INTRODUCTION:

Jatropha multifida is widely distributed in tropical regions including Southeast Asia. In Indonesia, Jatropha multifida is known as betadine plant. This plant is often used as medicine to treat fevers, wounds, and various skin infections¹.

Previous studies have reported several biological activities of Jatropha multifida. The sap of this plant stopped bleeding in coagulation disorders². It also displayed antibacterial properties against multidrug-resistant strains of Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter freundii, and Staphylococcus aureus³.

Jatropha multifida root bark was reported to have anti-inflammatory and analgesic activities⁴. Jatropha multifida stems exhibited antimelanotic effect in a melanoma cell line (B16-F10)⁵ and also anti-influenza activity¹.

Petioles are one part of Jatropha multifida that can be used for medicine. Traditionally, the sap of this plant is applied to skin wounds. Wound healing is the process of tissue reparation^6,7, associated with the reduction of oxidative reactions using antioxidants. Inflammation that occurs in the body stimulates the release of free radicals to fight infections caused by pathogenic microorganism, such as those caused by wounds in skin tissue⁸.

Screening for antioxidant activity of Jatropha multifida has focused on leaves, stems, and roots. To our knowledge, there are no studies on the investigation of antioxidant in petiole. Based on this, research was conducted to investigate the antioxidant properties of Jatropha multifida petiole and identify compounds that act as antioxidants using TLC bioautography.

Many TLC techniques have been developed for the analysis of antioxidants and DPPH free radicals is widely used for screening confirmation⁹. TLC bioautography offers several advantages, including flexible, simplicity, and high throughput¹⁰. Compounds that scavenge free radicals are present as yellowish spot on the purple background of TLC plates¹¹. To support antioxidant activity, the total phenol content was also determined. The results of this study might demonstrate the effectiveness of Jatropha multifida petiole extract in reducing DPPH free radicals.

MATERIALS AND METHODS:

Materials:

Jatropha multifida was collected from South Sumatra, Indonesia. Methanol was purchased from Merck Millipore. All other chemicals were purchased from local supplies.

Petiole Extraction:

Washed Jatropha multifida petioles were dried for 3 days. Fifty grams of dried petioles were extracted with methanol at a ratio of 1:10 (w/v). Extraction was done by maceration over 2x24. The macerate was concentrated using vacuum rotary evaporator at 50 ⁰C.

Assay of Antioxidant Activity:

A mL of DPPH reagent (400 M in methanol) was pipetted into test tube. The solution was added with 3 mL of methanol and 0.1 mL of each prepared sample (10, 50, 100, 150 and 200 ppm). The mixture was vortexed and the absorbances were measured after 25 minutes using UV-Visible spectrophotometer at wavelength of 517 nm^12,13.

Measurement of Total Phenolics:

Sample extracts were dissolved in distilled water. A mL solution was added by 0.8 mL Folin reagent and 5% Na₂CO₃. The solution was left to stand for 60 minutes and the absorbance was measured at a maximum wavelength of 750 nm.

TLC-DPPH Screening for Antioxidant Component:

The test was performed qualitatively using TLC by immersing a silica gel 60 F₂₅₄ plate in a chamber saturated with 20 mL of chloroform. Extracts were spotted onto the plate. The presence of flavonoids, phenols, alkaloids, and terpenoid were detected using AlCl₃, FeCl₃, Dragendorff’s, and anisaldehyde-sulfuric acid spray reagent, respectively. Antioxidant components were detected by spraying 0.05% (m/v) fresh DPPH-methanol solution onto the plate.

RESULT AND DISCUSSION:

Phytochemical Screening:

Phytochemical screening indicated that the methanol extract of petioles contained several active compounds such as alkaloids, flavonoids, sterols, and phenols. The complete results of phytochemical test can be seen in Table 1. The methanol extract of petiole was considered to have a high content of flavonoids and phenolic compounds, followed with the presence of alkaloids and sterols. It was also known that all parts of the petiole did not contain saponins, steroids and triterpenoids. Previous studies have reported a positive association between flavonoids and phenols with their antioxidant activity^14,15,16.

