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A new sesquiterpenoid from Scaphiummacropodum (Miq.) BeumeeL.M. Ramadhan Al Muqarrabuna, Norizan Ahmata, S. RuzainaS. Arisa, Nurdiana Shamsulrijala, Syarul N. Baharumb, RafidahAhmadb, A. Rifki Rosandyc, M. Nazip Suratmana & H. Takayamad

a Faculty of Applied Sciences, Universiti Teknologi MARA (UiTM),40450 Shah Alam, Selangor, Malaysiab Institute of System Biology, Universiti Kebangsaan Malaysia(UKM), Bangi, Selangor, Malaysiac Faculty of Science and Technology, Universiti KebangsaanMalaysia (UKM), Bangi, Selangor, Malaysiad Graduate School of Pharmaceutical Sciences, Chiba University,1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, JapanPublished online: 25 Feb 2014.

To cite this article: L.M. Ramadhan Al Muqarrabun, Norizan Ahmat, S. Ruzaina S. Aris,Nurdiana Shamsulrijal, Syarul N. Baharum, Rafidah Ahmad, A. Rifki Rosandy, M. NazipSuratman & H. Takayama (2014) A new sesquiterpenoid from Scaphium macropodum (Miq.)Beumee, Natural Product Research: Formerly Natural Product Letters, 28:9, 597-605, DOI:10.1080/14786419.2014.886211

To link to this article: http://dx.doi.org/10.1080/14786419.2014.886211

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A new sesquiterpenoid from Scaphium macropodum (Miq.) Beumee

L.M. Ramadhan Al Muqarrabuna, Norizan Ahmata*, S. Ruzaina S. Arisa,

Nurdiana Shamsulrijala, Syarul N. Baharumb, Rafidah Ahmadb, A. Rifki Rosandyc,

M. Nazip Suratmana and H. Takayamad

aFaculty of Applied Sciences, Universiti Teknologi MARA (UiTM), 40450 Shah Alam, Selangor, Malaysia;bInstitute of System Biology, Universiti Kebangsaan Malaysia (UKM), Bangi, Selangor, Malaysia;cFaculty of Science and Technology, Universiti Kebangsaan Malaysia (UKM), Bangi, Selangor, Malaysia;dGraduate School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku,Chiba 263-8522, Japan

(Received 30 November 2013; final version received 19 January 2014)

A new sesquiterpenoid, malayscaphiol (1), and three known compounds, lupeol (2),lupenone (3) and stigmasterol (4), were isolated from the methanolic extract of the stembark of Scaphium macropodum. The structures of the isolated compounds weredetermined using several spectroscopic methods, including UV–vis, FT-IR, 1D and 2DNMR, and mass spectrometer. Major isolated compounds were assayed for cytotoxicityand anti-acetylcholinesterase activities. The chemotaxonomy significance of this plantwas also discussed.

Keywords: Sterculiaceae; Scaphium macropodum; sesquiterpenoid; malayscaphiol;cytotoxicity; anti-acetylcholinesterase

1. Introduction

Scaphium macropodum (Miq.) Beumee (Sterculiaceae), also known as malva nut (English) or

kembang semangkuk jantung (Malay), is a common emergent tree of tropical rain forests in

Malaya, Cambodia, Thailand, Sumatra and Borneo (Kochummen 1972). The mature tree attains

a stature of 40m. This plant undergoes defoliation in only a brief period of time and the flowers

grow on its bare twigs after defoliation (Kostermans 1953). This species is shortly deciduous and

flowers on bare twigs before the flush of new leaves. It changes its leaf shape and crown form

conspicuously with tree size. It is very likely that these dramatic ontogenic changes are related to

the plant growth and survival (Yamada & Suzuki 1996). The dispersal distance of the fruit

seldom exceeds 50m from the base of the parent tree (Yamada & Suzuki 1997).

S. macropodum has been reported to possess some medicinal properties. The seed forms a

large quantity of gelatine which can be used to treat intestinal infections, diarrhoea, throat aches,

asthma, dysentery, fever, coughs, inflammation and urinary illness (Lim 2012). In China, it is

also used as a traditional drug to prevent pharyngitis and also to treat tussis and constipation.

