ANTIMICROBIAL AND IDENTIFICATION OF ACTIVE COMPOUND Curcuma xanthorrhiza Roxb

International Journal of Basic & Applied Sciences IJBAS-IJENS Vol: 12 No: 01 69 ANTIMICROBIAL AND IDENTIFICATION OF ACTIVE COMPOUND Curcuma xanthor...
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International Journal of Basic & Applied Sciences IJBAS-IJENS Vol: 12 No: 01

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ANTIMICROBIAL AND IDENTIFICATION OF ACTIVE COMPOUND

Curcuma xanthorrhiza Roxb. Wibowo Mangunwardoyo1), Deasywaty2) , Tepy Usia2) 1)Department of Biology, Faculty of Mathematic and Sciences, University of Indonesia, Depok, 16424. Indonesia. 2) Center of Food and Drug Research, National Agency of Drug and Food Control, Jalan Percetakan Negara 23, Jakarta, Indonesia. Correspondence: [email protected] ; [email protected] Abstract The ethanol 70% extract of Curcuma xanthorrhiza Roxb inhibited the growth of Gram positive bacteria S. aureus and S. mutans in a concentration of 1.0-5.0% (w/v), while B. cereus in a concentration of 2.0-5.0% (w/v). The Minimum Inhibitory Concentration (MIC) of ethanol 70% extract toward S. aureus and S. mutans were 0.1% (w/v), while against B. cereus it showed 2.0% (w/v). Phytochemistry analysis of ethanol 70% extract consists of alkaloid, quinone, and terpenoids. Thin Layer Chromatography (TLC) analysis showed five spots, the third spot effectively inhibited Staphylococcus aureus ATCC 25923, Streptococcus mutans type F (MUI) and Bacillus cereus ATCC 11778 and contain terpenoids. Analysis UV-Vis and Infra Red spectrophotometry showed that it contains phenolic group at absorbance 275.2 nm and have hydroxyl (-OH) and carbonyl (C-O) functional groups. The result of GC-MS indicated that the extract which contains one compound is xanthorrhizol m/z 218.

Index term: antimicrobial; Curcuma xanthorrhiza Roxb.; GC-MS; TLC; UV-Vis and infra-red spectrophotometry.

I.INTRODUCTION Curcuma xanthorrhiza Roxb is one of many medicinal plants that had been used for generations in Indonesia [1]-[2]-[3]. Most people use the rhizome of this plant as they believe it has medicinal effect. The rhizomes of Curcuma xanthorrhiza Roxb contain volatile oil, saponin, flavonoid and tannin [1]-[3]-[4] . Chemistry analysis showed that the main substances of Curcuma xanthorrhiza Roxb are starch (48.18-59.64%), fiber (2.58-4.83%), volatile oil such as, phelandren, camphor, tumerol, sineol, borneol, and xanthorrhizol (1.481.63%), and also curcuminoid like, curcumine and desmetoxicurcumine (1.6-2.2%) [1]-[3] . Some research about the chemical substances and the benefit of Curcuma xanthorrhiza Roxb had been conducted. One of those studies found that the extract of Curcuma xanthorrhiza Roxb in ethanol 96% could inhibit the growth of Staphylococcus aureus and Staphylococcus epidermidis as the cause of pimples [5]. Xanthorrhizol isolation from Curcuma xanthorrhiza Roxb methanol extract could inhibit the growth of bacteria Streptococcus mutans [6,7]. Through many studies, another benefit of Curcuma xanthorrhiza Roxb as antimicroba has also been found [1]-[6]-[8]-[9]. The volatile oil and curcuminoid are the main substances with antimicrobial effect [1]-[2]-[6][9]. Analysis method using thin layer chromatography (TLC) has been done by some researcher include. The detachment using chloroform: methanol (9:1) in seven ethanol extract and three extract of root, stem and leaves of Accacia (Acacia aroma Gill.) [10]. Mobile phase chloroform: methanol (99:1) was used to detach the ethanol extract of gambier leaves (Uncaria gambir) [11]. The TLC method also has been used by [12] with solution of n-hexan: ethyl acetate (10:1) to isolate xanthorhizol from curcuma rhizomes. Spectrophotometry method like, UV-Vis, Infra Red dan GC-MS have been used to identified natural ingredients by some researchers, include [13] who identified triterpenoid group in the rhizomes of Curcuma zedoaria (Berg.) Roscoe. Reference [12] identified xanthorrhizol in it. The purpose of research is to find out the antimicrobial effect of Curcuma xanthorrhiza Roxb. and to identify the compound class in the extract of ethanol 70% curcuma rhizomes. The findings could support the development of curcuma as antimicrobial element to enrich the traditional medicine in Indonesia.

