The Comparison of Spectrophotometric and TLC-Densitometric for DPPH Radical Scavenging Activity Analysis of Three Medicinal Plant Extracts

Bambang Cahyono, Christina Setyadewi M., M. Suzery, Agustina L. N. Aminin


In this research, Thin Layer Chromatography-Densitometry has proven to be a good method for analyzing 1, 1 diphenyl 2-picrylhydrazyl (DPPH) radical scavenging activity, since this approach displayed the similar trends with UV-Vis spectrophotometric method. Three medicinal plants collected from Semarang used to evaluate both methods.  The IC50 value ranged from 25.26 - 4913.74 ppm shown by UV-Vis spectrophotometric and 24.74 – 4674.61 ppm using TLC-Densitometric. Meniran Dechlorophyllated (Phyllanthus niruri) provides the strongest antioxidant activity and the weakest de-chlorophylated parsley (Petroselinum crispum). The paired sample t-test points from a non-dechlorophylation extract using the TLC densitometry test significantly gives a lower IC50 value than the UV spectrophotometry method-has. The maximum increase of peak area under the UV light 365 nm up to 56.08 %. This evidence supports the presumption that the scavenging radical DPPH caused not only decreasing the maximum absorbance under UV light 516 nm but also increasing the absorbance under UV light ± 365 nm.


spectrophotometric; TLC-Densitometric; scavenging DPPH)

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B. A. Oyekanmi and I.B Osho, "Antimicrobial, phytochemical and pharmacological properties of phyllanthus niruri Linn," The FASEB Journal, vol. 30, no. 1_supplement, pp. 1192.6-1192.6, 2016.


P. Wong and D.D. Kitts, "Studies on the dual antioxidant and antibacterial properties of Parsley (Petroselinum crispum) and Cilantro (Coriandrum sativum) Extracts," Food Chemistry, vol. 97, pp. 505-515, 2006.


O.F. C. Mogollón, R. E. G. Cuello, J. S. G. Lopez."In vitro Antibacterial and Antioxidant Activity of Muntingia calabura Fruits Extract," Contemporary Engineering Sciences, vol. 11, pp. 881-890, 2018.


N. L. Md. Nasir, N. E. Kamsani, N. Mohtarrudin, F. Othman, S. F. Md. Tohid & Z. A. Zakaria . "Anti-carcinogenic activity of Muntingia calabura leaves methanol extract against the azoxymethane-induced colon cancer in rats involved modulation of the colonic antioxidant system partly by flavonoids", Pharmaceutical biology, vol. 55, pp. 2102-2109, 2017.


R. F. de A. Júnior, T. P. de Souza, J. G. L. Pires, L. A. L. Soares, A. A. de Araújo, P. R. Petrovick, H. D.O. Mâcedo, A. L. C. de S. L.Oliveira, G. C. B. Guerra, "A dry extract of Phyllanthus niruri protects normal cells and induces apoptosis in human liver carcinoma cells," Experimental biology and medicine (Maywood, N.J.), vol. 237, pp. 1281-8, 2012.


M. N.Beidokhti, M. V. Andersen, H. M. Eid, M. L. S. Villavicencio, D. Staerk, P. S. Haddad, &A. K. Jäger, "Investigation of anti-diabetic potential of Phyllanthus niruri L. using assays for α-glucosidase, muscle glucose transport, liver glucose production, and adipogenesis," Biochemical and Biophysical Research Communications, vol. 493, pp. 869-874.2017.


H. A.Soliman, N. A. Eltablawy, & M. S.Hamed, " The ameliorative effect of Petroselinum crispum (parsley) on some diabetes complications.," Journal of Medicinal Plants Studies. , vol. 3, pp. 92-100., 2015.

Google Scholar

S.Syahara, U.Harahap, & T.Widyawati, "Activity of Muntingia calabura Leaves Ethanolic Extract on Glucose and Insulin Blood Levels in Streptozotocin-induced Rat," Asian Journal of Pharmaceutical Research and Development, vol. 7, pp. 8-11, 1970.

DOI: 10.22270/ajprd.v7i4.552.

Z. E.Pápay, A.Kósa, I.Boldizsár, A.Ruszkai, E.Balogh, I.Klebovich, & I. "Pharmaceutical and formulation aspects of Petroselinum crispum extract," Acta pharmaceutica Hungarica, vol. 82, pp. 3-14, 2012.

Google Scholar

M. H. Farzaei, Z. Abbasabadi, M. R. S. Ardekani, R. Rahimi, and F. Farzaei, "Parsley: a review of ethnopharmacology, phytochemistry and biological activities," Journal of Traditional Chinese Medicine, vol. 33, no. 6, pp. 815-826, 2013.

