Formulation and characterization of Sauropus androgynous (L) Merr leaf extract Gel in Combination with CMC-Na and Carbopol 940 Using DPPH Method

 

Ni NyomanYuliani¹, Jefrin Sambara¹_, Sandeep Poddar², Urmisha Das²

¹Department of Pharmacy, Kupang Health Polytechnic of Health Ministry, Indonesia

²Lincoln University College, Wisma Lincoln, No, 12-18, Jalan SS 6/12, 47301 Petaling Jaya, Selangor D. E., Malaysia

 

ABSTRACT:

Introduction: Katuk Leaf (Sauropus androgynus L. Merr.) from Kupang, East Nusa Tenggara, Indonesia is a type of plant that contains chalcone which is used as an antioxidant. This study aims to formulate a gel preparation from Katuk Leaf extract using various concentrations of CMC-Na and carbopol 940, to see the stability of the physical properties of the Katuk leaf extract gel Sauropus androgynus (L.) Merr. Methods: Katuk Leaf Extract was obtained by percolation method using 70% ethanol as solvent. The gels were made in formulas 1, 2, 3, 4, and 5 with a concentration of CMC-Na and carbopol 940 at 0:10; 1:9; 2:8; 9:1; 10:0 ratio. Antioxidant activity was tested with DPPH, observed for its physical stability including organoleptic, homogeneity, viscosity, pH dispersion, adhesion, and gel stability using the process of freeze thaw action. Results: The results showed that the concentration of carbopol 940 increased viscosity and adhesion and decreased dispersion and pH, while CMC-Na increased dispersion and pH and decreased viscosity and adhesion in the physical properties of the Katuk Leaf extract gel. Test results with IC₅₀ extracts of Katuk Leaves at 73.815 ppm, showed that the formula 3 with a concentration of carbopol 940 and CMC-Na at 2: 8 percent by ratio, is a gel with antioxidant activity being most effective with the value of the IC₅₀ 100.814. Conclusion:So, it can be concluded that the stability of the physical properties of the gel shows the formula 3 with concentration carbopol 940 and CMC-Na at 2%: 8% gel with the best stability of physical properties.

 

 

INTRODUCTION:

The skin is the most important part in the anatomical structure of the human body, especially the facial skin. Body care is something that is commonly done by everyone¹. The aging process in the skin occurs because the skin can no longer produce enough collagen and elastin, which function is to tighten and supple the skin². Sunlight is the main factor causing the aging process of the skin. Excessive sun exposure will produce free radicals that cause various damage to the skin structure and reduce the immune response³.

 

The negative effects of free radicals can be inhibited with antioxidants, one of which is the use of natural ingredients or traditional medicine.

 

The traditional medicine is still often used in society, especially in Indonesia for the purpose of prevention and treatment of disease. The extracts from Katuk Leaves (Sauropus androgynus L. Merr.) is one type of medicinal plant that has not been widely knownin Indonesia.But in Japan the Katuk Leaf plant is consumed as a popular vegetable.In Indonesia itself the Katuk plant is consumed as a tea drink from the dried Katuk leaves.

 

Previous research proved phytochemical properties of the leaves, stems and roots of Katuk plant. Studies showed that the Katuk plant contains chemical compounds belonging to the group alkaloids, saponins, flavonoids, triterpenoids, and glycosides that are quite strong. Especially in the leaves, there are chemical compounds belonging to the strongest tannin class which are also called polyphenols and prove the antioxidant activity of the leaf extract of Katukat IC₅₀ is 38.00 ppm⁴.

 

The gel preparation was chosen because it has the advantages of being non-sticky, easy to apply, easy to wash, does not leave oil film on the skin, and the viscosity of the gel does not change significantly during storage⁵. Gels containing antioxidants can be used as topical preparations to scavenge free radicals. Semisolid preparations are usually used on the skin and generally these preparations are used as protection from the sun's ultraviolet (UV) rays. Skin care cosmetic preparations are needed to protect the skin because the skin is very sensitive to inflammation. Cancer and premature aging are also caused by ultraviolet light which has the oxidative effect of free radicals⁶.

 

The base functions as a carrier, protector and skin softener, must be able to release the drug optimally and as much as possible be suitable for certain diseases and certain skin conditions. Selection of a suitable gelling base in a gel preparation is one of the most important things in formulating a gel preparation⁷.