Table 1. Phytochemical analysis of the methanol extract of Jatropha multifida petiole

No.	Analysis	Result
1.	Alkaloids · Mayer · Wegner · Dragendorff	+ + +
2.	Flavonoids	++
3.	Saponins	-
4.	Triterpenoids/Steroids	-
5.	Sterols	+
6.	Phenols	++

Description: (+) = Present (-) = Absent

Antioxidant Analysis using DPPH Method:

Antioxidant activity was analyzed to confirm the ability of the extract to reduce DPPH free radicals¹⁷ which are unstable molecules^12,18. The results are presented in Table 2. The percent inhibition of DPPH free radicals was directly proportional to the increase in extract concentration. Figure 1 shows a positive relationship among them with the equation y = 0.164x+3.1678 and the regression coefficient = 0.9989. Based on this equation, the IC50 of the extract was 569.69 ppm. IC50 is concentration that reduce free radicals by 50 percent. The extract was classified as a weak antioxidant as the IC50 value was between 500 and 1000 ppm. According to the limits of antioxidant activity, an extract is considered to have antioxidant activity if IC50 < 1000 ppm, strong activity < 100 ppm, medium 100-500 ppm and weak 500-1000 ppm^19,20,21.

Several other parts of Jatropha multifida from Pindamonhangaba, Sao Paulo State, Brazil was tested for antioxidant activity. IC50 values for latex, 0.01% leaves extract, and 1% leaves extract were 3.44 ppm, 0.0 ppm, and 1,530.75 ppm²². The ethanol extract of Jatropha multifida leaves from Agome-Seva, Togo was also tested for antioxidant properties. It has a weak activity with IC50 43.23 ppm²³. Antioxidant activity of plants could be associated to their secondary metabolites²⁴. Secondary metabolites are substances produced by plants to increase their competitiveness in their own environment²⁵. These metabolites can vary depending on the location or environmental conditions where the plants grow.

Figure 1. Curve of DPPH free radicals scavenging from the methanol extract of Jatropha multifida petiole

Table 2. Antioxidant activity of the methanolic extract of Jatropha multifida petiole

Assay	Result
Antioxidant IC50	569.69 ppm
Total phenolic content	25 mg GAE/g extract

Thin Layer Chromatography:

To determine the components responsible for antioxidant, a TLC test was performed using DPPH reagent. Additionally, TLC spray reagents such as Dragendorff, Lieberman-Burchad, and FeCl₃ were used to detect the phytochemicals in Jatropha multifida petiole extract. The extract components were previously separated using TLC. Several spots were obtained on the plate through visible light, UV 254 nm and UV 366 nm (figure 2). After TLC separation, analysis was continued using spray reagents. The TLC-DPPH produced yellowish colored spots on a purple background, indicating the presence of antioxidant components. Antioxidants react with DPPH to form DPPH-H. This radical state undergoes decolorization (yellow spot) due to the increase of gathered electron²⁶. Study²¹ reported that the components of Jatropha multifida leaves that act as antioxidants were phenols, flavonoids, terpenoids, and alkaloids, as evidence by the yellow discoloration when sprayed with DPPH. Results of TLC-DPPH analysis are shown in figure 2.

Phytochemical analysis using Dragendorff’s and Liebermann-Burchad spray reagent exhibited no color spots, suggesting the absence of alkaloids and triterpenoids for free radical reduction. Spraying the FeCl₃ reagent produced a black spot indicating the presence of phenolic compounds. It was suggested that the antioxidant component in the methanol extract of Jatropha multifida petiole was phenolics. As antioxidant, phenolics compounds react with various free radicals based on either hydrogen transfer, single electron transfer, or transition metal chelation^27,28.

Figure 2. TLC analysis of the methanol extract of Jatropha multifida petiole, a) visible light b) UV 254 nm and c) UV 366 nm d) TLC-DPPH e) FeCl₃ spray reagent

Determination of Total Phenol Content:

TLC-DPPH analysis revealed the presence of phenolics as the dominant component for free radical reduction, which could be substantiated by measuring total phenolics content. Tabel 2 shows the measurement results of total phenolics content.

Total phenolics content of the extract was equivalent to 25 mg GAE/g extract. According to previous studies, the highest total phenolics content of Jatropha multifida stems was produced at 98 mg GAE/g in samples extracted with semi-polar solvents, whereas in polar and non-polar solvents, the values ranged from 25 to 29 mg GAE/g²¹. The number of total phenols in the latex and hydroalcoholic leaves extract of Jatropha multifida were 4.808 mg GAE/g and 0.130 mg GAE/g, respectively²². While, the total phenolic content of ethanol extract of Jatropha multifida leaves was 1.09±0.26 mg GAE/g²³. Differences in total phenolics content may occur due to differences in plant parts that was analyzed.

CONCLUSION:

Phytochemical analysis of methanol extract of Jatropha multifida petioles showed the presence of alkaloids, flavonoids, sterols, and phenols. The IC50 value of the antioxidant evaluation was 569.69 ppm. Meanwhile, TLC-DPPH test suggested that the active antioxidant component in the extract was phenolic compounds, with a total concentration of 25 mg GAE/g extract.