DOI: 10.1016/S0254-6272(14)60018-2.

B. Mafuvadze, I. Benakanakere, F. R. L. Pérez, C. Besch-Williford, M. R. Ellersieck, & S. M. Hyder, "Apigenin prevents development of medroxyprogesterone acetate-accelerated 7,12-dimethylbenz(a) anthracene-induced mammary tumors in Sprague-Dawley rats", Cancer Prev Res (Phila), vol. 4, pp. 1316-24, 2011.

DOI: 10.1158/1940-6207.capr-10-0382.

L. Masuelli, M.Benvenuto, R. Mattera, E. D. Stefano, E. Zago, G.Taffera, I. Tresoldi, M. G. Giganti, G. V. Frajese, G. Berardi, A. Modesti, and R.Bei, "In Vitro and In Vivo Anti-tumoral Effects of the Flavonoid Apigenin in Malignant Mesothelioma," (in eng), Front Pharmacol, vol. 8, p. 373, 2017.

DOI: 10.3389/fphar.2017.00373.

T.Leonardi, J. Vanamala, S. S. Taddeo, L. A. Davidson, M.E Murphy, B.S. Patil, N. Wang, R. J. Carroll, R. S. Chapkin, J.R. Lupton, N. D. Turner, "Apigenin and naringenin suppress colon carcinogenesis through the aberrant crypt stage in azoxymethane-treated rats", Exp Biol Med (Maywood), vol. 235, pp. 710-7, 2010.

DOI: 10.1258/ebm.2010.009359

E. O. Farombi, A. C. Akinmoladun, and S. E. Owumi, "Anti-cancer Foods: Flavonoids," in Encyclopedia of Food Chemistry, L. Melton, F. Shahidi, and P. Varelis, Eds. Oxford: Academic Press, 2019, pp. 224-236

"> Google Scholar

X. Mao, L.F Wu, H.L. Guo, W.J Chen,Y.P. Cui, Q. Qi, S.Li, W.Y. Liang, G.H. Yang, Y.Y.Shao, D. Zhu, G.M. She, Y. You, L.Z.Z. Wu, L.Fan, Guo, H.Ling, Chen, W.Jing, Cui, Y.Ping, "The Genus Phyllanthus: An Ethnopharmacological, Phytochemical, and Pharmacological Review," Evidence-based complementary and alternative medicine : eCAM, vol. 2016, pp. 7584952-7584952, 2016.

DOI: 10.1155/2016/7584952

S. A. Tasaduq, K. Singh, S. Sethi, S. C. Sharma, K. L. Bedi, J. Singh, B. S. Jaggi, R. K. Johri, "Hepatocurative and antioxidant profile of HP-1, a polyherbal phytomedicine", Hum Exp Toxicol, vol. 22, no. 12, pp. 639-45, 2003.

DOI: 10.1191/0960327103ht406oa

L. Tona, N.P.Ngimbi, M. Tsakala, K.Mesia, K.Cimanga, S.Apers, T.De Bruyne, L.Pieters, J.Totté, A.J.Vlietin, "Antimalarial activity of 20 crude extracts from nine African medicinal plants used in Kinshasa, Congo", J Ethnopharmacol, vol. 68, pp. 193-203, 1999.


V. Murugaiyah and K. L. Chan, "Antihyperuricemic lignans from the leaves of Phyllanthus niruri", Planta Med, vol. 72, no. 14, pp. 1262-7, Nov 2006.

DOI: 10.1055/s-2006-947224

R. Bhattacharjee and P. C. Sil, "The protein fraction of Phyllanthus niruri plays a protective role against acetaminophen induced hepatic disorder via its antioxidant properties," Phytotherapy research:PTR, vol. 20, pp. 595-601, 2006.

DOI: 10.1002/ptr.1933

A.K. Khanna, F. Rizvi, and R.Chander. "Lipid lowering activity of Phyllanthus niruri in hyperlipemic rats," J Ethnopharmacol, vol. 82, pp. 19-22, 2002.


G. Bagalkotkar, S. R. Sagineedu, M. S. Saad, and J. Stanslas, "Phytochemicals from Phyllanthus niruri Linn. and their pharmacological properties: a review," (in eng), J Pharm Pharmacol, vol. 58, pp. 1559-70, 2006.

DOI: 10.1211/jpp.58.12.0001

G. Bagalkotkar, S. R. Sagineedu, M. S. Saad,J. Stanslas, "Phytochemicals from Phyllanthus niruri Linn. and their pharmacological properties: a review," Journal of Pharmacy and Pharmacology, vol. 58, pp. 1559-1570, 2006.

