Indones. J. Chem.

, 2015, 15 (2), 179 - 186 179

TWO ISOPHALERIN COMPOUNDS FROM ETHYL ACETATE OF LEAVE AND FRUIT

OF MAHKOTA DEWA (Phaleria macrocarpa (Scheff.) Boerl.)
AND ITS ANTIBACTERIAL ACTIVITY
Susilawati1,*, Sabirin Matsjeh2, Harno Dwi Pranowo2, and Chairil Anwar2
1
Department of Education of Mathematics and Natural Sciences, FKIP, Universitas Riau, Pekanbaru, Indonesia
2
Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Gadjah Mada,
Sekip Utara PO BOX BLS 21 Yogyakarta 55281, Indonesia

Received December 17, 2014; Accepted March 3, 2015

ABSTRACT

Mahkota dewa plant (Phaleria macrocarpa (Scheff.) Boerl.) which is belong to family of Thymelaeaceae is one
of Indonesia's traditional medicines. The aim of this research is to isolate secondary metabolites from ethyl acetate
extract of leave and fruit of mahkota dewa and to determine the molecular structure of isolated compounds using
spectroscopic method and to know the antibacterial activity of the isolated compound. Sample was extracted with
methanol, concentrated then extracted by n-hexane, chloroform and ethyl acetate. The compounds were separated
and purified with column chromatography. The compound 1 was isolated from ethyl acetate extract of leave as white
needle amorphous solid as 45 mg. The compound was identified by spectroscopic as 4,6-dihydroxy-4’-
methoxybenzophenon-2-O-β-D-glucopyranoside and named isophalerin B. From the test results of antibacterial
activity showed that the compound 1 (10 mg/mL) in ethanol has a weak activity against the bacteria S. aureus and E.
coli. The compound 2 was isolated from ethyl acetate extract of fruit as peach needle crystal as 10 mg. The
compound was identified by spectroscopic as 4,6-dihydroxy-4’-methoxybenzophenon-2-O-α-D-glucopyranoside and
named isophalerin A.

Keywords: mahkota dewa; Phaleria macrocarpa; isophalerin; antibacterial; benzophenon

ABSTRAK

Tumbuhan Mahkota dewa (Phaleria macrocarpa (Scheff.) Boerl.) yang termasuk dalam famili Thymelaeaceae
adalah salah satu tumbuhan obat tradisional Indonesia. Tujuan penelitian ini adalah untuk mengisolasi metabolit
sekunder dari ekstrak etil asetat daun dan buah mahkota dewa dan untuk menentukan struktur molekul senyawa
hasil isolasi menggunakan metode spektroskopi serta untuk mengetahui aktivitas antibakteri dari senyawa hasil
isolasi. Sampel diekstrak dengan metanol, dipekatkan kemudian diekstrak dengan n-heksana, kloroform dan etil
asetat. Senyawa dipisahkan dan dimurnikan dengan kromatografi kolom. Senyawa 1 diisolasi dari ekstrak etil asetat
daun sebagai padatan amorf jarum putih sebanyak 45 mg. Senyawa diidentifikasi dengan spektroskopi sebagai
4,6-dihidroksi-4’-metoksibenzofenon-2-O-β-D-glukopiranosida dan dinamai isofalerin B. Hasil test aktivitas
antibakteri menunjukkan bahwa senyawa hasil 1 (10 mg/mL) dalam etanol mempunyai aktivitas lemah melawan
bakteri Staphylococcus aureus dan Eschericia coli. Senyawa 2 diisolasi dari ekstrak etil asetat buah sebagai kristal
jarum krem sebanyak 10 mg. Senyawa diidentifikasi dengan spektroskopi sebagai 4,6-dihidroksi-4’-
metoksibenzofenon-2-O-α-D-glukopiranosida dan dinamakan isofalerin A.

