Introduction

ISOLATION AND IDENTIFICATION OF ACTIVE COMPOUNDS

TERPENOIDS ON ANTIBACTERIAL HERBA MENIRAN

(phyllanthus niruri linn)

I W. G. Gunawan, I G. A. Gede Bawa, dan N. L. SutrisnayantiJurusan Kimia FMIPA Universitas Udayana, Bukit Jimbaran

ABSTRACT

Isolation and identification of terpenoid, antibacterial compounds meniran

herb (Phyllanthus niruri Linn) by Gas Chromatography – Mass Spectroscophy

were carried out. Two kinds of extraction, i.e. maseration using methanol and the

sochlet using n-hexane were employed.

The extract obtained were contains terpenoids basedon fitochemical test of

Liberman-Burchard n-hexane extract was tested for antimicrobial activity against

Escherichia coli ATCC® 25292 and Staphylococcus aureus ATCC® 25293. In

this study we obtained that n-hexane extract by sochlet extraction showed greater

activity compared to the extract by maseration with methanol, as indiated by disc

diameter of inhibition zone. Diametric inhibition zone for these two extract are 1

mm for Escherichia coli and 0,5 mm for Staphylococcus aureus, for methanol

extract, and where are 10 mm for Escherichia coli and 12 mm for Staphylococcus

aureus for n-hexane extract.

The n-hexane extract was then purified using column chromatography. The

pure extract was analyzed using Gas Chromatography - Mass Spectroscophy. Gas

Chromatography - Mass Spectroscophy data indicated that the extract contains

two compounds, i.e. phytadiene [ M+ ] 278 and 1,2 seco – cladiellan m/z 335

[ M+ - H ].

Keyword : Phyllanthus niruri Linn, terpenoid, active againts bacteria

INTRODUCTION

The development of the use of traditional medicines especially from plants

to help improve the health of the community is quite widespread. One type of

plant that can be used as medicine is meniran (Osward, 1995).

Meniran is a herb that comes from the genus Phyllanthus niruri Linn

Phylanthus scientific name (Heyne, 1987). This herb was traditionally used as a

remedy kidney inflammation, inflammation of the mucous membranes of the

eyes, viral hepatitis, laxative sputum, laxative menstruation, epilepsy, tooth pain,

jaundice, mouth ulcer, antibacterial, cancer, and urinary tract infections (Anonim,

2005; Mangan, 2003).

Herba meniran plavonoid containing of secondary metabolites, terpenoids,

alkaloids and steroids (Kardinan and Kusuma, 2004). Some research indicates

terpenoid compounds have antibacterial activity as the monoterpenoid linalool,

diterpenoid (-) hardwicklic acid, phytol, triterpenoids and triterpenoid saponin

glycosides (Grayson, 2000; Bigham et al., 2003; Lim et al., 2006; Anonymous,

2007 ; Anonymous, 2007).

Based on the above background it is necessary to investigate whether

meniran herb (Phyllanthus niruri Linn) contains terpenoids antibacterial

compounds.

MATERIALS AND METHODS

material

The material used in this study are all part meniran fresh herb (Phyllanthus

niruri Linn) obtained from District Kelod Kerobokan, North Kuta district, Badung

regency, Bali. Meniran dried herbs then blend until powder. The chemicals used

in the study consisted of methanol (pa), acetic acid

anhydride (pa), concentrated H2SO4, chloroform (pa), nheksana (pa),

benzene (pa), KOH 10%, calcium chloride anhydrous, 4 M HCl, potassium

bromide, GF254 silica, silica G60, aquades.

equipment

The tools used in this study include: analytical balance, blender, erlenmeyer

flask, rotary vacuum evaporator, measuring pipette, volumetric flask, funnel,

reagent bottle, filter paper, glassware set, a set of tools thin layer chromatography,

column chromatography, chromatography gas-mass spectroscopy, reflux, sokhlet

and ultra violet light 254 nm and 366 nm.

How it Works

extraction

Terpenoid compounds extraction is done in two ways namely :

1. Sokletasi

As heavy as 1000 g powder of dried herbs meniran disokletasi with 5 L of

solvent n - hexane. Extract n-hexane and saponifiable concentrated in 50 mL

of 10% KOH. N-hexane extracts thickened and tested phytochemical and

antibacterial activity assay.