 

Carbopol with low concentrations can increase the viscosity⁸. Carbopol can form a clear gel base and has low irritation with a chemical stability that can maintain formulation stability and can increase the bioavailability of the active ingredient due to its bioadhesive properties⁹. CMC-Na is also widely used as a gelling base which can increase viscosity at low concentrations. CMC-Na is non-toxic but causes hypersensitivity reactions or allergic reactions to topical drug use. CMC-Na has an excess of stability at pH 2-10 and viscosity decreases at pH >10 and maximum viscosity with stability at pH 7-9⁸. In this study, the purpose of combining carbopol 940 and CMC-Na was done, so that the gel preparation had good viscosity and formed a clear gel consistency. The addition of carbopol base is expected to improve the deficiency of CMC-Na to produce a more optimal gel preparation.

 

MATERIAL AND METHODS:

Population and Sample:

The sample used in this study was a leaf extract gel of Katuk leaf (Sauropus androgynus L. Merr.) with a combination of carbopol 940 and CMC-Na (0% : 4%), (1% : 3%), (2 % : 2%), (3% : 1%), (4% : 0%).

 

Research variables:

The independent variables in this study were variations in the concentration of carbopol 940 and CMC-Na in the formulation of the antioxidant gel preparation of Katuk Leaf Extract. The controlled variables in this study were the gelling process, laboratory conditions and equipment used, 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radicals, and the conditions of the researchers in the study.The confounding variables in this study were the physical stability of the gel (organoleptic, viscosity, adhesiveness, spread ability, homogeneity, and pH), and the antioxidant activity of the gel extract of Katuk Leaf (Sauropus androgynus L. Merr.)

 

Work procedures:

1.     Katuk Leaf Plant Determination:

The correct sample of Leaf Katuk ( Sauropus androgynus L. Merr.) was selected by matching the characteristics and morphology of the plant to be studied to avoid errors. This process was done at the Pharmacognosy Laboratory of the Health Poltekkes, Kupang Ministry of Health. The young, fresh, and undamaged samples were obtained from Baumata Timur, Kupang Regency.

 

2.     Powder making:

The leaves were washed underrunning water to remove impurities attached to the leaves. After that they were dried in an oven at 45°C for 2-3 days. The dried leaf was powdered with a blender and then sieved using a number 40 Mesh until the powder was used up.

 

3.     Determination of moisture content:

The moisture content of Katuk Leaf powder was determined using atoolmoisture balance. The moisture content value appears on the device in percent.

 

4.     Making the Leaf Extract:

500 g of powder was put into a maceration bottle with 3750 mL of 70% ethanol, soaked for 5 days and then shaken 3 times a day. The maceration results were filtered using a sterile flannel cloth. The dark bottle was rinsed with 1250 mL 70% ethanol to wash the remaining extract left in the dark bottle. After that the pulp is separated from the filtrate. Then the extract wasconcentrated with a vacuum rotary evaporator at a temperature of 50°C to obtain a thick extract.

 

5.     Organoleptic determination:

The organoleptic determination of the Katuk Leaf extract was carried out by observing the shape, color, and odor of the 70% ethanol extract.

 

6.     Alcohol-free test of Katuk Leaf extract:

The ethanol-free examination of the concentrated extract of Katuk Leaf aims to ensure that the concentrated extract is free from ethanol by esterification reaction.

 

7.     Identification of the chemical content:

The flavonoids, polyphenols content in Katuk Leaf extract was determined.

 

8.     Gel preparations:

CMC-Na was dissolved in sufficient hot aquadest in a glass beaker andallowed to form a clear mass. Carbopol 940 was dispersed in 10 mL of hot aquadest in another glass beaker, stirred until homogeneous. Methyl paraben dissolved with distilled water in a mortar and added glycerin and stirred until homogeneous, then mixed with the base that has been developed, stirred rapidly until homogeneous. Then Triethanolamine was added while stirring continuously, finally the Katuk Leaf extract was added to the gel preparation and stirred until a homogeneous gel was obtained.

 

9.     Physical stability test:

Organoleptic homogeneity test and viscosity test was done to check the physical stability of the antioxidant gel. The spread ability, gel adhesion and pH test were also done.

