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Anti-proliferative activity of a novel tricyclic triterpenoid acid from Commiphora africana resin against four human cancer cell lines

Applied Biological Chemistry volume 63, Article number: 16 (2020) Cite this article

Abstract

Myrrh, a resin derived from the damaged bark of Commiphora genus, has traditionally been used for treatment of various human diseases, such as amenorrhea, ache, tumors, fever, and stomach pains. In spite of this widespread use of the myrrh in Ethiopia, the pharmacological activity and chemical composition have not been studied in detail. A new tricyclic triterpene acid (3S,4S,14S,7E,17E,21Z)-3,30-dihydroxypodioda-7,17,21-trien-4-carboxylic acid (commafric A) has been isolated from a crude methanolic extract of Commiphora africana (A. Rich.) Engl. resin along with the known pentacyclic triterpene α-amyrin. The structure of commafric A was characterized using different spectroscopic techniques such as 1D and 2D NMR, IR, and VCD combined with computations. The anti-proliferative activity of both isolated compounds was evaluated using SRB based colorimetric cellular assay against four human cancer cell lines. Etoposide was used as a positive control. Commafric A showed significant anti-proliferative effects against non-small cell lung cancer (A549) with IC50 values of 4.52 μg/ml. The pentacyclic triterpene α-amyrin showed a weak anti-proliferative activity against A2780 (ovarian cancer), MIA-PaCa-2 (pancreatic cancer), and SNU638 (stomach cancer) cell lines tested with IC50 values ranging 9.28 to 28.22 μg/ml. Commafric A possessed anti-proliferative activity against non-small cell lung cancer (A549), which suggests that commafric A has potential to be further optimized being a lead compound in the search for new drugs against cancer diseases.

Introduction

Natural products are very inspiring in the drug discovery process. Aziz et al. [1] reviewed several drugs that have been obtained from different plant sources. Some of them can be obtained from Commiphora africana (A. Rich.) Engl. (Burseraceae), which is a bush about 1.5 m tall and is wide-spread in African countries, such as Sudan, Ethiopia, Eritrea, Somalia, Kenya, Uganda, and Mozambique [2]. Roots, bark and leaves are traditionally used to treat measles, hyperlipidemia, and cardiovascular disorders [3, 4]. The leaves are also used as a sedative and soporific [3], and the resin is used for antiseptic washes and baths to treat skin infections, sores, and leprosy [5]. Ethnobotanical information has indicated that bark, resin, and leaves of C. africana are used to treat snakebite, skin wounds, tumor, stomachache, and as anti-ticks [6]. A fraction obtained after partitioning the crude ethanolic extract roots of C. africana exhibited a promising in vitro antimicrobial activity [4].

The resin of C. africana contains betulin [7], which has been shown to have antitumor activity, especially in combination with cholesterol [8]. Betulinic acid derivatives can inhibit HIV-1 [9]. Other Commiphora species have also provided pharmaceutically interesting compounds. Furanoeudesma-1,3-diene, the major compound of myrrh [10, 11], has been reported to exhibit analgesic activity in mice [12].

Previously, we have described oxygenated furanosesquiterpenes, curzerenone, and furanodienone from the resins of C. sphaerocarpa (Chiov.), C. holtziana (Engl.), and C. kataf [11, 13, 14]. Messina et al. [15] has shown that furanodienone has anti-inflammatory, antimicrobial, and anticancer activities. Additionally, the cytotoxicity of nordammarane triterpenes, such as vibusambucin A, 12β-hydroxy-3,15-dioxo-20,21,22-23,24,25,26,27-octanordammarane, and hupehenol A against KB, HepG-2, LU-1, and MCF-7 cell lines has been reported [16]. The resins of C. confusa contain triterpenes of the dammarane type [17] with moderate cytotoxic activity against HepG2 [18]. From the resin of C. erlangeriana we have previously isolated polygamain-type lignans named erlangerin A, and erlangerin B, and two lignans related to podophyllotoxin, erlangerin C, and erlangerin D [19]. The effects of the erlangerins C and D are closely related to those of podophyllotoxin. They induce concentration-dependent cytotoxicity in the murine macrophage cells (RAW 264.7) and a cytostatic effect in HeLa, EAhy926, and L929 cell lines [20].