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II. MATERIAL AND METHOD A.Plant materials The sample for this research is dried simplicias of Curcuma xanthorrhiza Roxb rhizomes from P.T. Vitaher, Semarang. The plants were cultivated in a high altitute about 75-100 m and were harvested after 10 months. The simplicias were dried by oven with the temperature started from 50-55º C for 7 hours.

B.Microorganisms Staphylococus aureus ATCC 25923, Bacillus cereus ATCC 11778, anaerob Streptococcus mutans type F (MUI), Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, anaerob Porphyromonas gingivalis ATCC 33277 and Candida albicans ATCC 10231. These microbes were supplied by Microbiology Laboratory, Control Laboratory of Drug and Food, NA-DFC (Badan POM) and Microbiology Laboratory, Faculty of Dentistry, University of Indonesia.

C.Media Brucella Broth was used as a medium for Porphyromonas gingivalis while TSB was used for growing Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, also Escherichia coli, and BHIB medium + yeast extract were used for growing Streptococcus mutans.

D. Extractions An amount of 100 g powder of Curcuma xanthorrhiza Roxb was added by aquadest till it reached 500 ml then boiled for 20 minutes [14] and was filtered by glass wool. Thick extract of aquadest was acquired by evaporating in a waterbath. The filtered residue was reflucted by ethanol 70% within one hour and then was filtered by glass wool. The ethanol was eliminated by evaporating with vacuum evaporator at 40º C. The residue of ethanol was being extracted back with dichloromethane maceratively with shaker at 120 rpm for 24 hours. The extract of dichloromethane was evaporated by vacuum evaporator at 40º C until thick extract of dichloromethane was acquired [13]-[15].

E.Antimicrobial and antifungal assay broth dilution methods The extract was dissolved by aquadest in various concentrations of 50%, 40%, 30%, 20% and 10% (b/v). Every single tube was filled by the mixture of medium and extract in various concentrations. Then, the bacterias were inoculated into the appropriate medium for about 200 µl with Mc Farland turbidity of 0.5 and then were incubated at 35-37º C for 18-24 hours. The anaerob bacterias were incubated using anaerobic jar [16][17].

F. Minimum Inhibitory Concentration Identification (MIC) This study used broth dilution method. The extract was dissolved until it reached concentration of 0.025%; 0.05%; 0.10%; 0.25%; 0.50% and 0.75% (b/v) The test was repeated for 3 times [16]-[17].

G.Identification Chemical Composition Using Thin Layer Chromatography (TLC) [18] Ethanol 70% extract was identified using TLC, silica gel 60F254 (20x20 cm) with 0.25 mm thickness. As the developer solvent was n-hexan: ethyl acetate (14:1). The eluent distance was 15 cm from the spotted starting point. The developer solvent was put into chromatography vessel, and let it till it became saturated. Then, an amount of 10 µl extract solution was used to spot silica gel plate. Stain observation has been done under UV light 254 nm to see fluorecensy and reactant CeSO4 spraying. Every spoted was scraped for being tested for its activity against microbes.

H. TLC Results identification using spectrophotometry UV-Vis The TLC scraped with Rf value showing antimicrobial activity then were put into chromatography column in the form of pipette, eluted with ethanol 70% and accumulated in vials. The results were measured by spectrophotometer UV-Vis [19]-[20].

