Optimization of Bioactive Compound Extraction from Propolis by Reflux, Maceration and Ultrasound-assisted Methods and Characterization of the Extracts

Abstract

The region, botanical origin, and bee species influence the raw propolis content and its bioactive properties. Additionally, the extraction methods, solvents, and various process parameters significantly affect the bioactive properties of propolis extract, which is consumed as a food supplement or pharmaceutical product. In this study, propolis with a chestnut botanical origin, obtained from the Black Sea region in Turkey, was used as the raw material. The process parameters of three basic extraction methods— maceration (M), reflux (R), and ultrasound-assisted (UA)—were optimised using response surface methodology. Antioxidant activity (AA) and total phenolic content (TPC) were used as response parameters. The optimised levels for M were 78.46% ethanol concentration and 71.05 hours for extraction time; for R, 80.64% ethanol concentration, 117.44 minutes for extraction time, and 38.38°C for temperature; and for UA, 82.49% ethanol concentration, 59.12 minutes for extraction time, and 40.53°C for temperature. The results were statistically validated using the t-test. The AA, TPC, and phenolic, volatile, and mineral contents were compared among the optimised chestnut propolis extracts. Chrysin, a flavone, and pinocembrin, a flavanone, along with ferulic and ellagic acid, among the phenolic acids, were identified as the most abundant phenolic compounds. Among the 11 elements, the highest macro elements were Na, K, and Ca, while the trace elements were Fe and Zn. The phenolic, volatile, and mineral compositions of the optimised propolis extracts exhibited heterogeneous distributions. However, fatty acids (e.g., 18:0, 18:1) were present at relatively high levels only in R; phenolic compounds were obtained in relatively high amounts via M extraction. Some minor volatiles were detected only by UA extraction. Following the characterisation of the optimised extracts, it was determined that each extraction method has its own unique advantages. The results indicate that all three methods should be optimised and used together to achieve the highest component composition and bioactivity.

Keywords

• Food supplement
• bee products
• Response surface methodology
• bioactivity

References

1. Abduh M Y, Ramdhani F, Setiawan A, Rifqialdi G, Rahmawati A & Zainudin I M (2023). Determination of productivity, yield and bioactivity of propolis extract produced by Tetragonula spp. Cultivated in Modular tetragonula hives. Heliyon: 9(6) https://doi.org/10.1016/j.heliyon.2023.e17304
2. Acun S & Gül H (2021). Usage of microencapsulated pine propolis in cupcake production. Journal of the Institute of Science and Technology 11(2): 1205-1217 (in Turkish). https://doi.org/10.21597/jist.855038
3. Al Khalifa A S & Ahmad D (2010). Determination of key elements by ICP-OES incommercially available infant formulae and baby foods in Saudi Arabia. African Journal of Food Science 4(7): 464 – 468
4. Al Dreini S, Fatfat Z, Abou Ibrahim N, Fatfat M Gali-Muhtasib H & Khalife H (2023). Thymoquinone enhances the antioxidant and anticancer activity of Lebanese propolis. World Journal of Clinical Oncology 14(5): 203–214. https://doi.org/10.5306/wjco.v14.i5.203
5. Anonymous (2021). Turkish Food Codex Bee Products Communiqué (draft), Ministry of Agriculture and Forestry of the Republic of Turkey, https://www.tarimorman.gov.tr/Duyuru/1346/Mevzuat-Taslagi-Turk-Gida-Kodeksi-Ari-Urunleri-Tebligi (24.11.2024).
6. Anonymous (2024). Delta 3 Carene: The Terpene for Healthy Bones (and Can Cause Cotton mouth), https://leafwell.com/blog/delta-3-carene (24.01.2024)
7. Arda G (2022). Determination of macro and micro element contents in propolis products by ICP-OES, MSc Thesis, Tekirdağ Namık Kemal University. Tekirdağ
8. Asem N, Abdul Gapar N A, Abd Hapit N H & Omar E A (2020). Correlation between total phenolic and flavonoid contents with antioxidant activity of Malaysian stingless bee propolis extract. Journal of Apicultural Research, 59(4): 437-442. https://doi.org/10.1080/00218839.2019.1684050

