ETHANOL EXTRACTS OF PROPOLIS (EEP) AGAINST LYMPHOCYTE ACTIVATION CELLS IN HEALTHY MICE (Mus Musculus) BALBC

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ETHANOL EXTRACTS OF PROPOLIS (EEP) AGAINST LYMPHOCYTE

ACTIVATION CELLS IN HEALTHY MICE (Mus Musculus) BALB/C

Emi Rohmawati1)*_{and Muhaimin Rifa’i}2)

1,2)_{Departement of Biologi, Faculty of Mathematics dan Science, Brawijaya University, Jl. Veteran 65145 Malang.} Email:1)_{emi.bioub10@gmail.com dan}2)_{rifa123@ub.ac.id}

ABSTRACT

Propolis is a substance like glue formed by honey bees from resin of plant which has the ability to stimulate immune system. The purpose of this study is to determine the ethanol extract of propolis on the activity of lymphocytes in healthy Balb/c mice and to asses the optimum dose administration of EEP for lymphocyte activation in Balb /c mice. Methods: mice was aclimate for two weeks, mice control without treatment EEP and a other was treated with EEP dependent dose, dose 1 (50 mg/ kgBW), dose 2 (100 mg/kgBW), and dose 3 (200 mg/kgBW). Spleen was isolated and to find out the amount of lymphocyte we analyzed with flow cytometry. Parameter measured in this experiment is quantitative by measuring relative number of T cells that consist of CD4+CD62L+, CD4+CD62Lˉ, CD8+CD62L+, dan CD8+CD62Lˉ _.

Then data was analyzed by SPSS 16.0 softwarefor windowswithANOVAtest and advanced byTukeytest andGomes-Howell with an interval 0.05 is used. The result showed that ethanol extract of propolis can activate CD4 T cells so that CD4 T cell lost CD62L molecule and turned into T cells CD4+CD62Lˉ .

Ethanol extract of propolis can enhance proliferation of naïve type of CD8 T cell, so that the number of T cell memory (TCM) decreased. Dose of 100 mg/kgBW of ethanolic propolis extract spatially act as immunostimulant for CD4+CD62Lˉ _{activation, while} _the _dose _of _{200 mg/kgBW} _{act as}

immunosuppressant in the same cells.

Key words: T cell activation, propolis, Balb/c mice.

INTRODUCTION

Indonesia is a rich country who have a greatest biodiversity. Various kinds of natural materials can be used to cure many diseases. In fact, many people use propolis as alternative drug for therapy to healing various diseases. Propolis is a substances like a glue collected by honey bees from buds and exudates of plants, processed by enzymes released by bees and mixed with wax present in the nest. Honey bees use propolis as a tool for self-defense to protect it nest from the environmental from other organisms and to prevent the development and spread of diseases caused by microbes. Propolis has a wide range of positive effects on health, as an immunomodulator, antioxidant, antibacterial, antitumor, antifungal, and anti-inflammatory (Muradet al., 2002). Bees wax is basically white but can change form the dark brown color due to contamination by contact with pollen and bees in the hive (Krell, 1996).

Based on the research propolis is able to assist homeostasis by enhancing the immune system to maintain the body's balance system.

Therefore, further research is necessary to determine the mechanisms and work of propolis in the immune system, so this research needs to show for all people are more certain to use herbal medicine like a propolis with the available scientific evidence. Parameter was measure by analyzed relative number of T cells CD4+CD62L+, CD4+CD62L-, CD8+CD62L+, CD8+CD62L- and this research is expected to improve usability herbal medicine in Indonesian has the potential in health.

METHODS

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Jurnal Biotropika | Vol. 2 No. 4 | 2014 204

Table 1. Group Treathment

Extraction Propolis. Prepara consists of 2 stages include extr evaporation processes. Extraction p from 200 grams of dried sample, glass erlenmeyer then soaked with e volume 1 L and allowed to Furthermore, the solution was fil filter paper to separate the ethanol sample with the active substa evaporated the extracted solut evaporation flask mounted on the Water bath filled with water until fu in pairs all series tools includi evaporator, water bath heater (set 90ºC). Ethanol solution was allowed the active substance in the flask.

Preparation Extracts and Treatment in Mice. Preparation depends on the average weight of extracts were weighed then dilution with ratio 1:10. Furthermore, mice i oral treatment with ethanol extract according to the dose in each group weeks.

Isolation of Lymphoid Organs

mice was dislocation. Sprayed w 70% and cut ventral using sectio s organs were taken and put in a containing sterile PBS, then using syringe crushed with rotated Homogenates pipetted until mix, t using a sterile wire. Propylene tub and filter again until the volume rea and then centrifuged.

