INTRODUCTION:

Free radicals are very reactive because they include molecules, atoms, or groups, and there is more than one unpaired electron in the outermost layer. They are radicals, such as free radicals (reactive oxygen species), which come from reactive oxygen species¹^,².

Antioxidants work as cell protectors against damage by free radical molecules. At low levels, antioxidants can inhibit the oxidation of proteins, fats, carbohydrates, and DNA in cells³^,⁴.

In the past, the Nelumbo nucifera plant was used to treat a wide range of illnesses⁵. Meanwhile, for the pharmacological aspect, many studies have shown that the Nelumbo nucifera plant has different pharmacological activities, one of which is antioxidant^6–8. The leaf and stems are used as a medicine for dizziness, diarrhea, beriberi, bleeding such as nosebleeds and hematemesis, vaginal discharge in women, and chest tightness. The flowers can be used as a medicine for skin blemishes, bleeding, and purulent inflammation ^9–11.

The results of several researchers are compounds resulting from secondary metabolism, namely C₁₈H₂₁NO₄, glycosides, saponins, tannins, flavonoids and steroids or triterpenoids found in Nelumbo nucifera leaf. From the results of secondary metabolites in the leaf and stem of Nelumbo nucifera, it has the potential to be an antioxidant ^12–14.

This research method uses in vitro activity testing for antioxidants on Nelumbo nuciferaleaf and stem, namely DPPH, ABTS, and FRAP. The DPPH method tests a component's ability to eliminate free radicals in components or extracts. The advantage of the DPPH method is that it is quick and easy to implement^15–18.

ABTS is a method for determining antioxidant activity obtained by oxidizing salt ABTS diammonium for potassium persulfate. Signs of antioxidants in the sample were shown by the disappearance of the blue color in the ABTS reagent. ^19,20

FRAP method as a test for antioxidants in plants. The FRAP method has the advantage of being relatively cheap, making reagents quickly, and being fast and straightforward ²¹. This method can determine the total antioxidant content of materials according to the antioxidant compounds in reducing Fe3⁺ ions to Fe²⁺, where the antioxidant capacity is the same as reducing these compounds ^22,23.

Researchers were interested in the antioxidant activity of ethanol extracts of bay leaf and Nelumbo nucifera stem and used the DPPH, ABTS, and FRAP methods to do so.

MATERIALS AND METHODS:

Materials:

Analytical balance, Erlenmeyer tube (Pyrex), test tube (Pyrex), measuring flask (Pyrex), measuring cup (Pyrex) 10 cc, 25 cc, 50 cc, 250 cc, blender, stirring rod, dark bottle, glass beaker (pyrex), micropipette, aluminum foil, cuvette, Rotary Evaporator, Incubator, Centrifuge and UV – Vis Spectrophotometer.DPPH method (1,1 diphenyl 2 picryhidrazil soja (Nelumbo nucifera Gaertn), Ethanol 70%, Ethanol p.a, Aquadest, DPPH, ABTS, Potassium Persulfate (𝐾₂𝑆₂𝑂₈), Vitamin C, 0.2 M phosphate solution with pH 6.6, 1% Oxalate solution, 1% potassium ferricyanide solution, 0.1% 𝐹𝑒𝐶𝑙3 solution, and 10% trichloroacetic acid (TCA) solution.

Making Simplisia Nelumbo nucifera Leaf and Stem:

Making simplicia begins with collecting raw materials, namely 5 kg of Nelumbo nucifera leaf and stem. Next, the Nelumbo nucifera leaf and stem are separated from unnecessary impurities and washed using running water until clean. Next, drain and chop the Nelumbo nucifera leaf and stem, then dry them in a drying cupboard at a temperature of ± 40⁰C; after drying the Nelumbo nucifera leaf and stem, grind them using a blender and put them on a sieve.

