Immunostimulating Study of Active Agent Fraction from Sungkai (Peronema canescens Jack.) Leaf from SARS-COV-2 Virus Antigen Exposure to NK and CD8+T Cells

The immune system or immune system is the body's defense mechanism that functions to respond to or respond to "attacks" from outside the body. When an attack occurs, the antigen will begin to stimulate the immune system. This mechanism will protect the body from attacks by various microorganisms such as bacteria, viruses, fungi, and various germs that cause disease. When the immune system does not work optimally, the body will be vulnerable to disease. Some things that affect the immune system for example food, environmental factors, daily lifestyle, age, stress, and hormones. Therefore, everyone is advised to maintain a healthy and good lifestyle with a healthy way of life, namely consuming healthy foods with balanced nutrition and regular exercise.1


INTRODUCTION
The immune system or immune system is the body's defense mechanism that functions to respond to or respond to "attacks" from outside the body. When an attack occurs, the antigen will begin to stimulate the immune system. This mechanism will protect the body from attacks by various microorganisms such as bacteria, viruses, fungi, and various germs that cause disease. When the immune system does not work optimally, the body will be vulnerable to disease. Some things that affect the immune system for example food, environmental factors, daily lifestyle, age, stress, and hormones. Therefore, everyone is advised to maintain a healthy and good lifestyle with a healthy way of life, namely consuming healthy foods with balanced nutrition and regular exercise. 1 An immunostimulant can increase body defense mechanism. Generally, an immunostimulant is defined as a compound that can increase body defense mechanism specifcally and nonspecifically through cellular or humoral response. Certain plants contains compound that show immunostimulatory activity. 2 The components of the body's immune system that are very important in protecting the body against the Covid-19 virus attack are Natural Killer (NK) and CD8+ T cells. These two cells protect the body against Covid-19 virus infection by producing a group of cytokines in the form of interferon, granzyme, and perforin compounds as well as pro-inflammatory cytokines known as cytokine storms.
The immune system can be divided into two types; the non-specific immune system (innate immunity) and the specific immune system (adaptive immunity). The non-specific immune system has a faster activity because it does not involve the memory cells. 3 One of components that are involved in the non-specific immune system is natural killer cells which protect the body from pathogen attacks so that the body eventually builds its defense system. The body's defense system can be activated by providing compounds that can increase the body's imune response. 4 Body protection against viral exposure is through the activity of NK cells, where NK cells will release interferon, perforin, and granzyme compounds. Interferon play a role in inhibiting the proliferation and differentiation of viruses, besides that they can also inhibit the attachment of the virus to receptors in normal cells, so that normal cells are not infected, perforin compounds play a role in disrupting permeability or leakage from the cytoplasmic membrane of cells infected with the virus so that the cells die. Granzyme compounds function in the process of apoptosis for cells experiencing viral infection so that the cells die. 2 CD8+T cells are a type of white blood cell that help regulate and carry out the body's immune response. CD8+T cells include suppressor T cells and cytotoxic T lymphocytes or T-8 cells (CD8+). In the regulation of cytokines in the immune response, CD8+ cells kill infected target cells by releasing lytic granules (perforin and granzyme) or by induction of production (FasL) or TNF-alpha, which by binding to their receptors initiates cell apoptosis. target. Some of the plants used by the community to increase endurance used by the community are Sungkai (Peronema canescens Jack.). All these plants are abundant in West Sumatra. This medicinal plant has properties as an immunomodulatory agent or immune system enhancer. In the study 5 that the dose of Peronema canesens Jack. for the antibacterial species of the protozoan parasite Babesia gibsoni, the dose was 0.7 g/kg BW and did not cause toxicity in the mice tested and in the study, 6 Peronema canesens Jack. a dose of 0.5625 mg/kg BW can increase the number of leukocytes by 36%. Ningsih's research results in 2013 show that the ethanol extract from the leaves of Peronema canescens Jack. has antiplasmodial activity in vivo with its ED50 value of 102.88 mg/kg BW. Several research findings of Sungkai plants have secondary metabolites in their leaves. The method of obtaining these compounds is by extracting Sungkai leaves using a solvent. The findings of secondary metabolites contained in the form of flavonoid, phenolic, and terpenoid compounds. The scientific process is very important so that the use of traditional medicine is not based on experience alone but has scientific evidence so that it can be used in a modern formal health care system. 6 Based on the description above, researchers are interested in researching the activity of standardized extracts from the leaves of the Sungkai Peronema canesens Jack. protection against SARS-CoV-2 virus infection by observing the activity of NK and CD8+ T cells.
Based on the description above, researchers are interested in researching the activity of standardized extracts from the leaves of the Sungkai Peronema canesens Jack. with several variations of the solvent fraction against the protection of SARS-CoV-2 virus infection by observing cell activity in the form of NK cells and CD8+T cell activity.

