Optimization of microwave-assisted extraction of total flavonoid content from red betel leaf (Piper crocatum Ruiz and Pav) and its correlation with antioxidant and antibacterial activities using response surface methodology

Ririn Astyka Poppy Anjelisa Zaitun Hasibuan Sumaiyah Sumaiyah Nur Aira Juwita Muhammad Fauzan Lubis   

Open Access   

Published:  Jun 08, 2024

DOI: 10.7324/JAPS.2024.170411

The optimization of microwave-assisted extraction (MAE) was conducted using response surface methods to improve the extraction of flavonoids from Piper crocatum Ruiz and Pav leaf. The optimization process included a Box–Behnken experimental design (BBD), which involved three variables at three levels. The present study aimed to evaluate the impact of varying ethanol concentrations (50%, 75%, and 100%), microwave power levels (180, 300, and 450 W), and extraction durations (3, 8.5, and 14 minutes) on the respective responses. The experimental data was subjected to fitting using a second-order polynomial model. Subsequently, an analysis of variance (ANOVA) and multiple regression analysis were employed to assess the adequacy of the model and determine the ideal settings. Taking into account the highest concentration of extracted total flavonoids, as well as the antioxidant and antibacterial properties. The experimental results indicate that the optimum conditions for all the reactions under investigation were an ethanol concentration of 78.48%, a microwave power of 327.96 W, and an extraction duration of 8.60 minutes. Under the ideal conditions, the anticipated outcomes of the sample indicate a total flavonoid content (TFC) of 229.647 mg QE/g dry weight (DW), a 1,1-diphenyl-2-picryhydrazyl (DPPH) scavenging activity of 73.915%, and an inhibition zone measuring 18.621 mm. The implementation of a concurrent MAE methodology for the isolation of total flavonoids, as well as the evaluation of antioxidant and antibacterial properties from P. crocatum, signifies the recognition of the extract as a significant reservoir of bioactive substances.

Keyword:     Piper crocatum Ruiz and Pav response surface methodology total flavonoids content antioxidant antibacterial


Astyka R, Hasibuan PAZ, Sumaiyah S, Juwita NA, Lubis MF. Optimization of microwave-assisted extraction of total flavonoid content from red betel leaf (Piper crocatum Ruiz and Pav) and its correlation with antioxidant and antibacterial activities using response surface methodology. J Appl Pharm Sci. 2024. Online First. http://doi.org/10.7324/JAPS.2024.170411

Copyright: © The Author(s). This is an open-access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

HTML Full Text


1. Alvin A, Miller KI, Neilan BA. Exploring the potential of endophytes from medicinal plants as sources of antimycobacterial compounds. Microbiol Res, 2014;169(7-8):483-95. https://doi.org/10.1016/j.micres.2013.12.009

2. Licea-Dominguez S, Estevez-Rioja A, Hernández-Lozano LC, Alvarado-Ponce GE, Asemota H, González-Cordova AF, et al. Market orientation and cuisine innovation as a driven sensory methodology to develop a sweet potato snack as an added-value food product. Int J Gastron Food Sci. 2023;32. https://doi.org/10.1016/j.ijgfs.2023.100750

3. Jam N, Hajimohammadi R, Gharbani P, Mehrizad A, Antibacterial activity of Punica granatum L. and Areca nut (P.A) combined extracts against some food born pathogenic bacteria. Saudi J Biol Sci. 2022;29(3):1730-36. https://doi.org/10.1016/j.sjbs.2021.10.057

4. Azmir J, Zaidul ISM, Rahman MM, Sharif KM, Mohamed A, Sahena F, et al. Techniques for extraction of bioactive compounds from plant materials: a review. J Food Eng. 2013;117(4):426−36. https://doi.org/10.1016/j.jfoodeng.2013.01.014

5. Jam N, Hajimohammadi R, Gharbani P, Mehrizad A, Evaluation of antibacterial activity of aqueous, ethanolic and methanolic extracts of areca nut fruit on selected bacteria. Biomed Res Int. 2021;6663399. https://doi.org/10.1155/2021/6663399

6. Bitwell C, Indra SS, Luke C, Kakoma MK, A review of modern and conventional extraction techniques and their applications for extracting phytochemicals from plants. Sci Afr. 2023;19(e01585). https://doi.org/10.1016/j.sciaf.2023.e01585

7. Wu C, Wang F, Liu J, Zou Y, Chen X. A comparison of volatile fractions obtained from Lonicera macranthoides via different extraction processes: ultrasound, microwave, Soxhlet extraction, hydrodistillation, and cold maceration. Integr Med Res. 2015;4(3):171−77. https://doi.org/10.1016/j.imr.2015.06.001