Analysis of Changes Num Cells Subset Using Flow C

Supernatant was discarded, the resul centrifugation were resuspended w sterile PBS and pippeting. Tak resuspension and incorporated in containing 500 ml of sterile PBS and Then centrifuged and the supernatant Pellet 2 obtained was added 50 m

aration extract l full and then luding rotary ct of propolis group until 2 sult of pellet 1 d with 1 ml of ake 70 mL in mikrotube S and pipeting. tant discarded. 50 ml of each

preparation antibodiBD Scienc ᵀᴹ

CD4 FITC conjugated, BD Scienc ᵀᴹ

CD4 FITC conjugated, PE-C ᵀᴹ

antimouse CD8 and PE-Cyᵀᴹ 7 CD62L for 15 minutes. Pippeting for 15 min in dark conditions. A of sterile PBS. Resuspended and a cuvette flow cytometer. Cuvet nozzla Caliburᵀᴹflow BD FACS c computer was set with BD Cells Q Proᵀᴹ and made connections cytometer (acquiring mode).

Data Analysis. The parame the relative number of T CD4+CD62L+, CD4+CD62L-, CD8+CD62L-, then data was a SPSS 16.0 for Windows. AN normality and homogeneity, homogeneous advanced Tukey t data are not homogeneous test Howell with the confidence interv

RESULTS AND DISCUS

Effect of ethanol extract healthy mice Balb/C by analy number of T cells, namely CD4+CD62L-, CD8+CD62L+, CD

Population T Cells CD4 CD4+CD62Lˉ on Spleen Organ. flow cytometry analysis showed treatment the relative number of CD62L+is 2.26%. Dose 1 is 2.24 1.07%. The relative number of c again at dose 3 that is 2.1% (fig activator molecules bound by ot so precentage was increas. B analysis of Gomes-Howell, relati T cells CD4+CD62L+ (Figure treatment showed no significant di dose 1 and dose 3. Dose 1 and dos no significant between treatment treatment had significantly differ The relative number of T cells (figure 1) control treatment amount Dose 1 have a similar value whe control as 5.6%. Dose 2 showe number of cells is highest amon treatment equal to 14.18%. The re of CD4+CD62L+cells decreased is equal to 2.5%. Based on the Gomes-Howell relative number CD4+CD62Lˉ (figure 3) control significant difference with a dose

enceᵀᴹantimouse nd transferred to uvette mounted in

ᵀᴹ S cytometer. The

ls Quest software

ᵀᴹ ons with a flow

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a significant difference with dose 2 a 2 was very significant value with dos

Figure 1. Profile Relative Number CD4+CD62L+ dan CD4+ Spleen Organ.

Figure 2. Changes of Propolis Extr Treatment Toward the Number Of T Cells CD K=Control; D1=Dose

mg/kgBW; D2=Dose

mg/kgBW; D3=Dose 3 200

ˉ

ˉ. Increasing the number of

IFNγ IFNγ

2 and 3. Dose h dose 3.

ber of T Cells 4+CD62Lˉ in

xtract Ethanol the Relative CD4+CD62L+;

1 50

e 2 100

3 200 mg/kgBW

Figure 3. Changes of Propolis E Treatment Toward

Number Of T Cells ˉ

K=Control; D1=Dose

mg/kgBW; D2=Dos

Comparison the number

CD4+CD62L+ dan CD4+CD ˉ

threathment by ethanol extract of that number of CD4+CD62L+les CD4+CD62Lˉ. CD62L is a m mediate naive T cells to periphe organs, this happens initiation response that decreased expressi also lead to the decline in the num cells (Rifa'i, 2011). It shows tha extract of propolis can activ CD4+CD62L+, so T cells CD4 CD62L molecules and change be

CD4+CD62Lˉ. Increasing the number of

memory cells happens at dose immunostimulatory effects on T When the number of memory T c the number of naïve T cells becom opposite. Taheri et al., (2005) propolis can respond the immune example can increase the macrophages, increasing IL-1, I Dose 100 mg/BW can increas pr lymphocyte. Gu (2005) explain can enhance the number of T l cells CD4+produce cytoknesused cells CD8+, so T cells CD8+dif cytotoxic T cells effector and m Increased proliferation of T cells triggered by saponin compounds saponins has the ability to incr

IFNγ (Cheeke, 2010). IFNγ can

s Extract Ethanol d the Relative ls CD4+CD62Lˉ;

ose 1 50

ose 2 100

3 200 mg/kgBW

ber of T cells CD62Lˉ after of propolis show less then T cells

ˉ molecule can

ipheral lymphoid ion of immune ssion of CD62L, number of naïve that the ethanol tivated T cells 4+CD62L+ lose become T cells

ˉ. Increasing the number of

dose 2 shows that on T cells CD4+. T cells increased ome less and the 2005) explain that une system, for he activity of 1, IL-2 and IL-4. s proliferation T in that propolis lymphocyte. T ed to activated T differentiate into nd memory cells. ells CD4+can be pounds, because ncrease cytokine

