Optimization of phenolic and flavonoid content from Graptophyllum pictum (L.) Griff. leaf under maceration extraction methods using response surface methodology and its antioxidant activity

Year 2025, Volume: 29 Issue: 5, 2077 - 2090, 01.09.2025

Sri Yuliasmi Jane Melita Keliat Lokot Donna Lubis , Muhammad Fauzan Lubis

https://doi.org/10.12991/jrespharm.1766338

Abstract

Graptophyllum pictum (L.) Griff. is a plant with many pharmacological effects and contains secondary metabolites which are phenolic and flavonoid compounds. This study aims to determine the optimum phenolic and flavonoid extraction conditions using a Box-Behnken design (BBD) and the correlation of optimized extract with antioxidant activity. The experimental design was conducted based on 3 factors and 3 levels: solvent concentration (X1; 50, 70, and 90% ethanol in water), extraction time (X2; 1, 2, and 3 days), and solid-to-liquid ratio (X3; 1: 5 – 1: 15). The phenolic and flavonoid compounds of the optimized extract were identified using LC-HRMS. Meanwhile, the antioxidant activity of the optimized extract was determined using DPPH radical scavenging activity. Statistical analysis results suggest that the model used is quadratic (p-value < 0.05) for the entire response. The R2 was obtained at a high figure of 97.96%, 98.59%, and 91.13%, respectively. The optimum yield, TPC, and TFC with the application of response surface methodology were obtained at 68.33% (X1), 2.10 days (X2), and 1: 10.36 (X3) and the experiment value of yield, TPC, and TFC was 48.712 ± 2.896 g, 99.937 ± 3.672 mg GAE/ g, and 37.562 ± 2.984 mg QE/ g, respectively. The optimized extract's phytochemical compounds included phenolics of p-coumaric acid, ferulic acid, cinnamic acid, and anacardic acid. Moreover, the optimized extract has the strongest antioxidant activity in radical DPPH inhibition with a dependent concentration manner. In summary, this method was successfully applied to optimize yield, TPC, and TPC in G. pictum leaves following the highest antioxidant activity. The optimized extraction method is reproducible and could be applied in the pharmaceutical industry to develop a product containing G. pictum extract.

Keywords

Graptophyllum pictum (L.) , Graptophyllum pictum (L. , maceration , phenolic content , flavonoid , antioxidant

References

• [1] Rahmi H, Artika IM, Azwar NR, Seno DSH, Nurcholis W. The activity of Wungu leaf (Graptophyllum pictum (L) Griff) extract in reducing blood glucose level of hyperglycemic mice. Curr Biochem. 2014; 1(2): 83 – 88. https://doi.org/10.29244/cb.11.1.2.
• [2] Juniarti DE, Kusumaningsih T, Juliastuti WS, Soetojo A, Wungsu ND. Phytochemical analysis and antibacterial activity of purple leaf extract [Graptophyllum pictum (L.) Griff] against Streptococcus mutans. Acta Med Philipp. 2021; 55(8): 802 – 806. https://doi.org/10.47895/amp.v55i8.2125.
• [3] Kurniawati A, Kristanti YD, Rahmat NA, Rahayu YC, Cholid Z, Sosiawan A. The role of purple leaves extract (Graptophyllum Pictum (L.) Griff) on the number of fibroblasts and blood vessels in the socket after tooth extraction. Dent J. 2024; 57(1): 56 – 61. https://doi.org/10.20473/j.djmkg.v57.i1.p56-61.
• [4] Kusumawati I, Rullyansyah S, Rizka A F, Hestianah EP, Matsunami K. Histomorphometric study of ethanolic extract of Graptophyllum pictum (L.) Griff. leaves on croton oil-induced hemorrhoid mice: A Javanese traditional anti- hemorrhoid herb. J Ethnopharmacol. 2022; 284: 114765. https://doi.org/10.1016/j.jep.2021.114765.
• [5] Ramdhan B, Chikmawati T, Waluyo EB. Ethnomedical herb from Cikondang indigenous village, district Bandung West Java Indonesia. J Bio Env Sci. 2015; 6(2): 277 – 288.
