21 © 2020 Adama Science & Technology University. All rights reserved Ethiopian Journal of Science and Sustainable Development e-ISSN 2663-3205 Volume 7 (2), 2020 Journal Home Page: www.ejssd.astu.edu.et ASTU Research Paper Chemical Constituents and Anti-proliferative Activity of Resin of C. sphaerocarpa against Four Human Cancer Cell Lines Worku Dinku1, Sang Un Choi2, Sang-Ho Lee2, Young-Sik Jung2, Aman Dekebo1,  1Department of Applied Chemistry, School of Applied Natural Science, Adama Science and Technology University, P. O. Box 1888, Adama, Ethiopia 2Bio and Drug Discovery Division, Korea Research Institute of Chemical Technology, Republic of Korea Article Info Abstract Article History: Received 1 February 2020 Received in revised form 14 March 2020 Accepted 24 March 2020 Myrrh, a resin derived from the Commiphora genus, has traditionally been used for treatment of various human diseases, such as amenorrhea, ache, tumors, fever, and stomach pains. Despite widespread traditional medicinal use, the pharmacological activity and chemical composition of many Commiphora species are not well known. The current study pursues to evaluate the cytotoxicity activity of the n-hexane and chloroform fractions of C. sphaerocarpa. Both the anti-proliferative effect and chemical compositions were correlated. The compositions of the chemical constituents were analyzed by gas chromatography coupled to mass spectrometry (GC-MS). The in vitro cytotoxicity activity of crude extracts were evaluated in cellular lines of non-small cell lung cancer A549, ovarian cancer A2780, pancreatic cancer MIA-PaCa-2, and stomach cancer SNU-638 by SRB assay. The n-hexane fraction showed better apparent cytotoxicity on A549, A2780, and SNU-638 cancer cell lines with IC50value ranging from 9.62 μg/mL to 10.30 μg/mL with dose-dependent relationship in vitro compared to the chloroform fraction. This efficacy might be correlated with the presence of pentacyclic triterpenes such as lupeol, urs-12-en-3-one, -amyrin, -amyrin, and sesquiterpenes: -copaene, caryophyllene, humulene, alloaromadendrene, -muurolene, - selinene, -humulene and caryophyllene oxides in resin of the plant. Our findings indicated components of n-hexane fraction of C. sphaerocarpa might be useful to treat aforementioned selected cancers after further studies. Keywords: Anti-proliferative activity Commiphora sphaerocarpa GC-MS ovarian cancer pancreatic cancer stomach cancer SRB assay 1. Introduction Cancer, a cellular malignancy that results in the loss of normal cell-cycle control, such as unregulated growth the lack of differentiation, can develop in any tissue of any organ, at any time (Chang and Kinghorn, 2001). The global cancer burden was estimated 18.1 million new cases and 9.6 million deaths in 2018. Cancers of the lung, female breast, and colorectal cancer are the top three cancer types in terms of incidence, are ranked within the top five in terms of mortality (first, fifth, and  Corresponding author, e-mail: amandekeb@gmail.com https://doi.org/10.20372/ejssdastu:v7.i2.2020.194 second, respectively). Together, these three cancer types are responsible for one third of the cancer incidence mortality burden worldwide (Bray et al., 2018). In Ethiopia, cancer has become the second leading cause of death next to cardiovascular diseases in the adult population (Memirie et al., 2018). The most common adult cancers were: cancers of the breast cervix, colorectal cancer, non-Hodgkin lymphoma, leukemia, cancers of the prostate, thyroid, lung, stomach, and liver (Memirie et al., http://www.ejssd.astu.edu/ mailto:amandekeb@gmail.com https://doi.org/10.20372/ejssdastu:v7.i2.2020.xxxxxx Worku Dinku et al. Ethiop.J.Sci.Sustain.Dev., Vol. 7 (2), 2020 22 2018). Natural products have proven to be an important source of anticancer drugs (Cragg and Newman, 2005), identification of phytochemical cytotoxic entities that are effective against a range of cancer cell lines, less