 

10. Testing of the antioxidant activity of Katuk Leaf extract gel:

Free radical scavenging activity test. Stock solution with five series of dilutions were made. From each solution 4 mL was taken, then 1 mL of 0.4 mM DPPH solution was added. The mixture was incubated for the operating time and the absorbance was read at the maximum wavelength of DPPH.

 

Analysis Techniques:

Research data was obtained in the form of organoleptic, homogeneity, viscosity, pH examination, adhesion, and spread ability. The data were analyzed using One Sample Kolmogorov Smirnov and One-Way ANOVA with SPSS17 program.

 

RESULTS:

Identification of the chemical content of Katuk Leaf extract:

This study aims to determine the chemical content in Katuk Leaf extract. The results of the chemical identification of Katuk Leaf extract can be seen in      table 1.

Table 1: Test Results for the identification of chemical constituents Katuk Leaf extract

Sl. No.	Chemical Content	Procedure	Results	Library	Comments
1	Flavonoid	Extract filtrate + Mg powder + ethanol solution: HCL (1:1) + amyl alcohol shaken vigorously	Color reddish orange on the amyl alcohol	Color formation red/yellow/orange an amyl layer alcohol	+
2	Polyphenols	Extract filtrate + solution Iron(III) chloride 1%	Color black  snippet	Color formation green/red/purple black snippet	+

 

Table 2: Test results for the physical properties of the gel

 

 

Figure 1: Gel viscosity test of Katuk Leaf Extract

 

Identification of the physical properties of the gel:

The physical properties test of the gel was carried out by organoleptic observation, gel homogeneity test, viscosity test, dispersion test, adhesion test, pH test, and gel stability test. Organoleptic examination was carried out to describe the color, odor, and consistency of the gel preparation.

 

Spread ability test:

The dispersion test is intended to determinethe distribution area of the gel preparation. A good gel is a gel that has the widest spread ability, easy to wash, and well absorbed by the skin, so that the contact between the active substance and the skin is better. The results of the dispersion measurement show that the dispersion is inversely proportional to the viscosity, the greater the viscosity, the smaller the dispersion and conversely a high viscosity will be difficult to flow because it has a large cohesive force between the base molecules, causing the gel to be difficult to spread.

 

The data in table 2 shows that the increasing concentration of carbopol causes the bonds between compounds to be stronger, also the gel is stronger, and the dispersion power is getting smaller. The increasing concentration of CMC-Na causes the bonds between compounds to be weaker, the gel is weaker, and the dispersion is wider. The results of the dispersion test can be seen in Figure 1.

 

Adhesion test:

The adhesion test was carried out to determinethe ability of a gel to adhere to the site of application. The greater the adhesive power of the gel,the longer it will remain attached to the skin so that it is better and more effective in delivering active substances into the skin.From the data in table 2it is seen that greater viscosity, the greater the adhesion, conversely the smaller the viscosity, the smaller the adhesion of a gel preparation.  The results of the adhesion test can be seen in Figure 2.

 

Figure 2: Adhesive Strength of Katuk Leaf Gel Extract

 

pH test:

The pH test was carried out to determine the pH of leaf extract gel in accordance with the pH of the skin. The pH test data above shows that formula 1 has the highest pH, while formula 5 shows the lowest pH, this low pH is due to the acidic nature of carbopol 940. In this study, the desired pH in the preparation is a neutral pH, a pH value that is close to 7, namely in formula 2, formula 3, and formula 4.The results of the pH test can be seen in Figure 3.

 

Table 3: Physical stability test results of Katuk Leaf extract gel

 

 

Figure 3: pH Test Results of Katuk Leaf Extract Gel

 

Physical stability:

The stability test of the physical properties of the gel was carried out by observing organoleptic test, gel homogeneity test, viscosity test, dispersion test, adhesion test, pH test, and gel stability test, aimed to determine whether there was a change in the storage period for 21 days. The results of the physical stability test of the gel can be seen in table 3.

 

Testing of antioxidant activity using the DPPH method:

The result of determining the maximum wavelength (λ max):

Determination of maximum wavelengththat reacted with the test solution (routine solution, extract, formula 1, formula 2, formula 3, formula 4, formula 5). The results of the measurement of the maximum wavelength in all test solutions obtained the maximum wavelength of DPPH which is 515 nm. The maximum wavelength measurement results can be seen in Figure 4.