• [6] Makkiyah F, Rahmi EP, Revina R, Susantiningsih T, Setyaningsih Y. Graptophyllum pictum (L.) Griff.(Syn: Justicia picta Linn.) and its effectiveness: a well-known indonesian plant. Pharmacogn J. 2021; 13(3): 835 – 838. https://doi.org/10.5530/pj.2021.13.106.
• [7] Metiefeng NT, Tamfu AN, Fotsing Tagatsing M, Tabopda TK, Kucukaydin S, Noah Mbane M, Atchade A de T, Talla E, Henoumont C, Laurent S, Anouar EH, Dinica RM. In Vitro and ın silico evaluation of anticholinesterase and antidiabetic effects of furanolabdanes and other constituents from Graptophyllum pictum (Linn.) Griffith. Molecules. 2023; 28(12): 4802. https://doi.org/10.3390/molecules28124802.
• [8] Jiangseubchatveera N, Liawruangrath B, Liawruangrath S, Teerawutgulrag A, Santiarworn D, Korth J, Pyne SG. The chemical constituents and the cytotoxicity, antioxidant and antibacterial activities of the essential oil of Graptophyllum pictum (L.) Griff. J Essent Oil Bear Plants. 2015; 18(1): 11-17. https://doi.org/10.1080/0972060X.2014.935036.
• [9] Tahseen MA, Bhatnagar A, Bhatnagar A. Phytochemical and pharmacological investigations of Graptophyllum pictum leaves. J Pharmacogn Phytochem. 2023; 12(6): 252-266. https://doi.org/10.22271/phyto.2023.v12.i6c.14791.
• [10] Azmir J, Zaidul ISM, Rahman MM, Sharif KM, Mohamed A, Sahena F, Jaharul MHA, Ghafoor K, Norulaini NAN, Omar AKM. Techniques for extraction of bioactive compounds from plant materials: A review. J Food Eng. 2013; 117(4): 426-436. https://doi.org/10.1016/j.jfoodeng.2013.01.014.
• [11] Aspé E, Fernández K. The effect of different extraction techniques on extraction yield, total phenolic, and anti-radical capacity of extracts from Pinus radiata Bark. Ind Crop Prod. 2011; 34(1): 838-844. https://doi.org/10.1016/j.indcrop.2011.02.002.
• [12] Tušek A J, Benković M, Cvitanović A B, Valinger D, Jurina T, Kljusurić J G. Kinetics and thermodynamics of the solid- liquid extraction process of total polyphenols, antioxidants and extraction yield from Asteraceae plants. Ind Crop Prod. 2016; 91: 205-214. https://doi.org/10.1016/j.indcrop.2016.07.015.
• [13] Lubis MF, Hasibuan PAZ, Syahputra H, Astyka R, Baruna I. Phytochemical profile and pharmacological activity of Vernonia amygdalina Delile stem bark extracts using different solvent extraction. Macedonian J Med Sci. 2022; 10(A). https://doi.org/10.3889/oamjms.2022.8921.
• [14] Norshazila S, Koy CN, Rashidi O, Ho LH, Azrina I, Zaizuliana NR, Zarinah Z. The effect of time, temperature and solid to solvent ratio on pumpkin carotenoids extracted using food grade solvents. Sains Malaysiana. 2017; 46(2): 231- 237. http://dx.doi.org/10.17576/jsm-2017-4602-07.
• [15] Mokrani A, Madani K. Effect of solvent, time and temperature on the extraction of phenolic compounds and antioxidant capacity of peach (Prunus persica L.) fruit. Sep Purif Technol. 2016; 162: 68-76. https://doi.org/10.1016/j.seppur.2016.01.043.
• [16] Chantigny M H, Harrison-Kirk T, Curtin D, Beare M. Temperature and duration of extraction affect the biochemical composition of soil water-extractable organic matter. Soil Biol Biochem. 2014; 75: 161-166. https://doi.org/10.1016/j.soilbio.2014.04.011.
• [17] Kaya M, Sousa A G, Crépeau M J, Sørensen S O, Ralet M C. Characterization of citrus pectin samples extracted under different conditions: influence of acid type and pH of extraction. Ann Bot. 2014; 114(6): 1319-1326. https://doi.org/10.1093/aob/mcu150.
• [18] Baiano A, Romaniello R, Giametta F, Fiore A. Optimization of process variables for the sustainable extraction of phenolic compounds from chicory and fennel by-products. Appl Sci. 2023; 17(7): 4191. https://doi.org/10.3390/app13074191.
