Cytotoxicity, antioxidant and phytochemical screening of propolis extracts from four different Malaysian stingless bee species


  • Nor Elani Mat Nafi Universiti Sultan Zainal Abidin
  • Nur Basyirah Md Zin Universiti Sultan Zainal Abidin
  • Norzilawati Pauzi Universiti Sultan Zainal Abidin
  • Ammira Shafiqha Abdul Khadar Universiti Sultan Zainal Abidin
  • Aulia Rani Anisava Universiti Sultan Zainal Abidin
  • Ainur Awanis Mohd Badiazaman Universiti Sultan Zainal Abidin
  • Khamsah Suryati Mohd Universiti Sultan Zainal Abidin



propolis, stingless bee propolis, cytotoxicity, TLC, antioxidant


Propolis is a plant-derived substance collected by stingless bee’s product from various sources, including plant resins with combination of bee’s saliva and wax. Propolis has been used to treat several diseases since ancient times and it is an important source of bioactive natural compound and drug derivatives. The aim of this study was to evaluate biological and chemical profiles of ethanolic extracts from propolis produced by Heterotrigona itama (HI), Geniotrigona thoracica (GT), Lepidotrigona terminate (LT), and Tretrigona apicalis (TA). Cytotoxicity activity was evaluated by using 3-(4,5-Dimethylthiazol-2-YI)-2,5-Diphenyltetrazolium Bromide (MTT) assay against three cancer cell lines. H. itama extracts showed the highest cytotoxicity effect with   of 5 µg/mL, 4 µg/mL and 8 µg/mL for MDA-MB-231, SK-UT-1 and HeLa, respectively. Other species only possessed moderate to weak cytotoxicity effect against tested cells. Phytochemical screening was carried out by thin layer chromatography (TLC) analysis and visualized by derivatives agents in order to detect the presence of terpenoids, steroids, saponins, essential oils and phenol. It  was found that H. itama (HI) possessed the highest antioxidant activity with the lowest  of 30 µg/mL with percetange of inhibition at 85.69 % evaluated by (2,2-diphenyl-1-picryl-hydrazyl-hydrate) DPPH scaveging assay. In conclusion, bee species was considered as important factor in selecting the quality of propolis. It was found that H. itama produced the most active extract compared to other species. The data obtained from this study would be the basis for further investigation on therapeutic application especially for cytotoxic activity, antioxidant and phytochemical screenings for four Malaysian stingless bees of propolis.

Author Biographies

Nor Elani Mat Nafi, Universiti Sultan Zainal Abidin

School of Agriculture Sciences and Biotechnology, Faculty of Bioresources and Food Industry

Nur Basyirah Md Zin, Universiti Sultan Zainal Abidin

School of Agriculture Sciences and Biotechnology, Faculty of Bioresources and Food Industry

Norzilawati Pauzi, Universiti Sultan Zainal Abidin

School of Agriculture Sciences and Biotechnology, Faculty of Bioresources and Food Industry

Ammira Shafiqha Abdul Khadar, Universiti Sultan Zainal Abidin

School of Agriculture Sciences and Biotechnology, Faculty of Bioresources and Food Industry

Aulia Rani Anisava, Universiti Sultan Zainal Abidin

School of Agriculture Sciences and Biotechnology, Faculty of Bioresources and Food Industry

Ainur Awanis Mohd Badiazaman, Universiti Sultan Zainal Abidin

School of Agriculture Sciences and Biotechnology, Faculty of Bioresources and Food Industry

Khamsah Suryati Mohd, Universiti Sultan Zainal Abidin

School of Agriculture Sciences and Biotechnology, Faculty of Bioresources and Food Industry


Ab Hamid, S., Salleh, M. S., Krishnan, K. T., Hashim, N. A. 2016. Distribution and morphometrical variations of stingless bees (Apidae: Meliponini) in urban and forest areas of Penang Island, Malaysia. Journal of Tropical Resources and Sustainable Sciences, 4(1), 1-5.

Agarwal, R. 2000. Synthesis & biological screening of some novel coumarin derivatives. Biochemical Pharmacology, 6, 1042-1051.

Azmi, W. A., Zulqurnain, N. S., Ghazi, R. 2015. Melissopalynology and foraging activity of stingless bees, Lepidotrigona terminata (Hymenoptera: Apidae) from an apiary in Besut, Terengganu. Journal of Sustainability Science and Management, 10(1), 27-35.

Bahuguna, A., Khan, I., Bajpai, V. K., Kang, S. C. 2017. MTT assay to evaluate the cytotoxic potential of a drug. Journal of Bangladesh Pharmacology, 12, 115-118.