active or non-toxic against the normal cell population is an effective method for selection of fractions. The genus Commiphora (Burseraceae) comprises over 150 species, most of which are confined to Eastern Africa, with few species also occurring in southern Arabia, India and South America (Shen et al. , 2012; Shen and Lou, 2008; Xu et al., 2011). According to Soromessa et al. (2013), South east lowland of Ethiopia is characterized by its high diversity of Acacia and Commiphora species and the same study revealed that out of total species in the genus Commiphora about half of them are endemic to the small area of south eastern Ethiopia, north eastern Kenya and Somalia. Over 50 Commiphora species are known to occur in Ethiopia, of which 14 (25%) species are endemic (Vollesen et al., 1989). The resin of C. sphaerocarpa is sold under the product name myrrh. Ethnobotanical notes on herbarium specimens of Commiphora species deposited at the National Herbarium in Addis Ababa University also revealed various traditional uses of resins by different communities in Ethiopia. For example, the resin of C. sphaerocarpa is used against cough, diarrhea, headache, and against ticks of cattle. Additionally, the note indicated fruits of this plant are edible. This resin is sometimes found in true myrrh as adulterants. Previous study revealed that petroleum ether extract of C. sphaerocarpa afforded six sesquiterpenes of which one of them i.e. (1E)-8,12- epoxygermacra-1,7,10,11-tetraen-6-one was novel (Dekebo et al, 2002). We hereby report chemical composition of n-hexane fraction of C. sphaerocarpa which was analyzed by GC-MS along with anticancer activity of crude methanol extracts, n-hexane, and chloroform fractions, obtained by liquid-liquid partitioning from methanol extracts, against four human cancer cell lines: A549, A2780, MIA-PaCa-2 and SNU- 638 using SRB assay under in vitro conditions. 2. Materials and Methods 2.1. Plant material collection Resin of C. sphaerocarpa Chiov (Burseraceae) and other botanical specimens were collected from Sof Omar Bale zone, Oromia regional state, Ethiopia in October, 2016. The specimens have been identified by botanist Shambel Alemu and deposited at the National Herbarium, Biology Department, Addis Ababa University, Ethiopia (Voucher number: 072820). 2.2. Extraction and Liquid-Liquid fractionation The resin sample was air dried left under shadow till experimental work. The powdered resin of C. sphaerocarpa (95.1 g) was extracted with methanol (1 L x 3) for three days at 25°C. The crude methanolic extract was evaporated under vacuum to afford a yellow solid (34.8 g, 36.6%) which was then suspended in water (500 ml) and successively partitioned with n- hexane (500 ml x 3) followed by chloroform (500 ml x 3) to afford 12.1 g (34.8%) and 2.5 g (7.1%) yields for n-hexane and chloroform fractions, respectively. 2.3. GC-MS analysis of n-hexane fraction GC-MS analysis was done by using a GC (7890B, Agilent Technologies, USA) coupled with an MS (5977A Network, Agilent Technologies). The GC had an HP 5MS column (non-polar column, Agilent Technologies), 30 m × 250 μm internal diameter (i.d.) 0.25 μm film thickness. The carrier gas was helium flowing at a rate of 1 mL/ min. The injector temperature was 230°C and the injection mode was split mode with split ratio 4:1. The initial oven temperature was 40°C held for 3 min. It was raised to 70°C at 4°C/min held at this temperature for 3 min. The oven temperature was then raised 80°C at 1°C/min with no holding time, 100°C at 4°C/min with no holding time then to 180°C at 10°C/min, then with no holding time 20°C/min until it reached 280°C held at this temperature for 2 min. The total run- time was 60 min. Mass spectra were recorded in EI mode at 70 eV, scanning the 33-500 m/z range. The identification of the volatile compounds was performed by comparing the mass spectra of the compounds with those in the database of NIST11 and literature