I. TLC Results identification using spectrophotometry Infra Red An amount of 1 mg chromatographed scraped that has been eluted with ethanol 70% and heated by waterbath was made into a thin disc with 100 mg Potasium Bromide addition. This thin disc was observed by Infra Red spectrophotometer [21]-[22].

J.TLC Results Identification using spectroscopy GC-MS The TLC scraped with Rf value showing antimicrobial activity then were put into chromatography column in the form of pipette, eluted with ethanol 70% and accumulated in vials. The results were identified using spectroscopy GC-MS to find out the compound class and its molecular weight [12]-[13]-[23].

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III. RESULTS AND DISCUSIONS A.Rhizome extraction (Curcuma xanthorrhiza Roxb.) The result of extraction 100 g powder of Curcuma xanthorrhiza Roxb with aquadest, ethanol 70% dan dichloromethane can be seen in the Table I. Table I. Yield from extraction of 100 g powder of Curcuma xanthorrhiza Roxb. Solvent Weight of yield (g) Characteristic of yield Aquadest

63.54 g

Yellow brownish, condensed

Ethanol 70%

13.33 g

Brown, condensed

Dichloromethane

3.01 g

Yellow brownish, condensed

The yield from aquadest has the highest result. This result could be because there are starch and phenol compounds in the extraction and also because aquadest is a polar solvent that could dissolve starch and phenol [18]. References [24] and [25] reported that the extraction in aquadest has second metabolite substances which are, alkaloid, saponin and quinone. Starch is the main component of Curcuma xanthorrhiza Roxb about 48.1859.64% in composition [1]-[2]. The high percentage of starch is influenced by the altitude where it cultivated. When it planted below 240 m, it will contain high percentage of starch[12]. Extraction with ethanol 70% was conducted since ethanol 70% is a versatile solvent for preliminary extraction and it has universal charactheristic [18] so it can attract polar compounds in the rhizomes especially alkaloid [4]-[26] curcuminoid and terpenoid [18] and also non polar compounds [27]. Another component as a result of the extraction using ethanol is phenol compound [25]-[26]-[28]-[29]. Extraction of the rhizomes with dichloromethane has the least amount of yield, since dichloromethane is a semi polar solvent. As a result, the extraction could only attract some of semi polar and non polar compounds such as, some of flavonoids [30], triterpenoid [22]-[25] alkaloid [30] and saponin[25].

B.Antimicrobial activity rhizome (Curcuma xanthorrhiza Roxb.) Rhizome extractions could inhibit the growth of all Gram positive tested as shown in the Table II. Table II. Antimicrobal activities in the extract of Curcuma xanthorrhiza Roxb in aquadest, ethanol 70%, and dichloromethane against Gram positive bacteria. Inhibitory capacity Microbes Concentration % (b/v) Aquadest Ethanol 70% Dichlorome5.0 4.0 3.0 2.0 1.0 5.0 Bacillus cereus 4.0 ATCC11778 3.0 2.0 1.0 5.0 Streptococcus mutans 4.0 Type F (MUI) 3.0 2.0 1.0 Note: +: the extract could inhibit the growth of bacteria - : the extract could not inhibit the growth of bacteria Staphylococcus aureus ATCC 25923

