Volume 02 Number 01 February 2024 p-ISSN: 2986-5662 e-ISSN: 2985-959X The Fatty Acid Composition and the MTT-Cytotoxicity Test of Commercially Available Commiphora Gileadensis Balsam Oil Arzu Özgen1 1 Istanbul Gelisim University, Istanbul, Turkey Correspondence should be addressed to: Arzu Özgen aozgen@gelisim.edu.tr Abstract: Article info: Commiphora gileadensis, an aromatic plant, is traditionally used in Middle Eastern Submitted: countries to prevent pain and inflammation. Fatty acid composition, peroxide number and 06-12-2023 free fatty acidity analyzes of commercially available C. gileadensis balsam oil and Revised: investigation of its effect on cell viability. Commercially available C. gileadensis balsam oil 17-01-2024 Accepted: fatty acid composition, peroxide number and free fatty acidity values were determined by 19-01-2024 the IUPAC IID19 method, and MTT cell viability tests were performed on L929 fibroblast and HeLa (human epithelial cervical carcinoma) cell lines. Commercially available C. gileadensis balsam oil did not exert cytotoxic effects on both L929 fibroblast and HeLa (human epithelial cervical carcinoma) cell lines and promoted the growth of cell lines. Due to its growth promoting feature on L929 fibroblast cells, which is a healthy cell line, this material can be used as a cell culture medium additive. Keywords: C. gileadensis; L929; HeLa; cytotoxicity DOI: https://doi.org/10.53713/htechj.v2i1.133 This work is licensed under CC BY-SA License. INTRODUCTION Commiphora gileadensis, also known as C. opobalsamum, a member of the Burseraceae family, is a small evergreen tree that grows widely in East Africa and tropical and subtropical regions such as Arabia and India (Hepper and Taylor, 2004; Iluz et al., 2010; Paparozzi, 2005; Vollesen, 1989; Almahbashi et al., 2019). C. gileadensis is still considered an important medicinal plant in the Middle East and particularly in Saudi Arabia, and is an aromatic herb traditionally used to treat pain, swelling and fever (Al-Sieni, 2014). It is also used to treat skin ulcers, empyrosis and wounds (Dai et al., 2001; Shang and Lu, 2007; Shen et al., 2012). Balsam oil is obtained from the trunk of this tree by wounding. In recent years, it has been reported that it contains cycloartane- type triterpenoid, an aliphatic alcohol glycoside, an eudesmane-type sesquiterpenoid, and a guaiane-type sesquiterpenoid (Shen et al., 2007; Shen et al., 2008; Shen et al., 2009; Xu et al., 2011; Xu et al., 2011a). Studies on the effects of commercially available C. gileadensis balsam oil on cell viability are limited. In this study, fatty acid composition, peroxide number and free fatty acidity analyzes of commercially available C. gileadensis balsam oil were performed, and MTT cell viability tests were performed on L929 fibroblast and HeLa (human epithelial cervical carcinoma) cell lines. Özgen 10 Volume 02 Number 01 February 2024 p-ISSN: 2986-5662 e-ISSN: 2985-959X METHOD C. gileadensis balsam oil C. gileadensis balm oil, obtained by wounding the trunk of the C. gileadensis tree, was purchased from the local herbal store. Determination of fatty acid composition Fatty acid composition, peroxide number and free fatty acidity were determined by TUBITAK- MAM. Cell culture L929 fibroblast and HeLa (human epithelial cervical carcinoma) cell lines were used. DMEM + 2mM Glutamine + 10% Fetal Bovine Serum (FBS) and DMEM (High Glucose) + 10% FBS (Biological Industries) were used as cell culture medium, respectively. Cells were grown at 37 °C, 5% CO₂. MTT-based cytotoxicity testing C. gileadensis balsam oil sample was mixed into DMEM at concentrations of 180, 90, 45, 22.5 and 9 µg/mL. It was sterilized through a 0.22 µm filter, and the L929 and HeLa cell lines were used for MTT-based cytotoxicity testing. The L929 cell line and the HeLa cell line were seeded at 105 cells/mL in 96 well cell culture dishes. Cells were allowed to attach to the bottom in an incubator at 37 °C, 5% CO₂ for 24 hours. At the end of 24 hours, 20 µl of C. gileadensis balsam oil sample prepared at the above-mentioned concentrations was added to each well. MTT viability test (BioFroxx) was performed in an incubator at 37 °C, 5% CO₂ for 24 hours. 