data. 2.4. Anti-proliferative assessment The cancer cell lines including the human non-small cell lung cancer cell line (A549), ovarian cancer cell line (A2780), pancreatic cancer cell line (MIA-Paca-2) stomach cancer cell line (SNU-638) were used for evaluation of anticancer activity. All cell lines were maintained using RPMI1640 cell growth medium (Gibco, Carlsbad, CA), supplemented with 5% fetal Worku Dinku et al. Ethiop.J.Sci.Sustain.Dev., Vol. 7 (2), 2020 23 bovine serum (FBS) (Gibco), grown at 37°C in a humidified atmosphere containing 5% CO2. The cytotoxicity of the crude methanol extract its organic solvent fractions of the plant against cultured human tumor cell lines were evaluated on the already developed sulforhodamine B (SRB) method (Schols et al., 1988). 2.5. Treatment of cell lines Stock solutions of the crude MeOH extract, n-hexane and chloroform fractions dissolved in DMSO were prepared in the corresponding medium at different concentrations of 0.1, 0.3, 1.0, 3.0, 10.0 and 30 μg/mL to determine percentage of growth inhibition the and for the 50% growth inhibition (IC50). Each tumor cell line was inoculated over standard 96-well flat-bottom micro plates then incubated for 24 h at 37°C in a humidified atmosphere of 5% CO2. The attached cells were then incubated with serially diluted each samples. After continuous exposure to the compounds for 72 h, the culture medium was removed from each well and the cells were fixed with 10% cold trichloroacetic acid at 4°C for 1 h. After washing with tap water, the cells were stained with 0.4% SRB dye incubated for 30 min at room temperature. The cells were washed again and then, the cell bound SRB was solubilized with 10 mM buffered Tris base solution of pH 10.5. The absorbance was measured spectrophotometrically at 520 nm with a micro titer plate reader. Each experiment was conducted in triplicate. The IC50 values of crude MeOH extract and fractions were calculated by the nonlinear regression analysis. The IC50 was expressed as the concentration of drug reducing the plaque number by 50% as compared to mock-treated controls. It was calculated from a dose– response line obtained by plotting the percentage plaque reduction, with respect to the control plaque count, versus the logarithm of compound dose. Triplicate wells were utilized for each drug concentration tested. 3. Result and Discussion Analysis of n-hexane fraction comprised of 38 components among which 23 constituents were identified (Figure 1 and Table 1). Pentacyclic triterpenes were the major compounds with a high content of which, 1: (1S,6R,9S)-5,5,9,10-tetramethyltricyclo[7.3.0.0(1,6)]dodec- 10(11)-ene, 2: 2(1H)naphthalenone, 3,5,6,7,8,8a- hexahydro-4,8a-dimethyl-6-(1-methylethenyl)-, 3: - amyrin (13.42 %), 4: urs-12-en-3-one (14.39%), 5: lupeol (15.91%), 6: hop-22(29)-en-3.β-ol (7.89%) and 7: -amyrin (10.96 %) (Figure 2). Terpenoids have been previously reported to show antimicrobial activity (Field and Lettingam 1992). For example, β-amyrin has been shown to exhibit various pharmacological activities in vitro and in vivo against inflammation, microbial, fungal, viral infections and cancer cells (Vázquez et al., 2012). Sesquiterpene hydrocarbons such as -copaene (1.16%), caryophyllene (0.96%), humulene (0.12%), alloaromadendrene (0.12%), -muurolene (0.03%), - selinene (0.12%), -humulene (0.04%) and oxygenated sesquiterpene caryophyllene oxide (0.50%) were found as minor constituents in the n-hexane fraction (Table 1). Figure 1: GC of n-hexane fraction of C. sphaerocarpa, 1: (1S,6R,9S)-5,5,9,10-Tetramethyltricyclo [7.3.0.0(1,6)] dodec- 10(11)-ene, 2: 2(1H)Naphthalenone, 3,5,6,7,8,8a-hexahydro-4,8a-dimethyl-6-(1-methylethenyl)-, 3: -amyrin (13.42 %), 4: urs-12-en-3-one (14.39%), 5: lupeol (15.91%), 6: Hop-22(29)-en-3.