INTRODUCTION:

Gouty arthritis is type of arthritis caused by increased serum uric acid levels or chronic hyperuricemia which results in the deposition of MSU crystals in the joints where Mono Sodium Urate (MSU) gets accumulated in synovial fluid, other tissues and multiple areas in the bones^1,2,3. Based on WHO data (2015), gout arthritis is estimated to occur in 840 people out of every 100,000 people. The number of cases of gout arthritis in Indonesia is 32% under 34 years of age and 68% over 34 years.

This disease can be triggered by degeneration of body cells with age, impaired metabolic processes, and decreased immune response⁴.

Gout can be prevented by the presence of antioxidants in the human body. Antioxidants can inhibit oxidation reactions by scavenging free radicals so that cell damage can be inhibited⁵. Antioxidants consist of enzymatic and non-enzymatic antioxidants. Enzymatic antioxidants are the main defense system against oxidative stress conditions such as superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), glutathione reductase (GR). Meanwhile, non-enzymatic antioxidants (secondary antioxidants) can be obtained from fruits and seeds that contain vitamin C, vitamin E, carotenoids, flavonoids, selenium, manganese, zinc, omega-3⁶.

Several plants have been investigated for their antioxidant content and anti-uric acid activity, such as the leaves of the suruhan plant (Peperomia pellucida [L.] Kunth)⁷. Antioxidants are chemical substances that can react with electrons as free radicals by donating one or more electrons so that they can prevent oxidation from other compounds as free radicals and play a significant role in several important biological processes such as immunity, protect ion against tissue damage, reproduction and growth or development^8,9,10. It has been reported that natural antioxidant contained in the leaves of Peperomia pellucida [L.] Kunth are compounds of alkaloids, tannins, saponins and polyphenol which are efficacious in reducing uric acid levels¹¹. Another plant that has the potential to have anti-uric acid activity is the Musa balbisiana Colla.

Musa balbisiana Colla is one of the world's banana cultivars spread in Indonesia¹². Musa balbisiana Colla has the characteristics of thick leaves, and a fairly thick waxy layer. Sometimes there are seeds on the fruit, the skin is hard and thick and the fruit cannot be eaten fresh. Musa balbisiana Colla has been reported contain flavonoids, polyphenols, tannins, monoterpenoids, sesquiterpenoids, quinones and saponins which play an important role in secondary antioxidant activity¹³. Anti-uric acid activity has not been previously reported in the ripe fruit seeds of Musa balbisiana Colla.

In this study, the Musa balbisiana Colla seeds were extracted using Ultrasonic Assisted Extraction (UAE) probe where the extraction was carried out with the help of ultrasonic waves with a frequency of 20-20,000 kHz ¹⁴. The working principle of this extraction is to increase the permeability of the cell wall with cavitation power as a dynamic stress so that the interphase phase occurs¹⁵. The UAE can increase the extraction yield in a short time and low temperature. Another advantage of the UAE method is the small volume of solvent required which reduces solvent usage¹⁶.

MATERIALS AND METHODS:

Materials:

Mortar and pestle, knife, petri dish, bottle, rotary evaporator, Brookfield viscometer, paper disc, centrifuge, hotplate, oven, incubator, erlenmeyer, analytical balance, vial, test tube, ose, Bunsen, tweezers, and other glassware.

Aloe vera, 70% ethanol, aquadest, Nutrien Agar media, Potaos Dextros Agar media, test suspension, Meyer reagent, chloroform, Carbopol 940, TEA, methyl paraben, glycerin, Fe(Cl)3, Magnesium powder, HCl (s).

Simplicia Setup:

The skin of the ripe stone banana was peeled, then the seeds were separated from the fruit flesh. The seeds were mashed and then dried in an oven at 45^oC. Simplicia powder was stored separately in dry container, closed, identified and protected from direct sunlight until extraction was carried out.

Extraction of Simplicia:

A total of 50grams of simplicia powder of ripe banana seeds were weighed, then 400mL of methanol pa solvent was added. The mixture was sonicated using a vibrating ultrasonic probe for 30 minutes at room temperature with an amplitude of 0.6m.

Phytochemical Test:

The phytochemical test using the Ciulei method was carried out on crude methanol extract. Phytochemical screening tests carried out included tests for alkaloids, flavonoids, phenols, saponins, tannins, glycosides, and sterols-triterpenoids¹⁷.