Malva nut also has cooling properties (Lamxay 2001). However, the chemical constituents and

pharmacology of this species have not been studied.

This paper describes the isolation and detailed characterisation of a new sesquiterpenoid,

malayscaphiol (1). The cytotoxicity against two cancer cell lines, HT-29 and MDA-MB, and

normal cell line 3T3, and anti-acetylcholinesterase activities of the isolated compounds are also

discussed.

q 2014 Taylor & Francis

*Corresponding author. Email: noriz118@salam.uitm.edu.my

Natural Product Research, 2014

Vol. 28, No. 9, 597–605, http://dx.doi.org/10.1080/14786419.2014.886211

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2. Results and discussion

2.1. Structure determination of malayscaphiol

The methanolic extract of the stem bark of S. macropodum was fractionated using vacuum liquid

chromatography (VLC) and purified using radial chromatography (RC) and recrystallisation.

Four compounds (1–4) were isolated (Figure 1), including a new sesquiterpenoid named

malayscaphiol (1). The structure elucidation of the compounds was conducted using several

spectroscopic methods including mass spectrometer, UV–vis, FT-IR, 1D and 2D NMR (HMQC,

HMBC, COSY and NOESY) and by comparison with reported data.

Compound 1, isolated as a pale yellow amorphous solid, is a new sesquiterpenoid derivative

isolated from plant for the first time. The molecular formula is C15H14O5, generated using HR-

ESI-MS [M þ H]þ at m/z 275.0978 (calc. for C15H15O5 275.09195). The UV spectrum displays

several absorbance peaks at 203, 233, 256 and 324 nm indicating the occurrence of a conjugated

system of double bonds as aromatic ring in the molecule. The FT-IR spectral data display a broad

absorbance peak at 3435 cm21 representing the stretching vibration of OZH bond. The Csp3–H

stretching bands appear at 2962, 2926 and 2850 cm21. Carbon–carbon double bond is detected

in this compound shown by an absorbance peak of symmetrical stretching CvC at 1643 cm21.

The presence of carbonyl group is characterised by several absorbance bands, i.e. CvO stretch

at 1734 cm21 while a band at 1262 cm21 indicates that the carbonyl is a ketone group. Bending

C–O at 1032 cm21 represents the hydroxyl. A conjugated system of double bonds is indicated

by a peak at 1557 cm21.

The attached proton test spectrum reveals that the compound contains 15 carbons consisting

of three methyls, one methylene, two methines and nine quaternary carbons. The most highly

deshielded signal at dC 193.7 (C-4) integrating for two carbons is attributed to two ketone

groups. The methylene group at dC 72.4 (C-2) indicates an oxygenated methylene. Two signals

at dC 155.4 (C-4) and 116.9 (C-3) provide information of the presence of an aliphatic double

bond in the structure, while the other six highly deshielded carbons represent the existence of an

aromatic ring in the molecule. In the aromatic ring, three carbons (C-6, C-7 and C-9) are

oxygenated carbons. The double bond equivalent value of C15H14O5 is 9, indicating that the

compound consists of six double bonds (four olefinics and two carbonyls) and three rings.

According to the 1H NMR spectrum, the oxygenated methylene protons, H-2eq (dH 4.60, dd,

J ¼ 5.1 and 10.8 Hz) and H-2axi (dH 4.19, dd, J ¼ 10.4 and 10.8Hz) are double doublet,

Figure 1. Compounds isolated from the stem bark of S. macropodum.

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indicating that C-2 is adjacent to a methine group (C-3). This C-3 binds a doublet methyl group

(C-14), making the multiplicity of the H-3 multiplet. The other two methyl proton signals (H-15

and H-16) are singlets, showing that they are attached to olefinic quarternary carbons,

considering their high chemical shifts (H-15 ¼ 2.23, H-16 ¼ 2.40). One singlet olefinic proton

is detected at dH 6.36 (H-12).