CONFLICT OF INTEREST:

There are no conflicts of interest concerning this study.

ACKNOWLEDGMENTS:

The authors thanks to Polytechnic of AKA Bogor for granting financial support and laboratory facilities.

REFERENCES:

1. Shoji M, Woo S, Masuda A, Win N, Ngwe H, Takahashi E, Kido H, Morita H, Ito T, Kuzuhara T. Anti-influenza virus activity of extract from the stems of Jatropha multifida Linn. Collected in Myanmar. BMC Compementary and Alternative Medicine. 2017; February 07;17(96). doi: 10.1186/s12906-017-1612-8.

2. Klotoé JR, Atègbo JM, Dougnon TV, Loko F, Dramane K. Hemostatic Effect of Jatropha multifida L. (Euphorbiaceae) in Rats Having Coagulation Disorders. Journal of Applied Biology & Biotechnology. 2017; 5(5): 26-9. doi: 10.7324/JABB.2017.50504.

3. Victorien D, Toussaint S, Jerrold A, Alidah A, Afoussato A, Jacques D, Lamine BM. Evaluation of The Antibacterial Activity of Jatropha multifida Sap and Artemisia annua Extract on Some Clinical Strains Responsible of Urinary Tract Infections. Indian Journal of Science and Technology. 2019; 12(36). doi: 10.17485/ijst/2019/v12i36/146792.

4. A. Falodun A, Igbe I, Erharuyi O, Agbanyim OJ. Chemical Characterization, Antiinflammatory and Analgesic Properties of Jatropha multifida Root Bark. Journal of Applied Sciences and Environmental Mangement. 2013; 17(3): 357-62. doi: 10.4314/jasem.v17i3.3.

5. Woo SY, Wong CP, Win NN, Woo NN B, Elsabbagh SE, Liu QQ Morita H. Anti-melanin Deposition Activity and Active Constituent of Jatropha multifida Stems. Journal of Natural Medicine. 2019; 73(4): 805-13. doi: 10.1007/s11418-019-01314-7.

6. Bura, AR. Effect of Wound Healing Potential of Plumera obtusa (Champa) Spray. Asian Journal of Pharmaceutical Research. 2018; November 27; 8(4): 231-35. doi: 10.5958/2231-5691.2018.00039.4.

7. Amminbavi D, Lakshmi NP. Assessment of In vitro Wound Healing Potential of Hibiscus Leaf Extract Emulgel. Asian Journal of Pharmaceutical Research. 2020; May 7; 10(2): 67-72. doi: 10.5958/2231-5691.2020.00013.1.

8. Soneja A, Drews M, Malinski T. Role of Nitric Oxide. Nitroxidative And Oxidative Stress In Wound Healing. Pharmacol Report. 2005; 57: 108-19.

9. Kusznierewicz B, Piekarska A, Mrugalska B, Konieczka P, Namieśnik J, Bartoszek A. Phenolic Composition and Antioxidant Properties of Polish Blue-berried Honeysuckle Genotypes by GPLC-DAD-MS, HPLC Postcolumn Derivatization With ABTS or FC, and TLC With DPPH Visualization. Journal of Agricultural Food Chemistry, 2021; February 22; 60(7): 1755-63. doi: 10.1021/jf2039839.

10. Wang J, Yue YD, Tang F, Sun J. TLC Screening for Antioxidant Activity of Extracts from Fifteen Bamboo Species and Identification of Antioxidant Flavone Glycosides from Leaves of Bambusa. textilis McClure. Molecules. 2012; October 19; 17(10): 12297-311. doi: 10.3390/molecules171012297.

11. Cieśla L, Kryszeń J, Stochmal A, Oleszek W, Waksmundzka-Hajnos M. Approach to Develop a Standardized TLC-DPPH Test for Assessing Free Radical Scavenging Properties of Selected Phenolic Compounds. Journal of Pharmaceutical and Biomedical Analysis. 2012; November; 70: 126-35. doi: 10.1016/j.jpba.2012.06.007.

12. Krishna KK, Karuppuraj V, Perumal K. Antioxidant Activity and Folic Acid Content in Indigenous Isolates of Ganoderma lucidum. Asian Journal of Pharmaceutical Analysis. 2016 December 20; 6(4): 213-15. doi: 10.5958/2231-5675.2016.00032.6.

13. Valli G, Jeyalakshmi M. Preliminary Phytochemical and Antioxidant Study of Odina woodier Leaf Extract. Asian Journal of Pharmaceutical Research. 2012; December 28; 2(4): 153-55.