extract

extract

thane extract

+ + + + + + + + + +

+ + + + + + + + + + + + + +

+ + + + + + + + + +

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The inhibitory process against Gram positive bacteria is possibly caused by the phenol compound in the extraction that inhibites the growth of the microbes. High phenol penetration into the cell could cause protein coagulation and cell membrane lysis [28]. The phenol inhibitory mechanism is by hydrogen binding between hydroxyl group in the phenol compound and cell membrane protein, which cause membrane permeability imbalance. This mechanism makes the essensial components of the cell leak out of the cell and cause cell death [28]-[29]-[31] . Simple cell wall construction with no outer layer makes antibacterial compound to permeate the cell wall and cause cell wall biosynthesis disturbance [5]- [25]- [32]-[33]. The extraction in aquadest, ethanol 70%, and dichloromethane could not inhibit the growth of Gram negative bacteria like, E. coli, P. aeruginosa, P. gingivalis and fungi C. albicans (Table III). This could possibly happen because the extract concentration could not penetrate the cell wall of Gram negative bacterias and fungi. The cell wall of Gram negative bacterias consist of more complex composition than the one in Gram positive bacterias. Not only peptidoglycan, but Gram negative bacterias also have another outer layer that consists of lipopolysacharide, lypoprotein, and periplasm which is binded to peptidoglycan [25]-[34] Lipopolysacharide in the outer layer has a function as a defence system with peptidoglycan and select foreign substances. Lipoprotein contains hydrophilic protein called porin. There is a big possibility that because of porin in the outer membrane of Gram negative bacterias, the extraction could not permeate into the cell since the extraction is hydrophobic [25]-[34]. Table III. Antimicrobal activity in the extract of Curcuma xanthorrhiza Roxb in aquadest, ethanol 70%, and dichloromethaneagainst Gram negative bacteria and fungi. Inhibitory capacity Microbes Concentration % (b/v) Aquadest Ethanol 70% Dichlorome

Escherichia coli ATCC 25922

Pseudomonas aeruginosa ATCC 27853

Porphyromonas gingivalis ATCC 33277

Candida albicans ATCC 10231

extract

extract

thane extract

5.0 4.0 3.0 2.0 1.0 5.0

-

-

-

4.0 3.0 2.0 1.0 5.0 4.0 3.0 2.0 1.0 5.0 4.0 3.0 2.0 1.0

-

-

-

Note: (-): the extract could not inhibit the growth of bacteria The cell wall of Gram negative bacterias has a high concentration of lipid that makes these bacterias more resistant against chemical compound and impermeable with limited diffusion [35]. Gram negative bacterias have more complex cell wall composition and it has non polar characteristic so all those three extracts which contain polar to semi polar compound have more difficulty in penetrating the bacterias cell wall [28]. This also has been reported by references [15]-[36] and reference [25] mentioned that Gram negative bacterias have resistency capacity against antimicrobes more than Gram positive bacteria. All those three extracts were tested against C. albicans showed that all of them could not inhibit the growth of C. albicans. This is possibly because there is not any inhibitory process since chemical substance within the extracts could not penetrate the sterol layer in the cell wall to cease the stiffness of chitine synthesis on cell wall [37]-[38] . Reference [39] has quoted that the fungi cell wall has a stiff characteristic and consists of chitine, glucan dan mannan, and mainly contain 80-90% polysaccharide. Reference [36] reported that 90%

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ethanol extract of Sida acuta Burm. also could not inhibit the growth of C. albicans. Also [34] reported that berry extract in acetone water solvent (70:30) also could not inhibit C. albicans growth.

C. Minimum Inhibitory Concentration (MIC) Minimum inhibitory concentration has been done with the extract concentration of 0.025%; 0.05%; 0.10%; 0.25%; 0.50%; and 0.75% (b/v) (Table IV). Table IV. The result of Minimum Inhibitory Concentration (MIC) of ethanol 70% extractagainst Staphylococcus aureus ATCC25923 and Streptococcus mutans Type F (MUI) Minimum Inhibitory Concentration (MIC) Concentration Staphylococcus aureus Streptococcus mutans (%) ATCC 25923 Type F (MUI) 0.025 0.05 0.10 + + 0.25 + + 0.50 + + 0.75 + + Note: + : Curcuma extract could inhibit the growth of tested microbes - : Curcuma extract could not inhibit the growth of tested microbes Ethanol 70% extract could inhibit the growth of S. aureus dan S. mutans in 0.10-0.75% (b/v) concentration. Antimicrobial activity was affected by the concentration of extract. The higher the concentration, the higher the inhibition capability will be. Reference [5] has reported that 95% Curcuma ethanol extract is 0.4% to S. aureus. Research of [23] on Cinnamomum iners leaves in methanol extract showed that it contains xanthorrhizol with MIC about 0.78 mg/ml to S. aureus. Also, reference [7] has reported that the isolated xanhorrhizol from Curcuma methanol extract (Curcuma xanthorrhiza Roxb.) could inhibit S. mutans in 5.0 µMol/l concentration.