In spite of the use of Commiphora africana in traditional medicine, to the best of our knowledge, there has been no study aimed at isolation and characterization of anti-proliferative constituents of this plant. As part of the exploration of the pharmacological potential of the resins from Commiphora genus, we herein report the isolation, structural elucidation, and anti-proliferative activity screening of a new oxygenated podioda-7,17,21-triene [21, 22] and α-amyrin obtained from C. africana.

Materials and methods

Plant material

Resins and botanical specimens of C. africana were collected from district of Yabello, Borena zone, Ethiopia in December 2016 and identified by a botanist Shambel Alemu at the Biology Department, Addis Ababa University. A voucher specimen has been deposited at the National Herbarium (number: 072801) of Addis Ababa University, Addis Ababa, Ethiopia. This tree is known by its vernacular name hammesa Dihro (Afaan Oromo). There is no permission required to collect samples from wild to investigate their chemical composition and evaluate their anti-proliferative effects.

Extraction and isolation

The resin (322.5 g) of C. africana was air dried and extracted with methanol (1 L × 3) for 3 days at 25 °C. The solvent was removed in vacuo to yield a yellow extract (151.2 g, 46.82%). The extract (80.0 g) was suspended in water (800 ml) and successively partitioned with n-hexane (800 ml × 3), chloroform (800 ml × 3), EtOAc (800 ml × 3), and n-BuOH (800 ml × 3) yielding an n-hexane (33.39 g, 40.22%), a chloroform (41.26 g, 49.7%), an EtOAc (0.11 g, 0.132%), and an n-butanol fraction (0.074 g, 0.093%). Part of the n-hexane fraction (7 g) was flash chromatographed on a silica gel column using n-hexane/EtOAc (7:3) as an eluent furnishing commafric A (1) (430 mg), and α-amyrin (2) (22 mg) (Fig. 1).

Fig. 1
figure 1

Chemical structures of compounds isolated from C. africana and a reference compound podioda -7,17,21-trienol (3)

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General experimental procedure

Melting points are uncorrected. TLC was performed on precoated plates (Silica gel 60 F254, Merck) using n-hexane/EtOAc/MeOH (10:10:0.1) as a developing solvent and with vanillin-H2SO4 as the detecting reagent. CC was performed on silica gel. IR spectra were measured on a Perkin Elmer 1600 instrument using KBr tablets. Optical rotation was measured on a Perkin-Elmer 241 polarimeter. UV spectra were recorded on a Shimadzu UV–VIS spectrophotometer. LC-Mass was acquired on Waters ACQUITY H-CLASS Liquid Chromatograph/SQD2 Mass Spectrometer; Injection mode: FIA, Eluent: Methanol/Water (10 mM ammonium acetate) = 70/30 flow rate: 0.3 ml/min, Ionization mode: ESI (positive), Capillary voltage: 3 kV, Cone voltage: 30 V. HRESIMS data were obtained on Waters ACQUITY UPLC I - Class Liquid Chromatograph/TQ-S micro Tandem Mass-Spectrometer, Ionization mode: electrospray ionization (ESI), Mass Analyzer, JMS-700 Mass High Resolution Mass Spectrometer (Jeol LTD, Tokyo, Japan): Tandem (triple) quadrupole at mass range of 5–2000 m/z and scan speed 15,000 Da/sec.