-ol (7.89%) 7: -amyrin (10.96 %). Worku Dinku et al. Ethiop.J.Sci.Sustain.Dev., Vol. 7 (2), 2020 24 Table 1: Compounds identified in the n-hexane fraction of C. sphaerocarpa and their percentage area Name of Compounds a RT Mol.Wt Mol. Formula % composition Copaene 21.1 204.19 C15H24 1.16 Caryophyllene 22.0 204.19 C15H24 0.96 Humulene 22.7 204.19 C15H24 0.12 Alloaromadendrene 22.9 204.19 C15H24 0.12 uurolene 23.2 204.19 C15H24 0.03 Selinene 23.3 204.19 C15H24 0.12 umulene 23.4 204.19 C15H24 0.04 α-Amorphene 23.9 204.19 C15H24 0.03 Panasinsen 24.0 204.19 C15H24 0.12 Cadinene 24.1 204.19 C15H24 0.18 Caryophyllene oxide 25.3 220.18 C15H24O 0.50 (+)-epi-Bicyclosesquiphellrene 26.4 204.19 C15H24 0.07 (1S,6R,9S)-5,5,9,10-Tetramethyltricyclo[7.3.0.0(1,6)]dodec- 10(11)-ene (1) 39.2 218.20 C16H26 9.59 2(1H)Naphthalenone, 3,5,6,7,8,8a-hexahydro-4,8a-dimethyl- 6-(1-methylethenyl)- (2) 39.3 218.17 C15H22O 4.83 Olean-12-ene 40.3 410.39 C30H50 1.23 Olean-12-en-3-one 40.4 424.37 C30H48O 2.39 .-Amyrin (3) 42.0 426.39 C30H50O 13.45 Urs-12-en-3-one (4) 42.3 426.39 C30H50O 14.39 Lupeol (5) 43.0 426.39 C30H50O 15.91 Hop-22(29)-en-3 -ol (6) 43.1 426.39 C30H50O 7.89 Amyrin (7) 43.8 426.38 C30H48O 10.96 Taraxasterol 44.2 426.39 C30H50O 3.72 A'-Neogammacer-22(29)-en-3-one 48.1 424.37 C30H48O 0.34 a Compounds listed in order of elution from a HP 5MS column; RT = Retention time; Mol.Wt = Molecular weight; Mol. Formula = Molecular formula. Figure 2: Structures of some major compounds identified in n-hexane fraction of the resin of C. sphaerocarpa Worku Dinku et al. Ethiop.J.Sci.Sustain.Dev., Vol. 7 (2), 2020 25 GC-MS analysis results indicated that -amyrin had a shorter retention time than -amyrin (Table 1) which was consistent with the earlier reports (Burnouf- Radosevich et al., 1985). Some constituents such as - copaene, -humulene, -selinene, and -cadinene were identified in our previous study from the essential oil of C. sphaerocarpa resin (Dekebo et al., 2002). Sesquiterpenes, which are one of the most common terpenes, are a class of natural products with a diverse range of attractive industrial properties (Wang et al., 2011; Scalcinati et al., 2012). Several biological activities are attributed to sesquiterpenes, such as antimicrobial (Wu et al., 2012), antibacterial (Stojanović-Radić et al., 2012) antioxidant, antifungal (Al-maskri et al., 2011; Conforti et al., 2008) and antigenotoxic (Anter et al., 2011) activities. Nishida et al. (2000) reported that -copaene is not genotoxic and it increases the antioxidant capacity in human lymphocyte cultures. Fernandes et al., (2007) reported pronounced oral anti-inflammatory effects for the sesquiterpenes isolated from the essential oil of Cordia verbenacea. Their oral anti-inflammatory properties are probably related to an important inhibition of the activation and/or release of different inflammatory mediators such as bradykinin, platelet activating factor, histamine, IL-1β, TNFα and PGE2. The anti- inflammatory effects of these compounds seem to be closely associated with their ability to inhibit the up- regulation of both COX-2 and iNOS enzymes. These workers suggest that sesquiterpenes isolated from the essential oil of C. verbenacea, α-humulene and (−)- trans-caryophyllene, might constitute a relevant therapeutic alternative for the treatment of inflammatory diseases (Fernandes et al., 2007). In vivo disease resistance assays, using ZmTps21 and Zmtps21 near- isogenic lines, supported the endogenous antifungal role of selinene-derived metabolites involved in the biosynthesis of nonvolatile antibiotics, ZmTps21 exists as a useful gene for germplasm improvement programs targeting optimized biotic stress resistance (Ding et al., 2017). The concentrations of crude methanol extract which inhibit 50% of cell growth (IC50) was found to be less than 30 g/mL (Table 2, Figure 3a) against most of the cancer cell lines tested, which is within the limit of criteria set by the American National Cancer Institute for further purification (Radovanovic, 2015). Our anti- proliferative activity data indicated that the crude extract and fractions