The structure is confirmed using 2D NMR. The HMBC experiment shows 2J couplings

between H-2 (H-2axi and H-2eq) and C-3 besides 2J couplings between H-2 and C-4, C-9 and

C-14, thus confirming the position of carbonyl (C-4) and C-14. The other methyl, H-15, has

correlations with C-13 and the sole olefinic methine (C-12), while H-16 generates 2J couplings

with C-8 and 3J couplings with C-7 and C-9. Correlation between H-12 and C-5 also appears in

the HMBC spectrum. In the COSY spectrum, H-3 shows strong 3J couplings with H-2 and H-14,

while a long range 4J coupling also appears between H-12 and H-15 (Figure 2(a)).

The relative configuration of the sole chiral carbon (C-3)was observed usingNuclearOverhauser

Effect Spectroscopy (NOESY) experiment which describes the H–H correlation through space

(Figure 2(b)). In the NOESY spectrum, correlation was observed between two geminal protons of

aliphatic methylene C-2 (H-2eq and H-2ax). Carbon C-2 binds two protons, i.e. H-2ax and H-2eq.

Since both of the protons are double doublets and are in geminal position, they have two coupling

constants each, which are 10.4 and 10.8Hz for H-2ax and 5.1 and 10.8 for H-2eq. According to

Silverstein et al. (2005), the vicinal coupling constant between an axial Hwith another axialH ranges

from6 to10Hz,while the vicinal couplingconstantbetween an equatorialHwith an axialH is around

0–5Hz. Thus, the proton with coupling constants of 10.4Hz and 10.8Hz (H-2ax) is the one in the

axial position. H-2ax and H-2eq are coupled with J of 10.8Hz. In the NOESY spectrum, H-2axgenerates cross peak with H-3, whichmeans they are in the same orientation, i.e. axial, confirmed by

theother JofH-2ax (10.4Hz), indicating thatH-2ax is in eclipse position (dihedral angle of 08) towardsH-3 according to Karplus curve, thus placing C-14 in the equatorial position.

The plausible biogenesis of this compound is proposed in this paper (Figure 3). As a

sesquiterpenoid derivative, malayscaphiol is suspected to be synthesised via the mevalonate

pathway. An isopentenylpyrophosphate reacts with its allyl isomer, dimethylallylpyrophosphate

(DMAPP), to form geranyl pyrophosphate which then undergoes a series of reactions including

hydroxylation, deprotonation, cyclisation, reduction and oxidation reactions to form A. Another

DMAPP which has undergone a rearrangement reaction to become B then reacts with A in a

condensation reaction. The reaction to form compound 1 occurs in several steps from

hydroxylation, oxidation, cyclisation and reduction (aromatisation) reaction.

2.2. Chemotaxonomy significance

There have been many reports regarding the phytochemical studies of the species from the

family Sterculiaceae. Various classes of compounds including alkaloids, phenyl propanoids,

flavonoids and terpenoids have been successfully isolated and identified from several genera,

such as Melochia, Waltheria, Heisteria, Sterculia, Theobroma, Mansonia and Pterospermum

(Jalal & Collin 1977; Bhakuni et al. 1987; Hammerstone et al. 1994; El-Seedi et al. 1999;

Figure 2. Malayscaphiol 2D correlation NMR; (a) HMBC ( ) and COSY ( ), (b) NOESY.

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Tiew et al. 2003; Wang et al. 2003; Dias et al. 2007; Dixit et al. 2011). Up until now, this

research is the first to cover the phytochemical investigation of the genus Scaphium. In this

study, a new three-ringed sesquiterpenoid named malayscaphiol was obtained from the stem

bark of S. macropodum. The occurrence of sesquiterpenoid in this family is not something new.

A handful of sesquiterpenoids were also reported from other genera. Tiew et al. (2003) managed

to isolate sesquiterpenoid mansonone C, E, G, H, R and S from the heartwood of Mansonia

gagei. In 2006, Chen, Tang, Lou and Zhao and his colleagues identified mansonone F, M, and

mansonone H methyl ester from the root bark of Helicteres angustifolia. The heartwood of

Heritiera ornithocephala was reported to contain 7-hydroxycalamenene, 8-hydroxy-

2,3,4,5-tetrahydro-2,7,11,11-tetramethyl-1-benzox-epin-4-one and a sesquiterpen dimer, 8-bis

(7-hydroxycalamenene) (Cambie et al. 1990). Since only these four genera were reported to

Figure 3. Plausible biogenetic pathway of malayscaphiol.