Nuclear magnetic resonance spectroscopy

1H and 13C NMR data for commafric A (1) was acquired on an Avance III HD spectrometer (BrukerBioSpin GmbH, Germany) equipped with an inverse detected TCI cryoprobe with a cryogenic enhancement for 1H, 2H, and 13C, operating at 600 MHz for 1H. All spectra were recorded using TopSpin 3.5pl7 at 298 K in dichloromethane-d2 and chloroform-d1, using gradient selected and adiabatic inversion versions of pulse sequences where applicable. Analysis of NOE buildup for compound 1 was performed by complete relaxation matrix analysis using Mspin 2.3.2-694 (MestreLab Research S. L., Spain). The results are presented as Boltzmann averaged NOE enhancement across the conformational ensemble generated for analysis of IR and VCD section. The sample for anisotropic NMR measurements (10 mg) was dissolved in CDCl3 with 0.03% TMS to which increasing amounts of poly-γ-benzyl-l-glutamate (PBLG, 150,000–300,000 Da, Sigma-Aldrich CAS 25014-27-1) was added to yield weight-to-volume ratios of 7.9%, 12.1%, and 16.2%. For compound 1 1D carbon, CLIP-HSQC, IPAP-HSQC, and J-res carbon was acquired to extract the 1JCH coupling constants and anisotropic carbon shifts. The data treatment is thoroughly described in additional file.

Experimental vibrational circular dichroism (VCD) spectra

VCD and absorption spectra of 1 were measured in CDCl3 solutions (~ 1 mg/100 μl) using a BaF2 cell of an optical path of 50 μm and a BioTools ChiralIR-2X instrument. Spectra of pure CDCl3 solvent were subtracted as a baseline; the accumulation time was ~ 12 h using blocks of 1200 scans and 4 cm−1 resolution.

Computations

Using the Gaussian suite of programs [23] a systematic conformer search of compound 1 was performed considering the three torsional angles in the vicinity of the tricyclic system. Other C–C–C–C dihedral angles were all-trans and all-extended (180°) at the beginning of the optimizations and left to optimize without constraints. The B3LYP [24] PCM [25] (chloroform)/6–31 ++G** level was used for all quantum chemistry. For stable conformers thus obtained the magnetic field perturbation theory [26, 27] was used to simulate IR and VCD intensities; final spectra were obtained as a Boltzmann average, using a convolution with Lorentzian lines (FWHM = 10 cm−1). Since the consideration of the full molecular flexibility was not possible with our computational means, atomic axial tensors of the linear side chain (from carbon number 16) were deleted for VCD generation. The chain itself is not chiral and thus supposedly the error introduced by this approximation is small, whereas the dominant signal from the chiral more rigid molecular part may help to assign the absolute configuration. Alternated simulations of the spectra based on molecular dynamics were attempted as well, but did not provide results significantly different from the limited conformer model. Boltzmann-averaged isotropic shielding and spin–spin coupling constants were computed at the same level as for VCD.

Tumor cell lines

The cell lines used in this study were the human non-small cell lung cancer cell lines (A549), ovarian cancer cell line (A2780), pancreatic cancer cell line (MIA-Paca-2), and stomach cancer cell line (SNU-638), and they were maintained using RPMI1640 cell growth medium (Gibco, Carlsbad, CA), supplemented with 5% fetal bovine serum (FBS) (Gibco), and grown at 37 °C in a humidified atmosphere containing 5% CO2 [28]. All the cancer cells used were adapted for 6 months at least to the RPMI1640 medium at Korea Research Institute of Chemical Technology, then used in the anti-proliferative assays. Cells were exposed to the isolated compounds at concentrations ranging from 0.1 to 30 μg/ml.

