Home Physical Sciences A norterpenoid and tripenoids from Commiphora mukul: isolation and biological activity
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A norterpenoid and tripenoids from Commiphora mukul: isolation and biological activity

  • Najeeb Ur Rehman , Hidayat Hussain EMAIL logo , Husain Yar Khan , René Csuk , Ghulam Abbas , Ivan R. Green and Ahmed Al-Harrasi EMAIL logo
Published/Copyright: November 22, 2016
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Zeitschrift für Naturforschung B
From the journal Zeitschrift für Naturforschung B Volume 72 Issue 1

Abstract

Bio-guided fractionation of the guggul gum resin of Commiphora mukul HOOK using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay for the breast cancer cell line MDA-MB-231 led to the isolation of a new C17 norditerpene named myrrhanone C (1) along with two known polypodane-type triterpenes, namely, myrrhanone B (2) and myrrhanol B (3). The structures of the isolated compounds were elucidated by means of 1D (1H and 13C) and 2D (correlation spectroscopy, heteronuclear single-quantum coherence, heteronuclear multiple-bond correlation, and nuclear Overhauser effect spectroscopy) NMR spectroscopy as well as mass (electrospray ionization-mass spectroscopy) spectral analyses. Interestingly myrrhanone C (1) was able to induce a substantial decline in cell proliferation. It reduced the viability of cancer cells by almost 81% and 87% at concentrations of 50 and 100 μg mL−1, respectively. Myrrhanone B (2) and myrrhanol B (3) showed a concentration-dependent growth inhibitory effect on cancer cells, with the latter being slightly more cytotoxic than the former at both the concentrations tested. Furthermore, myrrhanone C (1) and myrrhanone B (2) showed good α-glucosidase and urease inhibition.

Keywords: anticancer activity; burseraceae; Commiphora mukul; natural products; structure elucidation

1 Introduction

Commiphora (Balsamodendron) is one of the largest genera of the family Burseraceae and contains approximately 190 species distributed in southern Arabia, Sri Lanka, and India [1]. It has been reported that the Commiphora mukul HOOK guggul gum resin has been employed in traditional medicines of India and many Arabian countries as anti-inflammatory, anti-obese, anti-coagulant, anti-bacterial, and anti-atherosclerosis agents [2]. Previous phytochemical studies of the resin of C. mukul led to the isolation of several sterols, lignans, oligosaccharides, triterpenes, sesquiterpenes, and dipenoids [1], [2], [3], [4]. Interestingly, previously isolated compounds from the C. mukul resin demonstrated analgesic and anti-inflammatory effects. Moreover, some constituents have been employed as pain killers and have demonstrated inhibitory potentials on nitric oxide production [1], [3]. Our efforts in finding the bioactive components contained in C. mukul through a bio-guided fractionation resulted in the isolation of a new compound, myrrhanone C (1), along with two known compounds myrrhanone B (2) and myrrhanol B (3). All three compounds exhibited anticancer activity against the breast cancer cell line MDA-MB-231.

2 Results and discussion

2.1 Structure elucidation

One new nor-diterpene (1) and two known polypodane-type triterpenes named myrrhanone B (2) and myrrhanol B (3) (Fig. 1) were isolated from the resin of C. mukul (see Experimental section).

Fig. 1: Structures of compounds 1–3 isolated from the resin of C. mukul.
Fig. 1:

Structures of compounds 1–3 isolated from the resin of C. mukul.

Compound 1 had the molecular formula of C18H30O3 determined from the quasimolecular ion peak at m/z=317.0 [M+Na]+ in the electrospray ionization-mass spectroscopy (ESI-MS) spectrum. The IR spectrum of compound 1 displayed absorption bands for aliphatic C–H (2815 cm−1), carbonyl (1710 cm−1), and ether (1313, 1016 cm−1) functionalities. The NMR spectra of compound 1 displayed signals for four tertiary methyl groups, namely, δH=1.39 (3H, s, H-17), δC=22.5 (C-17); δH=1.08 (3H, s, H-14), δC=26.4 (C-14); δH=1.00 (3H, s, H-15), δC=20.9 (C-15); and δH=0.88 ppm (3H, s, H-16), δC=15.5 (C-16), and one dioxygenated methine at δH=4.65 (d, J=4.2 Hz, H-13), δC 99.0 ppm (C-13). Moreover, the 1H NMR spectrum of compound 1 demonstrated two non-oxygenated methines at δH=1.50 (m, H-5) and δH=1.30 ppm (m, H-9), one methoxy at δH=3.67 ppm (3H, s), and six methylenes at δH=2.51–1.40 ppm (m).

