Coumarins from the leaves ofMelicope denhamii

RatihDewiSaputria, TjitjikSrieTjahjandariea, and MulyadiTanjunga*

a
Natural Products Chemistry Research Group, Organic Chemistry Division, Department of
Chemistry, Faculty of Science and Technology, Universitas Airlangga, Surabaya, Indonesia.

Ratih dewi saputri

duffputri@gmail.com, Phone: 085730013365

Background : Melicope locally known ‘Ki Sampang’ belongs to the Rutaceae family,
consisting of about 280 species arewidely distributed in Asia, Australia, Africa and
Polynesia. Phytochemical studies have shown that the species produce a variety of alkaloids,
flavonoids, coumarins, and acylphloroglucinols, which exhibit various biological activities
including antioxidant, anticancer, and antiinflammatory. Here we report the isolation of three
coumarins, xanthotoxine (1), bergaptene (2) and isopimpinellin (3) from the leaves of M.
denhamii and its cytotoxicity against P-388 cells with MTT methods.
Objective : Melicope denhamii is used in Indonesia traditional medicine for fever,
hypertension, cough, and as a toxic. Previous studies have isolated fungicidal, antifeedant,
antiinflamatory, anticancer and antibacterial from Melicope denhamii. This study is aimed at
the isolation coumarins and biological activity cytotoxicity against P-388 cells from leaves
extracts of Melicope denhamii.
Methods : Coumarins was isolated from M. denhamii leaves by extraction and repeat
column and planar radial chromatography and characterized by UV, IR, HRESIMS,
1Dand2DNMR. The cytotoxicity activity of coumarins used P-388 cell lines in vitro and were
assayed by MTT [3-(4,5-di-methylthiazole-2-yl)-2,5-diphenyltetrazolium bromide] methods.
Results : Phytochemical study on the MeOH extract from the leaves of M. denhamii
yieldedthree coumarins, xanthotoxine (1), bergaptene (2) and isopimpinellin (3).
Thecytotoxic activityof compounds 1-3were evaluated for their cytotoxicity by MTT assay
againstmurine leukemia P-388. Those cytotoxic data for coumarinssuggested that compound
1and 2haveweak activity and compound 3 inactive. The presence of methoxy at C-5 and C-8
in compound 3 can bedecreased activity.
Conclusion : The phytochemical investigation of the leaves of M. denhamiito yielded
compounds, xanthotoxine (1), bergaptene (2) and isopimpinellin (3)showed low activity
against murine leukemia P-388 cells.
 1. Introduction
Cancer one of the diseases caused by abnormal growth of body tissue cells that turn
into cancer cells. In its development, these cancer cells can spread to other body parts that can
cause death. In Indonesia, cancer is the second leading cause of death after diabetes.
Melicope denhamii is an evergreen medicinal shrub belonging to the family Rutaceae.
It is widely distributed in Asia, Australia, Africa and Polynesia.M. denhamii leaves are
traditionally used against skin diseases and as larvacidal agents [1-2]. Phytochemical studies
have shown that the species produce a variety of alkaloids [3-5], flavonoids [6], coumarins
[7], and acylphloroglucinols [8], which exhibit various biological activities including
antioxidant, anticancer, and antiinflammatory. In continuation of our research for
investigation of coumarinsof Indonesian Melicope plants, we report the isolation of
xanthotoxine (1), bergaptene (2) and isopimpinellin (3) from the methanol extract of the
leaves of Melicope denhamii. The chemical structure of compounds1-4were established by
UV, IR, HRESIMS, 1D and 2DNMR, as well as by comparison with those related
compounds previously reported.The cytotoxic activityagainst murine leukemia P-388 cells of
isolated compound from this species are also briefly described.

2. Result and Discussion

Phytochemical study on the MeOH extract from the leaves of M. denhamii yielded
Xanthotoxine (1), bergaptene (2) and isopimpinellin (3).

Figure1.Compounds 1-3 isolated from the leaves of M. denhamii.