9. Atayoglu A T, Atik D S, Bölük E, Gürbüz B, Ceylan F D, Çapanoğlu E & Palabiyik I (2023). Evaluating bioactivity and bioaccessibility properties of the propolis extract prepared with l-lactic acid: An alternative solvent to ethanol for propolis extraction. Food Bioscience, 53: 102756 https://doi.org/10.1016/j.fbio.2023.102756
10. Bankova V, Trusheva B & Popova M (2021). Propolis extraction methods: A review. Journal of Apicultural Research, 60(5): 734-743. https://doi.org/10.1080/00218839.2021.1901426
11. Bayram N E (2020). A study on free-radical scavenging activity, individual phenolic compounds and element concentration of propolis. Uludağ Arıcılık Dergisi 20(2): 145-156 https://doi.org/10.31467/uluaricilik.778751
12. Calegari M A, Ayres B B, Dos Santos Tonial L M, De Alencar S M & Oldoni T L C (2020). Fourier transform near infrared spectroscopy as a tool for predicting antioxidant activity of propolis. Journal of King Saud University 32(1): 784-790. https://doi.org/10.1016/j.jksus.2019.02.006
13. Cottica S M, Sawaya A C, Eberlin M N, Franco S L, Zeoula L M & Visentainer J V (2011). Antioxidant activity and composition of propolis obtained by different methods of extraction. Journal of the Brazilian Chemical Society 22: 929-935. https://doi.org/10.1590/S0103- 50532011000500016
14. Do Nascimento Araújo C, Mayworm M A S, Yatsuda R, Negri G, Salatino MLF, Salatino A & Campos G B (2020). Chemical composition and antimycoplasma activity of a brown propolis from southern Brazil. Journal of Food Science and Technology 57(11): 4228–4235. https://doi.org/10.1007/s13197-020-04461-y
15. El-Guendouz S, Lyoussi B & Miguel M G (2019). Insight on propolis from mediterranean countries: chemical composition, biological activities and application fields. Chemistry & Biodiversity 16(7): e1900094. https://doi.org/10.1002/cbdv.201900094
16. Farré-Armengol G, Filella I, Llusià J & Peñuelas J (2017). β-Ocimene, a key floral and foliar volatile involved in multiple interactions between plants and other organisms. Molecules 22(7): 1148. https://doi.org/10.3390/molecules22071148
17. Golubkina N A, Sheshnitsan S S, Kapitalchuk M V & Erdenotsogt E (2016). Variations of chemical element composition of bee and beekeeping products in different taxons of the biosphere. Ecological Indicators 66: 452-457. https://doi.org/10.1016/j.ecolind.2016.01.042
18. Kara Y, Can Z & Kolaylı S (2022). What should be the ideal solvent percentage and solvent propolis ratio in the preparation of ethanolic propolis extract? Food Analytical Methods 15:1707–1719. https://doi.org/10.1007/s12161-022-02244-z
19. Kim S H, Kim I H, Kang B H, Lee K H, Lee S H, Lee D S & Lee J M (2009). Optimization of ethanol extraction conditions from propolis (a bee product) using response surface methodology. Korean Journal of Food Preservation, 16(6): 908-914
20. Kasote D, Bankova V & Viljoen A M (2022). Propolis: chemical diversity and challenges in quality control. Phytochemistry Reviews 21(6): 1887– 1911. https://doi.org/10.1007/s11101-022-09816-1
21. Lovakovic B T, Lazarus M, Karaconji I B, Jurica K, Semren T Z, Lusic D & Pizent A (2018). Multi-Elemental composition and antioxidant properties of strawberry tree (Arbutus Unedo L.) honey from the coastal region of Croatia: risk-benefit analysis. Journal of Trace Elements in Medicine and Biology 45: 85-92. https://doi.org/10.1016/j.jtemb.2017.09.022
22. Margeretha I, Suniarti D F, Herda E M & Alim Z (2012). Optimization and comparative study of different extraction methods of biologically active components of Indonesian propolis Trigona spp. Journal of Natural Products 5: 233- 242
23. Mele E (2023). Electrospinning of honey and propolis for wound care. Biotechnology and Bioengineering 120 (5): 1229–1240. https://doi.org/10.1002/bit.28341
