INTRODUCTION:

The lifestyle of back to nature is increasingly popular in this modern era. It causes people’s interest in traditional medicine to increase as well. With the increasing awareness, current scientific research is mostly directed to natural ingredients¹. Extensive research in natural medicine has provided substantial evidence for the safety of phytochemicals. This is why the use of natural products for the treatment of chronic diseases is accepted worldwide². Plants and their derivatives have long been used as both drugs and dietary supplements by man. Plants are rich sources of secondary metabolites which have been found to have antioxidant and antibacterial properties. Flavonoids and flavones are a wide range of secondary metabolites with antioxidant and antiradical properties.

Plants are powerful biochemical factories and have always been part of phytomedicine. Natural ingredients can come from any part of the plant, including bark, leaves, flowers, roots, fruits and seeds. This means that any part of the plant can contain the active ingredient³. Many of the natural antioxidants in foods are found in phenolic compounds, especially flavonoids⁴.

Betadine plant (Jatropha muitifida Linn.) is an ornamental plant. That is traditionally widely used as a wound medicine and anti-pain due to accidents or cuts. Previous research. showed that the phytochemical test results of betadine stem ethanol extract contained saponins, phenols, terpenoids, flavonoids, tannins, and alkaloids and had an antioxidant activity with an IC50 of 72g/mL⁵. Some research showed the antibacterial activity of the active fraction of betadine leaves against Staphylococcus aureus bacteria. Furthermore, research showed that betadine leaves can be anti-inflammatory which is thought to be due to the presence of flavonoid compounds⁶. Flavonoids are anti-inflammatory so they can reduce inflammation and help reduce pain if there is bleeding or swelling in the wound. In addition, phenolic compounds (flavonoids and phenolic acids) are antioxidant agents that act as free radical terminators and their bioactivity may be related to their ability to chelate metals, inhibit lipoxygenase and bind free radicals⁴. The benefits of flavonoid compounds for health can be an obstacle because it is feared to have toxic properties for humans because flavonoids as secondary metabolites for plants function as self-protection from disease and radiation⁵. Based on this, it is known that there are many potential flavonoid compounds as active substances in betadine plants, so it is necessary to conduct research related to the total flavonoid content in the leaves and stems of betadine plants and test the toxicity of these extracts so that they can be used for traditional medicine safely. Total flavonoids maintain significant antioxidant activity and inhibit lipid peroxidation caused by O2, H2O2, and UV irradiation⁷.

MATERIALS AND METHODS:

Materials:

Betadine (Jatropha muitifida Linn.) leaf and steam were obtain from Bogor, West Java, Indonesia. Other chemicals were obtained from Merck Chemicals.

Simplicia Preparation:

Betadine (Jatropha muitifida Linn.) leaf and steam were obtained from Bogor, Indonesia. They whereas sliced, dried at room temperature and kept in an airtight plasticware and protected from the sunlight.

Fig 1. Jatropha multifida Linn. Steam

Extraction:

Simplicia (Fig.1) was extracted by maceration methods using methanol with a ratio of 1:10 (b/v) for 48 hours. Finally, the macerate was evaporated at 40^oC and 100 mbar to get concentrated extract. The concentrated extract were used for the futher studies.

Phytochemical Screening:

Phytochemical screening was carried out on the crude petioles and stem methanolic extract. Phytochemical screening tests included the tests for alkaloids, flavonoids, phenols, saponins, tannins, glycosides, and sterols-triterpenoids.

Total Flavonoid Determination:

The total flavonoid content of crude extract was ascertained using the aluminum chloride colorimetry technique. In a nutshell, 4mL of purified water, 1mL of crude extract, and 0.3mL of a 5% NaNO2 solution were mixed together. 0.3mL of a 10% AlCl3 solution was added after 5 minutes of incubation, and the combination was left to stand for 6 minutes. Double-distilled water was used to dilute the mixture to a final amount of 10mL. After leaving the mélange to stand for 15minutes, the absorbance was measured at 510nm. A calibration curve was used to determine the total flavonoid concentration, which was then expressed as mg of rutin equivalent per g of dry weight⁸.

