INTRODUCTION:

Indonesia is a country with a high level of biodiversity¹. As many as 31,750 types of plants were discovered in Indonesia, and 25,000 were flowering plants. Also, around 15,000 plant species have potential medicinal properties, but only around 7,000 species are used as medicinal raw materials^2,3. The Indonesian community utilised this high level of biodiversity, including the Moringa oleifera Lam. It is called the Miracle Plant because of its natural richness in nutritional compounds and medicinal properties not found in most other plants^4,5. The Moringa oleifera Lam. leaves can be used as a traditional medicine⁶. They are an antitumor, lower blood pressure, antioxidant, antimicrobial, antifungal, antihypertensive, reduce inflammation, and have diuretic properties^{7,8,9,10,11,12}.

Based on phytochemical analysis, Moringa oleifera Lam. leaves contain antioxidant compounds⁸. Previous research stated that the ethanol extract of Moringa oleifera Lam. leaves, based on the results of phytochemical testing, contains secondary metabolite compounds of alkaloids, flavonoids, phenolics, terpenoids and steroids but does not contain saponin compounds^8,13,114. Apart from having antioxidant properties, the compounds found in Moringa oleifera Lam leaves also act as immune system regulators or immunomodulators¹⁵. The immunomodulatory properties in Moringa oleifera Lam. leaves show the same role as natural killer (NK) cells in white blood cells¹⁶.

The human body has a defence system called the immune system. This system fights foreign objects or pathogens (bacteria, viruses and other dangerous microorganisms) that can enter the body. If a pathogen manages to enter the body, the immune system will detect it as a threat. This is responded to by coordinating cells and compounds in the body to fight the pathogen¹⁷. One of the cells that play a role in the body's defence system is NK cells¹⁸.

NK cells are innate leukocytes vital in the body's defence against viral infections^19,20. When an infection caused by a pathogen occurs, NK cells will play an essential role in limiting the body's immune response. NK cells will destroy activated immune cells to avoid excessive damage to the body. Thus, NK cells act as immune regulators that help maintain balance and prevent excessive immune reactions^21,22.

Previous studies reported that entering the virus into the body causes the body's defence system to become active²³. If the pathogen successfully passes the opsonisation process in the nose and gastrointestinal tract's mucosal lining, NK cells will protect the body. The occurrence of contact between NK cells and pathogens will produce three types of cytokines, such as granzyme, porphyrin, and interferon, to prevent viruses from entering new cells²³. The immunomodulatory properties and antiviral activity of NK cells can be weakened when the body is infected with a virus, one of which is SARS-CoV-2, which causes NK cells to become exhausted, resulting in excessive activation of the immune system. This is the leading cause of pathogenesis in COVID-19 patients²².

COVID-19 is a disease caused by the coronavirus (SARS-CoV-2)²⁴. This virus first appeared in Wuhan, China, at the end of 2019 and became a worldwide pandemic^25,26,27. This virus has a higher transmission ability to spread quickly^21,26. However, in May 2023, the COVID-19 pandemic was declared to have ended²⁸. However, there are still active cases of COVID-19. Based on WHO data, in May 2023, there were 38,150 confirmed cases of COVID-19, with a total of 497 deaths²⁹. This is because the COVID-19 virus reduces the number of NK cells circulating in the body and increases the expression of cytolytic proteins such as perforin and granzyme²¹. Research from Hammer et al.indicated that NK cells are essential in responding to COVID-19 infection and can respond to infection by the SARS-CoV-2 virus³⁰.

The use of Moringa oleifera Lam. leaf extract has an impact on the immune system³¹. Previous research explains that administering extract ethanol from Moringa oleifera Lam. leaves can increase total leukocytes. It is proven by the percentage of white blood cells, including 2-3% eosinophil cells, 4.2-6.4% neutrophil cells, 38.8-47% lymphocyte cells, and 3-8.8% monocyte cells³².

Another research stated that Moringa oleifera Lam. leaves significantly improved the body's immune system during the COVID-19 pandemic³³. Before that, another study has proved that Moringa oleifera Lam. leaf extract provides immunostimulant and immunosuppressant effects, which can increase the number of CD4+ cells and CD 8+ T cells at doses of 14 mg/kg BW, 42 mg/kg BW, and 84 mg/kg BW³¹. Moreover, an explanation from another research showed that giving Moringa oleifera Lam. leaf extract can increase the number of leukocyte cells with the largest total average count of 12,017 /µL of blood. An increase in leukocyte cells indicates the body's natural (non-specific) defence system, in which NK cells also play a role in the non-specific immune system³⁴. However, no research has examined the effect of Moringa oleifera Lam. leaf extract on the activity of NK cells exposed to COVID-19 virus antigens. Therefore, it is necessary to conduct a study to test the effect of Moringa oleifera Lam. leaf extract on NK cells exposed to COVID-19 virus antigens.

