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

  

ETHANOL EXTRACTS OF PROPOLIS (EEP) AGAINST LYMPHOCYTE

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

_{1)* 2)} and Muhaimin Rifa’i _1,2) Emi Rohmawati

Departement of Biologi, Faculty of Mathematics dan Science, Brawijaya University, Jl. Veteran 65145 Malang.

_{1) 2)}

Email: emi.bioub10@gmail.com dan 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

_{ˉ + + + + + +}

relative number of T cells that consist of CD4 CD62L , CD4 CD62L , CD8 CD62L , dan CD8 CD62Lˉ .

Then data was analyzed by SPSS 16.0 software for windows with ANOVA test and advanced by Tukey test

₊

and Gomes-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 Therefore, further research is necessary to

  determine the mechanisms and work of propolis Indonesia is a rich country who have a in the immune system, so this research needs to greatest biodiversity. Various kinds of natural show for all people are more certain to use materials can be used to cure many diseases. In herbal medicine like a propolis with the fact, many people use propolis as alternative available scientific evidence. Parameter was drug for therapy to healing various diseases. measure by analyzed relative number of T cells

  Propolis is a substances like a glue collected by

  CD4 CD62L , CD4 CD62L , CD8 CD62L , honey bees from buds and exudates of plants,

  CD8 CD62L and this research is expected to processed by enzymes released by bees and improve usability herbal medicine in mixed with wax present in the nest. Honey bees Indonesian has the potential in health. use propolis as a tool for self-defense to protect it nest from the environmental from other

  METHODS

  organisms and to prevent the development and

  Description Treathment. Animals used

  spread of diseases caused by microbes. Propolis in this research were Balb/C mice (Mus has a wide range of positive effects on health, muscullus) male, age 8 weeks, body weight ± as an immunomodulator, antioxidant, 40 grams. This research used 4 treatments: antibacterial, antitumor, antifungal, and anti- control treatment, treatment I (dose 50 mg inflammatory (Murad et al., 2002). Bees wax is

  /BW), treatment II (dose of 100 mg/BW) basically white but can change form the dark treatment da III (dose 200 mg/BW) with 3 brown color due to contamination by contact replications in each treatment. 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.

  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).

  SPSS 16.0 for Windows. AN normality and homogeneity, homogeneous advanced Tukey t data are not homogeneous test Howell with the confidence interv

  1 L he evaporator. l full and then luding rotary et temperature ed to separate

  ᵀᴹ ᵀᴹ aration extract xtraction and on process start ple, put in two h ethanol until o precipitate. filtered using hanol from the stance. Then olution

  ᵀᴹ ᵀᴹ

  ᵀᴹ ᵀᴹ

  Data Analysis. The parame

  the relative number of T CD4

  CD8

  PE-Cyᵀᴹ

  7 Rat ᵀᴹ 7 Rat antimouse ng and incubated

  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.

  Extraction Propolis. Prepara

  Table 1. Group Treathment

  . Added 300 mL nd transferred to uvette mounted in

  ᵀᴹ S cytometer. The ls Quest software ᵀᴹ ons with a flow meters measured cells include

• CD62L
• , CD4
• CD62L
• ,
• CD62L
• , analyzed using

  , CD8

• CD62L
• , then data was a

  NOVA to test ity, the data y test, while the st using Games- erval of 0.05.

RESULTS AND DISCUS

  gans. Neck of

  with alcohol o sets. Spleen n a petri dish ng base of the d clockwise.

  Based on the lative number of ure 2) control nt difference with nd dose 3 showed ents, but control ferent on dose 2. lls CD4

  ed that control of T cells CD4

  D4 + CD62L + dan gan. The results of

  D8

  ct of propolis in nalyzing relative CD4

  USSION

  Preparation Extracts and Treatment in Mice. Preparation

• CD62L
• ,
• CD62L
• , CD8
• CD62L
• , CD
• CD62L
• .
• is 2.26%. Dose 1 is 2.24

  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.

• 2.24%. Dose 2 is of cells increased figure 1). Dose 3 other molecules

  (figure 3) control significant difference with a dose enceᵀᴹantimouse enceᵀᴹantimouse

  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

  flow cytometry analysis showed treatment the relative number of CD62L

  Population T Cells CD4 CD4 + CD62L ^ˉ on Spleen Organ.

  Effect of ethanol extract healthy mice Balb/C by analy number of T cells, namely CD4

  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.

• 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
• ounted to 5.96%. hen compared to howed the relative mong the other he relative number d back at dose 3 on the analysis of ber of T cells rol treatment no dose 1, but there is

  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

  and Oral

• CD62L
• cells decreased is equal to 2.5%. Based on the Gomes-Howell relative number CD4
• CD62L ^ˉ

a significant difference with dose 2 a 2 and 3. Dose 2 was very significant value with dos h dose 3.

  S and pipeting. tant discarded. 50 ml of each preparation antibodiBD Scienc ᵀᴹ

  tion extracts of mice. Total on use aquades e in groups of ct of propolis group until 2

  umber of T Cytometry.

