic and Flavonoid Contents and Antimicrobial activity of Acorus calamus L. Rhizome

Ethanol Extract

- Rita Wiwik Susanah, Kawuri Retno and Swantara I. Made Dira

65-70

12. Optimization of process parameters for inulinase production from endophytic fungi Fusarium solani

LBKURCC67, Humicola fuscoatra LBKURCC68 and Fusarium oxysporum LBKURCC69

- Devi Silvera, Illiyin Luthfin, Ardhi Aulia, Pratiwi Nova Wahyu and Saryono

71-78

13. Morphological identification and hydrolytic enzyme-producing abilities of fungi associated with

wilting banana plants (Musa sp.)

- Saryono, Piska Finna, Sari Nurmala, Pratiwi Nova Wahyu and Ardhi Aulia

79-86

14. 20-Epibryonolic Acid from Tetrameles nudiflora Leaves

- Darmawan Akhmad, Fajriah Sofa, Megawati and Lotulung Puspa Dewi N.

87-90

15. Synthesis of fluorescent compound, 7-Hydroxy-4-methyl-2H-chroman-2-one via Pechmann

condensation of citric acid and resorcinol

- Al Anshori Jamaludin, Rahayu Diah Siti, Hidayat Ace Tatang, Hidayat Ika Wiani and Zainuddin Achmad

91-96

16. Distinguishing of Oligosaccharide Linkage Isomers by Nanoparticle-assisted Fragmentation in

MALDI-TOF MS

- Primadona Indah, Dedi, Wang Yi-Sheng and Chen Yu-Ju

97-101

17. Recombinant Anti BNP-SCFV Production in Escherichia coli and its Application for the Detection of

Heart Failure by Electrochemical Immunosensor using Screen-Printed Carbon Electrode-Gold

Nanoparticles (SPCE-GNP)

- Gaffar Shabarni, Hartati Yeni Wahyuni, Susanti Rini and Subroto Toto

102-107

18. Chemical Compositions of Banana Peels (Musa sapientum) Fruits cultivated in Malaysia using

proximate analysis

- Hassan Pyar and Peh K.K.

108-113

19. Isolation and Identification of Quercetin Derivatives and their -Glucosidase Inhibitory Acitivities

from Bryophyllum pinnatum

- Fajriah Sofa, Darmawan Akhmad, Megawati Megawati and Hanafi Muhammad

114-119

20. Influence of synthesis time on Lanthanum Silicate Apatite (La9.33Si6O26) properties

- Noviyanti Atiek Rostika, Juliandri, Agustina Shofia Utari and Malik Yoga Trianzar

120-123

21. A comparison of the fermentation performance and stress tolerance of baker’s yeast cells grown in

media with or without magnesium addition

- Rachman Saadah D., Hidayat Risma W., Safari Agus and Ishmayana Safri

124-128

22. Investigating Aluminasilicate Source in Geopolymer Compressive Strength: Surface Morphology and

Crystal Phase Study

- Anggarini Ufafa and Sukmana Ndaru Candra

129-133

23.

Optimization Density of Geopolymer Concrete based on Taguchi Method

- Sukmana Ndaru Candra, Pambudi Andry Agung and Anggarini Ufafa

134-138

24. Synthesis of 5-(4’-Chlorobenzylidene) hydantoin and N-3 Substituted 5-(4’-Chlorobenzylidene)-3-

Methylhydantoin

- Naufal Muhammad, Hamdani Ulfi Dadan, Maharani Rani, Al Anshori Jamaludin and Hidayat Ika Wiani

139-145

25. In vitro antioxidant activity of Sonneratia ovata Backer extract

- Khumaidah Laili, Nurmalasari Fatati, Putri Fatmawati Inneke, Purnomo Adi Setyo, Santoso Mardi,

Ersam Taslim and Fatmawati Sri

146-150

iv

26. Effects of bacterium Ralstonia pickettii addition on DDT biodegradation by Daedalea dickinsii

- Purnomo Adi Setyo, Rizqi Hamdan Dwi, Fatmawati Sri, Putro Herdayanto Sulistryo and Kamei Ichiro