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biologically synthesise sesquiterpenoids, thus chemotaxonomically, it can be assumed that the

genus Scaphium is related to genera Mansonia, Helicteres and Heritiera. In addition,

S. macropodum can be suspected to have a closer relationship with genus Helicteres as the other

reported three-ringed sesquiterpenoids were all reported from Helicteres angustifolia.

2.3. Biological activity

Triterpene compounds have been reported to have potent cytotoxicity against several cancer cell

lines (Gao et al. 2002; Kuzuhara et al. 2006; Joshi et al. 2013). In this study, three triterpenes

isolated from this plant (2, 3 and 4) were assayed for anti-cancer activity against human

colorectal cancer (HT-29) and mammary breast cancer (MDA-MB) cell lines. Their toxicity

towards normal cell was also tested against 3T3 cell line. Compound 1 could not be tested due to

insufficient amount of the compound. The activity was observed as percentage of cell viability

after being treated with the tested compounds for 72 h with doxorubicin used as positive control

in the experiment.

The results indicated that all the tested compounds exhibited weak cytotoxic property against

HT-29 and MDA-MB cancer cell lines. Compound 2 demonstrated the strongest cytotoxicity

against HT-29 cells amongst the tested compounds with IC50 of 44.82mg/mL, while 3 showed

the strongest activity against MDA-MB cells with IC50 of 30.80mg/mL. Of all the tested

compounds, only 2 showed toxicity against normal cell line 3T3 with IC50 of 38.92mg/mL,

while the other compounds did not demonstrate any significant toxicity towards the cells,

indicated by IC50 of .100mg/mL. The cytotoxicity of the tested compounds was significantly

weaker than that of doxorubicin (IC50 ¼ 0.02mg/mL against HT-29, IC50 ¼ 0.05mg/mL against

MDA-MB).

Two isolated triterpenes were tested for anti-acetylcholinesterase (anti-AChE) activity, i.e. 2and 3. Among the isolated compounds, only the aforementioned compounds were able to be

dissolved in the mixture of dimethylsulphoxide (DMSO, solvent) and the buffer used for this

test. The anti-AChE activities of the compounds are presented as percent inhibition. The results

showed that the three compounds exhibited similar activities. They did not display any anti-

AChE activity at concentrations of 10, 50, 100, 250 and 500mg/mL. It was only when the

concentration of the compounds was 1000mg/mL that they showed some activities. Compounds

2 and 3 demonstrated similar anti-AChE activities with IC50 of 977.23 and 954.99mg/mL,

respectively. Compared with positive control used, tacrine (IC50 ¼ 25.12mg/mL), the

compounds were considered to be not active in inhibiting the activity of AChE.

3. Experimental

3.1. General experimental procedures

The spectrophotometer instruments used in this research were Perkin-Elmer Lambda 35 UV-Vis

(Waltham, MA, USA), Perkin-Elmer FT-IR (Waltham, MA, USA), Bruker NMR (Billerica,

MA, USA) 300MHz for 1H and 75MHz for 13C, and Agilent Technologies GC-MS (Santa

Clara, CA, USA). Chromatographic separation and purification were conducted using the

following adsorbents: silica gel 60 PF254 (Merck catalogue number: 1.07747.2500) for VLC,

silica gel 60 PF254 containing gypsum (Merck catalogue number: 1.07749.1000) for RC, silica

gel 60 (0.040–0.063mm) (Merck catalogue number: 1.09385.1000) for column chromatog-

raphy (CC) and silica gel 60 (0.2–0.5mm) (Merck catalogue number: 1.07733.1000) for the

sample. For thin layer chromatography, silica gel 60 PF254 (aluminium sheets) (Merck catalogue

number: 1.05554.0001) was used for analysis.

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3.2. Plant materials

The sample used in this research was the stem bark of S. macropodum which was collected from

Mount Ledang, Johor National Park, Johor, Malaysia. The plant sample was identified by a

botanist at the Forest Research Institute of Malaysia (FRIM) and a voucher specimen has been

deposited in the Herbarium of FRIM with voucher number FRI 56479.