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up-regulation of MHC II expression cells deferentiated become T cells C al., 2008 and Shi et al., 2008)

Population T Cells CD8+C Spleen Organ.The results of the ana flow cytometry showed relative num cells control treatment is 1.97%. dose 1 is 1.81%. Dose 2 showed relative number of cells is equal to 3.03 3 is 2.05%, relative number of cell again at dose 2 (figure 4). Based on of Gomes-Howell (figure 5) the relat of CD8+CD62L+ showed that the significant between control with dos But dose 2 are significantly differe treatments. Ethanol extract of propo different dose give different resul activity of CD8+CD62Lˉ (Figure 6 number T cells CD8+CD62Lˉ treathment is 11.84%. Dose 1 is 9.32% is 7.30%, and dose 3 is 16.34%. Tukey analysis (figure 6), relative num cells CD8+CD62Lˉ showed that the significant difference between cont dose 2 , but dose 3 has significantly the other. Based on the relative num CD8+CD62L+ known that ethanol propolis can increase the relative CD8+CD62L+at dose 2 that is 100 This indicates that there is a mechani homeostasis, because the activa produce cytokines IFNγ and IL cytokines were used CD8+ cells to Increase of T cells CD4+ inf activation of T cells CD8+.

ˉ

IFNγ

ion, it cause T CD4+ (Lee et

+

CD62L+ in

analysis using numbers of T . Treathment d the highest o 3.03%. Dose ells decreased on the analysis elative number there was no dose 1 and 3. erent with all propolis with 4 esults on the ˉ

e 6). Relative ˉ

in control 9.32%, dose 2 %. Based on e number of T ˉ

there was no ontrol, dose 1, ntly value from number T cells nol extract of ve number of 100 mg/kg BW. anism of cells tivated CD4+

IFNγ IL-2, which

to proliferate. influence the

Figure 4. Profile Relative Numbe

CD8+CD62L+ dan CD ˉ

Spleen Organ

Figure 5. Changes of Propolis E Treatment Toward Number Of T Cells K=Control; D1=Dose

mg/kgBW; D2=Dos

Figure 6. Changes of Propolis E Treatment Toward

Number Of T Cells ˉ

K=Control; D1=Dose

mg/kgBW; D2=Dos

T cells CD8+can increase a by the presence of CD4+ tha activated. Activated T cells CD previously can activated Th1 defe produce cytokine IFNγ and IL-2 2008). By utilizing IL-2 CD8+ affinity than the affinity of CD4 2008). IL-2 produced from CD used to regulate itself and also us

ˉ

IFNγ

umber of Cells T CD8+CD62Lˉ in

s Extract Ethanol d the Relative s CD8+CD62L+;

ose 1 50

ose 2 100

3 200 mg/kgBW

s Extract Ethanol d the Relative ls CD8+CD62Lˉ;

ose 1 50

ose 2 100

3 200 mg/kgBW

e also influenced that have been CD4+ had been deferentiated, Th1

IFNγ -2 (Rifa'i et al.,

+

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a stimulant for cells proliferate.T cells CD8+are activated due to the stimulus of IL-2, which defferentiated become T cells killer. T cells killer used to lyse a variety of cells that have been exposed antigen by lysing perforin. Perforin is a protein that causes lysis of target cells by forming pores on target cells membrane. After lysing the cells that have been infected with the antigen, then CD8+cells will lyse other cells infected by a similar antigen (Rifa'i, 2004).

Most of T cells that developed into memory T cells can survive in a long time. Memory cells is off and circulate in the body, it can be respond quickly in the event of recurring exposure to microbes. After the T cells effector successfully overcome the infection, the stimulus that triggers the expansion and differentiation of T cells also stopped anyway. T cells clones that have been formed will die and then returned to the basal state. This occurs in T cells CD4+ and CD8+, but there is difference in function of efektor (Abbas, 2005).

CONCLUSIONS

Based on the results of the discussion can be concluded that the ethanol extract of propolis is able to increase the activation of T cells memory CD4+CD62Lˉ. Ethanol extract of propolis can decrease the proliferation of T cells memory CD8+CD62Lˉ. Dose of 100 mg/kgBW of ethanol extract of propolis are immunostimulatory the activation of T cells memory CD4+CD62Lˉ, whereas at dose 200

mg/kgBW are immunosuppressants on

proliferation of T cells memory CD8+CD62Lˉ.

ACKNOWLEDGEMENTS

The author would like to thank Muhaimin Rifa'i, Ph.D. Med.Sc., Dr. Ir. Moch.Sasmito Djati, MS., Drs. Aries Soewondo, M.Si from Department of Biology, Brawijaya University, Malang who has provided guidance, criticism, and suggestions for this research.

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