Time and place research
The research was conducted from June 2021 to June 2022 at the Cell Culture Laboratory, Pharmacology Laboratory, Immunology, and

Sungkai leaf extraction and fractionation
Sungkai leaf powder (Peronema canesens Jack.) was extracted by multistage maceration method, to obtain non-polar, semi-polar, and polar fractions. Maceration was started by using a non-polar solvent n-hexane with a ratio of 1:10, maceration was carried out according to the standard method in the pharmacopeia. After the maceration solvent obtained was clear, the maceration was continued by using semi-polar ethyl acetate solvent in a ratio of 1:10, after the maceration solvent with ethyl acetate, was obtained clear, the maceration was continued by using butanol solvent in a ratio of 1:10. So that obtained fractions of n-hexane, ethyl acetate, butanol, and residual water. Then the solvent for each fraction was evaporated using a rotary evaporator to obtain a thick fraction. 7,8 Preparation of experimental animal The animals used were 60 male white mice aged 2-3 months with a bodyweight of 20-30 grams and had never been treated with drugs. Before being used as experimental animals, mice were adapted for 7 days to adjust to the environment and control health and body weight as well as uniform food. These experimental animals were grouped into 12 groups consisting of 5 mice in one group for 1 extract in 4 fractions and 3 dose variations. Mice were given the Sungkai leaf extract fraction for 14 days and on day 0 were given the Moderna vaccine blood was taken and serum was taken which was then checked for protein concentrations of NK and CD8+ T cells using the ELISA technique. The principle of the sandwich ELISA method is the occurrence of a complex bond between the antigen and 2 antibodies, the first antibody (capture antibody) is coated into the well on the microplate and the second antibody is conjugated with an enzyme (detection antibody).
The addition of the substrate will cause a color change which indicates the presence of antigen in the sample. Absorption measurements were carried out tosdetermine the amount of antigen in the sample. 9

Dosage determination
The dose of Sungkai leaf extract used in this study was given in 3 dosage variants, namely 1, 10, and 100 mg/k bw. 10

NK and CD8+T Cell Count
On the eighth day, blood was drawn by guillotine (neck artery). Then the blood was collected and then centrifuged for 30 minutes at 3000 rpm to obtain serum. Then the serum was used to test the levels of NK cells and CD8+T cells by the ELISA method. 11

Data analysis
The data obtained from the research results were analyzed statistically using the one-way Analysis of Variance (ANOVA) method and continued with Duncan's analysis using SPSS statistical software. 12 The process of obtaining Sungkai leaf extract (Peronema canescens Jack.) begins with collecting fresh Sungkai leaves, cleaning them from impurities, washed with running water, and then air-dried to become dry Simplicia, then after the dried Simplicia is mashed using a blender and obtained Simplicia powder, maceration sample is carried out by   8 From 2400 grams of Simplicia powder, 387.5 grams of thick extract were obtained and the yield value of the extract was 16.14%. Sungkai leaf powder (Peronema canesens Jack) was extracted by the graded maceration method, to obtain non-polar, semipolar, and polar fractions. Maceration was started by using a non-polar solvent n-hexane with a ratio of 1:10 where it was carried out according to the standard method in the pharmacopeia. After the solvent obtained from maceration was clear, the maceration was continued using semi-polar solvent ethyl acetate with a ratio of 1:10. After the solvent obtained from maceration with ethyl acetate was clear, the maceration was continued by using butanol solvent in a ratio of 1:10 to obtain the n-hexane fraction, ethyl acetate, butanol, and residual water. The solvent for each fraction was evaporated using a rotary evaporator to obtain a viscous fraction. Then, standardization of Sungkai leaf (Peronema canescens Jack.) extract drying shrinkage obtained was 8.91% which complies with the provisions of the Indonesian Herbal Pharmacopoeia where the drying shrinkage of the Gotu Kola herb extract should be not more than 10%. The determination results of the total ash content of Sungkai leaf (Peronema canescens Jack.) extract is 3.66% which also meets the provisions of the Indonesian Herbal Pharmacopeia where the total ash content of the thick extract of Sungkai leaf should be not more than 10%.