Kata Kunci: mahkota dewa; Phaleria macrocarpa; isofalerin; antibakteri; benzofenon

INTRODUCTION makutadewa, makuto rajo, makuto ratu or makuto

mewo [1].
Mahkota dewa plant (Phaleria macrocarpa Mahkota dewa is most frequently and empirically
(Scheff.) Boerl.) which is belong to family of utilized by Indonesian various diseases treatment with
Thymelaeaceae, is widely found in Indonesia. This plant satisfactory results [2]. Mahkota dewa is one of
has synonym of Phaleria papuana var warb wichnanmi Indonesia's traditional medicines which not yet owned
(val) Back. In English, it is known as crown of God. The enough information reference in order to be applied
name of this plant in Sumatra (Malay) and Depok (West optimally. The research on mahkota dewa has been
Java) is simalakama. In Java, it is also called as much conducted, mostly about the bioactivity tests
(antimicrobial, brine shrimp, cytotoxic, pharmacological
* Corresponding author. Tel/Fax : +62-81268731997
Email address : [email protected]

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180 Indones. J. Chem., 2015, 15 (2), 179 - 186

Fig 1. Structure of benzophenone glucopyranosides and benzophenone aglucon were isolated from mahkota dewa

and antioxidant activity) of the extract or fraction of seed on Fig. 1. Benzophenone glucoside has been isolated
and fruit [3]. from Gnidia invoclurata which also included into the
Mahkota dewa used in treating diseases, among family Thymelaeaceae (A) [12].
others: pain heart kidney, hypertension, heart disease, This paper reported the isolation of phenolic
diabetes, rheumatism, arthritis and bacterial infectious compounds from the ethyl acetate extract of leave and
disease such acne, eczema secondary infections, fruit of mahkota dewa and its antibacterial activity.
dysentery, cough and fever. Leaves of mahkota dewa Structural elucidation of isolated compounds was
effective as analgesic, antibacterial and antihistamines. performed by means of spectroscopy analyses. Test of
Extract of mahkota dewa leaves by tube dilution method antibacterial activity employed in this research was
has antibacterial activity against Staphylococcus aureus diffusion method.
[4]. In this research, It has been conducted antibacterial
activity assay of isolated compound from ethyl acetate EXPERIMENTAL SECTION
extract of mahkota dewa leaves by agar diffusion
method against Staphyllococcus aureus and Escheria Materials
coli.
Chemical contents of mahkota dewa fruit were Leave and fruit of mahkota dewa was collected
benzophenone glucoside (6,4'-dihydroxy-4-methoxy from campus of Universitas Gadjah Mada (UGM),
benzophenone-2-O-β-D-glucopiranoside) (A) from Yogyakarta Indonesia in January 2009. The plant was
chloroform extract of ripe fruit [5], from ethyl acetate identified by Plant Taxonomy Laboratory, Faculty of
extract of red fruit, from n-butanol extract of the fruit of Biology, UGM. Chemicals used were consisted of
mahkota dewa [6-7]. Compound (A) was also isolated methanol (technical, distilled) and pa (Merck),
from ethyl acetate extract of stem bark of mahkota dewa n-hexane (technical, distilled), ethyl acetate (technical,
[8]. distilled), chloroform pa (Merck), acetone pa (Merck),
Phalerin (4,5-dihydroxy-4'-methoxybenzophenone- ethanol pa (Merck), FeCl3 (Merck), Mg powder (Merck),
3-O-β-D-glucopiranoside) (B) from the methanol extract HCl (Merck), KOH (Merck), NH4OH (Merck),
of leave of mahkota dewa was obtained. This compound dragendorff, acetic anhydride (Merck), sulfuric acid
had LC50 values of small brine shrimp test. Phalerin (Merck), ethanol 70%, Nutrient Agar (NA) media
displayed also cytotoxic activity in uterine cells NS-1 [9]. 20 g/L, physiologist NaCl, bacteria of gram positive
From the seed of mahkota dewa, mahkoside A (phenolic S. aureus and bacteria of gram negative E. coli.
glycosides new) (C) has been isolated [10].
Benzophenone aglycon of compound (A) (2,6,4’- Instrumentation
trihydroxy-4-methoxy benzophenone (D) has been
isolated from ethyl acetate extract of leave of mahkota Melting point apparatus (Electrothermal 9100),
dewa [11]. Structure of benzophenone glucopyranosides UV-vis spectrophotometer UV-vis (Spectronic 3000),
and benzophenone aglycon from mahkota dewa is given Fourier Transformation-Infra Red (FT-IR) spectrophoto