8. Janicka P, P?otka-Wasylka J, Jatkowska N, Chabowska A, Fares MY, Andruch V, et al. Trends in the new generation of green solvents in extraction processes. Curr Opin Green Sustain. 2022;37:100670. https://doi.org/10.1016/j.cogsc.2022.100670

9. Chemat F, Abert-Vian M, Fabiano-Tixier AS, Strube J, Uhlenbrock L, Gunjevic V, et al. Green extraction of natural products. Origins, current status, and future challenges. TrAC Trends Anal Chem, 2019;118:248−63. https://doi.org/10.1016/j.trac.2019.05.037

10. Armenta S, Garrigues S, de la Guardia M. The role of green extraction techniques in green analytical chemistry. Trends Analyt Chem. 2015;71:2−8. https://doi.org/10.1016/j.trac.2014.12.011

11. Rizwan M, Gilani SR, Durrani AI, Naseem S. Low temperature green extraction of Acer platanoides cellulose using nitrogen protected microwave-assisted extraction (NPMAE) technique. Carbohydr Polym. 2021;272:118465. https://doi.org/10.1016/j.carbpol.2021.118465

12. Kaderides K, Papaoikonomou L, Serafim M, Goula AM. Microwave-assisted extraction of phenolics from pomegranate peels: optimization, kinetics, and comparison with ultrasounds extraction. Chem Eng Process. 2019;137:1-11. https://doi.org/10.1016/j.cep.2019.01.006

13. Alvi T, Asif Z, Khan MKI. Clean label extraction of bioactive compounds from food waste through microwave-assisted extraction technique-A review. Food Biosci, 2022;46:101580. https://doi.org/10.1016/j.fbio.2022.101580

14. Alara OR, Abdurahman NH, Olalere OA, Optimization of microwave-assisted extraction of flavonoids and antioxidants from Vernonia amygdalina leaf using response surface methodology. Food Bioprod. 2018;107:36-48. https://doi.org/10.1016/j.fbp.2017.10.007

15. Nana O, Momeni J, Boyom FF, Njintang NY, Ngassoum, MB. Microwave-assisted extraction as an advanced technique for optimization of limonoid yields and antioxidant potential from Trichilia roka (Meliaceae). Curr Res Green Sustain Chem. 2021;4:100147. https://doi.org/10.1016/j.crgsc.2021.100147

16. Setyawati A, Wahyuningsih MSH, Nugrahaningsih DAA, Effendy C, Ibeneme S, Piper Crocatum Ruiz and Pav as a commonly used typically medicinal plant from Indonesia: What do we know about it? Scoping Review. ICON J. 2023;7(2):61-78.

17. Fatmawaty, Anggreni NGM, Fadhil N, Prasasty VD, Potential in vitro and in vivo antioxidant activities from Piper crocatum and Persea americana Leaf Extracts. Biomed Pharmacol J. 2019;12(2):661-7. https://doi.org/10.13005/bpj/1686

18. Astuti P, Wahyono, Nababan OA, Antimicrobial and cytotoxic activities of endophytic fungi isolated from Piper crocatum Ruiz and Pav. Asian Pac J Trop Biomed. 2014;4(2):S592-6. https://doi.org/10.12980/APJTB.4.2014APJTB-2014-0073

19. Lister INE, Ginting CN, Girsang E, Nataya ED, Azizah AM, Widowati W, Hepatoprotective properties of red betel (Piper crocatum Ruiz and Pav) leaves extract towards H2O2-induced HepG2 cells via anti-inflammatory, anti necrotic, antioxidant potency. SPJ. 2020; 28(10):1182-89. https://doi.org/10.1016/j.jsps.2020.08.007

20. Wulandari N, Meiftasari A, Fadliyah H, Jenie RI, Red betel leaves methanolic extract (Piper crocatum Ruiz and Pav.) increases cytotoxic effect of doxorubicin on WiDr colon cancer cells through apoptosis induction. ISCC, 2018;9(1):1-8. https://doi.org/10.14499/indonesianjcanchemoprev9iss1pp1-8

21. Heliawati L, Lestari S, Hasanah U, Ajiati D, Kurnia D. Phytochemical profile of antibacterial agents from red betel leaf (Piper crocatum Ruiz and Pav) against Bacteria in Dental Caries. Molecules. 2022;27(9):2861. https://doi.org/10.3390/molecules27092861

22. Gharbani P, Javazi H, The antioxidant, general toxicity and insecticidal activities of Nepeta scrophularioides rech. f. extracts in different developmental stages. Pak J Pharm Sci, 2015;28(5 suppl):1905-9.