Xanthotoxine (1), was isolated as a yellow solid, m.p. 104-106oC, showed a

quasimolecular ion [M+H]+ at m/z216.1895 corresponding to the molecular formulaC12H8O4.
The UV maximum absorptionat max221 (3.75),242 (3.10), and 269 (3.42) nmtypical for a
furanocoumarins skeleton[9]. The IR spectrum of 1 indicated absorptions for conjugated
carbonyl (1718 cm-1),aromatic (1589 cm-1) and ether (1099 and 1149 cm-1) groups,
respectively.The 1H-NMR spectrum of compound 1showed a pair of doublets(J = 9.6 Hz) at
δH [7.77 (H-4); 6.37 (H-3)], a singlet aromatic region at δH 7.35 (H-5), and a pair of doublets
(J = 2.2Hz) at δH [7.69 (H-2’); 6.82 (H-3’)] suggested the signal of a furanocoumarin with
substitutent at C-5 or C-8.Themethoxyl group showed singlet proton signal at δH4.30 ppm
suggested that the methoxyl group is either at C-5 or C-8 of the furanocoumarin structure.
The one bond and two/three bonds 1H-13C correlations found in the HMQC andHMBC
spectra of compound 1(Table 1) unambiguously placed the methoxyl group at C-8 by the
followingobservations.The presence of long-range correlations in the HMBC spectrum of
1between the doublet protonsignals (J = 2.2 Hz) of a furano group at δH 7.69 (H-2’), and
6.82 (H-3’) with one oxyaryl carbon signal at δC 147.7(C-7), and the correlation between a
proton aromatic signal at δH 7.35 with two oxyaryl carbon signals δC 147.7 (C-7), 143.0 (C-
9) and one methine carbon signal δC 106.7 (C-3’) showed methoxyl at C-8. Based on data
from 1D and2D NMR, compound 1is 8-methoxy-psoralen or known as Xanthotoxine [9].
Other HMBC correlations consistentwith the structure 1are shown in Table 1.
Bergaptene (2), was isolated as a yellow solid, m.p. 184-186oC, showed a
quasimolecular ion [M+H]+ at m/z216.1895 corresponding to the molecular formulaC12H8O4.
The UV maximum absorptionat max309 (3.83), 268 (3.92), 259 (3.88), 249 (3.91), 217
(4.08)and IR spectrum (1718,1589,1099 and 1149 cm-1) absorptions were similarity to those
of 1.The 1H NMR spectrum of 2(Table 1) showed the presence a pair of doublets(J = 9.8 Hz)
at δH [8.16 (H-4); 6.28 (H-3)], a singlet aromatic region at δH 7.14 (H-5), and a pair
ofdoublets (J = 2.4Hz) at δH [7.59 (H-2’); 7.02 (H-3’)] suggested the signal of a
furanocoumarin with substitutent at C-5 or C-8. The 13C NMR spectrum of 2 showed12
carbon signals and the structure of 2 were confirmed by HMQC and HMBC spectra (Figure 2
and Table 1). The 1D and 2D NMR spectra data are consistent with publish data [10].
Isopimpinellin (3), was isolated as a yellow solid, m.p. 147-148oC, showed a
quasimolecular ion [M+H]+ at m/z247.0609corresponding to the molecular formulaC13H11O5.
The UV maximum absorptionat max223 (4.20), 241 (4.00), 248 (4.00), 268 (4.09)and311
(3.91).The IR spectrum of 3 indicated absorptions for conjugated carbonyl (1751 cm-1), and
aromatic (1606 and 1491 cm-1) and ether (1145 cm-1) groups, respectively.The 1H-NMR
spectrum of compound 3showed a pair of doublets(J = 9.8 Hz) at δH [8.12 (H-4); 6.29 (H-3)]
and a pair ofdoublets (J = 2.3Hz) at δH [7.62 (H-2’); 7.00 (H-3’)] suggested the signal of a
furanocoumarin cyclic at C-6 or C-7 in coumarins aromatic with substitutent at C-5and C-
8.Themethoxyl group showed singlet proton signal at δH4.17 and 4,16 ppm suggested that
the methoxyl group at C-5 and C-8 of the furanocoumarin structure. The 13C NMR spectrum
of 3 showed13 carbon signals andthe one bond and two/three bonds 1H-13C correlations
found in the HMQC andHMBC spectra of compound 3(Table 1) unambiguously placed the
methoxyl group at C-8 by the followingobservations.The presence of long-range correlations
in the HMBC spectrum of 2between the singlet protonsignals at δH4.17 and 4.16 ppm with
twoquartener carbon signal at δC 144.4(C-8) and δC128.3(C-5) ppm showed methoxyl group
at C-5 and C-8. Based on data from 1D and2D NMR, compound 3is 5,8-dimethoxy-psoralen
or known as Isopimpinellin[11]. Other HMBC correlations consistentwith the structure 3are
shown in figure2.
 Table-1. NMR spectroscopic data of compounds 1-3in CDCl3.
No Xanthotoxine (1) Bergaptene (2) Isopimpinellin (3)
δH δC HMBC δH δC HMBC δH δC HMBC
(mult, (mult, (mult,
Jin Hz) Jin Jin Hz)
Hz)
1 - - - - - - - -
2 - 160.5 - - 161.6 - - 160.6 -
3 6.37 (d, 112.9 C-2; C-10 6.28 (d, 112.5 C-10; C- 6.29 (d, 112.9 C-2; C-
9.6) 9.8) 2 9.8) 10
4 7.77 (d, 144.3 C-2;C- 8.16 (d, 139.3 C-5; C-9; 8.12 (d, 145.2 C-2; C-9
9.6) 3;C-6; C-9 9.8) C-2 9.8)