D. Phytochemical identification of rhizome. Classes of compound identification in ethanol 70% extract (Table V) Table V. Phytochemical analysis result of 70% ethanol extract (Curcuma xanthorrhiza Roxb.) No. Classes of compound Result Characteristic 1 Alkaloid + Bouchardat: brown sediment, Mayer: -Dragendorf: brick red sediment 2 Flavonoid 3 Saponine 4 Tanine 5 Quinone + NaOH: red 6 Terpenoid/Steroid + Lieberman-Bouchardat: purple-violet 7 Glikoside Class of compound identification resulting negative on flavonoid identification because of the absent of red colour, saponine was not foaming around 1-10 cm after 10 minute, tannine was not turned into blue, and glycoside was not changed from red to blue or purple after the addition of Keller Killiani reagent [18]-[21]-[39].

E. Thin Layer Chromatography analysis. The result of TLC ethanol 70% extract of Curcuma on 60F254 plate with n-hexan: ethyl acetate (14:1) developer, were 5 spots with variable Rf value (Table VI).

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Table VI. The result of TLC ethanol 70% extract of curcuma with n-hexan : ethyl acetate (14:1) developer. Silica gel 60F254 Colour Rf 254 nm CeSO4 Yellowish brown Yellowish brown 0.16 Light purple Brown 0.26 Light purple Brown 0.64 Dark purple Brown-purple 0.73 Dark purple Brown 0.86 Light purple Brown

According reference [40] Curcuma longa L. methanol extract with mobile phase chloroform: ethanol: acid acetate (48:2:0,1) has Rf value of 0.4; 0.23 and 0.19. Ethanol 70% extract of curcuma is assumed to have some terpenoid compounds, such as xanhorrhizol. This is supported by the existence of purple after TLC plate sprayed by H2SO4 reagent. According to reference [18] the terpenoid compounds on TLC plate that was sprayed with reagent will form purple colour. Reference [12] was succeed in get Rf value from curcuma rhizome extraction with ethanol 96% solvent and developer solvent n-hexan: ethyl acetate (10:1) on Rf 0.56 and 0.86. Reference[41] said that xanthorrhizol Rf value is 0.58. Xanthorrhizol is the main antimicrobe chemical compound in curcuma [1]-[6]-[7]-[23]-[42].

F.Antimicrobial activity and phytochemical analysis results of TLC Antimicrobial activity test from TLC ethanol 70% extract curcuma by dilution tube method on Gram positive Staphylococcus aureus, Streptococcus mutans, and Bacillus cereus inhibit on the third spot with 0.64 Rf value. The third spot on CeSO4 reagant showed terpenoid compound assumed by the purple colour. The purple thin layer of chromatography with reagent is terpenoid [18]. Reference [12] has shown that xanthorrhizol TLC from curcuma methanol extract with n-hexan: ethyl acetate gave spot on Rf 0.54 and 0.86. The absence of microbe inhibition on fraction 1, 2, 4, and 5 were assumed because the fraction concentration of chromatography is too small so that the compound in the extract could not inhibit the bacteria growth. TLC scrape confirmation by alkaloid, quinone, and terpenoid reagent showed that Rf 0.64 contain terpenoid compound after being tested by Lieberman and Bouchard reagent. It gave a positive result with purple violet formation. A test using alkaloid and quinone reagent showed negative in result [18]-[21]-[39].

G. Qualitative Identification using UV-Vis, Infra Red dan GC-MS Spectrophotometry Analysis result of active compound identification using spectrophotometry UV-Vis (Fig 1).

275.2 nm

Fig 1. UV-Vis Spectrophotometry chromatogram profile Identification using UV-Vis Spectrophotometry showed that the peak absorbent wave is 275.20 nm it means that there is the electron transition from phenol compounds. Reference [43] mention that the maximum of phenol group absorbent is in 210 – 280 nm, with electron transition of aromatic group π → π*. Reference

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[23] said that result that maximum absorption of Cinnamonum iners extract with Chloroform solvent is 276.0 nm, by phenol group indication. The maximum absorption ethanol extract of rhizome (Curcuma zadoria (Berg.) Roscoe) is 242 nm [13]. Active compound identification of ethanol 70% curcuma extract by infra red Spectrophotometry test is shown on Fig 2 and Table VII.