Making Ethanol Extract of Nelumbo nucifera Leaf and Stem:

We weighed 500 grams of powdered Nelumbo nucifera leaf and stem, soaked 75 parts in 5 liters of 70% ethanol solvent in a maceration vessel for five days, and stirred occasionally daily. Then, the juice is filtered, and the dregs are squeezed. The residue is washed with the remaining solvent.Twenty-five parts were stirred, left for two days, and filtered to obtain 100 parts of the filtrate. With a rotating evaporator set to 40–50⁰C, the filtrate is then evaporated to make a thick extract.

DPPH (1,1diphenyl2 picryhidrazil) Method:

Sample Preparation:

Twenty-five milligrammes of ethanol extract of Nelumbo nucifera leaf, stem and Ascorbic acid (Vitamin C) and dissolved in 250 ml of 70% ethanol to obtain 100 ppm (µ𝑔/𝑚𝐿) as a stock solution which has a concentration of 100 ppm. 5 test samples were made, each with a saturation of 10, 20, 40, 60, and 80 ppm, then the test samples were measured for free radical activity using the DPPH method (1,1 diphenyl two picryhidrazil).

Preparation of 100 ppm DPPH Solution:

Five milligrams of DPPH powder are weighed out and mixed with ethanol slowly into a fifty-milliliter volumetric flask until the concentration line is reached (concentration 100 ppm (µ𝑔/𝑚𝐿). Getting a 40 ppm DPPH solution ready. In a twenty-five millilitre volumetric flask, add 10 ml of a 100 ppm DPPH solution, and make sure thevolume is filled with ethanol p.a to the maximum mark (concentration 40 ppm (µ𝑔/𝑚𝐿). Measurement of DPPHFree Radical Scavenging using UV-Vis Spectrophotometry2 ml of each test sample solution of leaf extract, Nelumbo nucifera stem, and ascorbic acid mixed with a percentage content of 10, 20, 40, 60, and 80 ppm was put into each test tube, and 2 ml of 40 ppm DPPH working standard solution was added to each test tube. Next, pipet 2 ml of the 40 ppm DPPH working standard solution, put it in a test tube, and drop 2 ml of ethanol pa into the blank solution. The test sample solution and blank were then mixed together and left in a dark room at room temperature for an hour. The absorbance was then measured with a UV-Vis spectrophotometer^24,25.

ABTS method (2,2 azinobis(3- ethylbenzothiazoline)-6-sulfonic acid):

a. K2S2O8 solution: Measured 14 mg of K2S2O8 (2.45 mM) mixed in 20 ml of distilled water in a glass

b. ABTS Solution: Measured ABTS (7 mm) 18 mg, diluted with 5 ml of distilled water.

c. ABTS stock solution: Dark blue ABTS is obtained by adding 5 ml of potassium persulfate solution to 5 ml of ABTS solution (7 mM) and stored in a dark room at 22-24°C for 12-16 hours before use.

d. PBS reagent pH 7.4: Dissolve 8 gr NaCl, 0.2 gr potassium chloride, 0.2 gr disodium phosphate, and 0.24 potassium dihydrogen phosphate in distilled water to make 1 L.

e. Absorption measurement of ABTS blank solution. Pipette up to 0.1 ml of PBS pH 7.4 solution, then add 2 ml of ABTS stock solution. The solution was then incubated for 6 minutes, and its quantitative size was calculated at a wave of 734 nm using UV-Vis spectrophotometry.

Measurement of ABTS Free Radical Reducing Activity using UV-Vis Spectrophotometry:

A 0.1 sample solution was examined by adding ingredient content levels of 10, 20, 40, 60, and 80 ppm to each test tube. Subsequently, 1 ml of orientated ABTS 7 mM working standard solution was added. After mixing and leaving the test solutions at room temperature for six minutes, a UV-Vis spectrophotometer assessed absorbance^26,27.