The 13C NMR, distortionless enhancement by polarization transfer, and heteronuclear single-quantum coherence spectra of compound 1 displayed the presence of 18 carbon signals, attributed to 5 methyls (including one methoxy), 6 methylenes, 3 methines [including one oxymethine at δC=99.0 ppm (C-13)], and 4 quaternary carbons one of which is a carbonyl group δC=217.2 ppm (C-3) and another an oxygenated quaternary carbon δC=75.3 ppm (C-8). The 1H and 13C NMR data strongly support the configuration of the A and B rings of compound 1 and are furthermore in very good agreement with spectral data reported for myrrhanone B (2), also isolated from the same plant [1], [3], [5]. The particular structure in ring C of compound 1 was further established through important heteronuclear multiple-bond correlation (HMBC) correlations, namely, H-13 to C-8, C-11, and C-12; OMe to C-13; Me-17 to C-7, C-8, and C-9; H-12 to C-9, C-11, and C-13; H-11 to C-8, C-9, C-12, and C-13; Me-16 to C-1, C-9, and C-10; Me-14 and Me-15 to C-3, C-4, and C-5 (Fig. 2). In addition, the tricyclic ring structure of (1) was deduced on the basis of the 1H–1H correlation spectroscopy (COSY) (bold lines) and HMBC experiments illustrated in Fig. 2. The trans junction between rings A and B are the same as for myrrhanone B (2) and the stereochemistry at C-13 was ascertained from the nuclear Overhauser effect spectroscopy (NOESY) correlation between H-13 with H-12α. In addition, the lack of any NOESY correlations between H-13 with Me-17 or Me-16 further supported the assigned configuration at C-13. Consequently, compound 1 was identified as (3S,4aR,6aR,10aS,10bR)-3-methoxy-4a,7,7,10a-tetramethyl-octahydro-1H-benzo[f]chromen-8(4aH,9H,10bH)-one, and named myrrhanone C (1).

Fig. 2: Key COSY and HMBC correlations of myrrhanone C (1).
Fig. 2:

Key COSY and HMBC correlations of myrrhanone C (1).

The structures of two known compounds, namely, myrrhanone B (2) [1] and myrrhanol B (3) [1], were confirmed by the comparison of our NMR spectral data with the reported NMR data.

2.2 Biological activities

In order to assess the effect of different solvent extracts of C. mukul on the viability of cancer cells, MDA-MB-231 breast cancer cells were treated with different solvent extracts at concentrations of 50 and 100 μg mL−1 for 24 h (Fig. 3). It was noted that at a concentration of 50 μg mL−1 the methanolic, aqueous, and butanolic (BAMF, BAQF, and BABF, respectively) extracts were ineffective in causing any growth inhibition of MDA-MB-231 cells. Solvent extracts, namely, n-hexane (BAHF), dichloromethane (CH2Cl2), and ethyl acetate (BAEF), showed slight inhibition of cell proliferation at this concentration. At a higher concentration of 100 μg mL−1 the methanolic and aqueous extracts once again exhibited no significant effect on cell survival, whereas the rest of the solvent extracts were able to induce varying degrees of growth inhibition in cancer cells. In this case n-hexane and EtOAc extracts proved to be the most potent (approx. 60% cytotoxicity). This suggests that these extracts, which show cytotoxic effects on breast cancer cells, possess one or more of the bioactive constituent(s) having anti-proliferative properties.

Fig. 3: The effect of n-hexane (BAHF), CH2Cl2 (BADF), aqueous (BAQF), MeOH (BAMF), EtOAc (BAEF), and n-butanol (BABF) extracts on the proliferation of MDA-MB-231 breast cancer cells in culture.
Fig. 3:

The effect of n-hexane (BAHF), CH2Cl2 (BADF), aqueous (BAQF), MeOH (BAMF), EtOAc (BAEF), and n-butanol (BABF) extracts on the proliferation of MDA-MB-231 breast cancer cells in culture.

Subsequently, myrrhanone C (1), myrrhanone B (2), and myrrhanol B (3) isolated from the n-hexane extract were tested for their anti-proliferative effects on cancer cells. Results of this cytotoxicity assay (Fig. 4) demonstrate that treatment of MDA-MB-231 cells with each of these isolated compounds resulted in a significant reduction in cell survival. Myrrhanone B (2) and myrrhanol B (3) showed a concentration-dependent growth inhibitory effect on cancer cells, with the latter being slightly more cytotoxic than the former at both the concentrations tested. However, the new compound myrrhanone C (1) was able to induce a substantial decline in cell proliferation. It reduced the viability of cancer cells by almost 81% and 87% at concentrations of 50 and 100 μg mL−1, respectively (Fig. 4). Hence, myrrhanone C (1) emerges as the best cytotoxic compound toward breast cancer cells compared to the other two known compounds, myrrhanone B (2) and myrrhanol B (3), isolated from C. mukul. The reason for this greater cytotoxic activity of myrrhanone C (1) may lie in its structure, which differs from myrrhanone B (2) and myrrhanol B (3) both of which have an aliphatic carboxylic acid chain attached at C-12.

