Malays. J. Anal. Sci. Volume 29 Number 6 (2025): 1590

Research Article

Mechanisms of action of total phenolics and flavonoids in kalangkala (Litsea angulata Blume) leaf extract and fractions, and their correlation with in vitro antioxidant potential

Setya Enti Rikomah¹, Nurkhasanah², and Sapto Yuliani²

¹Al-Fatah Health College, Bengkulu, Indonesia

²Faculty of Pharmacy, Ahmad Dahlan University, Yogyakarta, Indonesia

^*Corresponding author: nurkhasanah@pharm.uad.ac.id

Received: 27 May 2025; Revised: 20 October 2025; Accepted: 29 October 2025; Published: 28 December 2025

Abstract

Phenolic and flavonoid compounds are widely found in a variety of plant species, including kalangkala (Litsea angulata Blume), a species endemic to South Kalimantan, Indonesia. Phenolic and flavonoid compounds are the compounds responsible for antioxidant activity. The objective of this study was to examine the mechanisms of action of total phenolic content (TPC) and total flavonoid content (TFC) in kalangkala leaves, as well as their correlation with in vitro antioxidant activity against ABTS free radicals. The study entailed the extraction of kalangkala leaves through maceration with 70% ethanol, followed by multilevel fractionation. Determination of the total phenolic content and total flavonoid content was also conducted, along with the assessment of antioxidant activity and the determination of their respective correlations. The results showed that the total phenolic content of the ethanol extract was 51.65 ± 4.04 mg GAE/g, the n-hexane fraction was 64.39 ± 7.63 mg GAE/g, the ethyl acetate fraction was 97.05 ± 1.46 mg GAE/g, and the methanol fraction 28.96 ± 2.22 mg GAE/g. The total flavonoid content of the ethanol extract was 22.19 ± 0.91 mg QE /g, the n-hexane fraction was 20.27 ± 2.38 mg QE /g, the ethyl acetate fraction was 88.07 ± 1.34 mg QE /g, and the methanol fraction was 99.62 ± 1.97 mg QE /g. The antioxidant activity of the ethanol extract was 51.23±3.86 ppm/ml, the n-hexane fraction was 159.44±2.93 ppm/ml, the ethyl acetate fraction was 136.01±6.47 ppm/ml, and the methanol fraction was 93.28±0.66 ppm/ml. The total phenolic content of the extract and fractions of kalangkala leaves exhibited a significant correlation (p<0.05) with the antioxidant activity of the leaves, as indicated by a correlation coefficient of 0.580. In contrast, the total flavonoid content of the extract and fractions of kalangkala leaves did not demonstrate a significant correlation with the antioxidant activity the plant material, as evidenced by a correlation coefficient of -0.224. The study concluded that a strong antioxidant is present in the 70% ethanol extract of kalangkala leaves, with a value of 51.65±4.04, as well as in the methanol fraction, with a value of 93.28±0.66 ppm/ml. Additionally, the study establishes a correlation between the total phenolic and flavonoid content present in the ethanol extract, n-hexane fraction, ethyl acetate fraction and methanol fraction of the kalangkala leaves and the antioxidant activity of the plant material.

Keywords: total phenolic, total flavonoid, antioxidant, ABTS, kalangkala leaves (Litsea angulata Blume)

References

1. Umeno, A., Horie, M., Murotomi, K., Nakajima, Y., and Yoshida, Y. (2016). Antioxidative and antidiabetic effects of natural polyphenols and isoflavones. Molecules, 21(6): 1-15.

2. Rakha, A., Umar, N., Rabail, R., Butt, M. S., Kieliszek, M., Hassoun, A., and Aadil, R. M. (2022). Anti-inflammatory and anti-allergic potential of dietary flavonoids: A review. Biomedicine and Pharmacotherapy, 156 (1): 1-10.

3. Lai, H. Y., and Lim, Y. Y. (2011). Evaluation of antioxidant activities of the methanolic extracts of selected ferns in Malaysia. International Journal of Environmental Science and Development, 6 (2): 442–447.

4. Hanin, N. N. F., and Pratiwi, R. (2017). Phenolic, flavonoid content and antioxidant activity of fertile and sterile sea fern (Acrostichum aureum L.) leaf extracts in the Kulon Progo mangrove area, Yogyakarta. Journal of Tropical Biodiversity and Biotechnology, 2 (2): 51-56.