151-156

27. The effect of Calcination Temperature of Sulfated Zirconia Catalyst for Simultaneous Reactions in

Biodiesel Production

- Andrijanto Eko and Brown Rob

157-162

28. Synthesis of Zirconia 1-D Nanomaterials from Local Zircon-Based Zr(OH)4 mediated by PEG-6000

- Septawendar Rifki, Nuruddin Ahmad, Maryani Eneng, Sutardi Suhanda and Purwasasmita Bambang

Sunendar

163-171

29. Effects of the Precipitation pH of Sodium Silicate on the Amorphous Silica Characteristics and its

capability in the Pb and Cd Adsorption

- Maryani Eneng, Septawendar Rifki and Suhanda

172-178

30. Critical Parameters of Silver Nanoparticles (AgNPs) synthesized by Sodium Borohydride Reduction

- Sholikhah Umi Nur, Pujiyanto Anung, Lestari Enny, Sarmini Endang, Lubis Hotman, Widyaningrum

Triani and Febriana Siska

179-183

31. Antibacterial Activity of Prenylated Xanthones from Pericarp of Garcinia mangostana against

Persistent Dental Infection Microorganism Enterococcus faecalis

- Setiawan Arlette Suzy, Herawati Mieke, Dewi Warta and Supratman Unang

184-188

32. Identification of a DNA Polymerase I Gene Fragment from a Local Isolate (PLS 80) from an

Underwater Hot Spring

- Febriani, Helwati H., Velayati M.A. and Iqbalsyah T.M.

189-192

33. Biohydrogen Production through Separate Hydrolysis and Fermentation and Simultaneous

Saccharification and Fermentation of Empty Fruit Bunch of Palm Oil

- Kusmardini Diah, Prasetyo Joni, Saepudin Endang and Hudiyono Sumi

193-197

34. Study Cytotoxicity of Haarlem Oil by Crystal Violet Staining Assay

- Khairan Khairan, Jamier Vincent and Jacob Claus

198-202

35. Determination of Optimum Pressure for Gd3+ Separation from Gd-DTPA with Nanofiltration

Membrane

- Rahayu Iman, Winanti Anti, Juliandri, Noviyanti Atiek Rostika, Eddy Diana Rakhmawaty, Anggraeni Anni

and Bahti Husein H.

203-205

36. Cytotoxic Effects of Cassava (Manihot esculenta Crantz), Adira-2, Karikil and Sao Pedro Petro

Varieties against P-388 Murine Leukemia Cells

- Widiastuti Diana, Mulyati Ade Heri, Eka Herlina, Siti Warnasih, Suchyadi Yudhie, Triastinurmiatiningsih

and Supratman Unang

206-208

37. Coating of Nanotube Ti-6Al-4V Alloy with Hydroxyapatite-Chitosan Composite by Electrophoretic

Deposition Method

- Charlena, Suparto Irma Herawati and Puspitawati Tuti

209-213

38. pH Effect of Gold Nanoparticles Synthesis Encapsulation in Polyamide Amine Generation 3.0

(AuNP-PAMAM G3.0)

- Pujiyanto Anung, Lestari Enny, Kurniasih Dede, Mujinah, Sholikhah Umi Nur, Gunawan Adang Hardi,

Amalia Sarah S. and Mutalib Abdul

214-219

39. Mode of action of an insecticidal oleanane glycoside on digestive enzymes of Crocidolomia pavonana

- Maharani Rani, Rizqiani Anggun Fuji, Ishmayana Safri, Hidayat Yusup and Dono Danar

220-225

v

40. Comparative assessment of yeast fermentation performance, ethanol tolerance and membrane fluidity

- Ishmayana Safri, Kennedy Ursula J. and Learmonth Robert P.

226-235

41. Preparation of ZnO/SiO2 Composite using Silica isolated from Rice Husk

- Eddy Diana Rakhmawaty, Ishmayana Safri, Noviyanti Atiek Rostika and Wahyuni Astri

236-239

42. Separation of Radioisotope Iodine-131 from Radiotellurium-131 using Dowex-1x8 resin column

chromatography

- Setiawan Duyeh, Nugraha Nabil Aulia and Hastiawan Iwan

240-244

43. Prelimimary Study of using Palm Oil Mill Effluent to produce Bio Hydrogen as Biofuel

- Prasetyo Joni, Febijanto Irhan, Murti S.D. Sumbogo and Dewi Eniya L.