Bankova, V. S., de Castro, S. L., Marcucci, M. C. 2000. Propolis: recent advances in chemistry and plant origin. Apidologie, 31(1), 3-15.

Biesmeijer, J. C., Giurfa, M., Koedam, D., Potts, S. G., Joel, D. M., Dafni, A. 2005. Convergent evolution: Floral guides, stingless bee nest entrances, and insectivorous pitchers. Naturwissenschaften, 92(9), 444-450.

Brayfield, A. 2014. Martindale: The Complete Drug Reference. United Kingdom: Pharmaceutical Press.

Brown, R. 1989. Hive products: pollen, propolis and royal jelly. Bee World, 70(3), 109-117.

Chang, H., Wang, Y., Yin, X., Liu, X., Xuan, H. 2017. Ethanol extract of propolis and its constituent caffeic acid phenethyl ester inhibit breast cancer cells proliferation in inflammatory microenvironment by inhibiting TLR4 signal pathway and inducing apoptosis an autophagy. BMC Complementary and Alternative Medicine 17(471), 1-9.

Chinh, T. X., Sommeijer, M. J. 2005. Production of sexuals in the stingless bee Trigona (Lepidotrigona) ventralis flavibasis Cockerell (Apidae, Meliponini) in northern Vietnam. Apidologie, 36(4), 493-503.

Dolečková, I., Rárová, L., Grúz, J., et al. 2012. Antiproliferative and antiangiogenic effects of flavone eupatorin, an active constituent of chloroform extract of Orthosiphon stamineus leaves. Fitoterapia, 83, 1000-7.

Estévez, S., Marrer, M. T., Quintana, J., Estévez, F. 2014. Eupatorin-induced cell death in human leukemia cells is dependent on caspases and activates the mitogen-activated protein kinase pathway. PloS One, 9(11), e112536.

Halim, N. H., Pauzi, N., Hamil, S. H. R., Shafaei, A., Ismail, Z., Mohd, K. S. 2017. Standardization of orthoshiphon stamineus raw material and extracts for anti-uterine fibroid. International Journal of Pharmacognosy and Phytochemical Research, 9(4), 512-515.

Huang, S., Zhang, C. P., Wang, K., Li, G. Q., Hu, F. L. 2014. Recent advances in the chemical composition of propolis. Molecules, 19, 19610-19632.

Ibrahim, N., Mohd Niza, N. F. S., Mohd Rodi, M. M., Zakaria, A. J., Ismail, Z., Mohd, K. S. 2016. Chemical and biological analyses of malaysian stingless bee propolis extracts. Malaysian Journal of Analytical Sciences, 20(2), 413-422.

Jalil, A. H. 2014. Beescape for Meliponines: Conservation of Indo-Malayan Stingless Bees. Singapore: Partridge Publishing.

Kelly, N., Farisya, M. S. N., Kumara, T. K., Marcela, P. 2014. Species diversity and external nest characteristics of stingless bees in meliponiculture. Pertanika Journal of Tropical Agricultural Science, 37(3), 293-298.

Marcucci, M. C. 1995. Propolis: Chemical composition, biological properties and therapeutic activity. Apidologie, 26(2), 83-99.

Marshall, M. E., Butler, K., Fried, A. 1991. Screening of synthetic new hetrocyclic derivatives of 3-formyl-4-hydroxy coumarin and evaluated for Anti-Inflammatory activity in albino rats. Molecular Biotherapy, 3, 158-170.

Nagamitsu, T., Inoue, T. 2002. Foraging activity and pollen diets of subterranean stingless bee colonies in response to general flowering in Sarawak, Malaysia. Apidologie, 33, 303-314.

Norowi, M. H., Mohd, F., Sajap, A. S., Rosliza, J., Suri, R. 2010. Conservation and sustainable utilization of stingless bees for pollination services in agricultural ecosystems in Malaysia. In Proceedings of International Seminar on Enhancement of Functional Biodiversity Relevant to Sustainable Food Production in ASPAC, November 9-11 2010 Tsukuba. Japan. (pp. 1-11).

Omar, W. A. W., Azhar, N. A., Fadzilah, N. H., Kamal, N. N. S. N. M. 2016. Bee pollen extract of Malaysian stingless bee enhances the effect of cisplatin on breast cancer cell lines. Asian Pacific Journal of Tropical Biomedicine, 6(3), 265-269.

Oun, R., Moussa, Y. E., Wheate, N. J. 2018. Correction: The side effects of platinum-based chemotherapy drugs: a review for chemists. Dalton Transactions, 47(19), 6645-6653.

