Sains Malaysiana 51(7)(2022): 2329-2337

http://doi.org/10.17576/jsm-2022-5107-31

Jumlah Fenol, Aktiviti Antioksidan dan Ketoksikan Ekstrak Bendalir Lampau Genting (SFE)

Buah Ziziphus mauritiana

(Total Phenol, Antioxidant Activity and Toxicity of Supercritical Fluid (SFE) Extract of Ziziphus mauritiana Fruit)

ARNIDA HANI TEH^1,2,*, MUHIYUDDIN MD NASIR¹, SAIDATUL HUSNI SAIDIN³

& HAFEEDZAABDUL RAHMAN^1,2

¹Department of Food Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia
²Innovation Center for Confectionary Technology (MANIS), Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia

³Herbal Product Development Programme, Natural Products Division, Forest Research Institute Malaysia (FRIM), 52109 Kepong, Selangor Darul Ehsan, Malaysia

Received: 25 February 2022/Accepted: 18 April 2022

Abstrak

Kajian ini menentukan jumlah fenol, antioksidan dan ketoksikan buah Ziziphus mauritiana yang diekstrak menggunakan pengekstrakan bendalir lampau genting (SFE). Ekstrak dianalisis untuk jumlah kandungan fenol (TPC) dan aktiviti antioksidan berdasarkan aktiviti penyingkiran radikal bebas, 2,2-difenil-1-pikrilhidrazil (DPPH). Kajian ketoksikan akut dilakukan pada tikus Sprague Dawley (24 jantan, 24 betina), dibahagikan kepada 4 kumpulan; diberikan 0 (kawalan), 1, 2.5 dan 5 g ekstrak/kg berat badan. Kajian kesitotoksikan dilakukan menggunakan ujian MTT pada titisan sel V79-4 dan kajian kegenotoksikandilakukan menggunakan Ujian Mikronukleus (MNA) dengan pewarnaan akridin jingga (AO). Ekstrak menghasilkan TPC sebanyak 539.18 mg setaraan asid galik (GAE)/100 g dan penyingkiran radikal bebas DPPH sebanyak 16.65%. Dalam kajian ketoksikan akut, semua tikus selamat tanpa bukti manifestasi klinikal atau ketoksikan. Kajian kesitotoksikan ekstrak menunjukkan kesan kesitotoksikan (IC₅₀ 0.48 mg/mL). Kajian kegenotoksikan menunjukkan bahawa ekstrak tidak mempunyai kesan kegenotoksikan pada tikus yang diuji. Secara keseluruhan, ekstrak bertindak dengan cara yang berbeza, in vivo dan in vitro. Sebagai kesimpulan, ekstrak adalah sumber fenol yang baik namun tidak menunjukkan aktiviti antioksidan yang tinggi. Ia tidak toksik dan tidak menyebabkan kegenotoksikan terhadap haiwan yang dikaji pada kepekatan sehingga 5 g ekstrak/kg berat badan. Walau bagaimanapun, ekstrak didapati sitotoksi terhadap titisan sel V79-4 yang dikaji.

Kata kunci: Antioksidan; kegenotoksikan; kesitotoksikan; ketoksikan akut; pengekstrakan bendalir lampau genting; Ziziphus mauritiana

Abstract

This study determined the total phenolics, antioxidant and toxicity of Ziziphus mauritiana fruit extracted using supercritical fluid extraction (SFE). The extract was analysed for the total phenolic content (TPC) and antioxidant activity based on radical scavenging activity, 2,2-diphenyl-1-picrylhydrazyl (DPPH). Acute toxicity studies were done in Sprague Dawley rats (24 males, 24 females), divided into 4 groups; receiving 0 (control), 1, 2.5 and 5 g extract/kg body weight. Cytotoxicity studies were done using MTT assay on V79-4 cell lines, and genotoxicity studies were done using Micronucleus Assay (MNA) with acridine orange (AO) staining. The extract produced TPC of 539.18 mg gallic acid equivalent (GAE)/100 g and DPPH scavenging of 16.65 %. In the acute toxicity studies, all rats survived with no evidence of clinical or toxic manifestation. Cytotoxicity studies showed that the extract demonstrated a cytotoxic effect (IC₅₀ 0.48 mg/mL). Genotoxicity studies showed that the extract had no genotoxic effect on the tested rats. Overall, the extract behaves in different manner, in vivo and in vitro. As a conclusion, the extract is a good source of phenolics, however, does not show high antioxidant activity. It is non-toxic and not causing genotoxicity towards the studied animals at the concentration up to 5 g extract/kg body weight. However, the extract is found to be cytotoxic towards the V79-4 cell lines studied.

