Pharmacological activity of the Ethanolic extract of Lingzhi to the Immune system
Khusnul1, Meti Kusmiati1, Rianti Nurpalah1, Yedy Purwandi Sukmawan2,
I Nyoman Pugeg Aryantha3, Tutus Gusdinar4
1Department of Medical Laboratory and Technology, Faculty of Health,
University of Bakti Tunas Husada, Tasikmalaya, West Java, Indonesia.
2Department of Clinical Pharmacy, Faculty of Pharmacy,
University of Bakti Tunas Husada, Tasikmalaya, West Java, Indonesia.
3School of Life Sciences and Technology, Institut Teknologi Bandung, Bandung, Indonesia
4Department of Pharmacochemistry, School of Pharmacy,
Institut Teknologi Bandung, Bandung, West Java, Indonesia.
*Corresponding Author E-mail: khusnul@universitas-bth.ac.id
ABSTRACT:
The immune system is a defense system of the human body that has a function to protect humans from foreign. One of the indicators of an increased immune system is an increase in the number and type of the leukocyte cell (lymphocytes and monocyte) and lymph weight. In Indonesia, many natural ingredients can be used as an alternative medicine to maintain the immune system, including the Lingzhi mushroom (Ganoderma lucidum). The purpose of this study was to determine the pharmacological activity of Lingzi mushroom extract on the number and type of leukocytes (lymphocytes and monocyte) as well as spleen weight in white male swiss webster mice. The method of this research is an experimental laboratory with a Completely Randomized Design (CRD) using a post-test control group design. The sample population taken was 25 mice divided into five groups including negative, positive, test 1 (5.6mg/20g BW of mice), test 2 (11.2mg/20g BW of mice), test 3 (16.8 mg/20 g BW of mice). Each group consists of 5mice. The data collection was obtained from the result of the cell count, leukocyte cell count, and lymph weight in mice which was carried out after the treatment with lingzhi mushroom extract for 6 days. The results showed that a test 2 (11.2mg/20g BW in mice) was effective to increase the number of a leukocyte, test 3 (16.8mg/20gr BW of mice) was effective to increase leukocyte, lymphocytes and lymph weight. However, test 1, 2, and 3 reduced amount of neutrophil segment. Lingzhi mushroom extract test 3 showed better than other groups due to increase of leukocyte, monocyte, lymphocyte and lymph.
KEYWORDS: Animal model, Immune system, Leukocyte, Lingzhi mushrooms, Lymph.
INTRODUCTION:
Infections is the fourth leading death worldwide1. One of the factor that excacerbate this conditions is antibiotic resistance2. Various attempts have been made to reduce this incidence, one of which is the discovery and development for compounds that increase immunity3.
Immunity is defined as the balanced state of multicellular organisms having sufficient biological defences against the infection4. The evidence showed immunostimulants able to reduce need of antibiotics, inflammation reactions and cancer5,6.7,8,9. In addition, the immunostimulants showed activity for Covid-19 infection10. Lingzhi mushrooms (Ganoderma lucidum) is one of the plants that act as an immunostimulants that affect to B cells, T cells, dendritic cells, macrophages and natural killer cells, with the promotion of the immune organ growth and cytokines releases11. Acute and subchronic toxicity study data suggest that this plant is safe12,13. Athough various study of lingzhi as an immunostimulant, however this study only focus to T cells, B cells, Natural Killer cells and cytokines. The pharmacological study of this plant to lymphocyte, polymorpnuclear cells, and lymph organ weight is still lack. Therefore, we conduct the study to determine lingzhi activity to lymphocyte, polymorpnuclear cells, and lymph organ weight.
MATERIAL AND METHODS:
Chemicals and reagents:
Ethanol 70% (Brataco chemical), aquadest (Brataco chemical), Na-CMC (PT. DPH), Swab-alcohol (Onemed), EDTA (Brataco chemica), TURK solution (Ayubi Medika), Giemsa (Ayubi Medika), Methanol (Brataco chemical), and Imboost® (Soho Global Health), Sulphuric Acid 98% (PT DPH), Phenol 5% (PT Brataco chemical) Animal Feed.
