Activity of Pegagan Embun ( Hydrocotyle sibthorpioides Lam.) Extracts in Capsule Dosage Form to IgM and IgG Levels in Humans

Objective: Traditionally, Pegagan embun ( Hydrocotyle sibthorpiodes Lam.) has been used to enhance the immune system. Previous studies confirmed that Hydrocotyle sibthorpiodes Lam. enhanced antibody titers and was practically non-toxic by animal tests. This study aimed to determine the effect of Hydrocotyle sibthorpioides Lam. extract on IgM and IgG levels in humans. Methods: The participants involved 20 healthy volunteers aged 20-25 years were divided into two groups with a placebo as a comparison. The capsules containing Hydrocotyle sibthorpiodes Lam extract were in the treatment group at a dose of 67 mg and the placebo group was given capsules containing lactose orally for three days. IgM and IgG levels were measured before and after consuming the capsules. The ELISA method tested IgM and IgG levels and detected them using specific antibodies that form complexes with enzymes and substrates. Results: The results showed the average baseline IgM level of 1.480 mg/mL and after consumption of 1.405 mg/mL for placebo. For Hydrocotyle sibthorpiodes Lam. extract capsules, the baseline was 1.492 mg/mL and 1.854 mg/mL after consumption. For IgG, before given 8.941 mg/mL and after given 8.710 mg/mL for placebo, while before given Hydrocotyle sibthorpiodes Lam. extract capsules 8.946 mg/mL and after given Hydrocotyle sibthorpiodes Lam. extract capsules was 10.234 mg/mL. Statistical results showed a significant increase in IgM and IgG levels after consuming Hydrocotyle sibthorpiodes Lam. extract capsules for three days (p<0.05). Conclusion: It can be concluded that 67 mg of Hydrocotyle sibthorpiodes Lam. extract capsules can increase IgM and IgG levels in humans.