1% phenol solution was used as the positive control, only DMEM and DMEM F12 media were used as the negative control, and the experiments were performed as three independent replicates and the averages are presented for both L929 fibroblast and HeLa cell line. Photometric reading was performed at 570 nm. Results were calculated assuming the negative control is 100% viable. RESULT Fatty acid composition of C. gileadensis balsam oil C. gileadensis balsam oil fatty acid composition, peroxide number and free fatty acidity values obtained as a result of the analysis are given in Tables 1, 2, 3, respectively. Cell viability test Analyzes of effect of C. gileadensis on cell viability were performed by MTT viability assay on L929 fibroblast and HeLa cell lines. Information on the mean absorbance and viability percentage values of the C. gileadensis balsam oil sample on L929 fibroblasts and HeLa cells at concentrations of 180, 90, 45, 22.5 and 9 µg/mL are given in Table 4, 5 and Fig 1, respectively. Özgen 11 Volume 02 Number 01 February 2024 p-ISSN: 2986-5662 e-ISSN: 2985-959X Table 1. C. gileadensis balsam oil fatty acid composition Analysis Result (%) Method Caproic acid (C6:0) 0.37 IUPAC IID19 Caprylic acid (C8:0) 12.60 IUPAC IID19 Capric acid (C10:0) 0.12 IUPAC IID19 Lauric acid (C12:0) 0.14 IUPAC IID19 Myristic acid (C14:0) 0.10 IUPAC IID19 Myristoleic acid (C14:1) 0.01 IUPAC IID19 Pentadecanoic acid (C15:0) 0.10 IUPAC IID19 Palmitic acid (C16:0) 15.50 IUPAC IID19 Palmitoleic acid (C16:1) 0.11 IUPAC IID19 Heptadecanoic acid (C17:0) 0.14 IUPAC IID19 Stearic acid (C18:0) 10.73 IUPAC IID19 Elaidic acid (C18:1n9t) 0.08 IUPAC IID19 Oleic acid (C18:1n9c) 33.36 IUPAC IID19 Linoleic acid (C18:2n6c) 0.56 IUPAC IID19 Arachidic acid (C20:0) 0.51 IUPAC IID19 γ-Linolenic acid (C18:3n6) 0.16 IUPAC IID19 cis-11-Eicosenoic acid (C20:1) 0.22 IUPAC IID19 α-Linolenic acid (C18:3n3) 0.01 IUPAC IID19 Behenic acid (C22:0) 0.22 IUPAC IID19 Lignoceric acid (C24:0) 0.09 IUPAC IID19 Table 2. C. gileadensis balsam oil peroxide number Analysis Result Method Peroxide value Not dedected meq active ISO 3960 oxygen/kg oil Table 3. C. gileadensis balsam oil free fatty acidity Analysis Result (%) Method Free fatty acidity 0.18 The acidity based on the oleic acid content ISO 660 Table 4. Viability of L929 after exposure to C. gileadensis balsam oil measured by MTT assay Concentration Negative Positive 180 90 45 22.5 9 (µg/mL) control control Average 2.44855 2.651 2.50895 2.07815 2.035775 1.0915 0.0859 absorbance Viability 224.336 242.884 229.870 190.400 186.517 100.000 7.870 percentage Table 5. Viability of HeLa after exposure to C. gileadensis balsam oil measured by MTT assay Concentration Negative Positive 180 90 45 22.5 9 (µg/mL) control control Average 3.877 3.643 3.636 3.634 2.035775 3.716 0.136 absorbance Viability 134.82 126.68 126.47 126.39 129.24 100.000 4.74 percentage Özgen 12 Volume 02 Number 01 February 2024 p-ISSN: 2986-5662 e-ISSN: 2985-959X Cell viability of L929 and HeLa cell line 300,000 250,000 200,000 150,000 100,000 50,000 0,000 Control + Control - 9 µg/mL 22.5 45 µg/mL 90 µg/mL 180 µg/mL -50,000 µg/mL concentration µg/mL L929 cell viability % HeLa cell viabilty % Figure 1. Viability of L929 and HeLa cell line following C. gileadensis balsam oil treatment of MTT viability assay According to the results obtained, when the balm oil L929 fibroblast cell line was compared with the negative control group, it showed 186.5% viability at 9 µg/mL concentration, 190.400% viability at 22.5 µg/mL concentration, 229.870% viability at 45 µg/mL concentration, 242.884% viability at 90 µg/mL concentration, respectively. It showed 224.336% viability at 180 µg/mL concentration. Therefore, it did not have a toxic effect on cell viability at all