• [19] Rahman S S A, Pasupathi S, Karuppiah S. Conventional optimization and characterization of microbial dextran using treated sugarcane molasses. Int J Biol Macromol. 2022; 220: 775 – 787. https://doi.org/10.1016/j.ijbiomac.2022.08.094.
• [20] Mousavi L, Tamiji Z, Khoshayand MR. Applications and opportunities of experimental design for the dispersive liquid–liquid microextraction method – A review. Talanta. 2018; 190: 35 – 356. https://doi.org/10.1016/j.talanta.2018.08.002.
• [21] Breig SJM, Luti KJK. Response surface methodology: A review on its applications and challenges in microbial cultures. Mater Today. 2021; 42 (5): 2277 – 2284. https://doi.org/10.1016/j.matpr.2020.12.316.
• [22] Hadiyat MA, Sopha BM, Wibowo BS. Response surface methodology using observational data: a systematic literature review. Appl Sci. 2022; 12(20): 10663. https://doi.org/10.3390/app122010663.
• [23] Khuri AI, Mukhopadhyay S. Response surface methodology. WIREs Interdisc Rev: Comput Stat. 2010; 2(2): 128-149. https://doi.org/10.1002/wics.73.
• [24] Chelladurai SJS, Murugan K, Ray AP, Upadhyaya M, Narasimharaj V, Gnanasekaran S. Optimization of process parameters using response surface methodology: A review. Mater Today. 2021; 37: 1301-1304. https://doi.org/10.1016/j.matpr.2020.06.466.
• [25] Yolmeh M, Jafari SM. Applications of response surface methodology in the food industry processes. Food Bioprocess Technol. 2017; 10(3): 413-433. https://doi.org/10.1007/s11947-016-1855-2.
• [26] De Oliveira LG, de Paiva AP, Balestrassi PP, Ferreira JR, da Costa SC, da Silva Campos PH. Response surface methodology for advanced manufacturing technology optimization: theoretical fundamentals, practical guidelines, and survey literature review. Int J Adv Manuf Technol. 2019; 104: 1785-1837. https://doi.org/10.1007/s00170-019- 03809-9.
• [27] Davoudpour Y, Hossain S, Khalil H A, Haafiz MM, Ishak ZM, Hassan A, Sarker ZI. Optimization of high pressure homogenization parameters for the isolation of cellulosic nanofibers using response surface methodology. Ind Crop Prod. 2015; 74: 381-387. https://doi.org/10.1016/j.indcrop.2015.05.029.
• [28] Moussa ID, Masmoudi MA, Choura S, Chamkha M, Sayadi S. Extraction optimization using response surface methodology and evaluation of the antioxidant and antimicrobial potential of polyphenols in Scenedesmus sp and Chlorella sp. Biomass Convers Bioreferin. 2023; 13: 7185-7198. https://doi.org/10.1007/s13399-021-01850-x.
• [29] Martinez S, Fuentes C, Carbalio J. Antioxidant activity, total phenolic content and total flavonoid content in sweet chestnut (Castanea sativa Mill.) cultivars grown in northwest Spain under different environmental conditions. Foods. 2022; 11: 3519. https://doi.org/10.3390/foods11213519.
• [30] Kefayati Z, Motamed S M, Shojaii A, Noori M, Ghods R. Antioxidant activity and phenolic and flavonoid contents of the extract and subfractions of Euphorbia splendida Mobayen. Pharmacogn Res. 2017; 9(4): 362 – 365. https://doi.org/10.4103/pr.pr_12_17.
• [31] Castro-Puyana M, Marina ML, Plaza M. Water as green extraction solvent: Principles and reasons for its use. Curr Opin Green Sustain Chem. 2017; 5: 31-36. https://doi.org/10.1016/j.cogsc.2017.03.009.
• [32] Tian Y, Liimatainen J, Alanne AL, Lindstedt A, Liu P, Sinkkonen J, Kallio H, Yang B. Phenolic compounds extracted by acidic aqueous ethanol from berries and leaves of different berry plants. Food Chem. 2017; 220: 266-281. https://doi.org/10.1016/j.foodchem.2016.09.145.