Susilawati et al.
 Indones. J. Chem., 2015, 15 (2), 179 - 186 181

meter (Shimadzu IRPrestige-21). Nuclear Magnetic Escherichia coli. Activity of isolated compound and
1 13
Resonance, H-NMR and C-NMR (JEOL JNM ECA- extract was tested separately using agar diffusion
1
500 spectrometer), operating at 500 MHz ( H-NMR) and method [13]. All the tools, the media were sterilized in
13
125 MHz ( C-NMR), using Tetramethyl silane (TMS) as an autoclave at a temperature of 121 °C, pressure
an internal standard. Column chromatography was 1 atm, for 15 min. Test bacteria were rejuvenated from
carried out using Merck silica gel 60 (70-230 mesh stock pure cultures by growing on italics NA agar and
ASTM). Thin Layer Chromatography (TLC) analysis was then incubated for 18-24 h at 37 °C. Preparation of test
conducted on precoated Silica gel plates (Merck silica bacterial suspension on adolescent culture stock by
gel GF 254), Autoclave Omron Auto type STMN-Y222, taking it with the needle of ose and suspended in
Petri dish, test tubes, beaker glass, discs of paper, physiological NaCl solution and then vortexed until
tweezers, needle ose, aseptic cabinet, magnetic stirrer, homogeneous. Then the turbidity is measured to obtain
spectronic 21. 25% T at 580 nm for bacteria with spectronic-21.
Concentration of the isolated compound was 10 mg/mL
Procedure ethanol, while ciprofloxacin was 1 mg/mL. Each test
bacteria (S. aureus, E. coli) was taken 0.1 mL by
Extraction, isolation and identification pipette of cooked, placed in the middle petri dish. Then
The dried leave (0.5 kg) of mahkota dewa was the agar media that was still liquid (50-55 °C) poured
extracted using macerator (drip pan) with methanol into petri dish as 15 mL, shaken until a homogeneous
(6.5 L) by heating (60 °C) for 7 h then stand up overnight mix with the test bacterial suspension and then allowed
at room temperature. The residue was macerated again to solidification. Then 10 L solution of the isolated
for 3 times and all the filtrates were combined (22 L) and compound (10 mg/mL) was placed on paper discs
concentrated using vacuum distillation and rotary using a micro pipette and grown in media seeding.
evaporator. The methanol extract was partitioned with Incubation for 24 h at 37 °C in an incubator in the
n-hexane (6 x 320 mL) to give n-hexane extract. inverted position. The reading of a positive result if
Residue of extract methanol was partitioned with around the paper disc there are areas transparent
chloroform (10 x 640 mL) to give chloroform extract. without growth of bacteria. The inhibition area
Residue was partitioned again with ethyl acetate measured with a shove. As a control used sterile paper
(13 x 200 mL) to give ethyl acetate extract (25 g). Ethyl disc that has been poured solvent [14].
acetate extract (18 g) was then separated by column
chromatography on silica gel using gradient elution RESULT AND DISCUSSION
(n-hexane, ethyl acetate and methanol) to give
5 fractions. The fourth fraction was recrystallized to give The ethyl acetate extract of the leave of mahkota
white needle amorphous solid (Compound 1). dewa was fractionated by column chromatography. The
The dried fruit (0.5 kg) of mahkota dewa was fraction containing the major component was purified
extracted using macerator (drip pan) with methanol by recrystallization to give compound 1 with white
(4.6 L) by heating (60 °C) for 7 h then stand up overnight needle amorphous solid as 45 mg (burning point
at room temperature. The residue was macerated again 187-195 °C). Its spot gave dark fluorosence at TLC
for 3 times and all the filtrates were combined (12.5 L) plate (UV366) with Rf of 0.65 at TLC chromatogram with
and concentrated using vacuum distillation and rotary eluent of ethyl acetate; 0.80 with ethyl acetate :
evaporator. The methanol extract was partitioned with methanol (7:3); 0.85 with ethyl acetate : methanol (5:5).
n-hexane (10 x 600 mL) to give n-hexane extract. The compound was dissolved in methanol.
Residue of extract methanol was partitioned with The ethyl acetate extract of the fruit of mahkota
chloroform (4 x 450 mL) to give chloroform extract. The dewa was fractionated by column chromatography. The
residue was fractionated with ethyl acetate (7 x 300 mL) fraction 1.5 was purified by recrystallization to give
to give ethyl acetate extract (6 g). Ethyl acetate extract compound 2 with peach needle crystal solid as 10 mg.
(2.5 g) was then separated by column chromatography Its spot gave dark fluorosence at TLC plate (UV366) with
on silica gel using gradient elution (n-hexane, ethyl Rf of 0.4 at TLC chromatogram with eluent of n-hexane
acetate and methanol) to give 11 fractions. The first : ethyl acetate; 0.90 with ethyl acetate. The compound
fraction was refractionated by column chromatography to was dissolved in methanol.
give 5 fraction. The fifth fraction was recrystallized to Compound 1 and 2 were tested with some
give peach needle crystal solid (Compound 2). chemical reagents (FeCl3 for phenolic, Shinode (Mg
and HCl) and NH4OH for flavonoid, Lieberman
Antibacterial activity Burchard (acetic anhydric, sulfuric acid) for triterpenoid
The antibacterial activity was carried out by and steroid, KOH in ethanol for coumarin). The results
employing 24h cultures of Staphylococcus aureus and were positive for FeCl3 (blue-black) and negative for