MATERIALS AND METHOD:

Material:

The materials used are Moringa oleifera Lam. leaves, ethanol distillate 70% (Andalas Chemical), Ethanol 70% (Andeska Laboratory), ethanol P (Supelco), 0.5% Na-CMC (PT. Dwilab Mandiri Scientific), Aqua Pro Injection, COVID-19 vaccine (Indovac), Giemsa, immersion oil, distilled water, filter paper, aluminium foil, Silica gel F254 TLC plate, standard mouse food, Turk solution, aluminium chloride (Muda Berkah), sodium acetate (Emsure), ash-free filter paper, hydrochloric acid LP (Medical and Laboratory Supplies), quercetin (Sigma), Mouse Natural Killer Cell ELISA Kit (BT Lab).

Tools:

The tools used are measuring cups (Herma®), analytical scales (Ohaus®), animal scales (Ohaus®), animal metabolite cages, sondes, syringes (One Med®), dropper pipettes, spatula, beaker glass (Pyrex®), animal feeding and drinking containers, serum racks and tubes, stamper mortar, rotary evaporator (Buchi®), blender, funnel (Pyrex®), volumetric flask (Pyrex®), vial, watch glass, Erlenmeyer (Pyrex®), volume pipette ( Pyrex®), rack and test tubes, centrifuge (Rotovix 32®), UV-Vis spectrophotometer (Thermo Scientific Genesys 10S UV-Vis®), incubator, oven, furnace, ELISA reader (Bio-Rad®), glass slide, cover glass, and light microscope, Hot Plate, Evaporating Cup.

Preparation of Extraction:

A total of 5 kg of fresh Moringa oleifera Lam. leaves samples were sorted and cleaned from impurities, then air-dried to obtain dry samples, provided the samples could be crushed. Furthermore, powder making is carried out with a grinder without causing damage or losing the required chemical content until a fine powder of Moringa oleifera Lam. was obtained³⁵. The powder was macerated using 70% ethanol solvent (1:10) for 24 hours with 3 repetitions. The powder was soaked for the first 6 hours while occasionally stirred and left undisturbed for 18 hours. All the macerates were collected and evaporated using a rotary evaporator to obtain a concentrated extract.

Characterisation of Moringa oleifera Lam. Extract:

Organoleptic:

Organoleptic parameters for describing the thick extract of Moringa oleifera Lam. using the five senses, namely describing the shape, colour, smell and taste in the form of thick extract, brownish green colour, distinctive odour and bitter taste. The organoleptic test observed the characteristic odour of a thick extract of Moringa oleifera Lam. It was carried out after the sample was exposed to air for 15 minutes. The 15 minutes start counting after the container containing no more than 25 g of material is opened. This is done for containers containing more than 25 g of material after approximately 25 g has been transferred into a 100 mL evaporating cup. The odour mentioned is only descriptive and is not considered a purity standard for the thick ethanol extract of Moringa oleifera Lam³⁵.

Determination of moisture content (gravimetric method):

Weigh approximately 10 g of thick Moringa oleifera Lam. extract in a tarnished container. Samples were dried at 105°C for 5 hours and weighed. Then, continue weighing at 1-hour intervals until the yield between two consecutive weighings was less than 0.25%. The water content requirement for the thick Moringa oleifera Lam. extract is 10.0%³⁵.

(W1 – W0) – (W2 – W0)

Moisture content = -------------------------------- x 100%

W1 – W0

Determination of total ash content:

Weigh 2 to 3 g of the extract carefully, then put it into a silicate crucible that has been ignited and tared. After that, the sample is slowly heated until the charcoal runs out, then cooled and weighed. If the charcoal cannot be removed using this process, add hot water, stir, filter through ash-free filter paper, then evaporate and incandescent until the weight remains at 800 ± 25o C in the same crucible. Calculate the ash content of the material that has been dried in the air with the following equation, and the requirement for the total ash content of the thick extract of Moringa oleifera Lam. is not more than 9.0%³⁵ :

W2 – W0

Total ash content = --------------

W1 – W0

Determination of acid-insoluble ash content:

Boil the ash obtained to determine total ash content with 25 mL of dilute hydrochloric acid LP for 5 minutes. The acid-insoluble part is collected, filtered using ash-free filter paper, washed with hot water, and then ignited in a crucible until the weight remains at a temperature of 800 ± 25o C. Calculate the acid-insoluble ash content of the air-dried material with the following equation the following and the requirements for the acid insoluble ash content of the thick extract of Moringa oleifera Lam. which is not more than 0.9%³⁵.