O-H C-O

Fig 2. Infra red spectrophotometry profile Table VII. Infra Red wave numbers of ethanol 70% curcuma extract (Curcuma xanthorrhiza Roxb) Wave number (cm-1) Bonds Silverstein et al. (1963) Ethanol 70% extract Rf 0.64 C-C, C-O, C-N 1300 - 800 1100.41 C=C, C=O, C=N, N=O 1900 - 1500 -C=C, C=N 2300 - 2000 -C-H, O-H, N-H 3800 - 2700 3387.06 Based on Fig 2 and Table VII we can conclude that fraction with Rf value 0.64 contains compound with hydroxyl functional group (-OH) with wave number 3387.06 cm -1, supported by strong absorption on wave number 1100.41 cm-1 from C-O alcohol. Hydroxyl group (-OH) is on wave number 3400-2700 cm-1 [13][43]. Reference [44] said that phenol group which has –OH dan C-O group strong absorption, has absorption level between 3550-3200 cm-1 and C-O on wave number 1260 – 1000 cm-1. Reference [23] identified xanthorrhizol in Cinnamomum iners leaves extract with wave number 3382.8 cm-1 for –OH group.Reference [12] also identified xanthorrhizol in curcuma with wave number 3400 cm-1 for hydroxyl group. GC-MS test on 70% curcuma rhizome ethanol extract with Rf value 0.64 resulted in peak mass spectrum as shown on Fig 3.

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Fig 3. Mass spectrum of curcuma 70% ethanol extract (Curcuma xanthorrhiza Roxb.) Based on the spectrum data, the compound on ethanol 70% extract is stated to have molecule weight 218 g/mol, and on library GC-MS data is shown to have a 99% identical characteristic with compound peak of the ethanol 70% extract which is xanthorrhizol with 218 g/mol. Reference [41] - [45] reported that the molecular weight of xanthorrhizol is 218 g/mol.

IV. CONCLUSION The extraction result of 100 g rhizome powder of Curcuma xanthoriza Roxb. with ethanol 70% aquadest solvent and dichloromethane resulting in yield with weight of 63.54 g, 13.33 g, 3.01 g. Curcuma rhizome extract effective in inhibiting Staphylococcus aureus ATCC 25923 and Streptococcus mutans type F (MUI) positive Gram bacteria growth, while Bacillus cereus can only be inhibited by ethanol 70%. Gram negative bacteria consist of Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Porphyromonas gingivalis ATCC33277 and Candida albicans ATCC 10231 fungi could not be inhibited by those three extractions. Ethanol 70% extract of curcuma rhizome effectively inhibited S. aureus and S. mutans on 1.0 – 5.0% (b/v) concentration, while B. cereus on 2.0 – 5.0% (b/v), with 0.1% (b/v) Minimum Inhibitory Concentration (MIC) to S. aureus and S. mutan. UV-Vis and Infra Red spectrophotometry analysis showed contains phenolic group at absorbance 275.2 nm and have hydroxyl (-OH) and carbonyl (C-O) functional groups. Result of GC-MS indicated that the extract contains one compound which is xanthorrhizol m/z 218. Further research on determination of molecular structure and purification of the compound should be carried out.

REFERENCES [1] E. Afifah. “Khasiat dan manfaat temulawak, rimpang penyembuh aneka penyakit”. Agromedika, Pustaka. pp. 1-19. 2005. [2] BPOM (Badan Pengawas Obat dan Makanan). “Gerakan Nasional Minum Temulawak”. Info POM vol 6, no 6, pp 1-4. 2005. [3] M.H. Siagian. “Temulawak sebagai tanaman obat dan budidayanya secara intensif”. Balitbang Botani, Puslitbang Biologi LIPI, Bogor. pp. 1-8 2006.

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