RESULT
The organoleptic examination was aimed at a simple initial identification of the Sungkai leaf extract used, namely in the form of a viscous fraction having a characteristic odor, blackish-brown color, and bitter taste. The chemical content test of the extract included a phytochemical test, which showed that the condensed fraction of Sungkai leaves was positive for flavonoids, phenolics, saponins, and terpenoids.
On the Thin Layer Chromatography of the extract, the eluent or mobile phase used was Ethyl acetate: methanol (2:3), and using a silica gel plate F254 as the stationary phase, the comparison used was quercetin and obtained an Rf value of 0.54 cm.
Based on the results of the test using the ELISA method, the increase in NK cells was found in group B1 which was 5,437 ng/ml, namely the butanol fraction at a dose of 1 mg/kgbw with treatment from days 1-7 given the Sungkai leaf extract fraction. Based on the one-way ANOVA statistical test, showed that there were significant differences between the 12 treatment groups (P-value < 0.05). The results of the calculation of the average protein concentration of NK cells in male white mice given several fractions of Sungkai leaf extract and exposed to Sars-Cov-2 virus antigen sequentially from group 1 to group 12 14 NK cells are components of innate immunity that act as killers (cytotoxicity) by secreting lysosomes containing perforins and granzymes and also producing cytokines IFN-γ, TNF-α, IL-5, IL-13. 15 NK cells also function as co-stimulatory that can stimulate macrophages, T cells, and B cells, thus bridging the interaction between innate immunity and adaptive immunity. 16 The results of the protein concentration calculation of male white mouse CD8 + T cells after administration of the highest Sungkai leaf extract fraction was in group A3, which was 29,193 ng/ml in the water fraction with a dose of 100 mg with the extract from days 1-7. Based on the one-way ANOVA statistical test, showed that there were significant differences between the 12 treatment groups (P-value <0.05). The results of the calculation of the average concentration of male white mouse CD8+T cells given several fractions of Sungkai leaf extract and exposed to Sars-Cov-2 virus antigen sequentially from group 1 to group 12 18 An effective viral response from the host is carried out by the innate and adaptive immune systems through the production of various proinflammatory cytokines, and activation of T cells, CD 4, and CD8+T cells. T cells are important for controlling viral replication, limiting virus spread, and clearing infected cells. However, tissue caused by viruses can lead to overproduction of proinflammatory cytokines, and recruitment of proinflammatory macrophages and granulocytes. This situation is known as a cytokine storm which can lead to more severe tissue damage. 19

SUMMARY
Sungkai leaf plant had been used as an immune system enhancer. In this study, the effect of Sungkai leaf extracts from 4 different fractions, namely n-hexane, ethyl acetate, butanol, and residual water, with 3 variations in doses of 1, 10, and 100 mg/k bw on the activity of NK and CD8+T cells in male white mice that have been exposed to SARS-Cov-2 virus antigen was investigated. The experimental animals used were 60 animals divided into 12 groups with 14 days of treatment which had previously been induced with SARS-Cov-2 virus antigen (moderna) and given with Sungkai leaf extracts for 14 days and evaluated on day 8. The results showed that there was an increase in the concentration of NK cells in the butanol fraction at a dose of 1 mg/kg BW and CD8+T cells in the aqueous fraction at a dose of 100 mg/kg BW. Dillasamola D, et al.: Immunostimulating Study of Active Agent Fraction from Sungkai (Peronema canescens Jack.) Leaf from SARS-COV-2 Virus Antigen Exposure to NK and CD8+T Cells GRAPHICAL ABSTRACT