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182 Indones. J. Chem., 2015, 15 (2), 179 - 186

13 1
Table 1. Data C-NMR (CD3OD, 125 MHz) and H-NMR (CD3OD, 500 MHz) of compound 1 and benzophenone
glucoside [5]
No. atom Compound 1 (CD3OD-d4) Benzophenone glucoside) [5]
13 1 13 1
C C-NMR H-NMR C-NMR H-NMR
δ (ppm) δ (ppm), J (Hz) δ (ppm) δ (ppm), J (Hz)
C1 111.8 - 112.4 -
C2 158.6 - 158.9 -
C3 96.8 6.17 (d, 1H, 2.0) 97.0 6.38 (d, 1H, 2.0)
C4 164.3 - 163.9 -
C5 94.9 6.40 (d, 1H, 2.0) 94.8 6.16 (d, 1H, 2.0)
C6 159.1 - 158.3 -
C7 197.2 - 196.7 -
C1’ 132.0 - 129.3 -
C2’/6’ 133.7 7.69 (dd, 2H, 8.2 2.0) 134.0 7.65 (d, 2H, 8.7)
C3’/5’ 116.0 6.79 (dd, 2H, 8.2 2.0) 117.5 6.68 (d, 2H, 8.7)
C4’ 163.9 - 169.0 -
C1” 102.6 4.87 (d, 1H, 12.4) 102.7 4.91 (d, 1H, 7.8)
C2” 74.9 3.31 (t, 1H) 74.8 3.85 (dd, 2H)
C3” 77.9 3.37 (t, 2H) 77.8 3.38 (m, 2H)
C4” 71.3 3.25 (t, 1H) 71.3 3.25 (dd, 1H)
C5” 78.4 3.37 (t, 2H) 78.4 3.38 (m, 2H)
C6” 62.7 3.84 (dd, 1H) 62.6 3.85 (dd, 2H)
3.64 (k, 1H) 3.64 (dd, 1H)
OCH3 56.0 3.81 (s, 3H) 55.9 3.80 (s, 3H)
OH - - - -
CD3OD 49.1(t) 3.3 (k) - -
H2O - 4.87 - -