• [33] Dirar AI, Alsaadi DHM, Wada M, Mohamed MA, Watanabe T, Devkota HP. Effects of extraction solvents on total phenolic and flavonoid contents and biological activities of extracts from Sudanese medicinal plants. South Afr J Bot. 2019; 120: 261-267. https://doi.org/10.1016/j.sajb.2018.07.003.
• [34] Vongsak B, Sithisarn P, Mangmool S, Thongpraditchote S, Wongkrajang Y, Gritsanapan W. Maximizing total phenolics, total flavonoids contents and antioxidant activity of Moringa oleifera leaf extract by the appropriate extraction method. Ind Crop Product. 2013; 44: 566-571. https://doi.org/10.1016/j.indcrop.2012.09.021.
• [35] Alara OR, Abdurahman NH, Olalere OA. Ethanolic extraction of flavonoids, phenolics and antioxidants from Vernonia amygdalina leaf using two-level factorial design. J King Saud University-Sci. 2020; 32(1): 7-16. https://doi.org/10.1016/j.jksus.2017.08.001.
• [36] González-Montelongo R, Lobo MG, González M. The effect of extraction temperature, time and number of steps on the antioxidant capacity of methanolic banana peel extracts. Sep Purif Technol. 2010; 71(3): 347-355. https://doi.org/10.1016/j.seppur.2009.12.022.
• [37] Masjedi M, Nateghi L, Berenjy S, Eshaghi MR. Optimization of extraction of flavonoid, total phenolic, antioxidant, and antimicrobial compounds from Ganoderma Lucidum by Maceration Method. Iran J Chem Chem Eng. 2022; 41(9).
• [38] Jovanović AA, Đorđević VB, Zdunić GM, Pljevljakušić DS, Šavikin KP, Gođevac DM, Bugarski BM. Optimization of the extraction process of polyphenols from Thymus serpyllum L. herb using maceration, heat-and ultrasound-assisted techniques. Sep Purif Technol. 2017; 179: 369-380. https://doi.org/10.1016/j.seppur.2017.01.055.
• [39] Wong KH, Li GQ, Li KM, Razmovski-Naumovski V, Chan K. Optimisation of Pueraria isoflavonoids by response surface methodology using ultrasonic-assisted extraction. Food Chem. 2017; 231: 231-237. https://doi.org/10.1016/j.foodchem.2017.03.068.
• [40] Wong WH, Lee WX, Ramanan RN, Tee LH, Kong KW, Galanakis CM, Sun J, Prasad KN. Two level half factorial design for the extraction of phenolics, flavonoids and antioxidants recovery from palm kernel by-product. Ind Crop Prod. 2015; 63: 238-248. https://doi.org/10.1016/j.indcrop.2014.09.049.
• [41] Sulaiman ISC, Basri M, Masoumi HRF, Chee WJ, Ashari SE, Ismail M. Efects of temperature, time, and solvent ratio on the extraction of phenolic compounds and the anti-radical activity of Clinacanthus nutans Lindau leaves by response surface methodology. Chem Centr J. 2017; 11: 54. https://doi.org/10.1186/s13065-017-0285-1.
• [42] Lima RC, de Carvalho APA, da Silva BD, Neto LT, de Figueiredo MR da S, Chaves PHT, de Almeida AECC, Conte- Junior CA. Green ultrasound-assisted extraction of bioactive compounds of babassu (Attalea speciosa) mesocarp: Effects of solid-liquid ratio extraction, antioxidant capacity, and antimicrobial activity. Appl Food Res. 2023; 3: 100331. https://doi.org/10.1016/j.afres.2023.100331.
• [43] Ryu D, Park H, Koh E. Effects of solid-liquid ratio, time, and temperature on water extraction of anthocyanin from Campbell early grape. Food Anal Method. 2020; 13: 637 – 646. https://doi.org/10.1007/s12161-019-01688-0.
• [44] Nawaz H, Shad MA, Rehman N, Andaleeb H, Ullah N. Effect of solvent polarity on extraction yield and antioxidant properties of phytochemicals from bean (Phaseolus vulgaris) seeds. Brazil J Pharm Sci. 2020; 56: e17129. https://doi.org/10.1590/s2175-97902019000417129.
• [45] Strati IF, Oreopoulou V. Effect of extraction parameters on the carotenoid recovery from tomato waste. Int J Food Sci Technol. 2011; 46(1): 23-29. https://doi.org/10.1111/j.1365-2621.2010.02496.x.