Anti-proliferative assessment

All experiments were conducted by the NCI’s protocol [28]. Experimental cultures were placed in 96-well microtiter plates (Corning) containing 0.15 ml of growth medium per well with a cell density of 5 × 103 (A549, A2780, and MIA-Paca-2) and 1 × 104 (SNU-638). The culture was incubated at 37 °C and humidified by 5% CO2 for 1 day. Then, media were aspirated off and added the test material in triplicate which was dissolved in media at varying concentrations. In case of necessity, the test material was dissolved in small amount of dimethyl sufoxide (DMSO), but the final concentration of DMSO in the medium did not exceed 0.5%. The culture was incubated for additional 3 days. After 3 days of continuous drug exposure, the medium was removed by flicking plates over a sink. The cells attached to the plastic substratum were fixed by gently layering with 0.1 ml of cold 10% trichloroacetic acid (TCA). Incubation at 4 °C for 1 h was followed by wash with tap water five times to remove TCA solution. After being washed and dried at RT overnight, TCA fixed cells were stained with 0.1 ml of 0.4% sulforhodamine B (SRB) in 1% acetic acid per well for 30 min. At the end of the staining period, SRB supernatant was removed and the remaining cells were rinsed with 1% acetic acid five times. It was dried until no standing moisture was visible, and then the bound dye in cells was extracted with 0.1 ml of l0 mM unbuffered Tris base (pH 10.5) per well by stirring on a gyratory shaker for 5-10 min, followed by measured the optical density at 520 nm by MR700 microplate reader (Dynatech Laboratories). Antitumor activity of the test material at varying concentrations was estimated as the net growth  % of cells compared with that of control (without test material, net growth = 100%). The dose–response curves of test material were constructed and the IC50 value was calculated as the concentration of the test material that caused 50% inhibition of cell growth against the cancer lines A549, A2780, MIA-PaCa-2, and SNU638. Etoposide, a standard drug was used as a positive control. IC50 values of etoposide were compared with those for crude extract, n-hexane fraction, and isolated compounds.

Results

Isolation of compounds

Flash column chromatography of the n-hexane fraction using n-hexane/EtOAc (7:3) as an eluent furnished two pure compounds: commafric A (1) the most abundant isolated molecule and the known compound α-amyrin (2), were obtained as white solids. The structures of the isolated compounds were elucidated employing 13C and 1H-NMR as well as 2D-NMR, IR, FT-ESI–MS mass fragmentation pattern, and comparing with literature data reported for structurally related compounds. The 1H NMR and 13C NMR data of compound 2 agreed with corresponding published data for α-amyrin (2) [29,30,31].

Characterization of compound 1

Compound 1 was obtained as a white powder (mp 126–127 °C), with [α]\( _{D}^{22} \): +4o (c 1.0, CHCl3) and its molecular formula was established on the basis of a high resolution positive-ion mode HR-ESI-MS2 scanned from 170.00 to 600.00 m/z. The molecular formula was established as C30H48O4 on the basis of the positive-ion mode HRESIMS data due to Na adduct formation (m/z 495.3442 [M+Na]+, calcd. 495.3445) and NMR data indicating seven degrees of unsaturation (Additional file 1: Fig. S1). The two peaks with less intensity at m/z 473.36 and m/z 455.3515 corresponds to protonation of the molecule [M+H]+and protonation of a dehydrate molecule respectively (Additional file 1: Fig. S1).

The UV spectrum of 1 (EtOH) exhibited an absorption band at 209 nm indicating the absence of conjugated double bonds. The experimental IR-spectrum agrees with the simulated spectrum of commafric A (Fig. 2); the most prominent features comprise free (3607 cm−1) and hydrogen-bonded (3400 cm−1) OH stretching bands, split (2956/2872 cm−1) CH stretching signal indicating the presence of both sp2 and sp3 carbons, and the C=O stretching band (1695 cm−1).

Fig. 2
figure 2

Calculated (upper panel, raw DFT frequencies) and experimental (lower panel) IR spectra of commafric A

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1H NMR resonances (Table 1) of 1 included two near triplets at δH 5.30 and 5.04, and one doublet of a triplet at δH 5.16, suggesting the presence of three olefinic protons. Two geminally coupled protons appearing as doublets at δH 4.17 and 4.07 indicated the presence one oxy methylene moiety. A signal integrated as one proton at δH 4.02 suggested the presence of an oxy methine group. Two moderately deshielded methyl signals were observed (1.57 and 1.77 ppm), indicating that two methyls are attached to sp2 hybridized carbons (see Additional file 1: Fig. S2). Additionally, the compound has four aliphatic methyl signals (0.75, 0.86, 0.91, and 1.18).