245-248

44. Analysis and Characterization of Complex Compound Gadolinium-(1,4,7,10-Tetraazacyclododecane,

1-4-7-10-Tetraacetic Acid)n-Poliamidoamine Generation 3-Trastuzumab as a Novel Contrast Agent

for Magnetic Resonance Imaging

- Kusuma Yenni Nugraha Wira, Mutalib Abdul, Anggraeni Anni, Ramli Martalena, Fauzia Retna Putri and

Bahti Husein H.

249-254

45. Comparison of Different Methods for Purification of Gold Nanoparticles– Polyamidoamine

(PAMAM) Generation 4 Dendrimer

- Narsa Angga Cipta, Pujiyanto Anung, Gunawan Adang H., Yusup Iyus Maolana, Listyowati Indrarini,

Fauzia Retna Putri, Bratadiredja Marline Abdassah, Mutalib Abdul, Soetikno Ristaniah D. and

Soedjanaatmadja R. Ukun M.S.

255-259

46. Utilization of Sericin on Modification of Cotton Dyeing using Acid Dyes

- Ramadhani Puri Awaliyah and Umam Khairul

260-266

47. Toxicity of Seed oil of Azadirachta indica, Calophyllum inophyllum and their Mixture against

Crocidolomia pavonana Larvae

- Kadarusman Ratna Puji Astuti, Widayani Neneng S., Dono Danar, Hidayat Yusup, Sunarto Toto,

Hartati Sri and Maharani Rani

267-273

48. Addition of mango essence to methyl eugenol more attracted to Bactrocera dorsalis Complex

(Diptera: Tephritidae) on mango plantation in Majalengka, West Java

- Susanto Agus, Djaya Luciana and Sudrajat Rian Aprianti

274-279

49. Solubility Improvement of Antimalaria Drug through Co-Crystalization with Malic Acid

- Gozali Dolih, Alatas Fikri and Sugandi Rani

280-284

50.

Simple method and environmentally friendly ash content removal process from rice husk char using

potassium carbonate solution

- Solihudin, Rustaman and Haryono

285-288

51. Polymer Electrolyte Membrane Fuel Cell based on Sulfonated Polystyrene and Phosphoric Acid with

Biocellulose as a Matrix

- Naumi Fadlinatin, Natanael Christi L., Rahayu Iman, Indrarti Lucia and Hendrana Sunit

289-293

52. A Voltammetric Immunosensor for Detection of HER2 using Gold Modified-Screen Printed Carbon

Electrode

- Hartati Yeni Wahyuni, Beladona Siti U. Misonia, Wyantuti Santhy and Gaffar Shabarni

294-301

53. Fast and Simultaneous Detection of Sm, Eu, Gd, Tb and Dy using combination of Voltammetry

Method and Multivariate Analysis

- Wyantuti Santhy, Pratomo Uji, Hartati Yeni Wahyuni, Anggraeni Ani and Bahti Husein H.

302-306

vi

54. Vannamei Shrimp (Litopenaeus vannamei, Boone, 1931) Performance with Bacillus and Lactobacillus

Probiotic Formulation Consortium in Dry Preparation

- Andriani Yuli, Safitri Ratu and Fauziah Shaiyanne

307-312

55. The Implementation of Natural Pigments of Pumpkin Meal to enhance the Color Quality of Koi Fish

(Cyprinus carpio)

- Yustiati Ayi, Arisya Yuni, Herawati Titin, Rachman Boedi and Andriani Yuli

313-318

56. Novel Mechanical Filter for reducing Ammonia Concentration of Silver Barb culture in a

Recirculating Aquaculture System (RAS)

- Hasan Zahidah, Rajibbusalam, Maulina Ine and Andriani Yuli

319-324

57. The Reduction of Lead in Fish Meat from Floating Net Cages in Cirata Reservoir by Citric Acid

- Junianto, Hasan Zahidah and Afrianto Eddy

325-329

58. Cytotoxic Sesquiterpenoid from the Stembark of Aglaia argentea (Meliaceae)

- Harneti Desi, Farabi Kindi, Nurlelasari, Maharani Rani, Supratman Unang and Shiono Yoshihito

330-333

59. Cellulose isolation from Gracilaria Genus and its Potential as Bioethanol Raw Material

- Satiyarti Rina Budi, Febrishaummy Indriani, Yun Yenny Febriyani and Mulyani Rahmaniar