3.3. Isolation and purification

The ground, air-dried stem bark of S. macropodum (3.75 kg) was extracted for 3 days with 10 L

of MeOH at room temperature. This extraction step was repeated five times. The methanol

extract was concentrated at 508C with rotary evaporator under reduced pressure to give 100 g of

dark-brown crude extract. The tannins were separated by dissolving the crude extract in MeOH

and then fractionating it with diethylether. The ether fraction was then concentrated again to give

35 g of tannin-less crude extract.

The crude extract was subjected to VLC with column diameter of 17 cm and eluted with the

mixtures of n-hexane and EtOAc with increasing polarity (starting with n-hexane–EtOAc, 9.5:

0.5) to give five fractions (fractions 1–5). A white yellow solid was formed during the VLC. The

solid was washed with acetone to give 2 (91mg). Fraction 2 (21 g) was subjected to VLC with

n-hexane and the polarity was increased using EtOAc (starting with n-hexane–EtOAc, 9.75:

0.25) to give eight fractions (fractions 2.1–2.8). Fractions 2.4–2.7 were combined and washed

with acetone to give 2 (13 g). Fraction 2.2 (3 g) was subjected to RC with the solvent system of

n-hexane–EtOAc (9.9:0.1) to give seven fractions (fractions 2.2.1–2.2.7). Fraction 2.2.3

(297mg) was purified using RC with n-hexane–EtOAc (9.9: 0.1) to give 3 (20mg).

Fraction 3 (6.7 g) was subjected to VLC using the n-hexane–EtOAc solvent system with

increasing polarity to give six fractions (3.1–3.6). Fraction 3.4 (268.3mg) was purified using RC

with n-hexane–EtOAc–CHCl3 (8:1:1) to give 4 (5mg). Fraction 3.6 was washed with acetone to

remove the chlorophyll and then subjected to RC using the same solvent system with 3.4 to give

additional 4 (21mg). Fraction 4 (0.9 g) was subjected to CC with sephadex as the stationary

phase and eluted with MeOH to separate the chlorophyll. The chlorophyll-less fraction was

subjected to RC with n-hexane–EtOAc–CHCl3 (7:2:1) for purification to give six fractions

(4.1–4.6). Fraction 4.3 (35mg) was purified further using RC with the same solvent system to

give 1 (4.2mg).

Malayscaphiol (1). Pale yellow amorphous solid. HR-ESI-MS [M þ H]þ at m/z: 275.0978

(calc. for C15H15O5 275.09195). IR ῡmax (KBr disc) cm21: 3435, 2962, 2926, 2850, 1734, 1643,

1557, 1262, 1032. 1H NMR (CDCl3, 300MHz): dH 4.60 (1H, dd, J ¼ 5.1, 10.8Hz, H-2a), 4.19

(1H, dd, J ¼ 10.4, 10.8 Hz, H-2b), 2.81 (1H, m, H-3), 6.36 (1H, s, H-12), 1.30 (3H,

d, J ¼ 2.4Hz, H-14), 2.23 (3H, s, H-15), 2.40 (3H, s, H-16). 13C NMR (CDCl3, 75MHz): dC72.4 (C-2), 42.2 (C-3), 193.7 (C-4), 158.9 (C-5), 139.3 (C-6), 147.4 (C-7), 116.92 (C-8),

161.1 (C-9), 117.2 (C-10), 193.7 (C-11), 116.94 (C-12), 155.4 (C-13), 12.5 (C-14), 23.5 (C-15),

8.6 (C-16).

3.4. Cytotoxicity

3.4.1. Cell lines and cell culture condition

Cancer cell lines, HT29 (colon colorectal adenocar, ATCC HTB-38) and MDAMB (mammary

gland/breast, ATCC HTB-26), were maintained in RPMI 1640 (Sigma), supplemented with 10%

of foetal bovine serum (FBS, PAA Laboratories) and 1% of penicillin/streptomycin. The normal

cell line, 3T3 (mus musculus embryo, ATCC CRL-1658) was maintained in DMEM (PAA

Laboratories), supplemented with 10% of foetal bovine serum (FBS, PAA Laboratories) and 1%

of penicillin/streptomycin. The number of cells was counted by a haematocytometer, and the

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viability of cells was determined using trypan blue reagent. Cells of 80–85% confluence were

harvested and plated onto 96 flat-bottomed well plates for experimental use. In all experiments,

the cells were incubated in a CO2 incubator at 378C with 5% CO2 overnight prior to treatment.