Alkaloide Identification:

A total of 0.25g of sample was put into a test tube, dissolved in 2mL of 2% HCl, heated while being shaked, then filtered. The filtrate obtained was partly added 2-3 drops of Meyer reagent and partly added 2-3 drops Dragendorf reagent. The presence of alkaloid compounds was indicated by the occurrence of white deposits with Meyer reagent and orange precipitate with Dragendorff reagent.

Flavonoids Identification:

A total of 0.25g of the sample was put into a test tube, added 1-2mL of 50% methanol, heated at 50^oC, after the cold solution was added magnesium metal and 4-5 drops of concentrated HCl. The presence of red or orange in the filtrate indicates the presence of flavonoids.

Phenol Identification:

A total of 0.25g of the sample was put into a test tube, added 2mL of distilled water and a few drops of 1% FeCl₃ solution, the formation of a purple color indicated the presence of phenol.

Saponin Identification:

A total of 0.25g of sample was put into a test tube, added with 3mL of distilled water, then shaken for 15 minutes to observe the occurrence of 1cm high foam that was lasted for 15 minutes.

Tannin's identification:

A total of 0.25g of sample was inserted into the test tube, added with 1-2mL of 1% FeCl₃. The occurrence of a blackish blue or green-black color indicated the presence of tannins.

Steroid Glycoside Identification:

A total of 0.25g of sample was inserted into the test tube, 0.5mL of anhydrous acetate and 0.5mL of chloroform were added, then the solution was transferred to the test tube, then added concentrated H₂SO₄ to the bottom of the tube, forming a reddish brown ring indicating the presence of steroid glycosides.

Sterol-Triterpenoid Identification:

A total of 0.25g of sample was inserted into the test tube, 0.5mL of acetic anhydride and 0.5mL of chloroform was added, then 0.2mL of concentrated H₂SO₄ was added dropwise to the bottom of the tube, forming a brownish or blue violet (purple) color showing triterpenoids and green the presence of sterols.

Total Phenolic:

The total phenolic content of the extract was determined by the Folin – Ciocalteu method. Briefly 400μL of crude extract (1mg/mL) was made up to 6mL with distilled water, mixed thoroughly with 1mL of Folin – Ciocalteu reagent for 3 min, followed by the addition of 2,5mL of 10% (w/v) sodium carbonate, measured with distilled water in a 10mL measuring flask, then homogenized. The mixture was allowed to stand for a further 60 min in the dark, and absorbance was measured at 650nm. The total phenolic content was calculated from the calibration curve of gallic acid (concentrations of 0, 2, 4, 6, 8mg/L), and the results were expressed as mg of gallic acid equivalent per g dry weight¹⁸

DPPH Method Antioxidant Activity Test:

A total of 5 mg of the extract was dissolved with methanol pa in a 5 mL measuring flask, resulting in a sample solution with a concentration of 1,000mg/L. Solution was pipetted 40µL; 80µL; 160µL; 320µL; 640 µL, then put each into five 5 mL measuring flasks, then added 1mL of DPPH 39mg/L solution, then measured with methanol pa, and homogenized (sample concentrations 8, 16, 32, 64, and 128mg/L). The solution was incubated for 30 minutes at room temperature (25^oC), then the absorption of the solution was measured using a visible light spectrophotometer at a wavelength of 516nm. The process was carried out in two repetitions. The same operation was carried out on the BHT comparators by pipetting 10µL; 20µL; 40µL; 80 µL; 160µL BHT solution 1,000mg/L (BHT concentrations of 2, 4, 8, 16, and 23mg/L)¹⁹

Antioxidant activity was measured as a decrease in DPPH solution uptake due to the addition of sample. The absorption value of the DPPH solution on the sample is called the percent inhibition (% inhibition) with the following equation [1]:

(A_blank-A_Sample)

% inhibition = ---------------------- X 100 ………[1]

A_blank

Explanation:

A_blank= Absorbance without sample

A_Sample = Absorbance of sample at t minutes

The calculated value is entered into a linear equation (Y = aX + b) with the concentration ppm (mg/L) as the abscissa (X axis) and the% inhibition value as the ordinate (Y axis). The IC₅₀ value was obtained from the calculation when the % inhibition was 50%.