INTRODUCTION
Medicinal plants have medicinal properties and are used to heal and prevent disease. 1 Indonesia has many medical plants; about 30,000 species of flora are found in Indonesia's tropical forests, and around 9,600 species have been recognised as medicinal.Since ancient times, medicinal plants processed into medicine have been widely used by the Indonesian people, both in the form of single fresh, mixed, and herbaceous, better known as traditional medicine. 2e immune system can protect humans from foreign substances and pathogens such as viruses, bacteria, parasites, and fungi. 3There are two types of immune responses: specific and non-specific. 4on-specific immune responses work quickly to protect humans from microorganisms, while specific immune responses show specific responses to certain microbes. 5Immunostimulants can boost the body's defence mechanisms.Immunostimulant compounds can enhance the body's specific and non-specific defence mechanisms through cellular or humoral responses. 6Certain plants contain compounds that exhibit immunostimulatory activity. 7One such plant is the herb Hydrocotyle sibthorpioides Lam. which is widely used in traditional Chinese medicine to boost human immunity. 8e ethanol extract of Hydrocotyle sibthorpioides Lam. has immunostimulant activity.The previous study showed that ethanol extract of Hydrocotyle sibthorpiodes Lam. can increase antibody titers at doses 10, 50, and 200 mg/kg BW against male white mice. 9Previous study related to the extract of Hydrocotyle sibthorpioides Lam.possessed pharmacological activity to improve the immune system, with doses of 10, 50, and 200 mg/kg BW showing immunostimulatory effects through increased activity and capacity of macrophages, the number of leukocytes, and the number of macrophages, macrophage capacity, total leukocyte count, lymphocyte percentage, and significantly reduced TNF-α and total macrophage levels tested in male white mice. 10The broad utilisation of Hydrocotyle sibthorpioides Lam. in the field of pharmacology, significantly as an immunostimulant, can also increase the activity of NK cells, CD8 cells increase the number of erythrocytes, the number of reticulocytes, hematocrit values, and haemoglobin levels. 12It concluded that Hydrocotyle sibthorpiodes Lam.extract has immunomodulatory activity.
In previous preclinical studies, acute and sub-acute toxicity tests of Hydrocotyle sibthorpiodes Lam.extract was also reported, where the results obtained from the doses given, 7 mg/kg BW, 35 mg/kg BW, and 150 mg/kg BW to the test animals, showed no damage to the histology of the liver and kidneys of male white mice. 13As well as carbon clearance, creatinine clearance, 15 and SGOT and SGPT levels after administration of Hydrocotyle sibthorpiodes Lam.extract to test animals also had no toxic effects.The lethal doses (LD50) value for the ethanol extract of Hydrocotyle sibthorpioides Lam.amounted to 128.83 g/kg BW, from the LD50 category, then the ethanol extract of Hydrocotyle sibthorpiodes Lam. is classified into the range of practically non-toxic (LD50 >15,000 mg/kg BW). 13 IgG, which makes up 80% of the body's antibodies, is the most abundant immunoglobulin. 17Antibodies secreted by the salivary glands include IgA, which is crucial in mucosal surface defence. 18IgM (6%) is an antibody that activates the complement system. 19IgD (1%) is involved in immune tolerance. 20IgE (1%) is involved in immediate hypersensitivity reactions; this antibody causes mast cells to release large amounts of histamine, causing severe vasodilation. 19M is the first antibody produced and expressed on the surface of B cells.IgM plays a role in the primary immune response to microbial antigens, as it is the first antibody produced during infection. 21IgM circulates in the blood, lymph, and B cell surfaces (in monomeric form). 21The percentage of this antibody in serum is 5-10%, which acts as an antigen receptor for these cells and is also a molecular solute in the blood. 21Due to its large size, IgM is found mainly in the intravascular space (in the bloodstream). 21Since IgM is produced in the immune response, its efficiency in binding to antigens is critical until sufficient amounts of IgG have been synthesised. 22Although IgM usually has low affinity binding sites for antigens, it has ten combining sites per molecule that can synergise with each other on the same molecule when binding to microbes. 22Thus, the overall tightness of binding of IgM molecules (avidity) to microbes is relatively high, making this class of antibodies very effective in eliminating microbes. 22IgG levels also rise higher and last longer due to the presence of B cells and memory T cells due to the first antigen exposure. 22sed on the explanation, the study aimed to examine the activity of Hydrocotyle sibthorpiodes Lam.extract on IgM and IgG levels in human serum.It was conducted in the form of capsule preparation because it is easy to prepare and easy to use.

Preparation of ethanol extract from Hydrocotyle sibthorpiodes Lam.
Hydrocotyle sibthorpioides Lam.extract was collected from 5000g of fresh Hydrocotyle sibthorpioides Lam.plants, then cleaned from dirt by washing using running water, then aerated to dry the simplisia.The dried simplisia was crushed using a blender and sieved using sieve no.48.The sample that has been finely ground, then macerated by inserting 1 part of the powder and adding 10 parts of the solvent into a dark bottle as a maceration container, soaked with 70% ethanol for the first 6 hours while occasionally stirring, then let stand for 18 hours.All macerates were collected, and the solvent was evaporated using a rotary evaporator until a thick extract was obtained.After obtaining the thick extract, the yield calculation was carried out.The yield must reach at least the number specified in each extract monograph. 23

Capsule formulation
All ingredients were weighed 335mg for each capsule.Each ingredient for the composition of the test capsule consisted of Hydrocotyle sibthorpiodes Lam.extract, aerosil, amylum Manihot, Mg stearate, and lactose.Put the extract of Hydrocotyle sibthorpiodes Lam.into a mortar, then add some lactose little by little, grind until homogeneous, continue with the addition of amylum Manihot, ground again until homogeneous, then aerosol and Mg stearate was added, grind again until homogeneous then remaining lactose was added little by little.Furthermore, ground it again until the powder was homogeneous and placed into an empty capsule shell with a size of 0, which had a 300-500 mg capacity.The capsule formulation is presented in Table 1.