are far less than etoposide (Figure 3d). The n-hexane fraction (Table 2, Figure 3b) showed better anti-proliferative activity than the crude methanol extract and CHCl3 fractions (Table 2, Figure 3c) against all cell lines tested. The chloroform fraction has comparable activity to that of the crude methanol extract (Figures 3a and 3c). The n-hexane fraction showed apparent bioactivities on A549, A2780, and SNU-638 cancer cell lines with IC50 value ranging from 9.62 μg/mL to 10.30 μg/mL with dose-dependent relationship in vitro (Figure 3b and Table 2). These results suggest that n-hexane fraction is responsible for the observed high anti-proliferative activity. The cytotoxicity of C. sphaerocarpa crude methanol extract, n-hexane and chloroform fractions have not been studied before, but the cytotoxicity of some compounds identified in the n-hexane fraction extracted from a variety of plant species showed different pharmacological activities. In vivo studies have identified that lupane type pentacyclic triterpenoid have strong antitumor and anti-inflammatory effects (Saleem, 2009). Experimental results showed that, in the mouse skin carcinogenesis model, local application of lupeol for 28 weeks can inhibit the growth of tumor prolongs the latency of tumor cells. The mechanism might be related to the nuclear factor kappaB (NF-κB)/phosphatidylinositol 3-kinase (PI3K)/proteinkinase B (Akt) signaling pathway (Saleem et al., 2004). Aratanechemuge et al (2004), reported that hypodiploid apoptotic peak can be detected after the lupane type pentacyclic triterpenoid treatment on HL-60 leukemia cells, with time-dose-dependency (Aratanechemuge et al., 2004). Gallo and Sarachine (2009) reviewed that lupeol and some of its analogues have been shown to possess a wide range of biological activities such as anti-cancer, hepatoprotective, anti- microbial, cardioprotective, anti-melanoma, etc. Therefore, lupeol and its derivatives have a potential to be consumed as a dietery supplement to prevent cancer, coronary and hepatic disease. Though, the sesquiterpenes such as caryophyllene and caryophyllene oxide found in trace amount in n- hexane fraction, essential oils or fractions with high amount of the sesquiterpenes possessed higher cytotoxic activity against animal human tumor cells (El Hadri et https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4082788/#CR52 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4082788/#CR39 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4082788/#CR55 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4082788/#CR41 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4082788/#CR2 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4082788/#CR9 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4082788/#CR4 Worku Dinku et al. Ethiop.J.Sci.Sustain.Dev., Vol. 7 (2), 2020 26 A07-Me [ g/ml] 0.1 0.3 1.0 3.0 10.0 30.0 N e t G r o w th a s % o f C o n tr o l -50 0 50 100 A549 A2780 MIA-PaCa-2 SNU638 A07-CH [ g/ml] 0.1 0.3 1.0 3.0 10.0 30.0 N e t G r o w th a s % o f C o n tr o l -50 0 50 100 A549 A2780 MIA-PaCa-2 SNU638 al., 2010). The sesquiterpene hydrocarbon caryophyllene and its corresponding oxygenated sesquiterpene caryophyllene oxide showed anti-inflammatory effect on LPS induced paw edema rat with a mechanism of action by reduction of neutrophil migration inhibition of NF-κB (Medeiros et al., 2007) and inhibition of 5-LOX catalyzed leukotrienes rat (Jin et al., 2011). The study reported by Da Silva et al. (2016) has shown that n- hexane extract of I. coccinea flowers led to the isolation of a mixture of α and β-amyrin. The mixture was found to be moderately cytotoxic towards B16-F10 and HepG2 cancer cell line with IC50 23.21 and 24.09 µg/mL, respectively, and non-cytotoxic with IC50> 25 µg/mL against HL60, K562 and PBMC cancer cell lines (Da Silva et al., 2016). The mixture of - and -amyrin from Protium heptaphyllum demonstrated peripheral and central analgesic