other reagents. This indicated compound 1 and 2 were meta position each other. The second ring had proton
classified to the phenolic compound. aromatics symmetrically at δ 7.69 and 6.79 (doublet-
UV(MeOH) spectrum of compound 1 and 2 showed doublet, 2H, J = 8.2 and 2.0 Hz) which respectively
maximum absorbances at λ 210, 290 and 210, 293 nm, derived from H2’/H6’ and H3’/H5’ which had the same
respectively. Absorbance at 210 nm was peak of the chemical environment.
methanol solvent, absorbance at 290/293 nm described Protons of glucose were indicated by signal at
the system was substituted aromatic ketone. Compound δ 4.87 ppm (doublet, 1H, J = 12.4 Hz which came from
1 could be considered as major constituent in the leave H1”, whose signal was closed by H2O proton signal.
of mahkota dewa, because the methanol extract of the Chemical shift at δ 3.31 ppm (triplet, 1H) was derived
leave has also absorbance in almost the same region. from the H2“ and the signal at δ 3.25 ppm (triplet, 1H)
There was wavelength 207 and 286 nm. was derived from H4". Signal at δ 3.37 ppm (triplet, 2H)
IR spectrum (KBr) of compound 1 showed absorption derived from the H3“ and H5". Doublet-doublet signal at
-1 -1
bands at 3363 cm and 3209 cm indicating the δ 3.84 ppm (1H) was derived from H6"a. Multiplet
-1
presence of OH group. Absorption band at 2924 cm signal at δ 3.64 ppm (1H) was derived from H6"b.
indicated the presence of saturated C-H group. Furthermore the value J H (12.4 Hz) between H1” and
-1
Absorption bands at 1504 and 1442 cm indicated the H2” indicated that the two protons coupling was axial-
present of C=C in the aromatic system. Strong axial (β) as sugar molecules tends to form a stable
-1
absorption bands at 1604 and 1278 cm represented conformation in which the OH groups were nearly
group of hydroxyl aryl ketone or aromatic ketone. equatorial. The structure was projected upwards
-1
Characteristic absorption band at 1651 cm strongly anomeric OH (cis to the CH2OH end) called β-anomer,
correlated with C=O group. Strong absorption band at thus bonding the sugar in this compound 1 was
-1
1087 cm came from the vibration of ether [15]. β-glucoside [16].
Analyses to elucidate for the structure of compound The signal at δ 3.81 ppm (singlet, 3H) was
1 13
1 were done by H-NMR and C-NMR spectrometers derived from OCH3 protons which has the same
1
(Table 1, Fig. 2 and Fig. 3). The H-NMR (CD3OD chemical environment. Proton signal of 6OH does not
1
500 MHz) data indicated the presence of 16 H atoms appear in the H-NMR spectrum. Signal at δ 3.3 ppm
where there were signals at δ 6.17 and 6.40 ppm (multiplet, 1H) came from the proton it CD3OD-d4
(doublet, 1H, J = 2.0 Hz) which respectively derived from solvent. The signal at δ 4.87 ppm (doublet, 2H) came
H3 and H5 of the first aromatic ring. H3 and H5 were in from dissolved water in deuterated solvent CD3OD-d4.