23. Nayaka NMDMW, Fidrianny I, Sukrasno, Hartati R, Singgih M, Antioxidant and antibacterial activities of multiflora honey extracts from the Indonesian Apis cerana bee. JTUMED. 2020;15(3):211-17. https://doi.org/10.1016/j.jtumed.2020.04.005

24. Vaquero MJR, Serravalle LRT, de Nadra MCM, de Saad AMS, Antioxidant capacity and antibacterial activity of phenolic compounds from argentinean herbs infusions. Food Control, 2010;21(5):779-85. https://doi.org/10.1016/j.foodcont.2009.10.017

25. Hadiyat MA, Sopha BM, Wibowo BS, Response surface methodology using observational data: a systematic literature review. Appl Sci. 2022;12:10663. https://doi.org/10.3390/app122010663

26. Brownlee AEI, Epitropakis MG, Mulder J, Paelinck M, Burke EK, A systematic approach to parameter optimization and its application to flight schedule simulation software. J Heuristics. 2022;28:509-38. https://doi.org/10.1007/s10732-022-09501-8

27. Kumari M and Gupta SK, Response surface methodological (RSM) approach for optimizing the removal of trihalomethanes (THMs) and its precursor's by surfactant modifed magnetic nano adsorbents (sMNP)-an endeavor to diminish probable cancer risk. Sci Rep. 2019;9:18339. https://doi.org/10.1038/s41598-019-54902-8

28. Mehrizad A, Gharbani P. Removal of methylene blue from aqueous solution using nano-TiO2/UV process: optimization by response surface methodology. PCCC. 2016;9(2):135-43.

29. Bezerra MA, Santelli RE, Oliveira EP, Villar LS, Escaleira LA, Response surface methodology (RSM) as a tool for optimization in analytical chemistry. Talanta. 2008;76(5):965-77. https://doi.org/10.1016/j.talanta.2008.05.019

30. Chen S, Zhang H, Yang L, Zhang S, Jiang H. Optimization of ultrasonic-assisted extraction conditions for bioactive components and antioxidant activity of Poria cocos (Schw.) Wolf by an RSM-ANN-GA Hybrid Approach. Foods. 2022;12(3):619. https://doi.org/10.3390/foods12030619

31. Zekovic Z, Vladic J, Vidovic S, Adamovic D, Pavlic B. Optimization of microwave-assisted extraction (MAE) of coriander phenolic antioxidants-response surface methodology approach. J Sci Food Agric. 2016;96(13):4613-22. https://doi.org/10.1002/jsfa.7679

32. Do QD, Angkawijaya AE, Tran-Nguyen PL, Huynh LH, Soetaredjo FE, Ismadji S, et al. Effect of extraction solvent on total phenol content, total flavonoid content, and antioxidant activity of Limnophila aromatica. J Food Drug Anal. 2014;22(3):296-302. https://doi.org/10.1016/j.jfda.2013.11.001

33. Vidovic SS, Zekovic ZP, Lepojevic ZD, Radojkovic MM, Jokic SD, Anackov GT, Optimization of the Ocimum Basilicum L. extraction process regarding the antioxidant activity. Acta Period Technol. 2012; 43:315-23. https://doi.org/10.2298/APT1243315V

34. Lubis MF, Kaban VE, Gurning K, Parhan P, Syahputra H, Juwita NA, et al. Phytochemicals and biological activities of ethanolic extract of garcinia atroviridis leaf grown in Indonesia. J Med Chem Sci, 2023;6(10):2456-69.

35. Lubis MF, Syahputra H, Illian DN, Kaban VE, Antioxidant activity and nephroprotective effect of Lansium parasiticum leaves in doxorubicin-induced rats. J Pharm Res. 2022;26(3):565-73. https://doi.org/10.29228/jrp.154

36. Wang X, Wu Y, Chen G, Yue W, Liang Q, Wu Q, Optimisation of ultrasound assisted extraction of phenolic compounds from Sparganii rhizoma with response surface methodology. Ultrason Sonochem. 2013;20(3):846-54. https://doi.org/10.1016/j.ultsonch.2012.11.007

37. Yang L, Cao Y, Jiang J, Lin Q, Chen J, Zhu L, Response surface optimization of ultrasound-assisted flavonoids extraction from the flower of Citrus aurantium L. var. amara Engl. J Sep Sci. 2010;33(9):1349-55. https://doi.org/10.1002/jssc.200900776