334-336

60. Sn (IV) doped lanthanum silicate apatite structure (La9.33Si6-xSnxO26; x: 0.1; 0.3; 0.5) as an electrolyte

- Noviyanti Atiek Rostika, Eddy Diana Rakhmawaty, Hastiawan Iwan, Dzulfikar Muhammad

and Syarif Dani Gustaman

337-341

61. Determination of Individual Spectra of Sm, Eu, Gd, Tb and Dy from the UV-Vis Spectrum of

Mixture Solution

- Anggraeni Anni, Avian Mohamad Sepril, Purnama Andrew and Mutalib Abdul

342-346

62. Secondary Metabolites from Steambarks of Dysoxylum alliaceum

- Mayanti Tri, Nurcahyanti Ois, Darwati, Julaeha Euis, Farabi Kindi, Sumiarsa Dadan

and Dinata Deden Indra

347-350

Research Journal of Chemistry and Environment_______________________________Vol. 22(Special Issue II) August (2018) Res. J. Chem. Environ.

65

Total Phenolic and Flavonoid Contents and Antimicrobial activity of Acorus calamus L. Rhizome

Ethanol Extract Rita Wiwik Susanah1*, Kawuri Retno2 and Swantara I. Made Dira1

1. Chemistry Department, Faculty of Mathematics and Natural Sciences, Udayana University, INDONESIA

2. Biology Department, Faculty of Mathematics and Natural Sciences, Udayana University, INDONESIA

*susanah.rita@unud.ac.id

Abstract The rhizomes of Acorus calamus L. are widely used as

medicinal plant for diseases caused by microbes. The

antimicrobial activity of rhizomes is suspected to have

a positive correlation to the content of phenolic and

flavonoid compounds. This study aims to determine the

total phenolic and flavonoid contents and to evaluate

the antimicrobial activity of ethanol extract of Acorus

calamus L. rhizome. An extraction process was carried

out at room temperature by maceration methods.

The determination of total phenolic and flavonoid

contents was conducted by a UV-Vis Spectrofotometer

using standard gallic acid and quercetin respectively.

Additionally, the antimicrobial activity was evaluated

by the agar disc diffusion method. The total phenolic

and flavonoid contents were successively 2398.40 mg

GAE/100g and 190.46 mg QE/100g dry rhizome

extract. The extracts demonstrated antibacterial

activity against Escherichia coli and Staphylococcus

aureus and antifungal activity against Candida

albicans. Minimum inhibitory concentration of the

extracts against Escherichia coli, Staphylococcus

aureus and Candida albicans was 2, 3 and 3%

respectively with the inhibition zone of 9.80, 9.50 and

8.67 mm.

Keywords: Acorus calamus L., Antimicobial Activity,

Flavonoid contents, Phenolic contents.

Introduction The environment plays a major role in improving public

health. However, poor public health conditions such as the

incidence of various diseases are also influenced by the

surrounding environment. An inadequate environment can

potentially have a direct impact on human health, it can

spread dangerous agents, or indirectly by disrupting the

ecosystems that sustain life1. A dirty environment is good for

breeding various disease vectors such as microbes, including

both bacteria and fungi. Microbial pathogens can cause

various diseases in living organisms by infection. The

microbes that often cause the disease are Escherichia coli, Staphylococcus aureus and Candida albicans.

Medicines such as tetracycline, amoxicillin, miconazole are

often applied to treat infectious diseases. However, these

medicines can lose effectiveness due to microbial

resistance2. Hence, new medicines from natural sources such

as antibiotics from plants are required. A variety of plant

species are traditionally used in some countries for treatment

of infectious diseases such as Allium sativum, Bunium persicum, Oryza sativa3, Vitex negundo, Piper nigrum,

Duranta repens and Acorus calamus4.