Susilawati et al.
 Indones. J. Chem., 2015, 15 (2), 179 - 186 183

1
Fig 2. H-NMR spectrum of compound 1 (CD3OD-d4, 500 MHz)

13
Fig 3. C-NMR spectrum of compound 1 (CD3OD-d4, 125 MHz)
13
Based on C-NMR (CD3OD 125 MHz) and δ 197.2 ppm was supported by IR peak at 1651, 1604
-1
Distortionless Enhancement Polarization Transfer cm and UV absorbance at λ 290 nm indicating the
(DEPT) analysis, isolated compound contained C=O from substituted aromatic ketone.
20 carbon atoms which consisted of one C=O (C7), Chemical shifts at 164.3, 163.9, 159.1, 158.6
six quaternary C, eleven CH (methine carbon or tertiary 132.0 and 111.8 ppm were specific for 6 quaternary
C), one methylene carbon (secondary C) and one carbon atoms. Chemical Shifts at 133.7 (2), δ 116.0
CH3 (methyl carbon/ primary C). The exist of C=O (C7) at (2), δ 96.8 and δ 95.0 ppm were specific for 6 methine

Susilawati et al.
184 Indones. J. Chem., 2015, 15 (2), 179 - 186

13 1
Table 2. Data C-NMR (CD3OD, 125 MHz) and H-NMR (CD3OD, 500 MHz) of compound 1 and compound 2
No. Compound 1 (CD3OD-d4) Compound 2 (CD3OD-d4)
13 1 13 1
atom C C-NMR H-NMR C-NMR H-NMR
δ (ppm) δ (ppm), J (Hz) δ (ppm) δ (ppm), J (Hz)
C1 111.8 - 111.8 -
C2 158.6 - 158.6 -
C3 96.8 6.17 (d, 1H, 2.0) 96.9 6.17 (d, 1H, 2.0)
C4 164.3 - 164.3 -
C5 94.9 6.40 (d, 1H, 2.0) 95.0 6.40 (d, 1H, 2.0)
C6 159.1 - 159.1 -
C7 197.2 - 197.2 -
C1’ 132.0 - 132.0 -
C2’/6’ 133.7 7.69 (dd, 2H, 8.2 2.0) 133.6 7.69 (dd, 2H, 8.5, 2.5)
C3’/5’ 116.0 6.79 (dd, 2H, 8.2 2.0) 116.0 6.79 (dd, 2H, 8.5 2.5)
C4’ 163.9 - 163.9 -
C1” 102.6 4.87 (d, 1H, 12.4) 102.6 4.87 (d, 1H, 2.8)
C2” 74.9 3.31 (t, 1H) 74.9 3.13 (t, 1H)
C3” 77.9 3.37 (t, 2H) 77.9 3.37 (t, 2H)
C4” 71.3 3.25 (t, 1H) 71.3 3.24 (t, 1H)
C5” 78.4 3.37 (t, 2H) 78.4 3.37 (t, 2H)
C6” 62.7 3.84 (dd, 1H) 62.7 3.85 (dd, 1H)
3.64 (k, 1H) 3.64 (k, 1H)
OCH3 56.0 3.81 (s, 3H) 56.0 3.80 (s, 3H)
OH - - - -
CD3OD 49.1(t) 3.3 (k) 49.1 3.3 (k)
H2O - 4.87 - 4.91