5. Nur, S., Sami, F. J., Awaluddin, A., and Afsari, M. I. A. (2019). Correlation between total flavonoid and phenolic contents of white teak leaf extract and fraction (Gmelina Arborea Roxb.) and antioxidant activity. Jurnal Farmasi Galenika (Galenika Journal of Pharmacy) (e-Journal), 5(1): 33–42.

6. Sundu, R., Supriningrum, Risa, Fatimah, and Nurul. (2022). Total content of phenolic compounds, total flavonoid compounds and antioxidant activity of ethanol extract of sekilang bark (Embelia borneensis Scheff.). Bivalen: Chemical Studies Journal, 5(2): 31–36.

7. Rohman, A., Riyanto, S., Yuniarti, N., Saputra, W. R., Utami, R., and Mulatsih, W. (2010). Antioxidant activity, total phenolic, and total flavaonoid of extracts and fractions of red fruit (Pandanus conoideus Lam). International Food Research Journal, 17(1): 97–106.

8. Anwar, Khoerul, Fadlillaturrahmah, Sari, and Puspita, D. (2017). Analysis of total flavonoid content of ethanol extract of binjai leaves and its effect on blood glucose levels of high fructose-fat induced rats. Jurnal Ilmiah Ibnu Sina, 2(1): 20–30.

9. Prawitasari, D. S. (2019). Diabetes mellitus and antioxidants. Keluwih: Jurnal Kesehatan Dan Kedokteran, 1(1): 48–52.

10. Marjenah, Matius, P., and Hura, A. (2021). Application of coconut water to the germination of kalengkala seeds (Listea garciae Vidal) with soaking and incubation treatments. Jurnal Agrifor, 1: 139–152.

11. Rikomah, S. E., Nurkhasanah, Yuliani, S., and Daipadli. (2025). Bioactive compound profile of extracts and fractions of kalangkala leaves (Litsea angulata) as antioxidants using thin layer chromatography. Jurnal Ilmiah Pharmacy, 12(1): 43–50.

12. Kuspradini, H., Wulandari, I., Putri, A. S., Tiya, S. Y., and Kusuma, I. W. (2019). Phytochemical, antioxidant and antimicrobial properties of litsea angulata extracts. F1000Research, 7(1): 1–11.

13. Rikomah, S. E., Mahfudh, N., and Yuliani, S. (2024). Review: potential pharmacological activity of kalangkala plant (Litsea angulata). Scientific journal of pharmaceutical medical science, 9(4): 877–886.

14. Susiani, E. F., and Saputri, R. (2020). Antioxidant activity of ethanol extract of kalangkala leaves and bark (Litsea angulata) from South Kalimantan. Ibnu Sina Scientific Journal (JIIS): Pharmaceutical and Health Sciences, 5(1): 149–155.

15. Aryadi, M dan Fauzi, H. (2013). Proceedings of the National Agroforestry Seminar “Agroforestry for Better Food and Environment.” November 2018, 347

16. Pawah, W. A., Suryanto, E., and Fatimah, F. (2023). Antioxidant activity and α-amylase enzyme inhibition of basil stem waste extract. Chemistry Progress, 16 (1): 9–19.

17. Nurulita, N. aries, Sundhani, E., Amalia, I., Rahmawati, F., and Utami, N. (2019). Testing the antioxidant and anti-aging activity of body butter with the active ingredient of moringa leaf extract. Jurnal Ilmu Kefarmasian Indonesia, 17 (1): 1–8.

18. Chandra, K., Singh, P., Dwivedi, S., and Jain, S. (2019). Diabetes mellitus and oxidative stress: A co-relative and therapeutic approach. Journal of Clinical and Diagnostic Research, 1 (1): 1-9.

19. Sembiring, E. N., Elya, B., and Sauriasari, R. (2017). Phytochemical screening, total flavonoid and total phenolic content and antioxidant activity of different parts of Caesalpinia bonduc (L.) Roxb. Pharmacognosy Journal, 10 (1): 123–127.

20. Escarpa, A., and González, M. (2001). Approach to the content of total extractable phenolic compounds from different food samples by comparison of chromatographic and spectrophotometric methods. Analytica Chimica Acta, 427(1): 119–127.