Among the plant widely used as traditional medicine to treat

diseases caused by microbials is A. calamus L. This plant has

many uses. Rahamoz-Haghighi et al5 reported that A.

calamus rizhome ethanol and methanol extracts could inhibit

the growth of S. aureus, Staphylococcus epidermidis, E. coli with similar effects. Rawal et al6 revealed that acetone,

aqueus, ethanol and petroleum ether extracts of A. calamus

rizhome could potentially inhibit the growth of Fusarium oxysporum f.sp. lycopersici with Minimum Inhibitory

Concentration (MIC) of 500, 750, 250, 250 mg/mL

respectively. Funde7 investigated anticancer, antioxidants

and antimicrobial activity of A. calamus and stated that A.

calamus was useful for multi-diseases therapeutic research.

Anisah et al8 reported that ethanol extract of A. calamus

rhizome contains alkaloids, flavonoids and polyphenols.

Rahamoz-Haghighi et al9 reported that A. calamus ethanol

extract contains phenyl propanoids, monoterpenes,

sesquiterpenes and -asarone. The compounds

demonstrated antibacterial activity. Based on preliminary

tests, the methanol extract of rhizome collected in Denpasar

contains triterpenoids, steroids, flavonoids, polyphenols and

alkaloids. The antimicrobial activity of A. calamus rhizomes

is probably caused by the content of the compounds in the

rhizomes such as essential oils, flavonoids and polyphenols.

Rita et al10 reported that the essential oils of A. calamus

could potentially inhibit the growth of C. albicans with the

minimum inhibitory concentration (MIC) of 1%. The

essential oils also inhibit the growth of Fusarium solani, a

pathogenic fungus causing stem rot diseases on dragon fruit

stems11,12. The results demonstrate that the oils strongly

inhibited F. solani at a concentration of 10% with an

inhibition zone of 10 mm, MIC of 2 %. The results also

indicate that the growth of colony, spores and fungal

biomass increased with the increase of essential oil

concentration.

Besides essential oils, the antimicrobial activity of A.

calamus rizhome was associated with flavonoid and

phenolic content. Mahboubi et al13 revealed that the

Research Journal of Chemistry and Environment_______________________________Vol. 22(Special Issue II) August (2018) Res. J. Chem. Environ.

66

antimicrobial efficacy of the plant extracts is correlated with

their phenolic and flavonoid contents. Hence, it is necessary

to investigate antimicrobial activity to ethanol extract of the

rhizomes collected in Bali and to determine the total content

of phenolic and flavonoid compounds.

Material and Methods Plant Material: A. calamus L. was identified at LIPI-UPT

Center for Plant Conservation Botanical Garden "Eka

Karya" Bali. Rhizomes of A. calamus were collected around

Denpasar Bali. The rhizomes were respectively cleaned, cut

and dried at room temperature for 15 days. Next, they were

powdered and stored for later analysis.

Microbial Agents: Two strains bacteria E. coli (Gram-

negative) and S. aureus (Gram-positive) and a fungal

pathogen, C. albicans, were applied to the antimicrobial

assay. These microorganisms were obtained from culture

collection of Laboratory of Microbiology, Department of

Biology, Faculty of Mathematics and Natural Sciences,

Udayana University. The isolates were purified and

maintained at 4°C until later use.

Extraction: Around 750 g of A. calamus rizhome powder

was extracted with 10 L of ethanol 96% for 24 h at room

temperature (25°C). The extract was filtered through a

Whatmann filter paper and then evaporated under vacuum

and stored at 4°C until further analysis.

Determination of Total Phenolic and Flavonoid Contents

Total Phenolic contents: Folin-Ciocalteu reagent was

applied for the determination of total phenolic contents14. A

total of 0.1158 gram extract was dissolved in 96% ethanol to

obtain a volume of 5 mL. Dilution was performed 100 times;

1 μL of filtrate was dissolved in ethanol to obtain 100 μL of

solution. 100 μL of Folin-Ciocalteu reagent and 800 μL of

5% sodium carbonate were added so the total solution

volume became 1000 μL. The mixture was then allowed to

stand for 90 minutes, before the absorbance was measured at

a wavelength of 760 nm. A series of gallic acid solutions

with various concentrations were also prepared.

The absorbance of each concentration was measured at a

wavelength of 760 nm. From the standard gallic acids

standards, a calibration curve was made to get the equation

line of y = ax + b. The total phenolic contents were expressed

as mg gallic acid equivalents /100 g of extract. The total

phenols can be determined by using the following formula:

where F1 = total phenol, C = equality of gallic acid (g/mL),

V = total volume of extract (mL), F = the dilution factor and m = weight of sample (g).