glucoside moiety. Chemical shift at 56.0 ppm was

specific for 1 methyl carbon atom (C primary). Solvent
of CD3OD-d4 appeared as triplet at δ 49.6, 49.4 and
49.2 ppm.
1 13
Data H- C HMQC spectrum could provide
correlation between proton with the carbon which it was
attached. Example: HOCH3 (3.81ppm) correlated with
COCH3 (56.0 ppm), H2” (3.13 ppm) correlated with
C2” (74.9 ppm) and so on (Table 3).
1 1
The spectrum of H- H COSY represents
autocorrelation spectra by connecting the dots signals
from protons contained in the spectra. As H3
(6.17 ppm) coupled each other with H5 (6.40 ppm) and
H2’/6’ (7.69 ppm) coupled each other also with H3’/5’
Fig 4. Correlation HMBC of isophalerin B
(6.79 ppm).
HMBC spectrum provided information about the
correlation between each proton with the neighboring
1 13
carbon atom to 2 bond ( H-C- C) or 3 bond
1 13
( H-CC- C). Example: H5 (6.40 ppm) correlated with
C3 (96.8 ppm), C1 (111.8 ppm), C2 (158.6 ppm) and
C4 (164.3 ppm). Like that also H2’/6’ (7.69 ppm)
correlated with the C2’/6’ (133.7 ppm), C4’ (163.9 ppm)
and C7 (197.2 ppm). H3” (3.37 ppm) correlated with
C4” (71.3) and C2’ (74.9 ppm).
From the spectroscopy analysis it could be
Fig 5. Structure of isophalerin B ascertained that the compound 1 was isophalerin B or
4,6-dihydroxy-4’-methoxybenzophenone-2-O-β-D-gluco
carbon atom (tertiary C). Chemical shifts at 102.6, δ pyranoside with molecular formula C20H22O10 (Fig. 5).
78.4, δ 77.9, δ 74.9, δ 71.3 and δ 62.7 ppm represented Benzophenone glucoside was more polar from its
6 methine and methylene carbon atoms specific for aglucon, this is caused by a number of OH groups

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 Indones. J. Chem., 2015, 15 (2), 179 - 186 185

13 1
Table 3. Data C-NMR (CD3OD 125 MHz) and H-NMR (CD3OD 500 MHz) and correlation HMQC, COSY and
HMBC compound 1
13 1
No. Atom C C-NMR δ (ppm) H-NMR δ (ppm), J (Hz) HM QC COSY HMBC
C1 111.8 - - - -
C2 158.6 - - - -
C3 96.8 6.17 (d, 1H, 2.0) C3 H5 C5, C1,C6, C4
C4 164.3 - - - -
C5 94.9 6.40 (d, 1H, 2.0) C5 H3 C3, C1, C2, C4
C6 159.1 - - - -
C7 197.2 - - - -
C1’ 132.0 - - - -
C2’/6’ 133.7 7.69 (dd, 2H, 8.2 2.0) C2’/C6’ H3’/H5’ C2’/6’, C4’, C7
C3’/5’ 116.0 6.79 (dd, 2H, 8.2 2.0) C3’/C5’ H2’/6’ C3’/5’, C1’, C4’
C4’ 163.9 - - - -
C1” 102.6 4.87 (d, 2H, 12.4) C1” H2” C2
C2” 74.9 3.31 (t, 1H) C2” H4”,H3” C3”, C1”
C3” 77.9 3.37 (t, 2H) C3” H2”, H6” C4”, C2”
C4” 71.3 3.25 (t, 1H) C4” H2” C6”, C5”
C5” 78.4 3.37 (t, 2H) C5” H6a”, H6”a C4”, C2”
C6” 62.7 3.84 (dd, 1H) C6” H6”b -
3.64 (k, 1H) H3”,H6”a COCH3
OCH3 56.0 3,81 (s, 3H) COCH3 - C4’
OH - - -
CD3OD 49.1(t) 3.3 (k) CCD3OD - -
H2O - 4.87 - -

Table 4. Measured result of growth inhibition diameter of test bacteria by extract and Compound 1 in ethanol
d growth inhibition of bacteria (mm)
No. Sample (in ethanol)
Stapyllococcus aureus Escheria coli
1. Blank 6.0 6.0
2. Crude methanol extract 9.0 10.0
3. Ethyl acetate extract 8.0 7.0
4. Compound 1 7.3 7.2
5. Ciprofloxacin 18.0 18.0