38. Alara OR, Nour AH, Mudalip SKA, Screening of microwave-assisted-batch extraction parameters for recovering total phenolic and flavonoid contents from Chromolaena odorata leaves through two-level factorial design, Indones J Chem. 2019;19(2):511-21. https://doi.org/10.22146/ijc.40863

39. Gil-Martín E, Forbes-Hernández T, Romero A, Cianciosi D, Giampieri F, Battino M, Influence of the extraction method on the recovery of bioactive phenolic compounds from food industry by-products. Food Chem, 2022;378:131918. https://doi.org/10.1016/j.foodchem.2021.131918

40. Liu X, Liu Y, Shan C, Yang X, Zhang Q, Xu N et al. Effects of five extraction methods on total content, composition, and stability of flavonoids in jujube. Food Chem. X. 2022;14:100287. https://doi.org/10.1016/j.fochx.2022.100287

41. Dairi S, Dahmoune F, Belbahi A, Remini H, Kadri N, Aoun O et al. Optimization of microwave extraction method of phenolic compounds from red onion using response surface methodology and inhibition of lipoprotein low-density oxidation. J Appl Res Med Aromat Plants. 2021;22:100301. https://doi.org/10.1016/j.jarmap.2021.100301

42. Simi? VM, Rajkovi? KM, Stoji?evi? SS, Veli?kovi? DT, Nikoli? NC, Lazi? ML et al. Optimization of microwave-assisted extraction of total polyphenolic compounds from chokeberries by response surface methodology and artificial neural network. Sep Purif Technol, 2016;160:89-97. https://doi.org/10.1016/j.seppur.2016.01.019

43. Ranic M, Nikolic M, Pavlovic M, Buntic A, Siler-Marinkovic S, Dimitrijevic-Brankovic S, Optimization of microwave-assisted extraction of natural antioxidants from spent espresso coffee grounds by response surface methodology. J Clean Prod. 2014;80:69-79. https://doi.org/10.1016/j.jclepro.2014.05.060

44. Ahmad I, Yanuar A, Mulia K, Mun'im A, Optimization of ionic liquid-based microwave-assisted extraction of polyphenolic content from Peperomia pellucida (L) kunth using response surface methodology. Asian Pac J Trop Biomed. 2017;7(7):660-65. https://doi.org/10.1016/j.apjtb.2017.06.010

45. Akbari S, Abdurahman NH, Yunus RM, Optimization of saponins, phenolics, and antioxidants extracted from fenugreek seeds using microwave-assisted extraction and response surface methodology as an optimizing tool. C R Chim. 2019; 22(11-12):714-27. https://doi.org/10.1016/j.crci.2019.07.007

46. Jha AK, and Sit N. Comparison of response surface methodology (RSM) and artificial neural network (ANN) modelling for supercritical fluid extraction of phytochemicals from Terminalia chebula pulp and optimization using RSM coupled with desirability function (DF) and genetic algorithm (GA) and ANN with GA. Ind Crops Prod. 2021;170(113769). https://doi.org/10.1016/j.indcrop.2021.113769

47. Kaanin-Boudraa G, Brahmi F, Wrona M, Nerín C, Moudache M, Mouhoubi K et al. Response surface methodology and UPLC-QTOF-MSE analysis of phenolic compounds from grapefruit (Citrus× paradisi) by-products as novel ingredients for new antioxidant packaging. LWT, 2021;151:112158. https://doi.org/10.1016/j.lwt.2021.112158

48. Zhao Y, Du S, Wang H, Cai M. In vitro antioxidant activity of extracts from common legumes. Food Chem. 2014;152:462-66. https://doi.org/10.1016/j.foodchem.2013.12.006

49. Alfarabi M, Bintang M, Suryani MS. The comparative ability of antioxidant activity of Piper crocatum in inhibiting fatty acid oxidation and free radical scavenging. Hayati J Biosci. 2010;17(4):201-04. https://doi.org/10.4308/hjb.17.4.201

50. Rahardjo M, Mangalik G, Sihombing M, da Costa JF. Effect of the extraction solvent polarity and the ratio of feed and solvent on the phytochemical content and antioxidant activity of red betel leaves (Piper crocatum). Indones J Agricultural Res. 2018;1(1):71-7. https://doi.org/10.32734/injar.v1i1.173

51. Kamaruzaman SRS, Kasim KF, Jaafar MN. The effect of harvesting time on the antioxidant and antidiabetic activity of Piper Crocatum (Sirih Merah) extract. IOP Conf Ser Mater Sci. 2020;864:012211. https://doi.org/10.1088/1757-899X/864/1/012211