Antioxidant Activity Test of the FRAP Method:

A total of 5mg of the extract was dissolved with methanol pa in a 5mL measuring flask, resulting in a sample solution with a concentration of 1,000mg/L. Solution was pipetted at 40µL; 80µL; 160µL; 320µL; 640µL, then each put into five 5mL measuring flasks, then added 0.4mL of 0.001M citric acid; 0.2mL of Fe³⁺ 0.002 M solution; 0.4mL o-phenanthroline 0.2%, then filtered with distilled water, and homogenized (sample concentrations 8, 16, 32, 64, and 128mg/L). The solution was incubated for 35 minutes at 37^oC, then the solution absorption was measured using a visible light spectrophotometer at a wavelength of 510nm. The process was carried out in two repetitions. The same operation was carried out with a comparator of gallic acid with a concentration of 0.5; 1.0; 1.5mg/L).

Reducing activity can be calculated with the following equation [2]:

Aktivitas pereduksi dapat dihitung dengan persamaan sebagai berikut:

(A_blank-A_Sample)

% Reduction = ---------------------- X 100 ………[2]

A_blank

Explanation:

A_blank= Absorbance without sample

A_Sample = Absorbance of sample at t minutes

The calculated value is entered into a linear equation

(Y = aX + b) with the concentration ppm (mg/L) as the absis (X axis) and the% value of the reduction as the ordinate (Y axis). The IC₅₀ value is obtained from the calculation when the % capacity is 50%.

Uric Acid Test:

A total of 5mg of extract was dissolved with methanol pa in a 5mL measuring flask, resulting in a sample solution with a concentration of 1,000mg/L. The solution was piped 40µL, then put into a 5ml measuring flask, added 40µL standard uric acid 6mg/dL and let stand for 5 minutes. The solution was added with 0.25 mL of reagent 1 and allowed to stand for 5 minutes, then 62.5mL of reagent 2 was added, allowed to stand for 30 minutes at 20-25^oC. The solution was measured using a visible spectrophotometer at 513nm absorption. The same treatment was carried out on uric acid standards, allupurinol as a positive control, and blanks. From the absorbance, it can be calculated anti uric acid activity based on equation [3] and [4].

Sample absorption

Uric acid (mg/dl) = ------------------- X standard concentration (mg/dl)

Standard absorption

[3]

Standard concentration – Uric Acid concentration

Anti acid activity % = -------------------------------------------------- × 100

Standard Concentration

[4]

RESULT AND DISCUSSION:

The sample used in this study was the seeds of Musa balbisiana Colla. A sample of 20g was extracted with methanol using the UAE. The use of methanol as a solvent is due to its high polarity, so that it can dissolve high levels of phytochemical compounds²⁰. The extraction yield was 2.7420grams with a yield of 13.71%. Extraction with the UAE over the last few years has grown due to its role in reducing the use of solvents and energy, so the application of this green technology is necessary to protect the environment from toxic substances. In addition, extraction with UAE can break down plant tissue and work well in the process and release of active compounds into solvents with high efficiency.

Phytochemical Test:

The phytochemical test of methanol extract from Musa balbisiana Colla seeds was carried out as an initial step to conduct on the plant extracts revealed the presence of constituents which are known to exhibit medicinal as well as physiological activities such as triterpenoids, fatty acids, steroids, vitamins, proteins, sugars, flavonoids, tannins, saponins, vanillin, ursolic acid^21,22. Qualitative phytochemical screening will help to understand a variety of chemical compounds produced by plants and quantification of those metabolites will help to extract, purify and identify the bioactive compounds²³. One of the important things that play a role in the phytochemical screening procedure is the solvent for extraction. This is due to the presence of other compounds that affect the solubility process of the compounds studied²⁴.

Table 1: Screening Data of Musa balbisiana Colla seeds extract

Test Parameters	Cooked Stone Banana Seed	Positive results based on theory
Alkaloids: Dragendrof Meyer	+ +	Meyer reagent forms a white or creamy precipitate
Flavonoids	-	-
Phenolic	+	Sediment formed in blue, green, purple and black
Saponins	++	Forms a foam that can last for 10 minutes
Tannin	++	Blackish blue deposits are formed
Steroid Glycosides	++	A reddish brown ring is formed
Triterpenoid steroids	-	-

Based on Table 1, it was found that Musa balbisiana Colla seeds contained alkaloid, phenolic, saponin, tannin, and steroid glycoside compounds. Identification of compounds with specific reagents produces a solution /precipitate that has a specific color. The positive test results for alkaloids with Meyer reagent are indicated by the formation of a white or creamy precipitate. The white deposit is thought to be a potassium-alkaloid deposit. The emergence of foam in the saponin test indicates the presence of glycosides which have the ability to form foam in water which is hydrolysed into glucose and other compounds as shown in Figure 1²⁵.