5 7.35 (s) 105.8 C-7;C-9; - 149.5 - - 128.3 -

C-10; C-3’
6 - 126.1 - - 112.6 - - 114.8 -
7 - 147.7 - - 158.3 - - 150.1 -
8 - 132.8 - 7.14 (s) 93.9 C-10; C- - 144.4 -
3; C-9;
C-6
9 - 143.0 - - 152.2 - - 143.8 -
10 - 116.5 - - 106.3 - - 107.7 -
1’ - - - - - - - - -
2’ 7.69 (d, 146.6 C-7;C-3’ 7.59 (d, 144.8 C-3’; C- 7.62 (d, 139.5 C-6; C-7,
2.2) 2.4) 6; C-7 2.3) C-3’
3’ 6.82 (d, 106.7 C-7; C-2’ 7.02 (d, 105.2 C-2’;C- 7.00 (d, 105.2 C-7, C-3’
2.2) 2.4) 6; C-7 2.3)
5-OCH3 - - C-8 4.27 (s) 60.1 C-5 4.16 (s) 61.8 C-5
8-OCH3 4.30 (s) 61.3 - - 4.17 (s) 60.9 C-8

Figure 2. Selected HMBCcorrelations for compounds 1-3

 The cytotoxic activity(Table 2) of compounds 1-3 were evaluated for their
cytotoxicity by MTT assay againstmurine leukemia P-388. Artonin E was used as the positive
control. The cytotoxic activity of coumarins (1-3) not active than positif control. Those
cytotoxic data for coumarinssuggested that compound 3was inactiveand compound 1 and 2
haveweak activity. Addition Methoxy at C-8 in aromatic of compound 3 can bedecreased
activity.

Table-2. Cytotoxicactivity data of compounds 1-3by MTT assay.

No Compound IC50 (µg/mL)
1 Xanthotoxine (1) 10.68 ± 0.02
2 Bergaptene (2) 8.86 ± 0.12
3 Isopimpinellin (3) 22.02 ± 0.05
4 Artonin E 1.33± 0.18

3. Experimental
3.1 Generalexperimental procedure
Column chromatography and planar radial chromatography were carriedout using
silica gel 60 and silica gel 60 PF254(Merck, Darmstadt, Germany). NMR spectra
weremeasured on a JEOLJNM-ECA400 MHz FTNMR spectrophotometer(Tokyo, Japan)
in CDCl3with TMS as the internal standard. Mass spectra were measured on an ESI-
TOFWaters LCT Premier XE producing pseudo-molecular ions, [M+H]+ positive ion
mode (Santa Clara, CA, USA).UV spectra were recorded in MeOH ona Shimadzu series
1800UV-VIS spectrophotometer (Kyoto, Japan). IR spectra were recorded in KBr ona
One Perkin Elmer instrument (Waltham, MA, USA).

3.2 Plant material

Theleaves of M.denhamii was collected from the conserved forest of Gunung
Salak, Bogor, West Java, Indonesia on Nov 2016 and was identified by Mr. Ismail
Rachman from the Herbarium Bogoriense, Bogor, Center of Biological Research and
Development, National Institute of Science, Bogor, Indonesia.