21. Gupta, M., Dey, A., Majumder, S., Ghosh, S., and Barui, A. (2022). Evaluation of antioxidant activity using by H₂O₂, DPPH, ABTS methods and phytochemical tests, TPC and TFC of commonly used medicinal plants of West Bengal. International Journal of Pharmaceutical Sciences Review and Research, 72 (1): 123–132.

22. Bhattarai, A., Shrestha, N., and Shrestha, S. (2016). Determination of ascorbic acid in different citrus fruits of Kathmandu Valley. Journal of Medical and Biological Science Research, 2(1): 9–14.

23. Tenore, G. C., Novellino, E., and Basile, A. (2012). Nutraceutical potential and antioxidant benefits of red pitaya (Hylocereus polyrhizus) extracts. Journal of Functional Foods, 4(1): 129–136.

24. Yusof, N., Munaim, M. S. A., and Veloo Kutty, R. (2021). Optimization of total phenolic compounds extracted from propolis by ultrasound- assisted extraction. Chemical Engineering Communications, 208(4): 564–572.

25. Albuquerque, M. A. C. de, Levit, R., Beres, C., Bedani, R., de Moreno de LeBlanc, A., Saad, S. M. I., and LeBlanc, J. G. (2019). Tropical fruit by-products water extracts as sources of soluble fibres and phenolic compounds with potential antioxidant, anti-inflammatory, and functional properties. Journal of Functional Foods, 52(1): 724–733.

26. Abbas, Mohamed, A., Abdulamir, A. S., and Abas, H. A. (2008). A review on supercritical fluid extraction as new analytical method. American Journal of Biochemistry and Biotechnology, 4(4): 345-353.

27. Osorio-Tobón, J. F. (2020). Recent advances and comparisons of conventional and alternative extraction techniques of phenolic compounds. Journal of Food Science and Technology, 57(12): 4299–4315.

28. Zuraida, Sulistiyani, Sajuthi, D., and Suparto, I. (2017). Phenols, flavonoids and antioxidant activity in pulai bark extract (Alstonia scholaris R.Br.). Jurnal Peneltiian Hasil Hutan, 35(3): 211–219.

29. Puspawati, N. L., and Rustini. (2025). Determination of total phenol and total flavonoid levels and the antioxidant potential of various giraffe leaf extracts (Leea angulata Korth. Ex Miq). Jurnal Kimia, 19(1): 20–26.

30. Mahfudh, N., Thoyyibah, Naqiyyah, R., and Utami, D. (2024). Screening of antioxidant active fraction of Uncaria gambir Roxb (Bajakah kalawit) wood extract and determination of total phenolics and flavonoids. African Journal of Biological Sciences, 6(15): 11645–11657.

31. Velickovic, I., Grujic, S., and Marin, P. (2016). Antioxidant properties of Rubus discolor leaf extracts. Zbornik Matice Srpske Za Prirodne Nauke, 131(1): 189–196.

32. Borges, G., Mullen, W., and Crozier, A. (2010). Comparison of the polyphenolic composition and antioxidant activity of European commercial fruit juices. Food and Function,1 (1): 73-81.

33. Hidayah, L. A., and Anggarani, M. A. (2022). Determination of total phenolic, total flavonoid, and antioxidant activity of India onion extract. Indonesian Journal of Chemical Science, 11 (2): 124–135.

34. Ambarsari, N., and Dayanti, R. (2023). Literature review: Antioxidant activity of extracts and fractions of matoa leaves (Pometia pinnata). Journal Syifa Sciences and Clinical Research, 5(3): 447–452.

35. Wulandari, L., Nugraha, A. S., and Azhari, N. P. (2020). Determination of antioxidant and antidiabetic activity of kepundung leaf extract (Baccaurea racemosa Muell.Arg.) in vitro. Jurnal Sains Farmasi and Klinis, 7(1): 60-66.

36. Hasanah, M., Apriyanti, D., Patmayuni, and Dewi. (2021). Comparison of antioxidants of ethanol extract of agarwood leaves (Aqularia malaccensis L.) and three fractions of various solvents (hexane, ethyl acetate, and water). Jurnal Penelitian Sains, 21(3): 163–167.