Total Flavonoid contents: Total flavonoids were

determined by the aluminum chloride method14. A total of

0.1035 grams of samples were dissolved in 96% ethanol to

obtain a volume of 5 mL. Dilution was performed 5 times.

100 μL of filtrate was dissolved in ethanol to obtain 500 μL

of solution. 500 μL of 2% aluminum chloride was added, so

the total volume of the solution became 1000 μL. The

mixture was then allowed to stand for 90 minutes before the

absorbance was measured at a wavelength of 415 nm.

A series of quercetin solutions with various concentrations

were also prepared. The absorbance of each concentration

was measured at a wavelength of 415 nm. From the

quercetin standards, a calibration curve was made to obtain

equation line of Y = ax + b. The total flavonoid contents

were expressed as mg quercetin equivalents/100 mg extract.

The total flavonoids can be determined by using the

following formula:

where F1 = total flavonoids, C = equality of quercetin

(g/mL), V = total volume of extract (mL), F = the dilution

factor and m = weight of sample (g).

Antimicrobial Activity Assay: Antimicrobial activity assay

of A. calamus rhizome ethanol extract was conducted by the

well diffusion method at various concentrations of 0

(negative control), namely, 0.5, 1, 2, 3, 4, 6, 8 and 10% with

three repetitions10. In order to determine the optimum

concentration, the assay was performed on the extracts with

various concentrations greater than 10. The concentrations

applied were 12, 14 and 16%.

Petri dish containing 10 mL of PDA (Potato Dextro Agar)

media and 200 μL of suspension of the microbes (E. coli, S.

aureus and C. albicans) were allowed to solidify. After the

suspension was solid, the diffusion wells were made using a

cork borer and each well was filled with 20 μL of the extract

and incubated at a temperature of 37°C for 48 hours. The

antimicrobial activity was determined by the diameter of the

inhibition zone.

Results and Discussion Determination of Total Phenolic and Flavonoid Contents

The curves of the calibration to determine total phenolic and

flavonoid contents are presented in figures 1 and 2. Based on

the calibration curves, the calibration equation of gallic acid

(figure 1) obtained was y=0.047x + 0.0635 (R2=0.9899),

while that of quercetin (figure 2) was y=0.0313x+0.0272

(R2= 0.9961). The calculation to determine the total phenolic

and flavonoid contents is summarized at table 1. According

to table 2, the total phenolic and flavonoid contents were 2398.40 mg GAE/100g and 190.46 mg QE/100g dry

rhizome extract successively.

F1C.V.F. 10-6

m100%= ..................................... (1)

F2C.V.F. 10-6

m100%= ..................................... (2)

(2)

Research Journal of Chemistry and Environment_______________________________Vol. 22(Special Issue II) August (2018) Res. J. Chem. Environ.

67

The data showed that the total phenol was higher than total

flavonoids, likely due to only a few phenol compounds being

flavonoids especially quercetin. The most likely compound

is tannin, especially hydrolysable tannis. According to

Khanbabaee and Ree15, tannins consist of hydrolysable and

condensed tannins. The hydrolysable tannins include both

the gallotannins and the ellagitannins. The condensed

tannins consist of cathecin (flavan-3-ol) units.

Antimicrobial Activity Assay: Antimicrobial activity assay

against E. coli, S. aureus and C. albicans of A. calamus

rhizome ethanol extract was performed at concentrations of

0 (negative control), namely, 0.5, 1, 2, 3, 4, 6, 8 10, 12, 14

and 16 %. The antimicrobial assay results are shown in

table 3.

The data showed that the inhibition towards the microbes

increased with the increase of the concentration applied.

Rhoades and Roller16 revealed that in general, the inhibition

tends to increase with the increase of the extracts

concentration. Minimum inhibitory concentration (MIC) of

the extract against E. coli, S. aureus and C. albicans was 2,

3 and 3% respectively with the inhibition zone of 9.80, 9.50

and 8.67 mm.

Overall, E.coli was more sensitive to the extract compared

to S. aureus and C. albicans, except at a concentration of 6%.

This indicates that gram-negatives bacteria are more

sensitive compared to gram positive. These findings are in

contrast to the findings of Okigbo and Mmeka17 who

reported that S. aureus is the most susceptible to the plant

extracts followed by E. coli and then C. albicans. The reason

why E.coli is more susceptible to the extracts than other

microbes is unclear.