concentrations and promoted cell viability. The same effects on the HeLa cell line, when compared with the negative control group, were 129.24% viability at 9 µg/mL concentration, 126.39% viability at 22.5 µg/mL concentration, 126.47% viability at 45 µg/mL concentration, and 126.68% viability at 90 µg/mL concentration, and 134.82% viability at180 µg/mL concentration, respectively. DISCUSSION Fatty acid content analysis of Commifera species in the literature has been reported as linoleic, oleic, stearic, and palmitic acids in the seed of C. mukul (Hanuš et al., 2005). In the core of C. wightii species, it is reported as capric acid 3.50%, myristic acid 14.51%, palmitic acid 6.68%, stearic acid 4.70%, arachidic acid 3.18%, behenic acid 14.05%, myristoleic acid 1.34%, palmitoleic acid 12.07%, oleic acid 14.15%, eicosenoic acid 0.11%, linoleic acid 22.34% and alpha linoleic acid 1.37% (Patel et al., 2009). In this study, caprylic, palmitic, stearic, and oleic acids were detected in the balsam oil of C. gileadensis at the rates of 12.60%, 15.50%, 10.73%, and 33.36%, respectively. Plants with a broad spectrum of chemical compounds have the potential to form many therapeutic components for various diseases and also serve as a pool for the development of alternative pharmacological products (Bomfim et al., 2021). Since ancient times, people have either obtained herbal extracts for various purposes by traditional methods or bought them from local stores as it is more common today. Among these purposes, skin care is at the forefront. The Özgen 13 cell viabilit % Volume 02 Number 01 February 2024 p-ISSN: 2986-5662 e-ISSN: 2985-959X reliability of these extracts, which are purchased and used in this way, is also important in terms of personal health. C. gileadensis has been used extensively in the Middle East for centuries, both as a perfume and in traditional medicine (Hepper and Taylor, 2004; Iluz et al., 2010; Wineman et al., 2015). In addition to being used as an antiseptic agent by some tribes in Oman, C. gileadensis is also used ethnobotanically in the treatment of skin diseases such as inflammation and eczema (Iluz et al., 2010). It has been reported that it has an anti-proliferative effect on cancer cell lines in studies carried out with C. gileadensis extracts prepared under laboratory conditions. In a study conducted by Shen et al. in 2007, it was shown that C. gileadensis secondary metabolites have an antiproliferative effect on human prostate cancer cells (Shen et al., 2007). In another study with C. gileadensis sap extract, immortalized human keratinocyte cells and in A431 cells were used and it was reported that it had a selective cytotoxic effect (Wineman et al., 2015). In another study, the cytotoxicity of C. gileadensis L bark methanolic extracts against HELa and A-549 was investigated and it showed moderate dose-dependent cytotoxic activity in these cells. It has been reported that it exhibits weak cytotoxic effects in MCF-7 HepG2, HCT-116, HEP-2 and PC-3 cell lines (Almahbashi et al., 2019). The commercially available balsam oil used in this study did not show cytotoxic effects on the HeLa cancer cell line. It did not show toxic properties on L929 healthy cell lines, and it promoted cell viability. When considering application areas such as tissue engineering or cell culture, materials that promote cell viability or that are not toxic to cell viability are important. As a result, C. gileadensis balsam oil, which has no toxic effect for maximum cell productivity and has been observed to promote cell proliferation, has the potential to be used for this purpose. CONCLUSION This study showed that commercially available C. gileadensis balsam oil has no toxic effect on L929 fibroblast and HeLa cell lines. Due to its growth promoting feature on L929 fibroblast cells, which is a healthy cell line, this material can be used as a cell culture medium additive by further research. C. gileadensis balsam oil is thought to have the potential