W2 - W0

Acid insoluble ash content = --------------- x 100%

W1 - W0

Preparation of Animals:

The experimental animals used were 25 male white mice aged 8-12 weeks with a body weight of 20-35 g. They had never been used for experiments and showed normal behaviour. Before the research, mice were acclimatised for 7 days to adjust to the environment, control health and body weight, and were given enough drink and food³⁶.

Preparation of test preparations:

Dose planning:

The dose of a thick Moringa oleifera Lam extract given to white male mice was 10 mg/kg BW, 30 mg/kg BW, and 100 mg/kg BW. The dose given came from testing the immunostimulant activity of Moringa oleifera Lam. leaves at doses of 10 mg/kg BW, 30 mg/kg BW, and 100 mg/kg BW in male white mice³⁶. There are similarities in the doses used because the researchers wanted to see the immunomodulatory activity of Moringa oleifera Lam. leaves on NK cells in male white mice exposed to the COVID-19 virus antigen.

Preparation of 0,5% Na CMC suspension:

Weigh 500 mg of Na CMC and sprinkle it on 10 mL of hot water in a heated mortar. Leave it for 15 minutes and then grind until homogeneous. Next, add aquadest until the volume is 50 mL.

Preparation of suspension of Moringa oleifera Lam. leaf ethanol extract:

Suspend 1 g of extract into 50 mL of 0,5% Na CMC. Then, dilute the test suspension according to the concentrations of each designed dose: 10 mg, 30 mg, and 100 mg. The volume of the test formulation injection should be adjusted based on the body weight of the test animals.

Administration of Test Preparations

Group I	:	Only 0.5% Na CMC is given for 7 days.
Group II	:	Given 0.5% Na CMC for 7 days and 0.13 ml / g BW vaccine on the first day and 7^th day
Group III	:	Given the extract at a dose of 10 mg/kg BW for 7 days and 0.13 ml / g BW vaccine on the first day and 7^th day
Group IV	:	Given the extract at a dose of 30 mg/kg BW for 7 days and 0.13 ml / g BW vaccine on the first day and 7^th day
Group V	:	Given the extract at a dose of 100 mg/kg BW for 7 days and 0.13 ml / g BW vaccine on the first day and 7^th day

Examination of NK cell activity using ELISA:

Blood samples were taken by guillotine (neck artery). Blood was collected and centrifuged for 30 minutes at 3000 rpm to obtain serum. Then, the serum obtained was used to test NK cell activity using the ELISA method 40 times µL³⁷.

Calculating the Total Number of Leukocyte Cells:

The total number of leukocyte cells was calculated after treatment, and the mice were sacrificed, namely by sucking fresh blood using a micropipette and a microtip of 0.5 µL, then Turk solution put into a microtip using a micropipette of 10 µL. Then, the Turk solution and sample were homogenised for 3 minutes, and 1-2 drops were dropped from the micropipette in the haemocytometer drip chamber. In the final step, the liquid is left for 2 minutes until the leukocytes settle and the white blood cells are counted in the four counting chambers³⁶.

Calculating the Percentage of Total Leukocyte Count:

The percentage of the total leukocyte count was calculated by placing one drop of fresh mouse blood on the centre line of the slide, approximately 2 cm from the tip. Then, place another slide to the left of the droplet and move it to the right until a thin smear forms. After the smear preparation is dry, it is continued with fixation using methanol for 3-5 minutes and allowed to dry in air. Next, the preparation was dipped with Giemsa solution, diluted with distilled water (1:20) and left for 20 minutes. Then, the preparations were washed with running water and allowed to dry on a rack. After drying, the preparations were examined under a microscope with 1000x magnification³⁸.

Data Analysis:

The results obtained from this research will be analysed statistically using the one-way Analysis of Variance (ANOVA) method. After that, the significant data was analysed with the Duncan Multiple Range Test (DMRT). It was analysed using the IBM SPSS 25 version.