FRAP (Ferric Reducing Antioxidant Power) Method:

pH 6.6 0.2 M Phosphate Buffer, this solution is prepared withprovide 2 grams of NaOH mixed into exactly 250 ml of free distilled water 𝐶𝑂2 in a volumetric flask. Then, 6.8 gr 𝐾𝐻₂𝑃𝑂₄ was put into 250 ml𝐶𝑂2 distilled water in a glass flask. Next, 16.4 cc of NaOH was pipetted and dropped into a measuring flask, mixed with 50 ml of 𝐾𝐻₂𝑃𝑂₄, the pH was measured at 6.6 and filled with 0.2 L of free 𝐶𝑂2 distilled water.

a. Potassium Ferricyanide Solution 1%

The solution is prepared by dissolving 1 gram of potassium ferricyanide in distilled water and then diluting it in a 100 ml volumetric flask.

b. FeCl30 1% Solution

The solution was produced by combining 0.1 grams of 𝐹𝑒𝐶𝑙3 in distilled water and diluting it in a 0.1 L volumetric flask.

c. Trichloroacetic acid (TCA) solution 10%

Prepare 10 g of TCA in distilled water and dilute in a 0.1 L volumetric flask.

d. Making Blanks

After adding 1 ml of ethanol and 1 ml of phosphate buffer (pH 6.6), 1 ml of 1% K3Fe(CN)6 was added and A total volume of 1.5 ml consisting of 1 ml of distilled water and 0.5 mlof 0.1% FeCl3 were added and the logarithmic ratio was seen at 705 nm wavelength on a UV-Vis spectrophotometry^28,29.

Measurement of FRAP Radical Reducing Activity using UV-Vis Spectrophotometry:

One milliliter of the experimental solution was combined with saturation levels of 10, 20, 40, 60, and 80 ppm and 1 ml of ascorbic acid solution as a comparison with levels of 5, 10, 15, 20, and 25 ppm, each of which was poured into a test tube, Next, 0.2 ml of buffer solution was added to each test tube. After that, deep incubation was carried out.Twenty minutes using a temperature of 50°C, followed by adding 1 ml of TCA, then separated using a speed of 3000 rpm within 10 minutes. Proceed with extracting 1 ml of the uppermost layer of skin and transferring it into a test tube and add distilled water1.5 ml and 𝐹𝑒𝐶𝑙3 0.1% 0.5 ml. Then, leaf it for 10 minutes and calculate the absorbance at 705 nm^28,29.

RESULT:

Table 1. Phytochemical Screening Results of Ethanol Extract of Nelumbo nucifera Leaf and Stem

Metabolite Coumpounds	Reagents	Results
Metabolite Coumpounds	Reagents	*Nelumbo nucifera* Leaf	*Nelumbo nucifera* Stem
Alkaloids	Bouchardart	+	-
	Maeyer	-	-
Flavonoids	Fecl3	+	-
	Mg.HCl	+	-
	H2SO4	+	+
Terpenoids	Liebermann-Burchard	-	-
	Salkowsky	+	+
Steroids	Liebermann-Burchard	-	-
	salkowsky	-	-
Saponins	Aquadest	-	+
Tanins	Fecl3	+	-

Information:

+ : Positive

- : Negative

Results of Antioxidant Activity Analysis using the DPPH Method:

Table 2. Antioxidant Activity of Ethanol Extract of Leaf, Nelumbo nucifera Stem and Ascorbic Acid

Sample	Consentrations	% Reducing	^IC50 Values ^(µg/ml)
Leaf Ethanol Extract Nelumbo nucifera	10	34.3208
	20	50.8671
	40	64.5644	16.7473
	60	75.6296
	80	85.1982
Stem Ethanol Extract Nelumbo nucifera	10	39.2240
	20	48.4414
	40	58.1954	25.2170
	60	61.5195
	80	64.4095
Ascorbic Acid	10	37.2216
	20	51.0664
	40	78.8660	12.6013
	60	89.8601
	80	92.5955

^Tabl^e^{3. Regression Function Calculation Results
and IC50 Values from Test Samples and Comparative DPPH Methods}

Test Solution	Regression Equations	^IC50 Values ^(µg/ml)
Leaf Ethanol Extract Nelumbo nucifera	y=19.0930x+2.8182	16.7473
Stem Ethanol Extract Nelumbo nucifera	y=14.8354x+2.1184	25,2170
Ascorbic Acid	y=21.9943x–5.6002	12.6013

Results of Antioxidant Activity Analysis by ABTS Method:

Table 4. Antioxidant Activity of Ethanol Extracts of Nelumbo nucifera Leaf, Stem and Ascorbic Acid

Sample	Consentrations	% Reducing	^IC50 Values ^(µg/ml)
Leaf Ethanol Extract Nelumbo nucifera	10	26.0236
	20	30.2996
	40	54.8434	22.8995
	60	74.8661
	80	88.7063
Stem Ethanol Extract Nelumbo nucifera	10	14.4757
	20	16.5638
	40	18.5111	15.29
	60	20.7127
	80	23,7449
Ascorbic Acid	10	18.7426
	20	58.8243
	40	99.4825	11.1927
	60	99.7685
	80	99.9092

^Tabl^e^{5. Regression Function Calculation Results
and IC50 Values of Test Samples and Comparator ABTS Method}

Test Solution	Regression Equation	^IC50Values ^(µg/ml)
Leaf Ethanol Extract Nelumbo nucifera	19.0920x– 9.7792	22.8995
Stem Ethanol Extract Nelumbo nucifera	5.1052x+0.8087	15.29
Ascorbic Acid	25.8167x –2.3541	11.1927

Results of Antioxidant Activity Analysis by FRAP Method:

Table 6. Antioxidant Activity of Ethanol Extracts of Nelumbo nuciferaLeaf, Stem and Ascorbic Acid

Sample	Consentration	% Reducing	^IC50 Values ^(µg/ml)
LeafEthanol Extract Nelumbo nucifera	10	89.5516
	20	90.3846	5,0972
	40	94.4557
	60	95.8143
	80	96.3229
Stem Ethanol Extract Nelumbo nucifera	10	82.6747
	20	87.4064
	40	91.7792	5.7069
	60	93.3237
	80	95.1164
Ascorbic Acid	5	98.9648
	10	99.4013
	15	99.5980	2.8420
	20	99.6666
	25	99.7012

^Tab^l^{e 7. Regression Function Calculation Results and IC50 Values
of Test Samples and Comparator FRAP Method}

Test Solution	Regression Equation	IC50 Values (µg/ml)
Leaf Ethanol Extract Nelumbo nucifera	21.6513x+14.7366	5,.972
StemEthanol Extract Nelumbo nucifera	21.4302x+12.6754	5.7069
Ascorbic Acid	30.0466x+18.6156	2.8420

^{Table 8. Categories of IC50 Value as Antioxidant}

No.	Categories	IC50Values (µg/ml)
1.	Very Strong	<50
2.	Strong	50 – 100
3.	Medium	100 – 250
4.	Weak	250 – 500
5.	Inactive	>500

Sumber: Lungdan Destiani(2007)

DISCUSSIOIN:

This test reveals Phytochemical Screening of Nelumbo nucifera leaf and stem ethanol extract chemical components. Table 1 shows phytochemical screening results. Flavonoids, saponins, tannins, terpenoids, and steroids are in Nelumbo nucifera leaf extract. Flavonoids, terpenoids, and saponins are in Nelumbo nucifera stems. Nelumbo nucifera leaf and stem may be antioxidants due to phytochemical substances like flavonoids ^30,31.

Nelumbo nucifera ethanol extract antioxidant propertiesDPPH analysis of Nelumbo nucifera leaf, stem, and ascorbic acid antioxidant activity: The antioxidant activity of Nelumbo nucifera leaf ethanol extract and ascorbic acid was tested over 60 minutes. Then test solution was applied.

Antioxidant activity of ethanol extract of leaf, Nelumbo nucifera stems, and ascorbic acid using the DPPH method:

The antioxidant activity of Nelumbo nucifera leaf and stem ethanol extract and ascorbic acid was tested after 60 minutes. At saturation levels of 10, 20, 40, 60, and 80 ppm, the test solution was added. Nelumbo nucifera leaf ethanol starch antioxidants donate electrons to DPPH radicals to fight them. This electron transfer changes the solution's colour from purple to yellow or reduces its intensity. Thus, this colour change reduces DPPH free radical absorption³².