Table 1 NMR parameters of commafric A in CD2Cl2:1H (600 MHz) and 13C (150 MHz) NMR, HMBC, H2BC, ADEQUATE-1,1, and ROESY
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The 13C NMR (Table 1 and Additional file 1: Fig. S3) and multiplicity edited HSQC/DEPT confirmed the presence of thirty carbons, of which seven were quaternary, ten methylene, seven methine, and six methyl groups. A set of 2D NMR experiments, HSQC, HMBC, H2BC, HSQC-TOCSY, DQF-COSY, and 1,1-ADEQUATE (Additional file 1: Fig. S4–S9), was used to establish the molecular framework of commafric A as a tricyclic triterpenoid with structural similarity to podioda-7,17,21-trienol [21]. All carbon–carbon connections were unambiguously confirmed by 1,1-ADEQUEATE, except for the two quaternary to quaternary carbon bonds (C4 to C23 and C8 to C13), which did not correlate in this pulse sequence because of the absence of protons on both sides of the C–C bond (Fig. 3b). The C23 carboxyl group could be placed by HMBC correlations to H3, H5, and H24 from C23, while the C8 to C13 bond was confirmed by HMBC correlations between H26 and H12, and C8. The location of the hydroxymethylene group as well as the position of the 21, 22 double bond was confirmed by 2D HMBC (Fig. 3a), which showed three bond couplings between the olefinic proton H21 (δH 5.30) and the carbons at the C29 methyl group (δ21.4) and the C30 hydroxymethylene carbon (δC 61.5). Furthermore, H2BC two-bond couplings appeared between C22 and Me29 and hydroxymethylene protons H30′/″ (Fig. 3a).

Fig. 3
figure 3

2D NMR correlations through bonds, a COSY, HMBC and, b 1,1-ADEQUATE; and through space, c NOESY/ROESY of commafric A

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Determination of the relative configuration

Based on 3JCH couplings measured by selective CLIP-HSQMBC [32], as well as NOESY/ROESY analysis, the C17/C18 configuration was determined as E (3JC28H17 = 8.0 Hz and 3JC19H17 = 6.0 Hz) and the C21/C22 configuration was determined as Z (3JC29H21 = 6.0 Hz and 3JC30H21 = 9.3 Hz, Additional file 1: Figs. S10–S12). These conclusions are based on the fact that the trans-3JCH is expected to be stronger than the corresponding cis-3JCH. This was also consistent with the direct observations of the presence of the NOEH21H29 and ROEH21H29correlations, and the absence of the NOEH21H29 and ROEH21H29 correlations. The Z-configuration of the C21 to C22-double bond is also consistent with the 13C NMR chemical shifts of C21, C22, C29, and C30 in previously reported compounds with similar side chain configurations [33, 34].

The relative configuration of the tricyclic system was established by NOESY and ROESY correlations (Additional file 1: Fig. S12) supplemented with some 3JHH and 3JCH couplings. Most notably, an N/ROE correlation trace between H3–H5–H9–H11″–H12″ place these protons below the ring plane while the trace between H24–H25–H11′–H12′–H26 place these above the ring plane. The key correlations are displayed in Fig. 3c. The configuration at the C14 stereocenter could not be unambiguously determined from the N/ROE correlations without a full conformational analysis because of relatively free rotation about the C13–C14 bond.

Determination of the C14 configuration

Vibrational circular dichroism spectroscopy

In order to relate the measured VCD spectra to the structure, theoretical VCD spectra were simulated computationally. The theoretical modelling focused on the tricyclic system supposedly dominating in VCD spectrum, because a full conformer analysis (> 106 possible conformers) was not possible with available computational means. A reduced ensemble of 27 conformers was generated by 120° rotations around the C13–C14, C4–C23, and C3–O3 bonds only, and relative conformer energies were obtained for both the R and S C14 isomers. The molecular tail (C14–C30) was kept in the extended conformation and its contribution to VCD was not considered. The equilibrium geometries and spectral properties were calculated using the Gaussian software [24] and the B3LYP/6-311 ++G**/PCM (CHCl3) level of theory, relative enthalpies were used for property averaging. The simulation reproduced some spectral features observed experimentally (Fig. 4), although the theoretical R and S VCD sign patterns of 1 are similar. The 1150–1000 cm−1 spectral region is however predicted to be significantly different for the R- and S configurations, and comparison of the calculated and experimental VCD signs and relative intensities for this region strongly suggests the S configuration on C14.