Materials:
Digital compact balance scales (ACIS Series BC-5000), syringe 1mL, sode oral, glass object, filter paper (whatmann), beaker glass (pyrex), micropippete (Soccorex), counting booth (Improved Neubauer), dissecting set (Secheron), microscope (Olympus CX 23), rotary evaporator (IKA RV-10), waterbath (Memmert WTB-6).
Lingzhi mushrooms:
Plant preparation:
Ganoderma lucidum. was obtained from Singaparna, Tasikmalaya district, West Java, Indonesia. This plant is subjected to wet sortation, washing, chopping, drying, and followed by dry sorting processes. The botanical determination was conducted by Padjadjaran University, Bandung with the identification number 92/HB/02/2021.
Extract preparation:
The 100gram of Lingzhi mushroom was macerated using 70% ethanol until submerged, which was repeated 3 times. Maceration result was filtered with whatmann paper, prior to concentration using a rotary evaporator to obtain the concentrated extract.
Qualitative Determination of Beta-Glucan:
The test by adding 1mL of sample in a test tube followed by the addition of 1mL of 5% phenol and 5mL of sulfuric acid, then allowed to stand for 10minutes and stored in a water bath for 15minutes. Change in color to green, brown or purple indicate contains of beta-glucan.
Immune activity procedures:
Animals:
The male wistar mice (20-30gram) were used in this test. The animals were placed at room temperature 250C, 40-60% humidity and free access to water. All of the procedure was according to the guideline for the care and use of laboratory animals and approved by the Bakti Tunas Husada, Health Science College ethical committee with no. 045/ec.02/kepk-bth/VI/2022.
Wound healing test:
We divided the animals into five groups (5 mice in each group), namely negative (placebo), positive (imboost® 0.65mg/20g BW of mice), test 1(Lingzhi mushroom extract 200mg/kg BW), test 2(Lingzhi mushroom extract 400mg/kg BW), and test 3(Lingzhi mushroom extract 600mg/kg BW). All of these groups were given the test preparation for 6 days. Afterward, the blood of these animals were taken through retro-orbital plexus for the calculation of the leukocyte types. Then followed by euthanasia of the animal through dislocation procedures for the lymph weighing.
The lymph weighing calculation using equation: L = (LW/TW) x 100
L: Lymph weight percentage, LW: Lymph weight, TW: Total body weight.
Determination of the amount of the leukocyte:
The calculation of the number of leukocytes was carried out using improved neubauer counting booth. The first stage is taking 380 microliters of turk fluid into a test tube, then adding 20 microliters of blood that has been mixed with EDTA. Then the calculations were carried out using a microscope with a four-fold magnification on four leukocyte boxes.
Determination of leukocyte type count:
The 10 microliters of blood was taking on the slide, then place and pull the eraser glass at an angle of 30-40 degrees to the slide in front of the blood drop until 3-4 cm long blood smear is formed on the slide and dry. The blood smear was placed on a staining bath and fixed with absolute methanol, then soaked in Giemsa solution for 20-30 minutes and rinsed with running water. After that, the leukocyte type was examined with a magnification of 10x and looked for leukocytes that were evenly distributed and then the magnification was increased to 40x and 100x. After that, each nucleated cell in each field of view was classified up to 100 cells.
Data Analysis:
The data were analyzed using statistical tests One-Way ANOVA. If the results of normality are not normally distributed, then the analysis is carried out using non-parametric test, namely the Wilcoxon Test and Kruskal-Wallis. Meanwhile, if the results of the One-Way Anova showed significant differences, then it is continued with the Post-Hoc Test LSD.
RESULTS AND DISCUSSION:
The study showed if all the test groups such as test 1, test 2, test 3, and positive group exhibited significant difference compared to negative group (table 1). However, the activity of test 1, 2, and 3 were not similar compared to positive group and showed significantly declined of the neutrophil segment compared to negative group (p<0.05). The positive control showed better activity than test 1, 2, and 3, may because the combination composition of the positive group that contains echinaceae purpurea, elderberry and zinc picolinate compared to the test group that only lingzhy mushroom14. In addition, the study also showed no difference for the animal body weight between groups, therefore taking into this considerations, all the groups did not affect to the body weight. The elevation of the leukocyte of the test groups may be due to the elevation of the monocyte and lymphocyte (table 2), it was difference if we compared to the positive groups that increase only for neutrophil rod and monocyte. Meanwhile, for the weight lymph elevation was occured only to the test 3.