3.3 Extraction and isolation

The air-dried leaves of M.denhamii(3.5 kg) was macerated with methanol at room
temperature for 24 h two timesand then evaporated under reduced pressure to give a dark
brown residue (250 g). The extract was redissolved in MeOH-water (9:1) and partitioned
with n-hexane (201 g) and ethyl acetate (73 g) fractions. A part of ethylacetate fraction (70
g)was subjected tovacum liquid chromatography over silica gel andeluted with n-hexane-
ethylacetate (from 9:1 to1:9) to give fractions A-C. Fraction B (975mg) was added to
column chromatography and eluted with n-hexane-ethylacetate (from 9:1 to6:4) to
produce subfractions B1-B3. Subfraction B3(105 mg) was purified by planar radial
chromatography using n-hexane-acetone (from 9:1 to8:2)to yielded compound 1 (12 mg)
and 2(10 mg). Fraction C (625 mg) was refractionated using column chromatography and
eluted n-hexane-ethyl acetate (from 9:1 to7:3) to yielded subfractions C1-C2. Subfraction
C1 was purified by planar radial chromatography using n-hexane-chloroform (from 9:1
to7:3)to yielded compound 3 (9 mg).
 3.4 Spectral Data

Xanthotoxin(1): yellow solid,UV/Vis (MeOH) max (nm) (log ε): 221 (3.75),242 (3.10),
and 269 (3.42). IR (KBr) νmax (cm-1):1718, 1589, 1099 and 1149. 1H-NMR (CDCl3) δH
ppm: 7.77(1H, d, J = 9.6 Hz, H-4), 7.69 (1H, d, J = 2.2 Hz, H-2’), 7.35 (1H, s, H-5),
6.82(1H, d, J = 2.2 Hz, H-3’), 6.37 (1H, d, J = 9.6 Hz, H-3),4.30 (3H, s, 8-OCH3).13C-
NMR (CDCl3) δC ppm: 160.5 (C-2), 147.7 (C-7),105.8 (C-5),144.3 (C-4),143.0 (C-9),
132.8 (C-8),116.5 (C-10),146.6 (C-2’),112.9 (C-3),106.7 (C-3’),126.1 (C-6),61.3 (8-
OCH3).
Bergapten(2): yellow solid, UV/Vis (MeOH) max (nm) (log ε): 309 (3.83), 268 (3.92), 259
(3.88), 249 (3.91) and 217 (4.08). IR (KBr) νmax (cm-1):1718, 1589, 1099 and 1149.1H-
NMR (CDCl3) δH ppm: 8.16 (1H, d, J = 9.8 Hz, H-4), 7.59 (1H, d, J = 2.4 Hz, H-2’), 7.14
(1H, s, H-8), 7.02(1H, d, J = 2.4 Hz, H-3’), 6.28 (1H, d, J = 9.8 Hz, H-3), 4.27 (3H, s, 8-
OCH3). 13C-NMR (CDCl3) δC ppm: 161.6 (C-2), 158.3 (C-7), 152.2 (C-9), 149.5 (C-5),
144.8 (C-2’), 139.3 (C-4), 112.6 (C-6),112.5 (C-3), 105.2(C-3’), 106.3 (C-10), 93.9 (C-8),
60.1 (5-OCH3).
Isopimpinellin (3): yellow solid,UV/Vis (MeOH) max (nm) (log ε): 223 (4.20), 241 (4.00),
248 (4.00), 268 (4.09)and311 (3.91). IR (KBr) νmax (cm-1):1751, 1606, 1491 and 1145.1H-
NMR (CDCl3) δH ppm: 8.12 (1H, d, J = 9.8 Hz, H-4), 7.62 (1H, d, J = 2.3 Hz, H-2’), 6.29
(1H, d, J = 9.8 Hz, H-3), 7.00 (1H, d, J = 2.3 Hz, H-3’), 4.17 (3H, s, 8-OCH3), 4.16 (3H, s,
5-OCH3). 13C-NMR (CDCl3) δC ppm: 160.6 (C-2), 150.1 (C-7), 145.2 (C-4), 144.4 (C-8),
143.8 (C-9), 139.5 (C-2’), 128.3 (C-5), 114.8 (C-6), 107.7 (C-10), 112.9 (C-3), 105.2 (C-
3’), 61.8 (5-OCH3), 60.9 (8-OCH3).