• [46] Vuong QV, Hirun S, Roach PD, Bowyer MC, Phillips PA, Scarlett CJ. Effect of extraction conditions on total phenolic compounds and antioxidant activities of Carica papaya leaf aqueous extracts. J Herb Med. 2013; 3(3): 104-111. https://doi.org/10.1016/j.hermed.2013.04.004.
• [47] Prasad KN, Yang E, Yi C, Zhao M, Jiang Y. Effects of high pressure extraction on the extraction yield, total phenolic content and antioxidant activity of longan fruit pericarp. Innov Food Sci Emerg Technol. 2009; 10(2): 155-159. https://doi.org/10.1016/j.ifset.2008.11.007.
• [48] Prasad KN, Kong KW, Ramanan RN, Azlan A, Ismail A. Determination and optimization of flavonoid and extract yield from brown mango using response surface methodology. Sep Sci Technol. 2012; 47(1): 73-80. https://doi.org/10.1080/01496395.2011.606257.
• [49] Metrouh-Amir H, Duarte CMM, Maiza F. Solvent effect on total phenolic contents, antioxidant, and antibacterial activities of Matricaria pubescens. Ind Crop Prod. 2015; 67: 249 – 256. http://dx.doi.org/10.1016/j.indcrop.2015.01.049.
• [50]Lohvina H, Sándor M, Wink M. Effect of ethanol solvents on total phenolic content and antioxidant properties of seed extracts of fenugreek (Trigonella foenum-graecum L.) varieties and determination of phenolic Composition by HPLC-ESI-MS. Diversity. 2021; 14(1): 7. https://doi.org/10.3390/d14010007.
• [51] Park HY, Kim JH, Kim YH, Kim JW. Optimization of ultrasound-assisted extraction conditions for extraction of bioactive compounds from purple carrot (Daucus carota L.) using response surface methodology. Food Sci Technol. 2023; 43: e000523. https://doi.org/10.1590/fst.000523.
• [52] Shi L, Zhao W, Yang Z, Subbiah V, Suleria HAR. Extraction and characterization of phenolic compounds and their potential antioxidant activities. Environ Sci Pollut Res. 2022; 29(54): 81112-81129. https://doi.org/10.1007/s11356- 022-23337-6.
• [53] Abu Bakar FI, Abu Bakar MF, Abdullah N, Endrini S, Fatmawati S. Optimization of extraction conditions of phytochemical compounds and anti-gout activity of Euphorbia hirta L.(Ara Tanah) using response surface methodology and liquid chromatography-mass spectrometry (LC-MS) Analysis. Evid-Base Complement Altern Med. 2020: 4501261. https://doi.org/10.1155/2020/4501261.
• [54] Zhao S, Shan G, Yang X, Ma K, Li W, ZJ. Study of liquid-liquid extraction and mass transfer process with solid particles in the inline teethed high shear mixer. Sep Purif Technol. 2023; 314: 123559. https://doi.org/10.1016/j.seppur.2023.123559.
• [55] Do QD, Angkawijaya AE, Tran-Nguyen PL, Huynh LH, Soetaredjo FE, Ismadji S, Ju Y. Effect of extraction solvent on total phenol content, total flavonoid content, and antioxidant activity of Limnophila aromatica. J Food Drug Anal. 2014; 22: 296 – 302. https://doi.org/10.1016/j.jfda.2013.11.001
• [56] Sun C, Wu Z, Wang Z, Zhang H. Effect of ethanol/water solvents on phenolic profiles and antioxidant properties of Beijing propolis extracts. Evid-Base Complement Altern Med. 2015: 595393. https://doi.org/10.1155/2015/595393.
• [57] Bucić-Kojić A, Planinić M, Tomas S, Bilić M, Velić D. Study of solid–liquid extraction kinetics of total polyphenols from grape seeds. J Food Eng. 2007; 81(1): 236-242. https://doi.org/10.1016/j.jfoodeng.2006.10.027.
• [58] Vo TP, Le NPT, Mai TP, Nguyen DQ. Optimization of the ultrasonic-assisted extraction process to obtain total phenolic and flavonoid compounds from watermelon (Citrullus lanatus) rind. Curr Res Food Sci. 2022; 5: 2013-2021. https://doi.org/10.1016/j.crfs.2022.09.021.