at this compound is α-glucoside (Silverstein, et al.,

2005). Based on spectroscopy data analysis contended
that compound 2 is isophalerin A with sugar bending at
this compound is α-glucoside. This compound is isomer
of isophalerin B. Structure of isophalerin A presented at
Fig. 6.
From the results of testing the antibacterial
activity of compound 1 by agar diffusion method
showed that this compound had activity against
Fig 6. Structure of isophalerin A bacteria used. Diameter of inhibition of compound 1
against bacteria (average of 3 times measurements) in
contained in glucose. ethanol showed at Table 4.
1
Value of chemical shift of compound 2 at H and Tests for antibacterial activity used 2 types of
13
C-NMR very similar with isophalerin B from leave of bacteria that represent the class of bacteria of gram
mahkota dewa, except value of J proton anomeric of its positive and negative. From the test results of
sugar is different by significantly (Table 2). Proton antibacterial activity from leave of mahkota dewa
anomeric of glucose shown by signal at δ 4.87 ppm (d, (Table 4) shows that crude methanol extract, ethyl
1H, J = 2.8 Hz),. Value of J H (0-5 Hz) between H1” and acetate extract and the compound 1 (10 mg/mL) in
H2” showing 2 proton which coupling equatorial- ethanol has a weak activity against the bacteria S.
equatorial (α) as sugar molecule having conformation aureus and E. coli. This happened because compound
form which OH group at axial position. Structure 1 is compound of polar (dissolve in methanol/ethanol)
projecting OH anomeric group downwards (trans to so it does not easy to penetrate to cell membrane of
CH2OH end) referred α-anomer, so that sugar bending bacterium which having character of non polar. So

Susilawati et al.
186 Indones. J. Chem., 2015, 15 (2), 179 - 186

activity of compound 1 against both the bacteria is weak. Gadjah Mada, http://www.tempo.co.id/medika/
th
Compound of bactericide come from compound of arsip/122002/art-3.htm, accessed on May 08
phenol will have big activity if the compound more 2006.
dissolves in fat or less dissolves in water. 3. Lisdawati, V., 2007, Mahkota Dewa Fruit-toxicity,
Antibacterial activity category to be 'weak' if it has a Antioxidant Effects, and Anticancer Effect by
7-11 mm inhibition zone and 'medium' if it has a 12-16 Pharmacological Screening Tests,
mm inhibition zone with diameter of discs 6 mm [17-18]. http://www.mahkotadewa.com/Indo/info/makalah/Vi
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The presence of ciprofloxacin antibiotic aims to vi201002.htm, accessed on October 14 2008.
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isophalerin A (4,6,-dihydroxy-4’-methoxybenzophenone- Anwar, C., 2011, Indones. J. Chem., 11(2),
2-O-α-D-glucopiranoside) in the form of peach needle 180–185.
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ACKNOWLEDGEMENT Phytochemistry, 54(8), 883–889.
13. Ramakrishnan, G., Kothai R., Jaykar B., and
This work was supported by Director General of Rathnakumar, T.V., 2011, Int. J. PharmTech. Res.,
Higher Education of Indonesia through scholarship of 3(2), 1000–1004.
BPPS. The authors thanks to Mrs. Fitni and Mr. 14. Dey, P.M., and Harborne, J.B., 1991, Methods in
Suratmanto, the staff lab of Organic Chemistry of Plant Biochemistry: Assay for Bioactivity, Vol. 6,
Department of Chemistry UGM for analysis of UV and Academic Press Limited, London.
IR. We are also thankful to Mrs. Sofa Fajriah from 15. Creswell, C.J., Runquist, O.A., and Campbelll,
Research Center for Chemistry, Indonesian Institute of M.M., 2005, Spectral Analysis of Organic
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Science for analysis of NMR and 2 dimension NMR, to Compounds, 10 ed., ITB, Bandung.
Miss Mega, student of Department of Chemistry, Faculty 16. Silverstein, R.M., Bassler, G.C., and Morrill, T.C.,
of Mathematic and Natural Science UR for test of 2005. Spectrometric Identification Organic
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antibacterial activity and to Mr. Idham Darussalam for his Compounds, 7 ed., John Wiley and Sons Inc.,
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Susilawati et al.