14. Ghasemzadeh A, Jaafar HZE, Rahmat A. Antioxidant Activities, Total Phenolics and Flavonoids Content in Two Varieties of Malaysia Young Ginger (Zingiber officinale Roscoe). Molecules. 2010; June 14; 15(6): 4324-33. doi: https://doi.org/10.3390/molecules15064324.

15. Tiwari P. Phenolics and Flavonoids and Antioxidant Potential of Balarishta Prepared by Traditional and Modern Methods. Asian Journal of Pharmaceutical Analysis. 2014 March 28;4(1): 05-10.

16. Tiwari P, Patel RK. Estimation of Total Phenolics and Flavonoids and Antioxidant Potential of Ashwagandharishta Prepared by Traditional and Modern Methods. Asian Journal of Pharmaceutical Analysis. 2013; December 28; 3(4): 147-52.

17. Muthukumaran P, Shanmuganathan P, Malathi C. In vitro Antioxidant Evaluation of Mimosa pudica. Asian Journal of Pharmaceutical Research. 2011; June 28; 1(2): 44-6.

18. Samal PK. Antioxidant Activity of Strobilanthes asperrimus in Albino Rats. Asian Journal of Pharmaceutical Research. 2013; June 28; 3(2): 71-4.

19. Jun M, Yu HY, Hong J, Wan X. Comparison of Antioxidant Activities of Isoflavones from Kudzu Root (Pueraria lobata Ohwi). Journal of Food Science. 2006; July 20; 68(6): 2117-22. doi: https://doi.org/10.1111/j.1365-2621.2003.tb07029.x.

20. Akar Z, Kucuk M, Dogan H. 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 Medical Chemistry, 2017; December; 32(1): 640-7. doi: 10.1080/14756366.2017.1284068.

21. Hanafi, Rocheny H, Sirait SM, Rahmatia L, Setyawati SR. Gas Chromatography-Mass Spectrometry (GC-MS) Analysis of Volatile Components from Coral “BETADINE” (Jatropha multifida Linn) Leaves. Research Journal of Pharmacy and Technology. 2022; October; 15(10): 4633-6. doi: 10.52711/0974-360X.2022.00777.

22. de Carvalho C, Mariano LV, Negrao VS, Goncalves CP, Marcucci MC, 2018, Phenols, Flavonoids and Antioxidant Activity of Jatropha multifida L. collected in Pindamonhangaba, Sao Paulo State, Brazil, Journal of Analitycal Pharmacy Research. 2018; October 16; 7(5); 581-4. doi: 10.15406/japlr.2018.07.00286.

23. Anani K, Adjrah Y, Ameyapoh Y, Karou SD, Agbonon A, de Souza C, Gbeassor M. Antimicrobial, Anti-inflammatory and Antioxidant Activities of Jatropha multifida L. (Euphorbiaceae). Pharmacognosy Research. 2016; April-June; 8(2): 142-6. doi: 10.4103/0974‑8490.172657.

24. Rangasamy P, Hansiya VS, Maheswari PU, Suman T, Geetha N. Phytochemical Analysis and Evaluation of In vitro Antioxidant and Anti-urolithiatic Potential of Various Fractions of Clitoria ternatea L. Blue Flowered Leaves. Asian Journal of Pharmaceutical Analysis. 2019; May 4; 9(2): 67-76. doi: 10.5958/2231-5675.2019.00014.0.

25. Teoh, ES. Secondary metabolites of Plants. Medicinal Orchids of Asia. 2015; November 5: 59-73. doi: 10.1007/978-3-319-24274-3_5.

26. Baliyan S, Mukherjee R, Priyadarshini A, Vibhuti A, Gupta A, Pandey RP, Chang C. Determination of Antioxidants by DPPH Radical Scavenging Activity and Quantitative Phytochemical Analysis of Ficus religiosa. Molecules. 2022; February 16; 27(1326). doi: https://doi.org/10.3390/molecules27041326.

27. Zeb A. Concept, Mechanism, and Application of Phenolic Antioxidant in Foods. Journal of Food Biochemistry. 2020; July 20; 44(9). doi: https://doi.org/10.1111/jfbc.13394.

28. Narasimhan R, Sasthiyamoorthy M. Phytochemical Screening and Antioxidant Studies in The Pulp Extracts of Cucurbita maxima. Asian Journal of Phytochemical Research. 2016; March 28; 6(1): 01-04. doi: 10.5958/2231-5691.2016.00001.0/.

Received on 17.01.2024 Revised on 11.06.2024

Accepted on 18.09.2024 Published on 20.01.2025

Available online from January 27, 2025

Research J. Pharmacy and Technology. 2025;18(1):285-288.

DOI: 10.52711/0974-360X.2025.00044

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.