Fig. 4: The effect of myrrhanone C (1), myrrhanone B (2), and myrrhanol B (3) on the proliferation of MDA-MB-231 breast cancer cells in culture.
Fig. 4:

The effect of myrrhanone C (1), myrrhanone B (2), and myrrhanol B (3) on the proliferation of MDA-MB-231 breast cancer cells in culture.

Thus, myrrhanone C (1), which has been shown to possess potent cytotoxic activity against breast cancer cells in culture, may possibly exhibit similar effects on other types of cancer cell lines. However, the mechanisms of action of this compound need to be further studied using different cancer cell lines and in vivo tumor models, before being considered as a potential lead for the future development of novel anticancer drugs.

Different extracts of C. mukul and pure compounds 1–3 were also tested for DPPH antioxidant, urease enzyme inhibition, α-glucosidase enzyme inhibition activities in which case only the methanolic extract demonstrated weak antioxidant activity with 40% inhibition (Table 1). On the other hand, myrrhanone C (1) and myrrhanone B (2) showed good α-glucosidase (1: 50%; 2: 40%) and urease inhibition (1: 40%; 2: 65%). Interestingly, the reduced myrrhanol B (3) was inactive.

Table 1:

α-Glucosidase, urease, and antioxidant activities of different extracts and compounds 1–3.

Fractions/Compoundsα-GlucosidaseaUreaseaAntioxidant
n-ButanolNAbNANA
AqueousNANANA
n-HexaneNANANA
Methanol (whole)NANA40±2.00
DichloromethaneNANANA
Ethyl acetateNANANA
150±2.0040±1.00NA
240±1.0065±1.00aNA
690±2.50c
3NANANA

a% inhibition (1 mg mL−1).

bNA: not active.

cIC50 (μg mL−1).

3 Experimental section

3.1 General

The 1H and 13C NMR spectra were recorded on Bruker NMR spectrometers operating at 600 MHz (150 MHz for 13C). The chemical shift values are reported in ppm (δ scale) and the coupling constants J are given in Hz. IR spectra were recorded on a Bruker, ATR-Tensor 37 spectrophotometer. Optical rotations were measured on a KRUSS P P3000 polarimeter (A. Kruss Optronic, Germany). ESI-MS was recorded on a Waters Quattro Premier XE Mass Spectrometer (Waters, Milford, MA). For thin-layer chromatography (TLC), pre-coated aluminum sheets (silica gel 60F-254, E. Merck) were used. Visualizations of the TLC plates were achieved under the UV light at 254 and 366 nm and also by spraying with the ceric sulfate reagent.

3.2 Sample collection and identification

The resin of C. mukul was purchased from Souq (Nizwa, Sultanate of Oman), identified by the Plant Taxonomist at the Department of Biological Sciences and Chemistry, University of Nizwa, Oman. A voucher specimen (No. CMS-03/2014) was deposited in the herbarium of the Department of Biological Sciences and Chemistry.

3.3 Extraction and isolation

The resin of C. mukul (550 g) was finely minced and extracted with methanol (3.5 L) at room temperature (3 times×3 days). Evaporation of the solvent under reduced pressure gave the methanol extract (438.5 g). Fractionation of this residue on the basis of increasing polarity of organic solvents produced the following subfractions: n-hexane (31.8 g), CH2Cl2 (270.8 g), ethyl acetate (129.5 g), n-butanol (3.3 g), and aqueous (4.0 g). The n-hexane fraction (31.8 g) was subjected to silica gel column chromatography (70–230 mesh; Merck), using the n-hexane–CH2Cl2 (70:30) system as an eluent to give the new compound 1.

In order to check if myrrhanone C is an artifact resulting from the extraction with methanol, the resin of C. mukul was extracted with ethanol at room temperature for 20 days. The crude ethanol extract was subjected to column chromatography and careful isolation resulted in the purification of myrrhanone C (1). In conclusion, this experiment confirmed that myrrhanone C is not an artifact.