• [59] Makkiyah FA, Rahmi EP, Mahendra FR, Maulana F, Arista RA, Nurcholis W. Polyphenol content and antioxidant capacities of Graptophyllum pictum (L.) extracts using in vitro methods combined with the untargeted metabolomic study. J Appl Pharm Sci. 2024; 14(3): 055-063. https://doi.org/10.7324/JAPS.2024.153548.
• [60] Stagos D. Antioxidant activity of polyphenolic plant extracts. Antioxidants. 2019; 9(1): 19. https://doi.org/10.3390/antiox9010019.
• [61] Mašek T, Perin N, Racané L, Cindrić M, Čipčić Paljetak H, Perić M, Matijasic M, Verbanac D, Radic B, Suran J, Starčević K. Chemical Composition, antioxidant and antibacterial activity of different extracts of poplar type propolis. Croat Chem Acta. 2018; 91(1): 81-88. https://doi.org/10.5562/cca3298.
• [62]Zduńska K, Dana A, Kolodziejczak A, Rotsztejn H. Antioxidant properties of ferulic acid and its possible application. Skin Pharmacol Physiol. 2018; 31(6): 332-336. https://doi.org/10.1159/000491755.
• [63] Ji M, Gong X, Li X, Wang C, Li M. Advanced research on the antioxidant activity and mechanism of polyphenols from Hippophae species—A review. Molecules. 2020; 25(4): 917. https://doi.org/10.3390/molecules25040917.
• [64] Hu RS, Yu L, Zhou SY, Zhou HF, Wan HT, Yang JH. Comparative study on optimization of NADES extraction process by dual models and antioxidant activity of optimum extraction from Chuanxiong-Honghua. LWT. 2023; 184: 114991. https://doi.org/10.1016/j.lwt.2023.114991.
• [65] Fitri K, Lubis MF, Syahputra H, Astyka R, Kaban VE. Phytochemıcals analysıs of Baccaurea motleyana Mull. Arg. extracts and antıproliferatıon effect agaınst PANC-1 cell through p53 and bcl-2 expressıons. Rasayan J Chem. 2023; 16(3): 1516 – 1524. https://doi.org/10.31788/RJC.2023.1638326.
• [66] Lubis MF, Sumaiyah S, Lubis LD, Fitri K, Astyka R. Application of Box-Behnken design for optimization of Vernonia amygdalina stem bark extract in relation to its antioxidant and anti-colon cancer activity. Arab J Chem. 2024; 17(4): 105702. https://doi.org/10.1016/j.arabjc.2024.105702.
• [67] Lubis MF, Syahputra H, Illian DN, Kaban VE. Antioxidant activity and nephroprotective effect of Lansium parasiticum leaves in doxorubicin-induced rats. J Res Pharm. 2022; 26(3): 565-573. https://dx.doi.org/10.29228/jrp.154.
• [68] Hasibuan PAZ, Syahputra RA, Hey-Hawkins E, Lubis MF, Rohani AS, Pahlevi SA. Phytochemical composition and safety of Vernonia Amygdalina ethanolic extract with anti-colon cancer properties. J Agric Food Res. 2024; 101205. https://doi.org/10.1016/j.jafr.2024.101205.
• [69] Hasibuan PAZ, Keliat JM, Lubis MF. Mıcrowave-assısted extractıon ımpacts on pharmacologıcal actıvıty of Vernonia amygdalina Delile leaf extracts and FT-IR applıcatıon for phytochemıcals analysıs. Rasayan J Chem. 2023; 16(3): 1677 – 1685. http://doi.org/10.31788/RJC.2023.1638330.
• [70] Lubis MF, Hasibuan PAZ, Kaban VE, Astyka R. Phytochemıcals analysıs and cytotoxıc actıvıty of Lansium domesticum Corr extract-cısplatın combınatıon agaınst PANC-1 cell lıne. Rasayan J Chem. 2023; 16(1): 32- 37. http://doi.org/10.31788/RJC.2023.1617074.
• [71] Hasibuan PAZ, Keliat JM, Lubis MF. Combination of cisplatin and ethyl acetate extract of Vernonia amygdalina Delile induces cell cycle arrest and apoptosis on PANC-1 cells via PI3K/mTOR. J Pharm Pharmacogn Res. 2024; 12(5): 870- 880. https://doi.org/10.56499/jppres23.1748_12.5.870.