DOI: 10.1211/jpp.58.12.0001

R. S. Harish & T. Shivanandappa, "Antioxidant activity and hepatoprotective potential of Phyllanthus niruri," Food Chemistry, vol. 95, pp. 180-185, 2006.

Google Scholar

K. Ramandeep, A.Nahid, C.Neelabh, & K. Navneet,“Screening of Phyllanthus niruri collected from Kerala Region and its Antioxidant and Antimicrobial Potentials’, Journal of Pharmaceutical Sciences and Research, vol. 9, pp. 1312-1316, 2017

Google Scholar

D.Rusmana, R. Wahyudianingsih, M.Elisabeth, B. Balqis, M. Maesaroh, & W. Widowati, "Antioxidant Activity of Phyllanthus niruri Extract, Rutin and Quercetin," Indones Biomed, vol. 9, pp. 84-90J. 2017.

DOI: 10.18585/inabj.v9i2.281.

C.Ramalakshmi, A. Kalirajan, A. J. A.Ranjitsingh, & K. Kalirajan, Bioprospecting of Muntingia calabura: Bioactive Compounds and Its Antioxidant, Antimicrobial and Anthelmenthic Activity " Innovare Journal of Sciences, vol. 05, pp. 7-11, 2017.

Google Scholar

N. D. Mahmood, N. L. M.Nasir, M. S.Rofiee, S. M.Tohid, S. M. Ching,L. K. Teh, & Z. A. Zakaria, "Muntingia calabura: A review of its traditional uses, chemical properties, and pharmacological observations," Pharmaceutical Biology, vol. 52, pp. 1598-1623, 2014.

DOI: 10.3109/13880209.2014.908397.

D. Triswaningsih, S. Kumalaningsih, & P. Wignyanto "Identification of chemical compounds cherry leaves (Muntingia calabura) powder as a natural antioxidant," International Journal of Agronomy and Agricultural Research (IJAAR)” Vol. 10, p. 84-91, 2017

Google Scholar

K. Preethi, N. Vijayalakshmi, R.Shamna, & J. M. Sasikumar, "In Vitro Antioxidant Activity of Extracts from Fruits of Muntingia calabura Linn. from India," Pharmacognosy Journal, vol. 2 pp. 11-18, 2010.


A.M. Sindhe, Y. Bodke, & A. Chandrashekar, "Antioxidant and in vivo anti-hyperglycemic activity of Muntingia calabura leaves extracts " Der Pharmacia Lettre. Vol. 5. Pp. 427-43, 2013.

Google Scholar

G.Clarke, K. N.Ting, C. Wiart, & J. Fry, "High Correlation of 2,2-diphenyl-1-picrylhydrazyl (DPPH) Radical Scavenging, Ferric Reducing Activity Potential and Total Phenolics Content Indicates Redundancy in Use of All Three Assays to Screen for Antioxidant Activity of Extracts of Plants from the Malaysian Rainforest," Antioxidants, vol. 2, pp. 1-10, 2013.


M. N. Sarian, Q. U.Ahmed, S. Z.Mat So’ad, A. M. Alhassan, S. MurugesuV., Perumal, & J. Latip, "Antioxidant and Anti-diabetic Effects of Flavonoids: A Structure-Activity Relationship Based Study,", BioMed research international, vol. 2017, pp. 8386065-8386065, 2017.

DOI: 10.1155/2017/8386065.

N. B. Pigni, S. Berkov, A.Elamrani, M. Benaissa, F.Viladomat, C. Codina, & J. Bastida, "Two new alkaloids from Narcissus serotinus L," Molecules vol. 15, pp. 7083-7089, 2010.


A. F. H. Marker, "The use of acetone and methanol in the estimation of chlorophyll in the presence of phaeophytin," Freshwater Biology, vol. 2, pp. 361-385, 1972.


M.Chen, M.Schliep, R. D.Willows, Z. L. Cai, B. A.Neilan, & H. Scheer, "A red-shifted chlorophyll," Science, vol. 329, pp. 1318-9, 2010.


A. Escarpa, & M C. González, "Approach to the content of total extractable phenolic compounds from different food samples by comparison of chromatographic and spectrophotometric methods," Analytica Chimica Acta, vol. 427, pp. 119-127, 2001.


D. Chandrasekar, K.Madhusudhana, S.Ramakrishna, & P. V. Diwan, "Determination of DPPH Free Radical Scavenging Activity by RP-HPLC, Rapid Sensitive Method for the Screening of Berry Fruit Juice Freeze Dried Extract," Nat Prod Chem Res vol. 6, 1000341, 2018.