Standardisation of condensed extract Hydrocotyle sibthorpioides Lam.
The ethanol extract of Hydrocotyle sibthorpioides Lam. that has been produced underwent extract characterisation, including testing nonspecific and specific parameters and analysing the extract's chemical content.It was done to ensure that the extract obtained meets safety and quality standards.Characterisation of non-specific parameters were drying shrinkage of 5.72%, total ash content of 2.25%, acid insoluble ash content of 0.07% and water content of 5.56%.On the other hand, the specific characterisation consists of organoleptic tests, identity parameters, chemical content tests, thin layer chromatography, and determination of total flavonoid content.

Evaluation of powder contents of Hydrocotyle sibthorpiodes ;''''
The formulated powder was subjected to evaluation tests before being put into capsules to ensure the effectiveness of the powder.The evaluation tests conducted were flow rate, compressibility, and angle of repose tests.The evaluation of the capsule showed in Table 2.

Capsule evaluation of Hydrocotyle sibthorpiodes Lam. extract
The organoleptic test (weight uniformity and dissolution time) was used to evaluate the effectiveness of the capsules.The characterisation of the capsule is shown in Table 3.

Clinical trial protocol
Of the 20 research subjects, 10 healthy volunteers were given the test preparation of Hydrocotyle sibthorpiodes Lam.extract capsules and 10 healthy volunteers were given a placebo capsule preparation containing only lactose, which was consumed orally at a dose of 1 capsule per day after meals in the morning for 3 days.

Blood specimen collection and testing
Blood specimens were collected by health professionals under the supervision of the doctor in charge of the study after healthy volunteers had fasted for 6-12 hours.A volume of 3 mL of blood was taken through the mediana cubital vein at the elbow fold of the hand, which was first cleaned above the stab site with 70% alcohol and allowed to dry.Next, a damming tie (tourniquet) was attached to the upper arm, and the mediana cubital vein was pierced at a 45-degree angle.The aspirated blood will flow into the syringe, then remove the tourniquet and pull the needle while still pressing the puncture hole with alcohol cotton and the former puncture is covered with plaster.The blood obtained was tested for lymphocyte levels and then centrifuged for 30 minutes at 3000 rpm.Then, the serum was used to test IgM and IgG levels using an ELISA kit.

Data analysis
The data was analysed statistically using the paired T-test analysis method between pre and post-data, then continued with the Independent T-Test.

Ethical approval
Ethical approval was obtained from the Faculty of Medicine Ethics Committee, Universitas Andalas, with the contract number of the ethics letter: 1072/UN.16.2/KEP-FK/2022.

RESULTS
The measurement results of IgM levels of healthy volunteers are shown in Table 4, and the correlation of IgM levels with the administration of Hydrocotyle sibthorpioides Lam.extract capsules and placebo is shown in the bar chart in Figure 1.
The measurement results of IgG levels of healthy volunteers are shown in Table 5, and the correlation of IgM levels with the administration of Hydrocotyle sibthorpioides Lam.extract capsules and placebo is shown in the bar chart in Figure 2.
This study's results indicate a significant difference between the average IgM and IgG levels before and after the provision of Hydrocotyle sibthorpiodes Lam.extract capsule preparation.Based on the results obtained in this study, higher IgM levels were shown in the healthy volunteer group after consuming Hydrocotyle sibthorpiodes Lam.extract capsules.The average IgM levels before and after in the group of volunteers who consumed Hydrocotyle sibthorpiodes Lam.extract capsule preparation were 1.492 ± 0.388 mg/mL and 1.854 ± 0.198 mg/ mL.For the placebo group, they were 1.480 ± 0.294 mg/mL and 1.405 ± 0.222 mg/mL, respectively.In comparison, the average IgG levels before and after in the group of volunteers who consumed Hydrocotyle sibthorpiodes Lam.extract capsule preparation were 8.946 ± 0.466 mg/ mL and 10.234 ± 0.915 mg/mL.For the placebo group, they were 8.941 ± 0.843 mg/mL and 8.710 ± 0.528 mg/mL, respectively.
Based on the results of the paired T-test that has been carried out, it can be seen that there is a significant difference between the mean levels of IgM with a value of p<0.05 (Table 6) and IgG with a value of p<0.05 (Table 7) before and after consumed Hydrocotyle sibthorpiodes Lam.extract capsules.There was not a significant difference between the mean levels of IgM (Table 6) and IgG (Table 7) before and after administration of placebo capsules (p>0.05).Then, the results obtained from the two test groups were compared using an independent T-test.There was a significant difference between the mean levels of IgM (Table 8) and IgG (Table 9).after healthy volunteers consumed Hydrocotyle sibthorpiodes Lam.extract capsule preparation compared to placebo (p>0.05).