2. maceration

As heavy as 1000 g powder of dried herbs meniran macerated using methanol

solvent. Concentrated methanol extract and hydrolyzed in 100 ml of HCl 4

M. The results of hydrolysis was extracted with 5 x 50 mL n - hexane. Extract

n-hexane and saponifiable concentrated in 10 mL of 10% KOH. N-hexane

extracts thickened and tested phytochemical and antibacterial activity assay.

Antibacterial activity test

N-hexane extracts tested its activity against the bacteria Escherichia coli and

Staphyloccocus aureus with stages - stages as follows:

1. Taken as a colony of the bacteria Escherichia coli cultures by using a needle

ose performed aseptically.

2. Put into tubes containing 2 ml of Mueller-Hinton broth and then incubated for

24 hours at 35 º C.

3. Homogeneous bacterial suspension was incubated media ready to spread on

the surface of Mueller-Hinton agar, evenly using a sterile cotton stick.

4. Then placed the disk that contains the sample, the standard tetracycline and

solvent (n-hexane) were used as controls.

5. Then incubated for 24 hours at 35 º C.

6. Measurement of the inhibition of the bacterial agent.

7. For Staphylococcus aureus bacterial culture performed in the same manner as

Escherichia coli bacterial culture, but different temperature is at 37 º C.

Extracts were positive and most active antibacterial terpenoids separated by

column chromatography using silica gel 60 stationary phase and mobile phase

chloroform: methanol (3: 7). Fractions obtained from the chromatography column

was tested phytochemical and antibacterial activity assay. Positive fractions most

active antibacterial terpenoids and proceed to step purification using thin layer

kromatograi. Relatively pure isolates further identified using gas chromatography

- mass spectroscopy.

RESULTS AND DISCUSSION

Extracted by means sokletasi and maceration showed that n-hexane extract

both ways are positive for terpenoid compounds. This is evidenced by the

formation of a purple color after nheksana extract reacted with Lieberman

Burchard reagent. The test results of the antibacterial activity of n-hexane extract

sokletasi results provide power resistor greater than n-hexane extract of

maceration results. N-hexane to extract the results sokletasi separated using

column chromatography produced three fractions are presented in Table 1.

Table 1. Group results column chromatography fractions

No faction Number of Noda Rf color Extract 1 A (1-27) 1 0,725 Yellow

2 B (28-33) 2 0,690 and 0,600 Flaxen

3 C (34-) 1 0,580 Flaxen

The test results showed that the phytochemical fractions A and C positive

terpenoid fraction is giving pink (positive diterpenoid) on fractions A and

lavender (positive triterpenoids) in fraction C after reacted with pereksi

Lieberman-Burchard. These results are presented in Table 2.

Table 2. Phytochemical test results respectively - each fraction column

chromatography results

Name faction Color reagent Color reagent Information solution prior solution prior reacted with reacted with Lieberman-Burchard Lieberman-Burchard Fraksi A Flaxen Pink Positive terpenoids

(diterpenoid) Fraksi B Flaxen bluish green Negative terpenoid

(steroid)

Fraksi C Yellow orchid color Positive terpenoid(triterpenoid)

Further terpenoid fractions tested positive antibacterial activity. The test

results antibacterial activity against fraction A and fraction C presented in Table 3.

Table 3. The test results antibacterial activity fraction A and fraction C

N

oN-hexane extracts

Barriers Diameter Each Zone

Bacteria (mm)

Staphyloccocus aureus Escherichia coli ATCC® 25923 ATCC® 25922

1. Control n-hexana 0

1.

2. Aquades 0

2.

3. Standard tetracycline 30 μg 42

3.

4. Faction A 30 μg 19

4.

5. Faction C 30 μg 12

5.

From the test results of the antibacterial activity of fractions of A gives a

better power resistor so that the fraction of A proceeds to the stage of

purification. The results of the purification showed a single stain. It can be

said to be a relatively pure fractions of TLC. Relatively pure isolates were

identified using gas chromatography - mass spectroscopy. Gas

chromatograms of n-hexane fraction positive and active antibacterial

terpenoids shown in Figure 4 which shows the presence of two peaks with

retention times in a row: 25,74 and 21,93 minutes. Based on the above data

compound containing two compounds. Identification of compounds in peak I

tr 25,74 min.