The data in table 2 shows that the optimum concentration of

the extract to inhibit the growth of E. coli was 8.0 % because

the inhibition zone was not significantly different from the

concentration of 8.0 to 16%. Meanwhile, the optimum

concentration of that to inhibit S. aureus and C. albicans was

12.0 and 10.0% respectively. Figure 3 shows the inhibitory

zone of the extract with various concentrations towards E.

coli, S. aureus and C. albicans. The extract at a concentration

of 10% could strongly inhibit the growth of all microbes

used (figure 4). The inhibition zone less than 5 mm was

categorized as weak inhibition, between 5 and10 mm was

moderate, larger than 10 to 20 mm was strong and higher

than 20 mm was very strong inhibition14.

Camargo et al18 reported the antimicrobial effects of

phenolic acids and flavonoids of peanut by-products. Their

results showed that phenolic acid-rich extracts showed the

lowest minimum inhibitory capacity (MIC) which means

that the antibacterial effect is highest. Meanwhile, Xuan

et al19 studied the total phenolic and flavonoid contents of

commercial vegetable edible oils marketed in Japan. All the

oils studied possess antimicrobial activity on both S. aureus

and E. coli.

The total phenolic and flavonoid contents contribute to the

antimicrobial activity of this A. calamus rhizome. The

antimicrobial activities of the phenolic compounds are

associated to the ability to bind extracellular and soluble

proteins, thus enabling complexation with bacterial cell

walls20.

Figure 1: Calibration Curve of Standard Gallic Acid

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Figure 2: Calibration Curve of Standard Quercetin

Figure 3: Graph of Inhibitory Activity of A. calamus Rizhome Ethanol Extract against

E. coli, S. aureus and C. albicans

Figure 4: Inhibitory Activity of A. calamus Rizhome Ethanol Extract against

A) E. coli, B) S. aureus and C) C. albicans at concentration of 10% with three repetitions

(0: negative control; 1: the 1st repeat; 2: the 2nd repeat; 3: the 3rd repeat)

3 3 3

2

2

1 0 1 0 1 2

0

3

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Table 1

Total Flavonoid and Phenolic Contents of A. calamus Rhizome Ethanol Extract

Comp. Sample

Weight Abs

(Y)

Cons(x) Volume Dilution

Contents

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

Phenols 0.1158 0.325 5.5651 5.00 10 2.3984 2398.40

Flavonoids 0.1035 0.274 7.8850 5.00 5 0.1905 190.46

Table 2

Inhibition Zone of The Growth of E. coli, S. aureus and C. albicans of A. calamus Rhizome Ethanol Extract

at Various Concentrations

Treatment Avarage of Inhibition Zone (mm)

(%) E. coli S. aureus C. albicans

0 (negative control) 0a 0a 0a

0.5 0a 0a 0a

1.0 0a 0a 0a

2.0 9.80b 0a 0a

3.0 10.17b 9.50b 8.67b

4.0 11.17bc 11.00c 11.43c

6.0 12.17c 12.33d 13.60d

8.0 15.70d 13.27e 14.10d

10.0 16.60d 13.60e 15.27e

12.0 16.67d 14.53f 15.70e

14.0 16.70d 14.60f 15.73e

16.0 16.73d 14.63f 15.77e

* Values followed by the same letters in the same column are not significantly different according to the

Duncan’s Multiple Range Test at P<5%.

Conclusion The total phenolic and flavonoid contents of A. calamus

rizhome ethanol extract were successively 2398.40 mg

GAE/100g and 190.46 mg QE/100g dry rhizome extract.

The extract possessed the ability to inhibit the three

pathogens. Minimum inhibitory concentration of the extracts

against E. coli, S. aureus and C. albicans was 2, 3 and 3%

respectively with the inhibition zone of 9.80, 9.50 and 8.67

mm. The optimum concentration to inhibit E. coli, S. aureus

and C. albicans was 8, 12 and 10% respectively.

Acknowledgement We thank Udayana University for providing the financial

grant to conduct the research work. We express our gratitude

to the Head of Research and Community Institutions of

Udayana University, facilitating all the needs in the

disbursement of the research funds.

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