Pangestika, N. W., Atmowidi, T., Kahono, S. 2017. Pollen load and flower constancy of three species of stingless bees (Hymenoptera, Apidae, Meliponinae). Tropical Life Sciences Research, 28(2), 179-187.

Park, Y. K., Alencar, S. M., Aguiar, C. L. 2002. Botanical origin and chemical composition of Brazilian propolis. Journal of Agricultural and Food Chemistry, 50(9), 2502–2506.

Pierrot, L. M., Schlindwein, C. 2003. Variation in daily flight activity and foraging patterns in colonies of uruçu-Melipona scutellaris Latreille (Apidae, Meliponini). Revista Brasileira de Zoologia, 20(4), 565-571.

Pietta, P. G., Gardana, C., Pietta, A. M. 2002. Analytical methods for quality control of propolis. Fitoterapia 73(1), 7-20.

Primeau, A. J., Rendon, A., Hedley, D., Lilge, L., Tannock, I. F. 2005. The distribution of the anticancer drug Doxorubicin in relation to blood vessels in solid tumors. Clinical Cancer Research, 11(24), 8782-8788.

Rasmussen, C., Cameron, S. A. 2009. Global stingless bee phylogeny supports ancient divergence, vicariance, and long distance dispersal. Biological Journal of the Linnean Society, 99(1), 206-232.

Roubik, D. W. 2006. Stingless bee nesting biology. Apidologie, 37(2), 124-143.

Rosli, N. L., Roslan, H., Omar, E. A., Mokhtar, N., Abdul Hapit, N. H., Asem, N. 2016. Phytochemical analysis and antioxidant activities of Trigona Apicalis propolis extract. American Institute of Physics, 1791, 020018.

Sajap, A. S., Adam, N. A., Hamid, M. N. 2015. Flight Intensity of two species of stingless bees Heterotrigona Itama and Geniotrigona Thoracica and its relationships with temperature, light intensity and relative humidity. Serangga, 20(1), 35-42.

Sakagami, S. F., Inoue, T., Salmah, S. 1990. Stingless bees of central Sumatra. Stingless bees of central Sumatra, 125-137.

Salim, H. M., Dzulkiply, A. D., Harrison, R. D., Fletcher, C., Kassim, A., Potts, M. D. 2012. Stingless bee (Hymenoptera: Apidae: Meliponini) diversity in dipterocarp forest reserves in Peninsular Malaysia. The Raffles Bulletin of Zoology 2012, 60(1), 213–219.

Sanpa, S., Popova, M., Bankova, V., Tunkasiri, T., Eitssayeam, S., Chantawannakul, P. 2015. Antibacterial compounds from propolis of Tetragonula laeviceps and Tetrigona melanoleuca (Hymenoptera: Apidae) from Thailand. PLoS One, 10(5), e0126886.

Saufi, N. F. M., Thevan, K. 2015. Characterization of nest structure and foraging activity of stingless bee, geniotrigona thoracica (Hymenopetra: Apidae; meliponini). Jurnal Teknologi, 77(33), 69-74.

Sawatthum, A., Jitake, P., Rangyai, O., Prangprayong, R., Pimboon, P., Suparit, K. 2017. Efficacy of stingless bee lepidotrigona terminata as insect pollinator of F1 hybrid cucumber. International Journal, 13(37), 102-106.

Schwarz, H. F., Hobby, B. M., Moore, A. W. 1937. Results of the Oxford University Sarawak (Borneo) Expedition: Bornean stingless bees of the genus Trigona. Bulletin of the AMNH; 73, article 3.

Szliszka, E., Zenon, P., Czuba, Domino, M., Mazur, B., Zydowicz, G., Krol, W. 2009. Ethanolic extract of propolis (EEP) Enhances the apoptosis- inducing potential of TRAIL in cancer cells. Journal of Molecules, 14, 738-754.

Wagh, V. D. 2013. Propolis: A wonder bees product and its pharmacological potentials. Journal of Advances in Pharmacological Sciences, 2013, 308249.

Xuan, H., Li, Z., Yan, H., Sang, Q., Wang, K., He, Q., Wang, Y., Hu, F. 2014. Antitumor activity of chinese propolis in human breast cancer MCF-7 and MDA-MB-231 Cells. Evid Based Complement Alternat Medicine 2014, 280120.

Yadav, V. R., Prasad, S., Sung, B., Kannappan, R., Aggarwal, B. B. 2010. Targeting inflammatory pathway by triterpenoids for prevention and treatment of cancer. Toxins, 2, 2428-2466.






Special Issue on International Conference on Agriculture, Animal Sciences and Food Technology 2018 (Applied Sciences)