to be used in cell culture production of pharmacological products for both pets and humans. ACKNOWLEDGEMENT The authors thank the participant for the support to the study. FUNDING STATEMENT There was no financial support for this research. CONFLICT OF INTEREST The authors declare no conflict of interest. Özgen 14 Volume 02 Number 01 February 2024 p-ISSN: 2986-5662 e-ISSN: 2985-959X REFERENCES Almahbashi, L. H., El Shibany, A., Al-Massarani Sh., Salama, A., & Abudunia, A.M. (2019). Preliminary Phytochemical Composition and Biological Activities of Methanolic Extract of Commiphora Gileadensis. ISESCO JOURNAL of Science and Technology, 23. Al-Sieni, A. I. (2014). The antibacterial activity of traditionally used Salvadora persica L.(miswak) and Commiphora gileadensis (palsam) in Saudi Arabia. African Journal of Traditional, Complementary and Alternative Medicines, 11(1), 23-27. Bomfim, E. M. S., Coelho, A. A. O. P., Silva, M. C., Marques, E. J., & Vale, V. L. C. (2021). Phytochemical composition and biological activities of extracts from ten species of the family Melastomataceae Juss. Brazilian Journal of Biology=Revista Brasleira de Biologia, 82, e242112- e242112. Dai, L., Ma, S., & Qi, Y. (2001). Frankincense prescription used for the treatment of 386 skin ulcer cases. Hebei Medical Journal, 23, 587. Hanuš, L. O., Řezanka, T., Dembitsky, V. M., & Moussaieff, A. (2005). Myrrh-commiphora chemistry. Biomedical papers, 149(1), 3-28. Hepper N. F., & Taylor J. E. (2004). Date palms and opobalsam in the Madaba mosaic map. Palestine exploration quarterly, 136(1), 35-44. Iluz D., Hoffman M., Gilboa-Garber N., & Amar Z. (2010). Medicinal properties of Commiphora gileadensis. African Journal of Pharmacy and Pharmacology, 4(8), 516-520. Paparozzi E. T. (2005). Plant Resins-Chemistry, Evolution Ecology and Ethnobotany. Hort Science, 40(3), 508-508. Patel, B. H., Thakore, S., & Nagar, P. S. (2009). Chemical composition and characteristics of Commiphora wightii (Arnott) Bhandari seed oil. Journal of the American Oil Chemists' Society, 86(5), 497-498. Shang, Y., & Lu, X. (2007). 0.5%povidone-iodine solution plus frankincense and myrrh for the treatment of sores. Journal of Practical Training of Medicine, 35, 250-252. Shen, T., Li, G. H., Wang, X. N., & Lou, H. X. (2012). The genus Commiphora: a review of its traditional uses, phytochemistry and pharmacology. Journal of ethnopharmacology, 142(2), 319-330. Shen, T., Wan, W., Yuan, H., Kong, F., Guo, H., Fan, P., & Lou, H. (2007). Secondary metabolites from Commiphora opobalsamum and their antiproliferative effect on human prostate cancer cells. Phytochemistry, 68(9), 1331-1337. Shen, T., Wan, W. Z., Wang, X. N., Sun, L. M., Yuan, H. Q., Wang, X. L., ... & Lou, H. X. (2008). Sesquiterpenoids from the resinous exudates of Commiphora opobalsamum (Burseraceae). Helvetica Chimica Acta, 91(5), 881-887. Shen, T., Wan, W. Z., Wang, X. N., Yuan, H. Q., Ji, M., & Lou, H. X. (2009). A triterpenoid and sesquiterpenoids from the resinous exudates of Commiphora myrrha. Helvetica Chimica Acta, 92(4), 645-652. Vollesen K. In Burseraceae. Flora of Ethiopia. Hedberg I., Edwards S. (1989). Eds. Addis Ababa University Press, Addis Ababa, 3, 442-478. Wineman, E., Douglas, I., Wineman, V., Sharova, K., Jaspars, M., Meshner, S., ... & Shtevi, A. (2015). Commiphora gileadensis sap extract induces cell cycle-dependent death in immortalized keratinocytes and human dermoid carcinoma cells. Journal of herbal medicine, 5(4), 199-206. Xu, J., Guo, Y., Zhao, P., Xie, C., Jin, D. Q., Hou, W., & Zhang, T. (2011). Neuroprotective cadinane sesquiterpenes from the resinous exudates of Commiphora myrrha. Fitoterapia, 82(8), 1198- 1201. Özgen 15 Volume 02 Number 01 February 2024 p-ISSN: 2986-5662 e-ISSN: 2985-959X Xu, J., Guo, Y., Li, Y., Zhao, P., Liu, C., Ma, Y., ... & Zhang, T. (2011a). Sesquiterpenoids from the resinous exudates of Commiphora myrrha and their neuroprotective effects. Planta medica, 77(18), 2023-2028. Özgen 16