3.3.1 Myrrhanone C (1)

White solid. – [α]D25=+ 9.1 (c=1.00, MeOH). – IR (KBr): 2815, 1710, 1313, 1016 cm−1. – 1H NMR (600 MHz, CDCl3, TMS): δ=4.65 (d, J=4.2 Hz, 1H, H-13), 3.67 (s, 3H, 13-OMe), 2.51 (m, 1H, H-2a), 2.45 (m, 1H, H-2b), 1.90 (m, 2H, H-1a, H-12a), 1.80 (m, 1H, H-7a), 1.70 (m, 1H, H-12b), 1.67 (m, 1H, H-6a), 1.59 (m, 1H, H-11a), 1.50 (m, 1H, H-5), 1.46 (m, 2H, H-6b, 11b), 1.44 (m, 1H, H-1b), 1.40 (m, 1H, H-7b), 1.39 (s, 3H, H-17), 1.30 (m, 1H, H-9), 1.08 (s, 3H, H-14), 1.00 (s, 3H, H-15), 0.88 (s, 3H, H-16). – 13C NMR (150 MHz, CDCl3): δ=217.2 (C-3), 99.0 (C-13), 75.3 (C-8), 57.2 (C-9), 54.6 (C-5), 55.2 (13-OMe), 47.2 (C-4), 41.5 (C-7), 37.9 (C-1), 36.5 (C-10), 33.7 (C-2), 32.5 (C-12), 26.4 (C-14), 22.5 (C-17), 20.9 (C-15), 20.4 (C-11), 15.5 (C-16), 14.6 (C-6). – MS ((+)-ESI): m/z=317.0 [M+Na]+ (C18H30NaO3).

3.4 Anticancer activity

3.4.1 Cell line and reagents

Breast cancer cell line MDA-MB-231 was maintained in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA). The media was supplemented with 10% fetal bovine serum and 1% antimycotic antibiotic (Invitrogen). Cells were cultured in a 5% CO2-humidified atmosphere at 37°C. Stock solutions of compounds 1–3, extracts, and doxorubicin were made in DMSO at a final concentration of 2 mg mL−1 and were always prepared freshly just prior to the experiments.

3.4.2 Cell growth inhibition studies by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay

Cells were seeded at a density of 1×104 cells per well in 96-well microtiter culture plates. After overnight incubation, normal growth medium was removed and replaced with either fresh medium (untreated control) or different concentrations of compounds 1–3, and extracts in growth medium were diluted from a 10 mg mL−1 stock. After 24 h of incubation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg mL−1 in phosphate-buffered saline) was added to each well and incubated further for 4 h at 37°C. Upon termination, the supernatant was aspirated and the MTT formazan, formed by metabolically viable cells, was dissolved in a solubilization solution containing DMSO (100 μL) by mixing for 5 min on a gyratory shaker. The absorbance was measured at 540 nm on an Ultra Multifunctional Microplate Reader (Bio-Rad, USA). Each treatment had four replicates wells and each experiment was repeated at least three times.

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Received: 2016-3-14
Accepted: 2016-6-7
Published Online: 2016-11-22
Published in Print: 2017-1-1

©2017 Walter de Gruyter GmbH, Berlin/Boston

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  3. Invariom-based comparative electron density studies of iso-sildenafil and sildenafil
  4. A norterpenoid and tripenoids from Commiphora mukul: isolation and biological activity
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  11. Hydrogenium-bis-hydrogensulfate anions adjacent to [S2O7]2− in Rb3[S2O7][H(HSO4)2]: a structural evidence of the increasing acidity of polysulfuric acids with growing chain length
  12. High-pressure synthesis and crystal structure of In3B5O12
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https://doi.org/10.1515/znb-2016-0062
Keywords for this article
anticancer activity; burseraceae; Commiphora mukul; natural products; structure elucidation

Articles in the same Issue

  1. Frontmatter
  2. In this Issue
  3. Invariom-based comparative electron density studies of iso-sildenafil and sildenafil
  4. A norterpenoid and tripenoids from Commiphora mukul: isolation and biological activity
  5. Formiside and seco-formiside: lignin glycosides from leaves of Clerodendrum formicarum Gürke (Lamiaceae) from Cameroon
  6. Morpholinium hydrogen sulfate (MHS) ionic liquid as an efficient catalyst for the synthesis of bio-active multi-substituted imidazoles (MSI) under solvent-free conditions
  7. A highly selective fluorescent chemosensor based on naphthalimide and Schiff base units for Cu2+ detection in aqueous medium
  8. Synthesis and crystal structure of a cyanido-bridged copper(II)–silver(I) bimetallic complex containing a trimeric {[Ag(CN)2]−}3 anion, [Cu(Dach)2-Ag(CN)2-Cu(Dach)2][Ag(CN)2]3 (Dach=cis-1,2-diaminocyclohexane)
  9. Two highly acetylated sterols from the marine sponge Dysidea sp.
  10. New oxaphenalene derivative from marine-derived Streptomyces griseorubens sp. ASMR4
  11. Hydrogenium-bis-hydrogensulfate anions adjacent to [S2O7]2− in Rb3[S2O7][H(HSO4)2]: a structural evidence of the increasing acidity of polysulfuric acids with growing chain length
  12. High-pressure synthesis and crystal structure of In3B5O12
  13. The high-pressure phase of CePtAl
  14. Note
  15. Synthesis and structural characterization of Mn(II) and Cu(II) complexes with bis(4-(1H-imidazol-1-yl)phenyl)methanone ligands
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