(a)

(b)

Figure 1. (a) Estimated reaction of the Meyer test (b) Reaction of saponins in water ¹⁷

It has been reported that tannins are responsible for high immunomodulatory activity²⁶. Phenolic compounds in plants have a very important role related to the presence of hydroxyl groups in aromatic rings that can reduce/ trap free radicals. Phenolic-rich plant material can slow down the oxidative degradation of lipids as well as improve the quality and nutritional value of food, so it is increasingly used in the food industry. Alkaloids have been reported to have various pharmacological activities including anticancer and antibacterial activities.

Total Phenolic Test:

Phenolic compounds are secondary metabolite compounds found in plants with the characteristic of having aromatic rings containing one or two hydroxy (OH) groups. Analysis of total phenolic content was carried out to determine the potential Musa balbisiana Colla seeds extract as an antioxidant in warding off free radicals²⁷. The total phenolic content of Musa balbisiana Colla seeds extract was determined by the Folin – Ciocalteu method. The principle of the Folin-Ciocalteu method is the formation of complex blue compounds due to the reaction between phenolic compounds and Folin-Ciocalteu. This reagent oxidizes the phenolic-hydroxy reducing heteropoly acids (phosphomolybdate-phosphotungstate) present in the folin ciocalteu reagent to form a blue molybdenum-tungsten complex which can be detected by spectrophotometer²⁸. The total phenolic content is expressed as mg gallic acid/sample extract using the linear equation for gallic acid standard in Figure 2.

Figure 2. Regression analysis of the total phenolic of Musa balbisiana Colla seeds extract

The use of gallic acid is as a standard because this compound is very effective in forming complex compounds with the Folin-Ciocalteu reagent, so that the reaction that occurs is more stable and pure²⁹. From the graph, it was obtained the standard liner equation for gallic acid y = 0.0996x + 0.0456 with R² = 0.9829. With the absorbance of the sample extract of 0.1777, the total phenolic content of Musa balbisiana Colla seeds extract was 375mg/mg gallic acid. Phenolic content in plants has a direct correlation with antioxidant activity³⁰.

Antioxidant Activity:

DPPH method:

One of the most commonly used methods to test antioxidant activity is to use the free radical 1,1-diphenyl-2- picrylhydrazil (DPPH). In this method, DPPH which is a radical itself with a purple color, changes into a stable compound with a yellow color by reacting with an antioxidant and the extent of the reaction depends on the hydrogen donating ability of the antioxidant³¹. DPPH solution acts as a free radical that will react with antioxidant compounds so that the DPPH will change to become 1,1-diphenyl-2-picrylhydrazin which is non-radical. An increase in the amount of 1,1-diphenyl-2-picrylhydrazin will be indicated by a change in dark purple to pink or pale yellow and can be observed and seen using a spectrophotometer so that the free radical reduction activity of the sample can be determined³². The advantage of measuring antioxidants using the DPPH method is that it is simple, fast and does not require a lot of reagents. The DPPH method provides information on the reactivity of the tested compound with a stable radical³³. The test for antioxidant of Musa balbisiana Colla seeds extract using the DPPH method was carried out using a visible spectrophotometer and the% inhibition of each concentration was obtained and BHT compound was used as a positive control as in Figure 3.

Figure 3.Regression analysis of % inhibition against (a) concentration Musa balbisiana Colla seeds extract (b) BHT

From the linear regression equation in Figure 3, the IC₅₀value of the sample and BHT compound can be obtained with y = 50 and x = IC₅₀ value. The IC value of 50 determines the ability of an antioxidant to reduce 50% of DPPH free radicals. BHT compound was used as a positive control because it has a very strong IC 50 value, which is <50ppm. Molyneaux (2004) explains that the classification of antioxidants is divided into 5, namely <50ppm (very strong), 50-100ppm (strong), 100-150 ppm (moderate), 150-200ppm (weak) and> 200ppm are very weak. The smaller the IC50 value of a sample was obtained, the higher the antioxidant activity was occurred. The results of the IC₅₀ calculation of ripe stone banana seeds (Musa balbisiana Colla) using the regression equation y = 8.1927x - 10.98, obtained IC₅₀ tilapia of (94,265±0.216)mg/L and classified as strong. Meanwhile, the BHT compound used as a positive control had an IC 50 value of (7.44±0.03)mg/L³⁴.