Figure 1 A fraction gas chromatograms

The mass spectrum of peak I compound shown in Figure 2. Based on

spectral data, compound I has a peak molecular weight of m / z 278. Based on the

data base of gas chromatography - mass spectroscopy has shown that compounds

83% similarity with the compound in peak I. Compound is phytol molecular

weight m / z 296 [M +], the mass spectrum shown in Figure 2 and the structure

shown in Figure 3. Phytol can dehydrate naturally become phytadiene in group B

of Botryococcus braunii Botryococcus braunii which is a species of green algae

(Zang and Sachs, 2006; Fukushima et al., 1992; Grossi et al., 1996). Phytadiene

mass spectroscopic data of the m / z 278 [M +], 263, 179, 123, 109, 95, 82, 68, 57

(Nguyen et al., 2002). Phytadiene mass spectrum resembles the mass spectra of

compounds I peak at m / z 278 [M +]. In the mass spectrum base peak dodekane

contained m / z 57 is flanked by other high peaks are the peak m / z 43 and m / z

71 (Baker, 2000) which is the typical peak dodekane. I peak in the mass spectrum

contained peaks at m / z 71 as the base peak, and there are also other typical peaks

of dodekane the peak m / z 43 and m / z 57 with a fairly high abundance. This

means I have a group of compounds such as peak dodekane. Dodekane has 20 C

atoms and a double bond (Baker, 2000), it is also seen in phytadiene structure that

is composed of 20 C atoms and two double bonds are shown in Figure 4. Once

fragmented, the structure follows the pattern of fragmentation phytadiene

compounds in peak I. Phytadiene compound fragmentation pattern shown in

Table 4 and Figure 5. Thus the compound at the height of I m / z 278 compounds

suspected Mass Spectroscopy phytadiene based on the data, and the fragmentation

pattern of the relationship between peak I compound with phytol.

Ho

Figure 3 Structure of compound phytol

Figure 4 Structure of compound phytadiene

Table 4. Pattern beheading mass spectra of compounds in peak I

The spectrum of the compound in peak II shown in Figure 6. From the

spectral data, peak II compound has a molecular weight of m / z 335. Based on

internet search results, there are several pieces of the compound with m / z 335 of

them Leucyl-glycyl-DL-DLphenylalanine, 4-methoxy-4-methyl-1-(4-

Nitrophenyl) - Decane-1 ,3-dione, 2 - {1 - [2 - (3,4 - dimethoxyanilino)-2-

oxoethyl} Cyclohexyl} acetic acid, 2 - (acetylamino) -3 - {3 -

(cyclopentylmethoxy) -2 - Methoxyphenyl} propanoic acid. These compounds do

have a molecular weight of m / z 335 corresponding to the m / z peak II

compound but the fragmentation pattern of these compounds do not meet the

fragmentation pattern of the compound in peak II. Therefore traced compound

having a molecular weight of m / z 336 which has a fragmentation pattern that

meets the fragmentation pattern of the compound peak II with the assumption that

compounds with a molecular weight of m / z 336 is a compound that has a

molecular weight of m / z 335 [M + - H] .

Based on data from internet search results, there is a structure of compounds

having a molecular weight m / z 336 and a fragmentation pattern with groups that

meet cluster and fragmentation patterns of compounds in peak II. These

compounds are 1,2-seco-cladiellan (Friedal et al., 2005), the structure shown in

Figure 7. The compound 1,2-seco-cladiellan formed from karvon (Friedal et al.,

2005) which is a compound class karvon group-containing monoterpenoid

ketones (Fish, 1976). The presence of a ketone group at a mass spectrum of a

compound seen in the peak m / z 55 and the breakdown that occurs in the C - C

next to the oxygen atom (Silverstain et al., 1986). At the peak of compound II

seen the peak of m / z 55 and solving the C - C next to the oxygen atoms can be

seen at m / z 292 (M + - H - 43) a Loss of C3H7 molecules. Based on the above

data drawn a conclusion that peak II compound suspected compound 1,2-seco-

cladiellan, because the structure of these compounds meet the fragmentation

pattern of the compound peak II.

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