3.4.2. Cell viability assay: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

(MTT) assay

Stock solutions of 10mg/mL were made in DMSO and serial dilutions were made in culture

media prior to use. The final concentrations of DMSO did not exceed 0.1% (v/v), a concentration

which is non-toxic to the cells (Li et al. 2007). The cells treated with extracts, fractions and pure

compounds (0.01–100mg/mL) were incubated for 72 h after which the MTT assay was carried

out as described by Mosmann (1983), but with slight modifications. After 72 h, supernatants

were discarded and 50mL of MTT stock solution (5mg/mL) was added to each well and the

plates were further incubated for 4 h. DMSO (100mL) was added to each well to solubilise

the water-insoluble purple formazan crystals. The amount of MTT–formazan is directly

proportional to the number of living cells and was determined by measuring the optical density

(OD) at 570 nm using a microplate reader (Tecan). The percentage of cytotoxic activity

compared with the untreated cells was determined using the following equation:

Cell viability ¼ ODof treated cell

OD of control cell£ 100%:

Data generated were used to plot a dose response curve. Cytotoxic activity was expressed as

the mean concentration of the test sample required to kill 50% of the cell population. The

selectivity index (SI) of the extracts is defined as the ratio of cytotoxicity on normal cells to

cancer cells. Low SI indicates that the cytotoxic activity is probably due to cytotoxicity rather

than activity against the cancerous cells themselves (Saetung et al. 2005). In contrast, high SI

should offer the potential of safer therapy. Doxorubicin was used as the positive reference in this

study.

3.5. Anti-AChE activity

AChE and butyrylcholinesterase inhibitory activities were assessed by slightly modifying the

spectrophotometric method developed by Ellman et al. (1961). Electric eel AChE was used,

while acetylthiocholine iodide was employed as substrates of the reaction. Briefly, 125mL of

5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB) (50mM Tris–HCl, pH 8, 0.1M sodium chloride,

0.02M MgCl2·6H2O), 25mL of AChE (0.2U/mL in buffer), 25mL of test solution (samples

were dissolved in DMSO) and 50mL of buffer (50mM Tris–HCl, pH 8, 0.1% BSA) were mixed

and incubated for 30min at 258C. For controls, test solutions were replaced by the correspondingvolume of DMSO or buffer. The reaction was then initiated by the addition of 25mL of

acetylthiocholine iodide (0.25mmol/L) or the final volume was 250mL. The hydrolyses of thesesubstrates were monitored spectrophotometrically by the formation of yellow 5-thio-2-

nitrobenzoate anion, as the result of enzyme-catalysed reaction of DTNB with thiocholine,

released by the enzymatic hydrolysis of acetylthiocholine iodide, using a 96-well microplate

reader (Model 680, Biorad, USA) at a wavelength of 412 nm. The rates of the reaction were

obtained over 180 s, with a 20 s interval. Percentage of inhibition of AChE was determined by

comparison of the reaction rates of the samples relative to blank sample (DMSO in Tris–HCl

buffer, pH 8.0) using the formula (E2S)/E £ 100, where E is the activity of enzyme without test

sample, and S is the activity of enzyme with test sample. The experiments were carried out in

triplicate. Tacrine was used as reference compound.

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4. Conclusions

The phytochemical investigation of the stem bark of S. macropodum (Miq.) Beumee resulted in

the isolation of a new sesquiterpenoid named malayscaphiol (1) along with three known

compounds including lupeol (2), lupenone (3) and stigmasterol (4). Compounds 2, 3 and 4

exhibited weak cytotoxicity towards HT-29 and MDA-MB cell lines. Compounds 2 and 3 did

not display anti-acetylcholinesterase activity.

Supplementary material

Supplementary material relating to this article is available online, alongside Figure S1,

Tables S1 and S2 are available online.

Acknowledgement

The authors would like to thank the Faculty of Applied Sciences, Universiti Teknologi MARA for financingthis research project.

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