Fig. 4
figure 4

VCD spectra calculated for the R and S C14 isomers (upper and middle panels, frequency scaled by 0.967) and the experiment (lower panel). Experimental wavenumbers are denoted in black, and selected bands are assigned identifiers denoted in red. Positive experimental bands are traced with red lines and negative experimental bands are traced with blue lines

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Isotropic NMR parameters

For a more detailed NMR data analysis of distances and coupling constants localized near the C14, only the three Boltzmann averaged rotamers around the C13–C14 bond were used. The H14 proton is preferentially pointing towards the edge of the ring system (and H-7) in both the R- and the S C14 configurations (Table 2). This was problematic for using the otherwise very informative 3JCH couplings originating from the only proton (H14) directly attached to the rotating C13–C14 bond since the dihedral angles formed between the H14–C14 vector and the C13–C8, C12, C26 vectors will be very similar for R- and S. The predicted differences between the two configurations were smaller than 0.5 Hz. The experimental couplings (Table 3) are in agreement with the calculated rotamer populations of both configurations. These couplings were very challenging to measure accurately experimentally as the signal of H14 overlaps with that of H15s. That made it necessary to use heavily chemical shift filtered selective methods and there is unavoidable phase modulation from J-coupling. Coupling sums were utilized because of significant second order/phase contributions. For all these reasons, the experimental error is estimated to at least 1 Hz.

Table 2 Calculated C13–C14 angular distribution (p, %), for the C14 enantiomers
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Table 3 Calculated and experimental 3JCH scalar couplings for the C14 enantiomers
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Furthermore, the possibility to determine the relative configuration from NOE buildup rates involving the C27 methyl group were investigated. The C27 methyl group is predicted to be predominantly anti to the C26 methyl in the S-configuration and gauche to the C26 methyl in the R-configuration. NOE buildups are intrinsically difficult to quantify for flexible molecules due to the fact that they depend on the distance as r−6. A scarcely populated rotamer can contribute significantly to the observed NOE if the two protons are positioned very close to each other. The measured NOEs around and across the C13–C14 bond (Additional file 1: Fig. S13) are not conclusive alone as both theoretical configurations result in r2 values in the order of 0.7. However, the most important NOE between H26 and H27 that is expected to be the most sensitive to the C14 configuration indicates the S-configuration. The weighted experimental NOE was determined to η = 0.0054 (corresponding to r = 2.9 Å), i.e., closer to the value predicted for the S-model (η = 0.0032, r = 3.2 Å), than that predicted for the R-model (η = 0.0144, r = 2.5 Å).

Confirmation of the C14 configuration by anisotropic NMR parameters

Anisotropic NMR parameters have emerged as powerful tools in structural elucidation [35, 36]. Residual dipolar coupling (RDC) depend on the relative orientation of the 13C-1H bond vectors, while residual chemical shift anisotropy (RCSA) depend on the relative orientations of the carbon chemical shielding tensors. When combined, they can provide the configuration of stereogenic centers that are difficult to establish by traditional methods. A method described by Liu et al. [35] utilizing poly(γ-benzyl-l-glutamate) (PBLG) to form a liquid crystal that induces anisotropy in chloroform-d1 was employed in the present study. 13C residual chemical shifts were referenced to TMS, and combined with RDC data from HSQC-IPAP spectra (Additional file 1: Figs. S14–S16, Tables S1 and S2). In the comparison of the experimental and theoretical anisotropic parameters for the commafric A the S-model provided lower quality factors (Q = 0.19) than the R-model (Q = 0.33, Fig. 5). Based on the anisotropic parameters, together with the VCD and NOE data, we conclude that commafric A’s stereocenter C14 has the (S)-configuration.