Toxicity Determination:

The BSLT Methods,^9,10 were used for toxicity determination. Eggs of the Artemia salina crustacean were incubated in dark and light vessels. The dark zone is where the eggs and aerator are located, whereas the light zone is where the lamps for hatching and separating the cysts are located. The vessel is filled with 50-100mg of shrimp embryos to be hatched, then divided into two sections, the dark zone and the light zone, each of which receives 48 hours of illumination.

Ten Artemia salina shrimp larvae were exposed to test solutions with concentrations of 1,000, 100, 50, and 10 ppm, and a control was conducted with a solution containing no substance. Each test was repeated three times and compared to 24-hour control observations of Artemia salina crustaceans. The mortality rate of Artemia salina crustaceans was determined by applying the following equation:

Number of dead larvae

% Mortality = ------------------------------ × 100 %

Number of test larvae

The LC50 is calculated using the linear regression equation y = ax + b. The y value represents the probit value at 50% larval mortality based on the Miller Tainter probit method, while the x data represents the logarithmic value of the test solution concentration. The IC50 value is derived from the solution concentration that kills fifty percent of the larvae.

RESULT AND DISCUSSION:

Phytochemical Screening:

The phytochemical screening result in this research provided scientific answers to the efficacy of betadine leaves and stems which are traditionally used as wound healing drugs. Table-1 shows phytochemical contents in Betadine (Jatropha muitifida Linn.) leaf and steam methanol extract. Alkaloid, saponine, and sterol were found in all extract. Tannin and triterpenoid test gave negative result in betadine leaves and stem. The flavonoid level were higher in betadine leave extract than in betadine stem extract. The same results were shown in the phenol screening. Phenolic and flavonoid compounds are common in plants and known to have various biological activities. These beneficial properties include antimicrobial, antiviral, antioxidant, and anti-inflammatory activity. Many of these biological functions were known to have beneficial health. Phenolic compounds (flavonoids, phenolic acid, and Phenols) have antioxidant activity that could reduce free radicals, have the ability to chelate metals, inhibit lipoxygenase enzymes, reduce free radicals¹⁰ and maintain a healthy body from oxidative stress¹¹. Phenolic compounds and flavonoids are known to be antioxidants and free radical scavengers associated with oxidative damage, which justifies their ethnic use in folk medicine¹².Alkaloid, flavonoid, and phenol were dominant phytochemical compound that find in betadine leaf. Otherwise, alkaloid and saponine were dominant phytochemical compound in betadine stem.

Table 1: Phytochemical analysis of Betadine leaf and stem extract

Phytochemical test	Leaf	Stem
Alkaloid (Mayer)	+	++
Alkaloid (Wagner)	++	+
Alkaloid (Dragendorf)	+++	++
Flavonoids	+++	+
Phenols	+++	+
Tannins	-	-
Saponins	++	++
Triterpenoids	-	-
Sterols	+	+

^*Key: (+) = Low, (++)= Medium, (+++)= High, (-) = Absent

Determination of Total Flavonoid:

Graph-1 shows total flavonoid content in Betadine (Jatropha muitifida Linn.) leaf and stem methanol extract. Determination of total flavonoids of each extract was performed by the aluminum chloride colorimetric method¹³. Quercetin was used to make a standard calibration curve^8,
14. determination of total flavonoid showed that total flavonoids of Betadine leaves extract was higher than Betadine stems extract. Betadine leaves extract has 10,36±0,03mg EQ/g and otherwise, Betadine stems extract has 1,13±0,02mg EQ/g of total flavonoids. It was in line with the result of phytochemical screening. Generally, flavonoids and phenols content in leaves is higher than in the stem of the plant¹⁵. The presence of total flavonoid content usually indicates the antioxidant potential of the plant. Excessive production of free radicals causes oxidative stress. Plant phytochemicals are natural antioxidants that reduce free radicals and disease caused by oxidative stress¹⁶. Flavonoids are polyphenols, common natural substances in the plant kingdom¹⁷.