Keywords: Acute toxicity; antioxidant; cytotoxicity; genotoxicity; supercritical fluid extraction; Ziziphus mauritiana

REFERENCES

Akanda, M.K.M. & Hasan, A.H.M.N. 2021. Characterization of pharmacological properties of methanolic seed and stem bark extracts of Ziziphus mauritiana (BAU Kul) using in-vitro and in-vivo animal (Swiss albino male mice) model. Clinical Phytoscience 7: 8. https://doi.org/10.1186/s40816-020-00246-0

Al-Saeedi, A.H., Al-Ghafri, M.T.H. & Hossain, M.A. 2017. Brine shrimp toxicity of various polarities leaves and fruits crude fractions of Ziziphus jujuba native to Oman and their antimicrobial potency. Sustainable Chemistry and Pharmacy 5(December 2016): 122-126.

Azman, N.F.I.N., Azlan, A., Khoo, H.E. & Razman, M.R. 2019. Antioxidant properties of fresh and frozen peels of citrus species. Current Research in Nutrition and Food Science Journal 7(2): 331-339.

Beattie, K.D., Ulrich, R., Grice, I.D., Uddin, S.J., Blake, T.B., Wood, K.A., Steele, J., Iu, F., May, T.W. & Tiralongo, E. 2011. Ethanolic and aqueous extracts derived from Australian fungi inhibit cancer cell growth in vitro. Mycologia 103(3): 458-465.

Bhatia, A., Mishra, T. & Khullar, M. 2011. Anticancer potential of aqueous ethanol seed extract of Ziziphus mauritiana against cancer cell lines and Ehrlich ascites carcinoma. Evidence-based Complementary and Alternative Medicine 2011: 765029.

Blois, M.S. 1958. Antioxidant determinations by the use of a stable free radical. Nature 181: 1199.

Bonassi, S., Znaor, A., Ceppi, M., Lando, C., Chang, W.P., Holland, N., Kirsch-Volders, M., Zeiger, E., Ban, S., Barale, R., Bigatti, M.P., Bolognesi, C., Cebulska-Wasilewska, A., Fabianova, E., Fucic, A., Hagmar, L., Joksic, G., Martelli, A., Migliore, L., Mirkova, E., Scarfi, M.R., Zijno, A., Norppa, H. & Fenech, M. 2007. An increased micronucleus frequency in peripheral blood lymphocytes predicts the risk of cancer in humans. Carcinogenesis 28(3): 625-631.

Cardozo, M.L., Ordoñez, R.M., Alberto, M.R., Zampini, I.C. & Isla, M.I. 2011. Antioxidant and anti-inflammatory activity characterization and genotoxicity evaluation of Ziziphus mistol ripe berries, exotic Argentinean fruit. Food Research International 44(7): 2063-2071.

Elbatrawy, E.N., Ghonimy, E.A.A., Alassar, M.M. & Wu, F.S. 2015. Medicinal mushroom extracts possess differential antioxidant activity and cytotoxicity to cancer cells. International Journal of Medicinal Mushrooms 17(5): 471-479.

Fenech, M. 2000. The in vitro micronucleus technique. Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis 455(1-2): 81-95.

Ikram, E.H.K., Eng, K.H., Jalil, A.M.M., Ismail, A., Idris, S., Azlan, A., Nazri, H.S.M., Diton, N.A.M. & Mokhtar, R.A.M. 2009. Antioxidant capacity and total phenolic content of Malaysian underutilized fruits. Journal of Food Composition and Analysis 22(5): 388-393.

Islam, M.B. & Simmons, M.P. 2006. A thorny dilemma: Testing alternative intrageneric classifications within Ziziphus (Rhamnaceae). Systematic Botany 31(4): 826-842.