effects independent of the opioid system, and also showed a potent anti-inflammatory activity. The anti-inflammatory activity was potentiated by both indomethacin and thalidomide, suggesting a potential involvement of prostaglandins and TNF-alpha inhibitions (Aragao et al, 2008). Urs-12-en-3-one,(3β)- 27-[(Z)-feruloyloxy]-3-hydroxyurs-12-en-28-oic acid reported form the stem bark of Plumeria obtusa showed anti-mutagenic activity (Siddiqui et al., 2004). In conclusion, our results for the n-hexane fraction revealed moderate cytotoxic activity, which might be attributed to the presence of pentacyclic triterpenes and sesquiterpenes. The cytotoxicity to the three cancer cell lines by the n-hexane fraction needs further research on the underlying molecular mechanisms of action. Table 2: Cytotoxic activity of the crude MeOH extract and solvent fractions from C. sphaerocarpa resin against four human cancer cell lines using SRB assay Extract/Fractions Cell lines IC50 (g/ml) A549 A2780 MIA-PaCa-2 SNU-638 MeOH 11.03 11.32 23.51 11.56 n-Hexane 9.64 9.62 17.21 10.3 CHCl3 11.23 11.06 24.57 10.84 Etoposide, M 0.34 0.58 0.72 0.24 (Inhibition of cell growth by 50%). Data was generated by experiments performed in triplicates. Etoposide [ M] 0.1 0.3 1.0 3.0 10.0 30.0 N e t G r o w th a s % o f C o n tr o l -50 0 50 100 A549 A2780 MIA-PaCa-2 SNU638 A03-Hx [g/ml] 0.1 0.3 1.0 3.0 10.0 30.0 N e t G r o w th a s % o f C o n tr o l -50 0 50 100 A549 A2780 MIA-PaCa-2 SNU638 CspR - MeOH [µg/ml] CspR - Hx [µg/ml] CspR- CHCl3 [µg/ml] Etopoisde [µM] a) b) c) d) Figure 3: Cytotoxic activity of methanol extract and fractions determined by the percentage of growth inhibition. CsR- MeOH, CsR-Hx, CsR-CHCl3 are methanol extract, hexane chloroform, fractions, respectively, from C. sphaerocarpa resin against A549, A2780, MIA-PaCa-2 SNU 638 cancer cell lies. Etoposide was used as a positive control. The values are means ± standard deviation (SD) from three replicates. Worku Dinku et al. Ethiop.J.Sci.Sustain.Dev., Vol. 7 (2), 2020 27 The net growth as percent control or cell viability expressed in percentage of the crude MeOH extract, n- hexane and chloroform fractions showed concentration- dependence (Figure 3), which decreased with an increase in crude extract and solvent fraction concentration. The A549 and A2780 cell line were the most sensitive to Commiphora sphaerocarpa resin n- hexane fraction with IC50 value 9.64 and 9.62 µg/mL The n-hexane fraction showed a significant dose dependent in reducing the proliferation of A549 and A2780 cancer cells at 0.1, 0.3, 1.0, 3.0, 10.0, and 30.0 µg/mL (Figure 3b). Particularly, the cell viability of the cancer cell line decreased sharply at higher concentration, i.e., at 10 and 30 g/ml with more than 60% and 100% inhibition. Slight inhibitory activity was observed by the MeOH crude extract and chloroform fraction in the range of 10.84 - 11.23 against A549, A2780 and SNU- 638 cancer cell line (Figures 3a and 3c). The activity presented by the crude MeOH extract, n-hexane and chloroform fractions was least potent against the MIA- PaCa-2 cell line with IC50 value 23.51, 17.21 and 24.57 g/mL respectively (Figures 3). 4. Conclusion Among the two solvent fractions tested for cytotoxicity activity, the n-hexane fraction showed better apparent cytotoxicity against A549, A2780 and SNU-638 cancer cells with dose-dependent relationship in vitro compared to the chloroform fraction. Chemical constituents of the most active n-hexane fraction was analyzed by GC-MS and the result revealed that pentacyclic triterpenes and sesquiterpenes were found to be major components of the fraction which might be responsible for the observed anti-proliferative effects. The findings of this study suggest that the components of n-hexane fraction of C. sphaerocarpa might be useful to treat aforementioned selected cancers after further in vivo and clinical studies. 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