RESULTS AND DISCUSSION:

Extraction Process and Yield of Purified Extract:

Making Moringa oleifera Lam. leaf extract begins with collecting a fresh sample. The sample used was 5 kg, then 600 g of Moringa oleifera Lam. leaf simplicia powder was obtained, and 153.22 g of Moringa oleifera Lam. leaf thick extract was obtained with a yield of 25.53%. The yield of the thick extract meets the Indonesian Herbal Pharmacopoeia requirements, which stipulates that the yield of thick extract of Moringa oleifera Lam. leaves is not less than 9.2%, so further research can be carried out.

Table 1. Moringa oleifera Lam. leaf extract yield

Sample weight (g)	Extract weight (g)	Yield (%)
600	153.22	25.53%

Organoleptic Test of Purified Extract:

The organoleptic test results showed that the ethanol extract of Moringa oleifera Lam leaves was thick, brownish green in colour, had a distinctive smell, and had a bitter taste. These results comply with the organoleptic standards for Moringa oleifera Lam. leaf extract in the Indonesian Herbal Pharmacopoeia³⁵.

Examination of moisture content, total ash content and acid-insoluble ash content:

The examination of moisture content in the ethanol extract of Moringa oleifera Lam. leaves resulted in 9.73%. This moisture content value meets the requirements specified in the Indonesian Herbal Pharmacopoeia, which should not exceed 10%³⁵. The examination of total ash content yielded 1.38%. The ash content value meets the requirements in the Indonesian Herbal Pharmacopoeia, which is no more than 9%³⁵. Meanwhile, the examination of acid-insoluble ash content yielded 0.63%. This acid-insoluble ash content value complies with the requirements in the Indonesian Herbal Pharmacopoeia, which should not exceed 0.9%³⁶.

Administration of test preparations:

Twenty-five male white mice were grouped into 5 groups, each group consisting of 5 mice. The first group was the group that was only given 0.5% Na-CMC suspension orally for 7 days. The second group, namely the group that was given 0.5% Na-CMC suspension orally for 7 days and was given the COVID-19 vaccine intramuscularly at 0.13 mL/20 g on day 1^st and day 7^th. The third, fourth, and fifth groups, namely the groups that were given Moringa oleifera Lam. leaf ethanol extract sequentially at doses of 10 mg/kg BW, 30 mg/kg BW, and 100 mg/kg BW, were then administered the COVID-19 vaccine intramuscularly at 0.13 mL/ 20 g BW on day first and seventh day.

Analysis of NK cell activity:

The sample used in the NK cell examination is serum from a centrifuge of blood samples taken by guillotine (neck artery). Blood was collected and centrifuged for 30 minutes at 3000 rpm to obtain serum. Then, the serum was used to test NK cell activity using the ELISA method with 40 ul of serum. Anti-KLRD1 antibodies, or CD94, are used to test NK cell activity. CD94 is part of a receptor essential for MHC-I recognition in NK cells. Suppose cells do not express MHC class I or express them in insufficient amounts (infected cells). In that case, CD94 can activate NK cells to respond and destroy these cells³⁹. The use of CD94 in ELISA may help understand the role of NK cells in disease pathogenesis or response to specific therapies. By obtaining CD94 absorbance and making it into a concentration, results can be obtained that show the activity of NK cells.

Table 2. Absorbance of CD94

Group	Absorbance of CD94
1	0.478	0.479	0.467	0.461	0.475
2	0.474	0.477	0.465	0.478	0.463
3	0.478	0.482	0.483	0.488	0.482
4	0.492	0.497	0.49	0.489	0.471
5	0.524	0.522	0.527	0.54	0.538

Based on the results of the average CD94 concentration, it shows that from several treatment groups given varying doses of Moringa oleifera Lam. leaf ethanol extract and induced with the COVID-19 vaccine, the highest NK cell activity was in group 5, as much as 2.9806 μg/mL and the lowest activity was in group 1, which was only given 0.5% Na-CMC without any extract, as much as 2.9462 μg/mL. After carrying out the one-way ANOVA statistical test, there was a significant difference in the values (p<0.05). The DMRT results showed that administering the vaccine with ethanol extract of Moringa oleifera Lam. leaves 100 mg/kg BW was more effective than the other groups. NK cells are vital cells in the body's non-specific immune system. NK cells function to kill infected cells and destroy activated immune cells to avoid excessive damage to the body. Thus, NK cells act as immune regulators that help maintain balance and avoid excessive immune reactions²¹.

Calculating the total number of leukocyte cells:

Based on the results of the average count of total leukocytes, it was found that leukocytes had increased in all groups compared to the group that was only given 0.5% Na-CMC.