Khairuddin, K. & Noor Azlin, Y. 2013. Ziziphus mauritiana: Tasty little fruits. https://www.frim.gov.my/colour-of-frim/ziziphus-mauritiana-tasty-little-fruits/wppaspec/oc1/cv0/ab35/pt113. Diakses pada 27 Februari 2021.

Khan, A.R., Awan, F.R., Najam, S.S., Islam, M., Siddique, T. & Zain, M. 2015. Elevated serum level of human alkaline phosphatase in obesity. JPMA. The Journal of the Pakistan Medical Association 65(11): 1182-1185.

Koley, T.K., Kaur, C., Nagal, S., Walia, S., Jaggi, S. & Sarika. 2011. Antioxidant activity and phenolic content in genotypes of Indian jujube (Zizyphus mauritiana Lamk.). Arabian Journal of Chemistry 9: S1044-S1052.

Krishna, G. & Hayashi, M. 2000. In vivo rodent micronucleus assay: Protocol, conduct and data interpretation. Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis 455(1-2): 155-166.

Kviecinski, M.R., Benelli, P., Felipe, K.B., Correia, J.F.G., Pich, C.T., Ferreira, S.R.S. & Pedrosa, R.C. 2011. SFE from Bidens pilosa Linné to obtain extracts rich in cytotoxic polyacetylenes with antitumor activity. Journal of Supercritical Fluids 56(3): 243-248.

Lowe, D., Sanvictores, T. & John, S. 2017. Alkaline Phosphatase. Treasure Island: StatPearls Publishing.

Lu, C.C., Hsu, Y.J., Chang, C.J., Lin, C.S., Martel, J., Ojcius, D.M., Ko, Y.F., Lai, H.C. & Young, J.D. 2016. Immunomodulatory properties of medicinal mushrooms: Differential effects of water and ethanol extracts on NK cell-mediated cytotoxicity. Innate Immunity 22(7): 522-533.

Machado, B.A.S., De Abreu Barreto, G., Costa, A.S., Costa, S.S., Silva, R.P.D., Da Silva, D.F., Brandao, H.N., Da Rocha, J.L.C., Nunes, S.B., Umsza-Guez, M.A. & Padilha, F.F. 2015. Determination of parameters for the supercritical extraction of antioxidant compounds from green propolis using carbon dioxide and ethanol as co-solvent. PLoS ONE 10(8): 1-26.

Marwat, S.K., Rehman, F., Khan, E.A., Khakwani, A.A., Ullah, I., Khan, K.U. & Khan, I.U. 2014. Useful ethnophytomedicinal recipes of angiosperms used against diabetes in South  East Asian Countries (India, Pakistan & Sri Lanka). Pakistan Journal of Pharmaceutical Sciences 27(5): 1333-1358.

Nasir, W.N.H.W., Ibrahim, N.N.A., Hao, W.K., Sajak, A.A.B., Sofian-Seng, N., Mustapha, W.A.W. & Rahman, H.A. 2021. Effects of different drying methods and solvents on biological activities of Curcuma aeruginosa leaves extract. Sains Malaysiana 50(8): 2207-2218.

Nyanga, L.K., Gadaga, T.H., Nout, M.J.R., Smid, E.J., Boekhout, T. & Zwietering, M.H. 2013. Nutritive value of masau (Ziziphus mauritiana) fruits from Zambezi Valley in Zimbabwe. Food Chemistry 138(1): 168-172.

OECD. 2001. OECD Guideline for Testing of Chemicals. Guideline 420: Acute Oral Toxicity, Fixed Dose Procedure(17th December): hlm. 1-14.

Othman, Z.S., Maskat, M.Y. & Hassan, N.H. 2020. Optimization of cinnamaldehyde extraction and antioxidant activity of Ceylon cinnamon extract. Sains Malaysiana 49(5): 995-1002.

Owolarafe, T.A., Salawu, K., Ihegboro, G.O., Ononamadu, C.J., Alhassan, A.J. & Wudil, A.M. 2020. Investigation of cytotoxicity potential of different extracts of Ziziphus mauritiana (Lam) leaf Allium cepa model. Toxicology Reports 7(June): 816-821.

Pareek, S., Kitinoja, L., Kaushik, R.A. & Paliwa, R. 2009. Postharvest physiology and storage of ber. Stewart Postharvest Review 5(5): 1-10.