DOI: 10.4172/2329-6836.1000341.

K.Csepregi, M. Kocsis, & É. Hideg, "On the spectrophotometric determination of total phenolic and flavonoid contents," Acta Biol. Hung. vol. 64, pp. 500-9, 2013.

DOI: 10.1556/ABiol.64.2013.4.10.

A. Gitelson, & A. Solovchenko, "Non-invasive quantification of foliar pigments: Possibilities and limitations of reflectance- and absorbance-based approaches," Journal of Photochemistry and Photobiology B: Biology, vol. 178, 2017.


C. E. Sando, "The Isolation and Identification of Quercetin from Apple Peels," Journal of Agricultural Research, vol. 28, pp. 1243-1245, 1924.

Google Scholar

P. Molyneux, "The use of the stable radical Diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity," Songklanakarin Journal of Science and Technology, vol. 26, pp. 211-219. 2003.

Google Scholar

E. Abourashed, "Thin-Layer Densitometry as an Alternative Tool in the Quantitative Evaluation of the Free Radical Scavenging Activity of Natural Antioxidants," Zeitschrift fur Naturforschung - Section B Journal of Chemical Sciences, vol. 60, 1212– 1218, 2005.

DOI: 10.1515/znb-2005-1116.

M.M. Kasprzak, A. Erxleben, & J. Ochocki "Properties and applications of flavonoid metal complexes," RSC Advances, vol. 5, pp. 45853-45877, 2015.


N. Liang & D. D. Kitts, "Antioxidant Property of Coffee Components: Assessment of Methods that Define Mechanisms of Action," Molecules, vol. 19, pp. 19180-19208, 2014.

DOI: 10.3390/molecules191119180.

W. Nurcholis, B. P. Priosoeryanto, E. D. Purwakusumah, T.Katayama, & T. Suzuki, "Antioxidant, Cytotoxic Activities and Total Phenolic Content of Four Indonesian Medicinal Plants" Jurnal Kimia Valensi, Vol 2, pp. 501-510, 2012.


E. L. H.Tang, J. Rajarajeswaran, S. Fung, & M. S. Kanthimathi, "Petroselinum crispum has antioxidant properties, protects against DNA damage and inhibits proliferation and migration of cancer cells ".Journal of the Science of Food and Agriculture, vol. 95, pp, 2763–2771, 2015.


J. Stroka, B. Spangenberg, E.Anklam, "New Approaches in TLC-Densitometry," Journal of Liquid Chromatography & Related Technologies, vol. 25, pp. 1497-1513, 2002.

DOI: 10.1081/JLC-120005700.

R. S. Shivatare, D. H. Nagore, & S. U. Nipanikar, "HPTLC' an important tool in standardization of herbal medical product: A review." Journal of Scientific and Innovative Research, vol. 2, pp. 1086-1096 2013,

Google Scholar

M. C. Foti, C. Daquino, G. A. DiLabio, & K. U. Ingold, "Kinetics of the Oxidation of Quercetin by 2,2-Diphenyl-1-picrylhydrazyl (dpph•)," Organic Letters, vol. 13, pp. 4826-4829, 2011.


Z.Akar, M. Küçük, & H.Doğan, “A new colorimetric DPPH• scavenging activity method with no need for a spectrophotometer applied on synthetic and natural antioxidants and medicinal herbs”, Journal of Enzyme Inhibition and Medicinal Chemistry, vol. 32, pp. 640-647, 2017.


E.A. Abourashed "Thin-Layer Densitometry as an Alternative Tool in the Quantitative Evaluation of the Free Radical Scavenging Activity of Natural Antioxidants. Zeitschrift fur Naturforschung-Section B". Journal of Chemical Sciences. 60. pp. 1212-1218, 2005.

DOI: 10.1515/znb-2005-1116

M.Natic, D. Dabic, D. Milojković-Opsenica, B. Dojčinović, G. Roglić, D. Manojlović, & Ž. Tešić, "Development and validation of a simple thin-layer chromatographic method for the analysis of p-chlorophenol in treated wastewater", J. Serb. Chem. Soc. Vol. 77, pp. 1649–1659, 2012


M. Dołowy, K. Kulpińska-Kucia, & A. Pyka "Validation of a Thin-Layer Chromatography for the Determination of Hydrocortisone Acetate and Lidocaine in a Pharmaceutical Preparation". The Scientific World Journal Article ID 107879, 10 pages. 2014.