DISCUSSION
Pegagan embun (Hydrocotyle sibthorpioides Lam.) has many pharmacological activities, traditionally to relieve swelling, diuretic, expectorant, inflammatory, and neutralise toxins (detoxification). 14The immunostimulant activity of Hydrocotyle sibthorpiodes Lam. is evident from the flavonoid chemical content in its ethanol extract. 24Previous preclinical studies in animal tests have proven that Hydrocotyle sibthorpiodes Lam.extract has immunomodulatory effects, and toxicity tests that have been conducted state that Hydrocotyle sibthorpiodes Lam.extract is practically non-toxic. 9,25Therefore, this study was conducted to see the activity of Hydrocotyle sibthorpiodes Lam.extract capsules on IgM levels through an immunomodulatory approach in humans.The implementation of clinical trials of herbal medicines must be based on benefit claims describing the usefulness or benefits that promise a positive change for its users, which must be under empirical history supported by sufficient scientific evidence through relevant preclinical trials.The required scientific support evidence is safety data, at least the LD50 value of herb simplistic, and to reduce bias, it is recommended to use subject randomisation. 26e Immunomodulatory activity test of Hydrocotyle sibthorpiodes Lam.extract was conducted on humans.The average IgG and IgM levels in the placebo group decreased because the placebo capsule only contained excipients that did not increase IgG and IgM levels in the body.As for the treatment group given Hydrocotyle sibthorpioides Lam.capsules, it increased because Hydrocotyle sibthorpioides Lam. was proven preclinically to be efficacious as an immunomodulator that can increase the body's immune system, one of which is antibodies that play a role in the body's defence. 14Immunomodulators are materials that can modulate the body's immune system, both immunostimulants and immunosuppressants.In addition, this founding is also comparable to other studies conducted on bengkoang tuber extract, which has an immunostimulant effect which can also increase antibody levels. 27 Tables 6 and 7, the paired T-test showed significant results in the test group given Hydrocotyle sibthorpioides Lam.This result showed a significant difference in IgG and IgM levels before and after treatment. 28his result can be used as a reference to assess how the effect of giving Hydrocotyle sibthorpioides Lam extract, while in the placebo group, the results given did not have significant differences in results, so it can be concluded that by giving a placebo, there is no effect on changes in IgG and IgM levels observed. 29ter observing the IgG and IgM levels before and after treatment, the IgG and IgM levels were compared with the placebo group.It can be observed in Tables 8 and 9 that there is a significant difference in IgG and IgM levels after being compared with a placebo.The result indicates that the test capsules significantly changed IgG and IgM levels compared to placebo capsules. 28,29

CONCLUSION
Based on the results of the study to determine the immunomodulatory effect of Hydrocotyle sibthorpiodes Lam.extract in capsule dosage form consumed by healthy humans at a dose of 67 mg, the effect was a significant increase in IgM and IgG levels.

Figure 1 :
Figure 1: Chart of IgM level measurement results in healthy volunteers before and after consuming Hydrocotyle sibthorpiodes Lam.extract capsules and placebo for 3 days.

Figure 2 :
Figure 2: Bar chart of IgG level measurement results in healthy volunteers before and after Hydrocotyle sibthorpiodes Lam.extract capsules and placebo for 3 days consumed.

Table 3 : Capsule evaluation results.
Afriwardi A, et al.Activity of Pegagan Embun (Hydrocotyle sibthorpioides Lam.) Extracts in Capsule Dosage Form to IgM and IgG Levels in Humans

Table 9 : Independent T-test results of IgG levels of healthy volunteers in placebo and Hydrocotyle sibthorpiodes Lam. groups. Pharmacognosy
Journal, Vol 15, Issue 5, Sep-Oct, 2023