37. Effendy, Sanezea, Neldi, V., Ramadhani, and Putri. (2024). Determination of total flavonoid and total phenol levels and antioxidant activity test of ethanol extract of rosella flowers (Hibiscus sabdariffa L.). Jurnal Farmasi Higea, 16(1): 71-80.

38. Dias, M. C., Pinto, A., D. C. G., and Silva, A. M. S. (2021). Plant flavonoids: Chemical characteristics and biological activity. Molecules, 26(17): 1-16.

39. Zeb, A. (2021). Concept, mechanism, and applications of phenolic antioxidants in foods. J Food Biochem, 9(1): 44-53.

40. Haryoto. (2019). Antioxidant activity of ethanol extract, polar, semipolar and non-polar fractions from mangrove leaves using DPPH and FRAP methods. Jurnal Sains Kesehatan, 2(2): 1-10.

41. Farida, E., Betty, S., Nuraida, L., and Giriwono, P. E. (2019). Antioxidant Activity and Α-glucosidase inhibition by ethanol extract of indigenous lactic acid bacteria. Jurnal Teknologi Dan Industri Pangan, 30(1): 56–63.

42. Dolongtelide, J. I., Fatimawali, F., Tallei, T. E., Suoth, E. J., Simbala, H. E. I., Antasionasti, I., and Kalalo, M. J. (2023). In vitro antioxidant activity of chrysanthemum indicum flowers extract and its fraction. Malacca Pharmaceutics, 1(2): 43–47.

43. Hilma, R., Gustina, N., and Syahri, J. (2020). Measurement of total phenolic, flavonoid, antioxidant and antidiabetic activity of ethyl acetate extract of katemas leaves (Euphorbia heterophylla L.) in vitro and in silico through inhibition of α-glucosidase enzyme. ALCHEMY Jurnal Penelitian Kimia, 16 (2): 240-249.

44. Suleria, H. A. R., Barrow, C. J., and Dunshea, F. R. (2020). Screening and characterization of phenolic compounds and their antioxidant capacity in different fruit peels. Foods, 9 (9): 1-9.

45. Dahlan, M. S. (2018). Regresi linear. Epidemiologi Indonesia, Jakarta: 1. 30-35.

46. Wardani, Y. K., Kristiani, E. B. E., and Sucahyo. (2020). Correlation between antioxidant activity and phenolic compound content and growing location of Celosia argentea Linn. Bioma: Berkala Ilmiah Biologi, 22(2): 136–142.

47. Nainggolan, R. M., Rahayu, M. P., Rejeki, E. S., Total, F., and Daun, F. (2024). Antioxidant activity test, flavonoid content, and total phenolics of tamarind leaf extracts and fractions (Tamarindus indica L.). Jurnal Mandala Pharmacon, 10(2): 397–410.

48. Wang, X., Zhao, W., Zhang, X., Wang, Z., Han, C., Xu, J., Yang, G., Peng, J., and Li, Z. (2023). An integrative analysis to predict the active compounds and explore polypharmacological mechanisms of Orthosiphon stamineus Benth. Computers in Biology and Medicine, 163(1): 1-21.

49. Sookying, S., Duangjai, A., Saokaew, S., and Phisalprapa, P. (2022). Botanical aspects, phytochemicals, and toxicity of Tamarindus indica leaf and a systematic review of antioxidant capacities of T. indica leaf extracts. Frontiers in Nutrition, 9(1): 1-26

50. Pulido, R., Bravo, L., and Saura-Calixto, F. (2000). Antioxidant Activity of dietary polyphenols as determined by a modified ferric reducing/antioxidant power assay. Journal of Agricultural and Food Chemistry, 48(8): 3396–3402.

51. Tiranakwit, T., Puangpun, W., Tamprasit, K., Wichai, N., Siriamornpun, S., Srisongkram, T., and Weerapreeyakul, N. (2023). Phytochemical screening on phenolic, flavonoid contents, and antioxidant activities of six indigenous plants used in traditional Thai medicine. International Journal of Molecular Sciences, 24(17):1-20.

52. Ahda, M., Jaswir, I., Khatib, A., Ahmed, Q. U., Mahfudh, N., Ardini, Y. D., and Rohman, A. (2023). FTIR fingerprinting profiling, antioxidant activity, and α-glucosidase inhibitory activity of Orthosiphon stamineus leaf ethanolic extracts. International Journal of Food Properties, 26(1): 2025–2038.