24. Mokhtar S U, Hooi H S, Lene D T T & Jayaraman S (2019). Comparison of total phenolic and flavonoids contents in Malaysian propolis extract with two different extraction solvents. International Journal of Engineering & Technology, 6(2): 1–11 http://dx.doi.org/10.15282/ijets.6.2.2019.1001
25. Özdal H R, Nakilcioğlu E & Ötleş S (2023). Effects of extraction methods and extraction variables on bioactive compounds of propolis. Gıda, 48(6): 1123-1131 (in Turkish). http://dx.doi.org/10.15237/gida.GD23074
26. Pobiega K, Kraśniewska K, Derewiaka D & Gniewosz M (2019). Comparison of the antimicrobial activity of propolis extracts obtained by means of various extraction methods. Journal of Food Science and Technology 56(12): 5386-5395. https://doi.org/10.1007/s13197-019- 04009-9
27. Rocha V M, Portela R D, Dos Anjos J P, De Souza C O & Umsza-Guez M A (2023). Stingless bee propolis: composition, biological activities and its applications in the food industry. Food Production, Processing and Nutrition 5(29): 1-13. https://doi.org/10.1186/s43014-023-00146-z
28. Ruiz-Hurtado P A, Garduño-Siciliano L, Dominguez-Verano P, Martinez-Galero E, Canales-Martinez M M & Rodriguez Monroy M A (2021). Evaluation of the gastroprotective efects of Chihuahua propolis on indomethacin-induced gastric ulcers in mouse. Biomedicine & Pharmacotherapy 137: 111345. https://doi.org/10.1016/j.biopha.2021.111345
29. Saitoh J & Saya H (2016). Benzaldehyde suppresses multiple signal pathways in cancer cells by regulating 14-3-3ζ-mediated protein-protein interactions. Cancer Research 76(14): 4758-4758. https:// doi.org/10.1158/1538-7445.am2016-4758
30. Shahbaz M, Naeem H, Imran M, Ul Hassan H, Alsagaby S A, Al Abdulmonem W, Waqare A B, Ghorabf A H, Abdelgawadg M A, Ghoneimh M M, Hussainj M, Jbawik E A & Ihsan A (2023). Chrysin a promising anticancer agent: recent perspectives. International Journal of Food Properties 26 (1): 2294-2337. https://doi.org/10.21767/2248-9215.100057
31. Shahidi F & Naczk M (1995). Food phenolics: Sources, chemistry, effects and applications. Technology Publishing Company. 44(12): 287- 293
32. Sharifi-Rad M, Varoni E M, Iriti M, Martorell M, Setzer W N, Del Mar Contreras M, Salehi B, Soltani-Nejad A, Rajabi S, Tajbakhsh M & Sharifi-Rad J (2018). Carvacrol and human health: a comprehensive review. Phytotherapy Research 32: 1675–1687. https://doi.org/10.1002/ptr.6103
33. Silici S & Kutluca S (2005). Chemical composition and antibacterial activity of propolis collected by three different races of honeybees in the same region. Journal of Ethnopharmacology 99: 69–73. https://doi.org/10.1016/j.jep.2005.01.046
34. Sulaiman G M, Al-Amiery A A & Bagnati R (2014). Theoretical, antioxidant and cytotoxic activities of caffeic acid phenethyl esterand chrysin. International Journal of Food Sciences and Nutrition 65(1): 101–105. https://doi.org/10.3109/09637486.2013.832174
35. Ulanowska M & Olas B (2021). Biological properties and prospects for the application of eugenol-A Review. International Journal of Molecular Sciences 22(7): 3671. https://doi.org/10.3390/ijms22073671
36. Woźniak M, Mrówczyńska L, Kwaśniewska-Sip P, Waśkiewicz A, Nowak P & Ratajczak I (2020). Effect of the solvent on propolis phenolic profile and its antifungal, antioxidant, and in vitro cytoprotective activity in human erythrocytes under oxidative stress. Molecules, 25(18): 4266. https://doi.org/10.3390/molecules25184266
37. Zin N B M, Azemin A, Muslim M, Rodi M & Mohd S (2018). Chemical composition and antioxidant activity of stingless bee propolis from different extraction methods. International Journal of Engineering & Technology 7 (4.43): 90–95. https://doi.org/10.14419/ijet.v7i4.43.25825