Perumal, S., Mahmud, R., Piaru, S.P., Cai, L.W. & Ramanathan, S. 2012. Potential antiradical activity and cytotoxicity assessment of Ziziphus mauritiana and syzygium polyanthum. International Journal of Pharmacology 8(6): 535-541.

Ramar, M.K., Dhayanandamoorthy, Y., Ramachandran, S.S. & Kandasamy, R. 2020. HPLC-ESI-QqQ based standardization, mutagenic and genotoxic potential of methanol extract of Ziziphus mauritiana Lam leaves. Journal of Ethnopharmacology 246(September 2019): 112216.

Rao, T.V.R., Baraiya, N.S., Vyas, P.B. & Patel, D.M. 2016. Composite coating of alginate-olive oil enriched with antioxidants enhances postharvest quality and shelf life of Ber fruit (Ziziphus mauritiana Lamk. Var. Gola). Journal of Food Science and Technology 53(1): 748-756.

Saidin, S.H., Ali, N.A.M., Jamil, M., Kar, P., Yong, A.A. & Patah, M.F.Z. 2014. Natural fragrance extract production using supercritical carbon dioxide. Proceeding of the 30th Annual Seminar of the Malaysian Natural Products Society. Moving Translational Research in Natural Products Forward. hlm. 129-134.

Saidu, Y., Muhammad, S., Yahaya, A., Onu, A., Mohammed, I. & Muhammad, L. 2017. In vitro screening for protein tyrosine phosphatase 1B and dipeptidyl peptidase IV inhibitors from Nigerian medicinal plants. Journal of Intercultural Ethnopharmacology 6(2): 154-157.

San, A.M.M., Thongpraditchote, S., Sithisarn, P. & Gritsanapan, W. 2013. Total phenolics and total flavonoids contents and hypnotic effect in mice of Ziziphus mauritiana Lam. seed extract. Evidence-Based Complementary and Alternative Medicine 2013: 835854.

Schmid, W. 1976. The micronucleus test for cytogenetic analysis. In Chemical Mutagens, edited by Hollaender, A. Boston, MA: Springer. hlm. 31-53.

Shahrim, Z., Puteri J Noor, M.B., Noral’ashikin, Y., Muhammad, H., Rohana, A.B. & Zakiah, I. 2006. The in vivo rodent micronucleus assay of Kacip Fatimah (Labisia pumila) extract. Tropical Biomedicine 23(2): 214-219.

Shair, R.M., Maskat, M.Y. & Rosmin Kasran, M.K.A. 2020. Protective effect of cocoa extract on ethanol induced liver injury in Sprague-dawley rats. Sains Malaysiana 49(1): 93-101.

Singleton, V.L. & Rossi, J.A. 1965. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. American Journal of Enology and Viticulture 16(3): 144-158.

Suriyavadhana, M., Pakutharivu, T. & Anglais, A.E. 2011. Evaluation of acute and sub acute toxicity of ethanol extracts of Entada pursaetha, Toddalia aculeata, and Ziziphus mauritiana. World J Life Sci. and Medical Research 1(2): 43-47.

Suzuki, Y., Nagae, Y., Li, J., Sakaba, H., Mozawa, K., Takahashi, A. & Shimizu, H. 1989. The micronucleus test and erythropoiesis. Effects of erythropoietin and a mutagen on the ratio of polychromatic to normochromatic erythrocytes (P/N ratio). Mutagenesis 4(6): 420-424.

Talmale, S., Bhujade, A. & Patil, M. 2015. Anti-allergic and anti-inflammatory properties of Zizyphus mauritiana root bark. Food Function 6(9): 2975-2983.

Thomas, P. 2007. Changes in buccal biomarker in relation to ageing and Alzheimer's Disease. PhD Thesis. School of Molecular Biomedical Science, University of Adelaide: Adelaide, South Australia, Australia (Tidak diterbitkan).

Zayapor, M.N., Abdullah, A. & Mustapha, W.A.W. 2021. Influence of sugar concentration and sugar type on the polyphenol content and antioxidant activity in spiced syrup preparation. Italian Journal of Food Science 33(1): 96-105.

^*Corresponding author; email: arnida@ukm.edu.my