FRAP method:

The FRAP method is one of the antioxidant tests based on the ability of antioxidants to reduce the Fe^{3 +} -2,4,6-tripyridyl-s-triazine complex to Fe²⁺ -2,4,6-tripyridyl-s-triazine which is characterized by changing the colorless solution to solution is blue and gallic acid was used as positive control³⁵. FRAP assay was an important indicator of reducing potential of an antioxidant compound, which can break free radical chain by donating the hydrogen atom³⁶. Using UV-Vis spectrophotometer, It was obtained regression analysis % capacity of Musa balbisiana Colla seeds extract as in Figure 4.

Figure 4.Regression analysis of % capacity on the concentration of ripe banana seed extract (Musa balbisiana Colla)

where X is variation concentration of ripe banana seed extract (Musa balbisiana Colla) and Y is % Capacity which measured by UV -Vis spectrophotometer at a wavelength of 510nm

From the linear regression equation, It can be calculated IC₅₀ value of Musa balbisiana Colla seed extract. The value of IC₅₀ determined effective concentration to inhibit or reduce 50% of the number of free radicals. It was obtained that inhibitory concentration of Musa balbisiana Colla seeds extract to reduce 50% of the number of free radicals was (409.91±2.35)mg/L²³. Meanwhile, the gallic acid compound used as a positive control had an IC₅₀ value of (2.411±0.004)mg/L. Antioxidant ability of Musa balbisiana Colla seeds extract was classified as a weak because antioxidants was very strong environmental if the value IC₅₀ <50 ppm, 50-100ppm (strong), 100-250ppm (middle), 250-500ppm (weak), and > 500 ppm was absent³⁷.

Anti-Uric Acid Capacity:

Uric acid is a crystalline acid which is the end product of purine metabolism, where purine is one of the components of nucleic acids found in the body's cell nucleus³⁸. Xanthine oxidase is the key enzyme which is involved in the oxidation of hypoxanthine to xanthine and then to uric acid. Allopurinol, a widely used xanthine oxidase inhibitor is the most commonly used drug for the treatment of gout³⁹. In this study, an anti-uric acid activity test was carried out of Musa balbisiana Colla seeds extract using a UV-Vis spectrophotometer at a wavelength of 513nm. Allopurinol was used to determine the anti-uric acid activity of the banana seed extract (Musa balbisiana Colla) as a positive control. The absorption value that appears on the UV-Vis spectrophotometer indicates the uric acid content in an extract. The higher the absorbance value in an extract was obtained, the smaller the anti-uric acid activity was occurred. Comparison of the anti-uric acid activity of allopurinol as a positive control with ripe stone banana seed extract (Musa balbisiana Colla) can be seen in Table 2.

Table 2.The results of the anti-uric acid activity test against ripe stone banana seed extract (Musa balbisiana Colla)

Extract type

Anti-gout capacity (%)

Allopurinol

2.80 ± 0.00

Banana seed extract

(Musa balbisiana Colla)

38.27 ± 0.00

From Table 2, it was found that the anti-uric acid activity of ripe stone banana seed extract (Musa balbisiana Colla) was higher than that of allopurinol as a positive control. The high anti-uric acid activity in the ripe stone banana seed extract (Musa balbisiana Colla) is due to the high content of antioxidant compounds in the form of phenolic compounds which can inhibit uric acid activity⁴⁰. This allows the ripe stone banana seed extract (Musa balbisiana Colla) to have the potential as an anti-gout herbal remedy.

CONCLUSION:

In this study, it was found that seeds extract of Musa balbisiana Colla contained alkaloid, phenolic, saponin, tannin and steroid glycoside compounds. The antioxidant activity test was carried out using the DPPH method and the FRAP method with the final result in the form of IC₅₀. The IC₅₀value of Musa balbisiana Colla seeds extract using the DPPH method was around (94,265 ± 0.216) mg/L which was classified as a very strong antioxidant activity. The IC₅₀ value of Musa balbisiana Colla seeds extract using the FRAP method was around (409.91±2.35)mg/L which was classified as weak antioxidant activity. The anti-uric acid activity Musa balbisiana Colla seeds extract was higher than the synthetic anti-uric acid drug allopurinol, which was around 38.27% and allopurinol was around 2.80. These results illustrate that the seeds of Musa balbisiana Colla have the potential material as an ingredient in treating diseases caused by gout.

CONFLICT OF INTEREST:

The authors declare no conflicts of interest.

ACKNOWLEDGEMENT:

This work was supported by grants from Politeknik AKA Bogor.

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Received on 03.01.2022 Modified on 08.04.2022

Research J. Pharm. and Tech 2023; 16(2):502-508.

DOI: 10.52711/0974-360X.2023.00085