 

 

Figure4: Maximum wavelength measurement results

 

Free radical scavenging activity test:

Katuk Leaf Extract Gel is expected to have an effect as an antioxidant. The IC₅₀ describes the strength of free radical scavenging which is then correlated as the concentration of the test solution capable of reducing 50% of the DPPH free radical solution. The smaller the ICvalue₅₀, the better the antioxidant activity. The results of the antioxidant activity test can be seen in table 4.

 

The results of testing the antioxidant activity of the Katuk Leaf extract showed that the Katuk Leaf extract had an ICvalue₅₀ of 16,794 ppm, meaning that the Katuk Leaf extract had a very active antioxidant activity because it had an ICvalue₅₀of less than 50.

Table 4: Antioxidant activity of Katuk Leaf Extract Gel preparations

 

The test results demonstrate that the antioxidant activity RutinhasIC₅₀was the smallest with a value of 4.592 ppm, it indicates that katuk leaf extract has antioxidant activity four times weaker than the Rutin as a comparison. Rutin has the greatest antioxidant activity because rutin is a pure compound that has groups that have strong potential to scavenge free radicals. The mechanism of action of antioxidant compounds in reducing radical compounds is one of them by donating electrons to these unstable compounds, so that they can change unstable free radicals into more stable compounds.

 

DISCUSSION:

The preparation of the Katuk Leaf extract gel was also tested for antioxidant activity to determine the antioxidant activity of each formula. The antioxidant activity test results showed IC₅₀ formula 1, formula 2 formula 3, formula 4, and formula 5 were 86,497 ppm, 82,604 ppm, 83,368 ppm, 87,096 ppm, 83,368 ppm.

 

The smaller the ICvalue₅₀, the better the antioxidant activity in capturing free radicals. Antioxidant test showed IC value₅₀was smallest in formula 2 with a concentration of carbopol base 940 and CMC-Na (1%: 3%) that is equal to 82.604 ppm. The antioxidant activity of the extract decreased after the gel preparation was made, the decrease in antioxidant activity of the extract could be caused by the gel base used, and in this case the bases used were carbopol 940 and CMC-Na¹⁰. It can be seen that the IC value₅₀ rutinis 4.592 ppm, while the ICvalue₅₀ rutin gel 29.174 ppm, this means that the base carbopol 940 and CMC-Na are not good at carrying substances containing antioxidants because they actually increase the ICvalue₅₀. In addition, the decrease in antioxidant activity can be caused because antioxidant compounds are unstable under the influence of light and heat¹¹, the gel can be exposed during the process decreasing the antioxidant property.

 

Firstly, variations in the concentration of carbopol 940 base will increase viscosity and adhesion and reduce dispersion and pH, while CMC-Na increases dispersion and pH and decreases viscosity and adhesion in the physical properties of the Katuk Leaf extract gel. Secondly, the antioxidant gel preparation of Katuk Leaf Extract with various concentrations of carbopol base 940 and CMC-Na show that formula 2 (1%: 3%) has the best activity against antioxidants by DPPH.  Thirdly, the antioxidant gel preparation of Katuk Leaf Extract with various concentrations of carbopol base 940 and CMC-Na show that formula 2 (1%: 3%) has good physical stability.

 

From long back people use so many plants for their medicinal values.^{12,13,14,15,16} The medicinal plant and their extract have been use for herbal or traditional drug from ancient days.^{17,18,19,20,21,22,23,24,25,26} Katuk is also widely popular for its medicinal value.²⁷ Further research needs to be done to optimize the formula under study in order to obtain cream preparations with the most stable physical properties. Moreover, it is necessary to do research on antioxidant gel extract of Katuk Leaf (Sauropus androgynus L. Merr.) using a method other than DPPH to find out the antioxidant potential against other types of radicals.

 

ACKNOWLEDGMENT:

The authors are thankful to the management of Kupang Health Polytechnic of Health Ministry, Indonesia for giving necessary support and permission to carry over the work and Lincoln University College, Malaysia for academic collaboration.

 

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Accepted on 10.02.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(11):5211-5216.