Fig. 5
figure 5

Stereochemical differentiation between the aR- and bS configurations of C14 in commafric A using RDC and RCSA data collected in PBLG, showing the best agreement between experimental and theoretical data for the S configuration

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Based on the above experimental evidence the structure of the novel compound commafric A was elucidated as (3S,4S,14S,7E,17E,21Z)-3,30-dihydroxypodioda-7,17,21-trien-4-carboxylic acid.

In vitro anti-proliferative effect of the MeOH extract, n-hexane fraction, and isolated compounds

The anti-proliferative effects of the crude MeOH extract, n-hexane fraction, and isolated compounds commafric A and α-amyrin were tested for four cancer cell lines, A549 (non-small cell lung cancer), A2780 (ovarian cancer), MIA-PaCa-2 (pancreatic cancer), and SNU638 (stomach cancer). The crude methanol extract showed a strong anti-proliferative activity against A549 (IC50 = 3.55 μg/ml) (Table 4, Additional file 1: Fig. S17). The crude extract exhibited weaker activity against A2780, SNU-638, and MIA-Paca-2 compared with A549 cell lines. Among the cell lines tested the methanol crude extract showed strong effect on A549 cell lines (3.55 μg/ml). The n-hexane fraction exhibited significant inhibition of cell proliferation on the all four cell lines with dose dependent relationship in vitro. However, it was less sensitive and had a weaker net growth effect on A549 cell lines compared with the crude methanol extract (Table 4, Additional file 1: Fig. S17).

Table 4 IC50 values of MeOH extract, n-hexane fraction, and isolated compounds of resin of Commiphora africana against four cancer cell lines using SRB assay
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Discussion

A number of bioactive compounds were previously reported from different Commiphora species. In this study, the n-hexane fraction of C. africana resin yielded a new compound commafric A. Cancer, cardiovascular diseases, chronic respiratory disorders and diabetes are the primary cause of deaths worldwide. The knowledge of herbal medications and anticancer activity tests assist in the development of important anticancer drugs [37].

In our study for in vitro anti-proliferative activity, we found that the crude extract and related n-hexane fraction is lower than 30 μg/ml, which is within the limit of criteria set by the American National Cancer Institute for further purification [38]. For compound 1, a moderate anti-proliferative activity was observed for the A549 cancer cell lines, with IC50 value of 4.52 μg/ml. This is comparable with the activity of the crude extract and more than twice better than for α-amyrin. On the other hand, compound 1 showed weak anti-proliferative effects > 9 μg/ml for the other cell lines. The compound exhibited about twice higher activity on MIA-PaCa-2 compared with the methanol extract. The presence of carboxylic acid or terminal hydroxyl group in the compound might be the reason for its bioactivity. α-Amyrin (2) having an ursane skeleton showed weaker activity on A549, A2780, and SNU638 cell lines compared with both compound 1 and the crude methanol extract. Compounds with similar skeleton to α-amyrin such as ursolic acid and its derivatives showed strong anti-proliferative activity against ovarian carcinoma, pancreatic carcinoma, prostate cancer, cervical carcinoma, hepatic cancer, breast cancer, colorectal cancer, leukemia, neuroma, and colon adenocarcinoma [39]. The weak activity of α-amyrin might be due to lack of carboxylic acid group. According to the literature on α-amyrin pentacyclic triterpenes stimulate proliferation of human keratinocytes but do not protect them against UVB damage [40,41,42]. A review on antitumor effect of triterpene acid compounds revealed that triterpene acid type compounds have many effects including anti-inflammatory, regulating blood sugar level, antiviral, and antitumor activity. More important, triterpene acid type compounds have become one of the most popular topics recently because of its selective toxic effects on cancer cells and harmless to normal cells [43].