Graph 1: Total Flavonoid Content of Betadine Plant Extract

^*The blue chart was leaves; the orange chart was Stem betadine extract

Toxicity Determination:

The Artemia lethal assay is considered a useful tool for preliminary toxicity assessment¹⁸. The brine shrimp lethality test is a simple, reliable, rapid and inexpensive bioassay for screening biological activity and determining cytotoxicity of test samples. Surviving brine shrimp larvae were counted after 24 hours and the LC50 determined. The Brine Shrimp Lethality Test (BSLT) is a simple, sensitive, reliable, economical and easy method to measure the cytotoxicity of plant extracts, fractions or pure compounds¹⁹.Artemia salina lethality bioassay results were assessed by LC50 (lethal concentration)²⁰.

Table-2, Graph-1, and Graph-2 showed BSLT result for identified toxicity of Betadine (Jatropha muitifida Linn.) leaf and stem methanol extract. Secondary metabolites in plants suspected of having biological activity need to be tested to determine the level of toxicity. A toxicity test was being to Artemia salina with BSLT ( Brine Shrimp Lethality Test) methods. The result showed that Betadine leaves and stem extract has high toxicity because their LC-50 was 29,29 ppm and 27,79 ppm. The plant extract has high toxicity if their LC-50 of bioactivity compound is more than 1000 ppm²¹. It was related to the content of saponin. Saponin has some biological effect on membrane cells and may be toxic The bioactive compound could be toxic in high doses, but in low or measured doses, it may be a drug. LC-50 was analyzed with the normal population assumption (probit analysis) method. Based on probit analysis, the curve of log concentration with probit value Betadine leaves and the stem is shown in Graph-2 and Graph-3. The higher the log concentration, the higher % probit value of larva mortality and tends to be stable after a concentration of 62.5 ppm.

Table 2: Betadine Leaf and Stem Toxicity with BSLT Method

Simplicia	Extract concentration	*% Mortality)**	Probit Value	LC-50
Leaf	control	-	-	29,29 ppm
	15,625 ppm	68,34	5,55
	31,25 ppm	89,45	6,41
	62,5 ppm	100	7,41
	125 ppm	100	7,41
	250 ppm	100	7,41
	500 ppm	100	7,41
Steam	control	-	-	27,79 ppm
	15,625ppm	60	5,31
	31,25 ppm	73,3	5,58
	62,5 ppm	100	7,41
	125 ppm	100	7,41
	250 ppm	100	7,41
	500 ppm	100	7,41

Graph 2: Graph of the relationship between Betadine Leaf Methanol Extract Concentration and Probit Value % mortality

Graph 3: Graph of the relationship between Betadine Stem Methanol Extract Concentration and Probit Value % mortality

CONCLUSION:

Analysis of the result showed that methanol extract leaves and stem Betadine contains alkaloids, flavonoids, saponins, and phenol. Betadine leaves methanol extract had total flavonoids at least 0,16±0,03mg EQ/g and Betadine stem methanol extract was 1,13±0,02mg EQ/g. Betadine leaves and stems methanol extract has high toxicity activity. There were 29, 29ppm and 27, 79ppm. The high toxicity may possibly relate to containing saponins in line to it is qualitative screening.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this research.

ACKNOWLEDGMENTS:

The authors would like to thank Polytechnic AKA Bogor for their kind support and funding this entire research.

REFERENCES:

1. Apriyanti M. Tanaman Obat Paling Berkhasiat & Paling Dicari. Yogyakarta: Pustaka Baru Pres. 2010

2. Shweta Singh Chauhan, Pramod Kumar Mahish. Flavonoids of the flame of forest- Butea monosperma. Research J. Pharm. and Tech. 2020. 13(11)

3. Rekha Rajendran, R Hemachander, T Ezhilarasan, C Keerthana, DL Saroja, KV Saichand and Mohamed Gasim Abdullah. Phytochemical Analysis and In-Vitro Antioxidant Activity of Mimosa pudica Lin., Leaves. Research J. Pharm. and Tech. 2010. 3(2)

4. Singh D, Sharma SK, Rani R, Mishra S and Sharma RA. A New Flavonoid and other Two Flavonoids Isolated from Different Plant Parts of Selected Cassia Species. Asian Journal of Research in Chemistry. 2011; 4(5): 818-821.