Based on this results, the test 3 showed the best dose as an immunostimulant. Moreover, the test 3 may be more better than positive control, it because this test group not only increase the innate immunity (monocyte) but also adaptive immunity (lymphocyte) and lymph weight (table 1, 2, 3 and figure 1). As known the innate immunity acts as a first line defense through pattern recognition receptors and activating inflammatory pathway, however this reaction limited only for 4-7 days15,16. After the innate immunity reaction, then followed to activation of the adaptive immunity such as lymphocyte (T lymphocyte and B lymphocyte) in lymph through the antigen presenting cells which give more robust reactions than innate immunity16,17,18,19,20. The elevation of these may be due to beta-glucans that has function to activate macrophages, dendritic cells, neutrophils, B cells, T Cells and natural killer cells21. In addition, beta-glucans also stimulate production of cytokines such as IL-12 and TNF-alpha21. The mechanism of action of beta-glucans induce immune system through act as Pathogen Associated Molecular Patterns and are recognized by Pathogen Recognized Receptors such as Toll-Like Receptors and Dectin-122.
Table 1: Animal body weight and leukocyte amount
Groups |
Animal body weight (g) |
Leukocyte amount (cell/mm3) |
Negative control |
24.7 |
4.300 |
Positive control |
24.1 |
6.640*t |
22.2 |
5.535* |
|
Test 2 |
23.3 |
6.020* |
Test 3 |
24.4 |
6.380* |
*showed significant difference compared to negative control (p<0.05); t showed significant difference compared to all groups (p<0.05).
Table 2: Leukocyte type count amount
Parameters |
Negative Control |
Positive Control |
Test 1
|
Test 2
|
Test 3 |
Basophil % |
0 |
0 |
0 |
0 |
0 |
Eosinophil % |
0.4 |
0.2 |
0 |
0 |
0,2 |
Neutrophil rod % |
1.6 |
2.2* |
1.2 |
1.6 |
1.4 |
Neutrophil segment % |
63.8 |
62.6 |
51.2* |
46.4* |
41.6* |
Lymphocyte % |
30.4 |
31.5 |
42* |
47.8* |
52* |
Monocyte % |
3.8 |
4.8* |
5.6* |
4.6* |
4.8* |
*showed significant difference compared to negative control (p<0.05)
Table 3: Relative lymph weight
Parameters |
Relative lymph weight (%) |
Negative control |
1.0644 |
Positive control |
0.6998 |
Test 1 |
1.1266 |
Test 2 |
1.2844 |
Test 3 |
1.7184* |
*showed significant difference compared to negative control
Figure 1: Beta-glucans qualitative test
CONCLUSION:
Lingzhi mushroom extract test 3 showed immunostimulant activity and better activity than other groups due to increase of leukocyte, monocyte, lymphocyte and lymph.
ACKNOWLEDGEMENT:
This research was supported by the Kementerian Pendidikan, Kebudayaan, Riset, Dan Teknologi Riset, Republik Indonesia with Direktorat Akademik Pendidikan Tinggi Vokasi through SIMLITABMAS as the research funder on Program Penelitian Kerjasama antar Perguruan Tinggi (PKPT) with master contract number 126/SPK/D4/PPK.01.APTV/VI/2022. The authors are grateful to the authorities of University of Bakti Tunas Husada and Institut Teknologi Bandung for the facilities.
CONFLICT OF INTEREST:
The authors declare no conflict of interest.
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Received on 20.10.2022 Modified on 23.03.2023
Accepted on 14.06.2023 © RJPT All right reserved
Research J. Pharm. and Tech 2023; 16(10):4907-4910.
DOI: 10.52711/0974-360X.2023.00795