The tricyclic triterpene acid (3S,4S,14S,7E,17E,21Z)-3,30-dihydroxypodioda-7,17,21-trien-4-carboxylic acid (commafric A) was isolated from the n-hexane fraction of the resin of C. africana and its structure was determined. It showed the variable IC50 values against the four cancer cell lines studied. The anti-proliferative effect against non-small cell lung cancer (A549) cells with IC50 values of 4.52 μg/ml was the highest among the cancer cells tested. The anti-proliferative effect suggests that commafric A has a potential in further investigations as a drug against different cancer lines. Further work is required to evaluate the mechanism of action of the commafric A as an antitumor agent against non-small cell lung cancer and evaluate its anti-proliferative on a number of cancer cell lines. Further isolation of compounds from the resin and other parts of this plant will be pursued which might yield some novel bioactive compounds.

Availability of data and materials

The datasets used and analysed during the current study are available from the corresponding author on reasonable request.

Abbreviations

HRESIMS:

High resolution electrospray ionization mass spectroscopy

SRB:

Sulphorhodamine B

VCD:

Vibrational Circular Dichroism

RDC:

Residual dipolar coupling

RCSA:

Residual chemical shift anisotropy

PBLG:

Poly(γ-benzyl-l-glutamate)

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Acknowledgements

We thank Prof. Ermias Dagne for initiating a research project work on the chemical composition of Commiphora resins especially those of Ethiopian origin in his research laboratory and his assistance in this study.

Funding

Financial support for this work such as research materials required for extraction, isolation of compounds and cytotoxicity tests was provided by National Research Foundation of Korea (No. 2016K1A3A1A09939937), RDA Troms: TFK2014-207, Norway provided support in materials required for NMR studies. Funding from Digital Life Norway/Research Council of Norway, project ID: 269425, is gratefully acknowledged. Adama Science and Technology University supported for some research materials required for extraction, isolation of compounds and salary of WD and AD. The Czech Grant Agency (18-05770S) provided salary for PB and EB, Ministry of Education (LTC17012 and CZ.02.1.01/0.0/0.0/16_019/0000729) provided computational resources. The funders of Grants play no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. All the responsibilities starting from the design of the study up to the write up of the manuscript were undertaken by the authors.

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Authors and Affiliations

  1. Department of Applied Chemistry, Adama Science and Technology University, Adama, Ethiopia

    Worku Dinku & Aman Dekebo

  2. Department of Chemistry, UiT the Arctic University of Tromsø, 9037, Tromsø, Norway

    Johan Isaksson, Fredrik Garnås Rylandsholm, John Sigurd Mjøen Svendsen & Arne Jørgen Aasen

  3. Institute of Organic Chemistry and Biochemistry, Academy of Sciences, Flemingovo Náměstí 2, Dejvice, 16610, Prague 6, Czech Republic

    Petr Bouř & Eva Brichtová

  4. Department of Analytical Chemistry, University of Chemistry and Technology, Technická 5, 16628, Prague, Czech Republic

    Eva Brichtová

  5. Bio & Drug Discovery Division, Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea

    Sang Un Choi, Sang-Ho Lee & Young-Sik Jung

  6. Institute of Pharmaceutical Science, Adama Science and Technology University, Adama, Ethiopia

    Zae Sung No

Authors
  1. Worku Dinku
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  2. Johan Isaksson
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  12. Aman Dekebo
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Contributions

WD conducted the experimental studies such as extraction, isolation, characterization of compounds. WD also contributed in drafting the manuscript. PB and EB were involved in VCD analysis. JI and FG performed NMR analysis. JSMS participated in the interpretation of the NMR data and substantially revised the manuscript draft. AJAa supervised the project. SU and SL conducted the bioassay and HRMS analysis. YJ and ZS contributed the crucial help in supervision of the bioassay tests. AD conceptualized, designed and directed the study. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Aman Dekebo.

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Dinku, W., Isaksson, J., Rylandsholm, F.G. et al. Anti-proliferative activity of a novel tricyclic triterpenoid acid from Commiphora africana resin against four human cancer cell lines. Appl Biol Chem 63, 16 (2020). https://doi.org/10.1186/s13765-020-00499-w

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