3.5 Cytotoxic assay

Cytotoxic properties of the isolated compounds 1-3 against murine leukemia P-388
cells was evaluated according to the MTT method as previously described [12-14].
Artonin E was used as the positive control.

4. Conclusion
The phytochemical investigation of the leaves of M. denhamiito yielded
compounds, Xanthotoxine (1), bergaptene (2) and isopimpinellin (3)showed moderate
activity against murine leukemia P-388 cells.

Acknowledgement

This research was supported by Universitas Airlangga through Hibah Riset Mandat

2018 research.

References
1. KP, Prathibha.; BS, Raghavendra,; Vijayan, VA. Evaluation of larvicidal effect of
Euodia ridleyi Hochr. leaf extract against three mosquito species at Mysore. Biol Sci.,
2010, 5, 452-455.
2. KI, Nakashima,; M, Oyama,; T, Ito,; JR, Witono,; D, Darnaedi,; T, Tanaka,; J,
Murata,; Linuma,M. Novel zierane- and guaiane-type sesquiterpenes from the root of
Melicope denhamii.Chem Biodivers., 2012, 9, 2195-2202.
3. Tanjung, M.; Saputri, R.D.; Wahjoedi, R.A.; Tjahjandarie, T.S. 4-Methoxy-3-(3-
methylbut-2-en-1-yl)-7-((3-methylbut-2-en-1-yl)oxy) quinolin-2(1H)-one from
Melicope moluccana T.G. Hartley.Molbank, 2017, M939.
4. Nakashima, K.; Oyama, M.; Ito, T.; Witono, J.R.; Darnaedi, D.; Tanaka, T.; Murata,
T.; Iinuma, M. Melicodenines A and B, novel Diels-Alder type adduct isolated from
Melicope denhamii.Tetrahedron. Lett., 2011, 52, 4694-4696.
5. Chen, J-J.; Chang, Y-L.; Teng, C-M.; Su, C-C.; Chen I-S. Quinoline alkaloids and
anti-plateletaggregation constituents from the leaves of Melicope
semecarpifolia.Planta Med.,2002, 68, 790-793.
6. Sultana, N.; Hartley, T.G.; Waterman, P.G. Two novel prenylated flavanones from the
aerial parts of Melicope micrococca. Phytochemistry,1999, 50, 1249-1253.
7. Kassim, N.K.; Rahmani, M.; Ismail, A.; Sukari, M.A.; Ee, G.C.L.; Nasir, N.M.;
Awang, K. Antioxidant activity-guided separation of coumarins and lignan from
Melicope glabra (Rutaceae). Food. Chem.,2013, 139, 87-92.
8. Kamperdick, C.; Van, N. H.; Sung, T. V.; and Adam, G. Benzopyran from
Melicopeptelefolia Leaves.Phytochem., 1997,45(5), 1049-1056.
9. S, Ittipon,; L. Surat, Chemical constituents fromFeronia limonia roots. Chem. Nat.
Compounds, 2012, 48(2), 308-309.
10. Muller,M,; Byres, M,; Jaspars, M,; Kumarasamy, Y,; Middleton, M,; Nahar, L,;
Shoeb, M,; Satyajit, S. 2D NMR spectroscopic analyses of archangelicin from the
seeds of Angelica archangelica. Acta Pharm, 2004, 54, 277-285.
11. Sridechakorn,; S, Ittipon,; Laphookhieo, Surat. Chemical Constituents From Feronia
Limonia Roots.Chemistry of Natural Compounds, 2012,48(2), 2-4.
12. Tanjung, M.; Rachmadiarti, F.; Saputri, R.D.; Tjahjandarie, T.S. Mesucalophylloidin,
a new isoprenylated 4-phenylcoumarin from Mesua calophylloides (Ridl.) Kosterm.
Nat. Prod. Res 2018, 32, 1062-1067.
13. Tanjung, M.; Saputri, R.D.; Tjahjandarie, T.S. 5,9,11-Trihydroxy-2,2-dimethyl-10-
(3'-methyl-2'-butenyl)-3-(2''-methyl-3''-butenyl)pyrano[2,3-a]xanthen-12(2H)-one
from the stem bark of Calophyllum pseudomole.Molbank. 2016 (3), M906.
14. Tanjung, M.; Hakim, E.H.; Syah, Y.M. Prenylated dihydrostilbenes from Macaranga
rubiginosa. Chem Nat Compd. 2017, 53:215-218.