The solution used as a comparison is Ascorbic Acid (Vitamin C). The use of ascorbic acid in the antioxidant activity test aims to obtain information on whether or not the potential for antioxidants to be extracted from Nelumbo nuciferaleaf. Its stems are vital if a comparison is made with ascorbic acid. If the IC50 value of the sample is ≤ almost the same as the IC50 value of ascorbic acid, then it can be concluded that this sample has a significant chance of being an optional part of a strong antioxidant³³.

Based on table 2. IC50 results for ethanol extract of Nelumbo nucifera leaf worth 16.74 µg/ml, ethanol extract of Nelumbo nucifera stem worth 25.21 µg/ml, and ascorbic acid of 12.60 µg/ml. Apart from that, it shows that the higher the concentration of the solution, the higher the free radical scavenging activity of DPPH because the more the concentration of the extract increases, the more hydrogen atoms are added, and the DPPH decreases, resulting in a lighter DPPH color ³⁴.

Antioxidant activity of ethanol extract of leaf, Nelumbo nucifera stems, and ascorbic acid using the ABTS method:

The radical form, which has a blue-green elemental nitrogen level, and if the antioxidant undergoes a non-radical form change from having color to being colorless, is the ABTS method (35). This method is sensitive to light and even forms ABTS; it takes 12-16 hours to incubate in dark conditions. The oxidant activity test was tested using the ABTS method by removing the color of the ABTS cation to determine the amount of antioxidant when it comes into contact with the ABTS cation radical ³⁵.

Ethanol extract from table 1 Nelumbo Nucifera leaf and stem antioxidant tests using ABTS showed a positive connection between solution concentration and reducing activity. The ethanol extract of Nelumbo nucifera stem lacks antioxidant components, which explains its low antioxidant action.

See table 4. The ethanol extract from Nelumbo nucifera leaf has an IC50 value of 22.89 µg/ml. The ethanol extract from Nelumbo nucifera stem has an IC50 value of 1529 µg/ml, placing it in the inactive antioxidant group. The IC50 for ascorbic acid is 11.19 µg/ml.

Antioxidant activity of ethanol extract of leaf, Nelumbo nucifera stems, and ascorbic acid using the FRAP method:

The FRAP approach is founded on the notion of reduction of the Fe³⁺ complex, which has a yellow color, to the Fe²⁺ complex, which has a blue-green color, due to the provision of electrons from antioxidant compounds in an acidic environment ³⁶.

Antioxidant activity was measured by FRAP testing using a reference solution, namely ascorbic acid. He added that TCA was intended to precipitate the potassium ferrocyanide complex. Apart from that, the addition of FeCl3 is also intended to make the green compound blue (Berlin blue) ³⁷.

Compared to DPPH and ABTS, Nelumbo nucifera stem and leaf ethanol extract has substantial antioxidant activity. The test solution's antioxidant activity increases with lower IC50³⁸.

As per table 6. The IC50 values for Nelumbo nucifera leaf and stem ethanol extracts are 5.09 and 5.70 µg/ml, respectively. Ascorbic acid has an IC50 of 2.84 µg/ml.

CONCLUSION:

Ethanol extract of Nelumbo nucifera leaves and stems in the DPPH and FRAP tests showed strong antioxidant activity seen from the highest IC50 value found in Nelumbo nucifera stems with values of 25.2170µg/ml and 5.7069µg/ml, while for the IC50 value based on the ABTS method, the extract Nelumbo nucifera leaf ethanol has strong and high antioxidant activity compared to stems with a value of 22.8995 µg/ml.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

ACKNOWLEDGMENTS:

The authors acknowledge the facilities, and scientific and technical support from Research Laboratory, Universitas Sumatera Utara. The research was supported by Universitas Sumatera Utara and Institut Kesehatan Helvetia, who provided the necessary facilities and equipment.

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Received on 19.03.2024 Revised on 06.07.2024

Accepted on 13.09.2024 Published on 24.12.2024

Available online from December 27, 2024

Research J. Pharmacy and Technology. 2024;17(12):6084-6090.

DOI: 10.52711/0974-360X.2024.00922