5. Franyoto YD, Kusmita L, Mutmainah , Angrena RD. Total Flavonoid Content and Formulation Antioxidant Cream Stem of Jatropha multifida. IOP Conf. Series: Journal of Physics: Conf. 2018. 1025

6. Nagaharika Y, Kalyani V, Rasheed S, Karthikeyan R, Anti-inflammatory activity of leaves of Jatropha gossypifolia L. by HRBC membrane stabilization method. Journal of Acute Disease. 2013. 2 (2): 156–158

7. Shlini P and Siddalinga MKR. Extraction of Phenolics, Proteins and Antioxidant Activity from Defatted Tamarind Kernel Powder. Asian Journal of Research in Chemistry. 2011; 4(6); 936-941.

8. Edeoga HO, Okwu DE, Mbaebre BO. Phytochemical Constituent of Some Nigerian Medicinal Plants. Afr Journal of Biotechnology. 2005; 4: 685 – 688

9. Al-Owaisi, Mooza, Al-Hadiwi N, Khan SA. GC-MS analysis, determination of total phenolics, flavonoid content and free radical scavenging activities of various crude extracts of Moringa peregrina (Forssk.) Fiori leaves. Asian Pac J. Trop Biomed. 2014; 4(12): 964-970

10. Pisutthanan S, Plianbangchang P, Pisutthanan N, Ruanruay S, Muanrit O. Brine shrimp lethality activity of Thai medicinal plants in the family Meliaceae. Naresuan Uni J. 2004; 12: 13-18.

11. Robards K, Prernzler PD, Tucker G, Swatsitang P, Glover W. Phenolic compounds and their role in oxidative processes in fruits. Food Chemistry. 1999; 66: 401-36

12. Tekeshwar Kumar, Vishal Jain. Phytochemical Screening, Phenolic, Flavonoids, Carotenoids contents and antioxidant activity of Folkloric Memecylon edule roxb. Research J. Pharm. and Tech. 2016; 9(10)

13. Chang CC, Yang MH and Wen HM. Estimation of total flavonoid content in propolis by two complementary colorimetric methods. Journal of Food and Drug Analysis. 2002; 10 (3); 178-182

14. Tuapattinaya, Prelly MJ, Rumahlatu D. Analysis of Flavonoid Levels of Enhalus acoroides in Different Coastal Waters in Ambon Island, Indonesia. International Journal of Applied Biology. 2019; 3(1)

15. Manurung H, Kustiawan W, Kusuma IW, Marjenah. Total flavonoid content and antioxidant activity of tabat Barito (Ficus deltoidea Jack) on different plant organs and ages. Journal of Medicinal Plants Studies. 2017; 5(6): 120-125

16. Som K. Madhvi, Manik Sharma , Javaid Iqbal , Mohd Younis , Razda Sheikh. Phytochemical analysis, Total Flavonoid, Phenolic contents and Antioxidant activity of extracts from the Leaves of Rhododendron arboretum. Research J. Pharm. and Tech. 2020; April 13(4).

17. Szliszka E et al. Chalcones Enhances TRAIL- Induced Apoptosis in prostate Cancer Cells. International Journal of Molecular Science. 2011; 11(1): 1-13

18. Md. Saif Uddin, Mohammad Shah Hafez Kabir, and Syed Md. Abdul Kader. Brine shrimp lethality bioassay, thrombolytic and antibacterial activities of methanol extract of Boehmeria platyphylla d don leaves Int J Pharm 2016; 6(1): 29-36.

19. Mamata, A. Brine Shrimp Lethality Test of Andrographis paniculate. Research J. Pharm. and Tech. 2014; 7(7).

20. Ashpak. M. Tamboli, Kiran. A. Wadkar. Anticancer activity of leaves extracts of Convolvulus pluricaulis and Mimosa rubicaulis (Lam.) by Brine Shrimp Lethality Bioassay. Research J. Pharm. and Tech. 2021; 14(10).

21. Meyer BN, Ferrigni NR, Putnam JE, Jacobsen LB, Nichols DE, Mc Laughlin JL. Brine Shrimp: A convenient general bioassay for active plant constituents. Planta Medica. 1982; 45: 31-34.

Received on 28.02.2023 Revised on 18.03.2024

Accepted on 10.10.2024 Published on 28.01.2025

Available online from February 27, 2025

Research J. Pharmacy and Technology. 2025;18(2):773-776.

DOI: 10.52711/0974-360X.2025.00114

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