52. Alide T, Wangila P, Kiprop A. Effect of cooking temperature and time on total phenolic content, total flavonoid content and total in vitro antioxidant activity of garlic. BMC Res Notes. 2020;13(564):1-7. https://doi.org/10.1186/s13104-020-05404-8

53. Kaneria M, Kanani B, Chanda S, Assessment of effect of hydroalcoholic and decoction methods on extraction of antioxidants from selected Indian medicinal plants, Asian Pac J Trop Biomed. 2012;2(3):195-202. https://doi.org/10.1016/S2221-1691(12)60041-0

54. Mannoubi IE, Effect of extraction solvent on phenolic composition, antioxidant and antibacterial activities of skin and pulp of Tunisian red and yellow-orange Opuntia Ficus Indica fruits. J Food Meas Charact. 2021;15:643-51. https://doi.org/10.1007/s11694-020-00673-0

55. Gonfa T, Teketle S, Kiros T. Effect of extraction solvent on qualitative and quantitative analysis of major phyto-constituents and in-vitro antioxidant activity evaluation of Cadaba rotundifolia Forssk leaf extracts. Cogent Food Agric. 2020;6:1853867. https://doi.org/10.1080/23311932.2020.1853867

56. Neto OSZ, Batista EAC, Meirelles AJA. The employment of ethanol as solvent to extract Brazil nut oil. J Clean Prod. 2018;180:866-75. https://doi.org/10.1016/j.jclepro.2018.01.149

57. Kusuma SAF, Hendriani R, Genta A. Antimicrobial spectrum of red piper betel leaf extract (Piper crocatum Ruiz and Pav) as natural antiseptics against airborne pathogens. J Pharm Sci Res. 2017;9(5):583-87.

58. Yuan G, Guan Y, Yi H, Lai S, Sun Y, Cao S, Antibacterial activity and mechanism of plant flavonoids to gram-positive bacteria predicted from their lipophilicities. Sci Rep. 2021;11(10471). https://doi.org/10.1038/s41598-021-90035-7

59. Jawhari FZ, Moussaoui AEL, Bourhia M, Imtara H, Saghrouchi H, Ammor K et al. Anacyclus pyrethrum var. pyrethrum (L.) and Anacyclus pyrethrum var. depressus (Ball) Maire: correlation between total phenolic and flavonoid contents with antioxidant and antimicrobial activities of chemically characterized extracts. Plants. 2021;10(1):149. https://doi.org/10.3390/plants10010149

60. Bouchelaghem S, Das S, Naorem RS, Czuni L, Papp G, Kocsis M. Evaluation of total phenolic and flavonoid contents, antibacterial and antibiofilm activities of Hungarian propolis ethanolic extract against Staphylococcus aureus. Molecules. 2022;27(2):574. https://doi.org/10.3390/molecules27020574

61. Sartini S, Djide MN, Amir MN, Permana AD. Phenolic-rich green tea extract increases the antibacterial activity of amoxicillin against Staphylococcus aureus by in vitro and ex vivo studies. J Pharm Pharmacogn Res. 2020;8(6):491- 500. https://doi.org/10.56499/jppres20.844_8.6.491

62. Chen Y, Liu T, Wang K, Hou C, Cai S, Huang Y et al. Baicalein inhibits Staphylococcus aureus biofilm formation and the Quorum sensing system in vitro. Plos One. 2016;11(4):e0153468. https://doi.org/10.1371/journal.pone.0153468

63. Musini A, Singh HN, Vulise J, Pammi SSS, Giri A. Quercetin's antibiofilm effectiveness against drug resistant Staphylococcus aureus and its validation by in silico modelling. Res Microbiol, 2023;104091. https://doi.org/10.1016/j.resmic.2023.104091

64. Nag D, Dastidar DG, Chakrabarti G, Natural flavonoid morin showed anti-bacterial activity against Vibrio cholera after binding with cell division protein FtsA near ATP binding site. Biochim Biophys Acta Bioenerg. 2021;1865(8):129931. https://doi.org/10.1016/j.bbagen.2021.129931

65. Dias MC, Pinto DCGA, Silva AS, Plant flavonoids: chemical characteristics and biological activity. Molecules. 2021;26(17):5377. https://doi.org/10.3390/molecules26175377

66. Gharbani P, Modeling and optimization of reactive yellow 145 dye removal process onto synthesized MnOX-CeO2 using response surface methodology. Colloids Surf. A: Physicochem. Eng. 2018;548:191- 197. https://doi.org/10.1016/j.colsurfa.2018.03.046

Article Metrics
14 Views 3 Downloads 17 Total



Related Search

By author names