Table 3. Average total number of leukocytes

Group	Average total amount (µL)
1	6,200
2	12,390
3	15,420
4	20,620
5	24,900

An increase in total leukocyte cells proves that the body's defence system is natural or non-specific⁴⁰. The results of the one-way ANOVA statistical test showed that there were significant differences (p<0.05). To understand these differences, statistical analysis was continued with the DMRT. The results show that each group is not in the same subset. This states that administering vaccines and Moringa oleifera Lam. leaf ethanol extract with varying doses influences increasing total leukocyte cells.

Figure 1. Leukocyte cells in a hemacytometer counting chamber

Based on Figure 1, it can be explained that this is one of the counting rooms in the hemacytometer. The total number of leukocytes was obtained by counting the four hemacytometer chambers using the L rule method. Therefore, leukocytes on the right and upper lines do not need to be counted to avoid counting twice.

Calculating the Percentage of Total Leukocyte Count:

The purpose of this analysis is to determine the effect of the extract on its ability to stimulate the immune response. The purpose of making thin smears is to make it easier to observe and count leukocyte cells, allowing leukocyte cells to spread evenly on a glass slide so that leukocyte cells can be observed clearly and accurately. Giemsa solution is used to clarify the differences between leukocyte cells, namely segment neutrophils, rod neutrophils, lymphocytes, monocytes and eosinophils. Meanwhile, basophils are alkaline and dissolve in Giemsa solution, so they cannot be seen during analysis. After drying, the preparations were examined under a microscope with 1000x magnification using immersion oil to determine the shape of leukocyte cells⁴⁰. It showed in Figure 2 below. Calculation of the percentage of leukocyte cells is done by cross-sectioning or counting leukocytes starting from the tail end of the blood smear by snaking until 100 leukocyte cells are obtained, then expressed as a percentage⁴¹.

From Table 4, the average percentage of leukocyte types found in each group with different treatments was obtained. The significant difference in the results of monocytes and eosinophils from white male mice between the groups given 0.5% Na-CMC, the COVID-19 vaccine, and varying doses of Moringa oleifera Lam. leaf ethanol extract was due to the group only being given 0.5% Na-CMC without vaccine does not cause significant immune stimulation, so the results of monocytes and eosinophils are relatively higher because there is no decrease due to the specific immune response to the vaccine. In the group given the COVID-19 vaccine together with 0.5% Na-CMC, the immune response induced by the vaccine could cause changes in the leukocyte population, including a decrease in eosinophils because vaccines usually trigger a specific inflammatory response. Meanwhile, when administering a vaccine with Moringa oleifera Lam. leaf ethanol extract at a dose of 100 mg/kg BW, which is the highest dose, the immune system may develop an adaptation; certain cells, such as monocytes and eosinophils, decrease in response to excessive stimulation. This is the body's protective mechanism to avoid cell activation that could damage the body's own tissue⁴².

CONCLUSIONS:

Based on the results of the research that has been carried out, it can be concluded that the administration of ethanol extract of Moringa oleifera Lam. influenced increasing NK cell activity in male white mice exposed to COVID-19 virus antigens. It influenced increasing the total number of leukocyte cells in male white mice exposed to the COVID-19 virus antigen. Administering of ethanol extract of Moringa oleifera Lam. affected increasing the percentage of segment neutrophil cells, stem neutrophils, and lymphocytes and did not increase the percentage of monocyte and eosinophil cells in male white mice exposed to COVID-19 virus antigens.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

ACKNOWLEDGEMENT:

The authors thank the Rector Universitas Andalas through the Institute for Research and Service Society (LPPM Unand). The research was made possible and financially supported under "Penelitian Unggulan Jalur Kepakaran (PUJK), Batch I, the Year 2024, with contract number: 381/UN.16.19/PT.01.03/PUJK/2024 signed on 17 July 2024.

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Received on 28.11.2024 Revised on 17.03.2025

Accepted on 11.05.2025 Published on 08.11.2025

Available online from November 13, 2025

Research J. Pharmacy and Technology. 2025;18(11):5447-5454.

DOI: 10.52711/0974-360X.2025.00785

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

Group	Segmental Neutrophils	Stem Neutrophils	Lymphocytes	Monocytes	Eosinophils
1	31.8	13.6	32.6	11.4	10.6
2	33.2	13.8	34	10	9
3	34.8	14.4	36.2	5	9.6
4	36.8	17.8	37.2	5.4	2.8
5	37.8	18	38	3.8	2,4