  , then filtered tubes inserted reaches 10 ml

  sult of pellet 1 d with 1 ml of ake 70 mL in mikrotube

  Figure 3. Changes of Propolis E s Extract Ethanol

  Treatment Toward d the Relative

• ˉ ˉ ˉ

  Number Of T Cells ls CD4 CD62L ; K=Control; D1=Dose ose

  1

  50 mg/kgBW; D2=Dos ose 2 100 mg/kgBW; D3=Dose 3 200 3 200 mg/kgBW

  Comparison the number ber of T cells

• _ˉ
• Figure 1. Profile Relative Number
• ber of T Cells

  CD4 CD62L dan CD4 CD62Lˉ CD ˉ ˉ after

  CD4 CD62L dan CD4

  4 CD62L in threathment by ethanol extract of of propolis show

• Spleen Organ.
• that number of CD4 CD62L les less then T cells
• ˉ ˉ

  CD4 CD62Lˉ. CD62L is a m molecule can mediate naive T cells to periphe ipheral lymphoid organs, this happens initiation ion of immune response that decreased expressi ssion of CD62L, also lead to the decline in the num number of naïve cells (Rifa'i, 2011). It shows tha that the ethanol extract of propolis can activ tivated T cells

  CD4 CD62L , so T cells CD4

  4 CD62L lose CD62L molecules and change be become T cells

• ˉ. Increasing the number of ˉ. Increasing the number of

  CD4 CD62Lˉ. Increasing the number of memory cells happens at dose dose 2 shows that

• immunostimulatory effects on T on T cells CD4 . When the number of memory T c T cells increased the number of naïve T cells becom ome less and the opposite. Taheri et al., (2005) 2005) explain that propolis can respond the immune une system, for

  Figure 2. Changes of Propolis Extr xtract Ethanol

  example can increase the he activity of Treatment Toward the the Relative macrophages, increasing IL-1, I 1, IL-2 and IL-4. + + Number Of T Cells CD CD4 CD62L ;

  Dose 100 mg/BW can increas pr s proliferation T K=Control; D1=Dose

  1

  50 lymphocyte. Gu (2005) explain in that propolis mg/kgBW; D2=Dose e 2 100 can enhance the number of T l lymphocyte. T mg/kgBW; D3=Dose 3 200 3 200 mg/kgBW

• cells CD4 produce cytoknesused ed to activat

  cells CD8 , so T cells CD8 dif differentiate into cytotoxic T cells effector and m nd memory cells.

• Increased proliferation of T cells ells CD4 can be triggered by saponin compounds pounds, because saponins has the ability to incr ncrease cytokine

  IFNγ (Cheeke, 2010). IFNγ can

  IFNγ

  IFNγ

  IFNγ an stimulate the

  IFNγ up-regulation of MHC II expression ion, it cause T Figure 4. Profile Relative Numbe umber of Cells T

  ˉ ˉ cells deferentiated become T cells C CD4 (Lee et CD8 CD62L dan CD CD8 CD62Lˉ in al., 2008 and Shi et al., 2008) Spleen Organ

• + + + Population T Cells CD8 CD62L C in

  Spleen Organ. The results of the ana analysis using

  flow cytometry showed relative num numbers of T cells control treatment is 1.97%. . Treathment dose 1 is 1.81%. Dose 2 showed d the highest relative number of cells is equal to 3.03 o 3.03%. Dose 3 is 2.05%, relative number of cell ells decreased again at dose 2 (figure 4). Based on on the analysis of Gomes-Howell (figure 5) the relat elative number

  of CD8 CD62L showed that the there was no significant between control with dos dose 1 and 3. But dose 2 are significantly differe erent with all treatments. Ethanol extract of propo propolis with 4 different dose give different resul esults on the

  ˉ ˉ + ˉ

  activity of CD8 CD62L (Figure 6 e 6). Relative

  ˉ ˉ ˉ Figure 5. Changes of Propolis E s Extract Ethanol

• number T cells CD8 CD62L in control

  Treatment Toward d the Relative treathment is 11.84%. Dose 1 is 9.32% 9.32%, dose 2

  Number Of T Cells s CD8 CD62L ; is 7.30%, and dose 3 is 16.34%. %. Based on K=Control; D1=Dose ose

  1

  50 Tukey analysis (figure 6), relative num e number of T

• ˉ ˉ ˉ

  mg/kgBW; D2=Dos ose 2 100 cells CD8 CD62L showed that the there was no mg/kgBW; D3=Dose 3 200 3 200 mg/kgBW significant difference between cont ontrol, dose 1, dose 2 , but dose 3 has significantly ntly value from the other. Based on the relative num number T cells

  CD8 CD62L known that ethanol nol extract of propolis can increase the relative ve number of

• CD8 CD62L at dose 2 that is 100 100 mg/kg BW.

  This indicates that there is a mechani anism of cells

• homeostasis, because the activa tivated CD4 produce cytokines IFNγ and IL

  IFNγ

  IFNγ

  IL-2, which

• cytokines were used CD8 cells to to proliferate.
• Increase of T cells CD4 inf influence the
• activation of T cells CD8 .

  Figure 6. Changes of Propolis E s Extract Ethanol

  Treatment Toward d the Relative

  ˉ ˉ ˉ +

  Number Of T Cells ls CD8 CD62L ; K=Control; D1=Dose ose

  1

  50 mg/kgBW; D2=Dos ose 2 100 mg/kgBW; D3=Dose 3 200 3 200 mg/kgBW

• T cells CD8 can increase a e also influenced
• by the presence of CD4 tha that have been
• activated. Activated T cells CD CD4 had been previously can activated Th1 defe deferentiated, Th1 produce cytokine IFNγ and IL-2

  IFNγ

  IFNγ -2 (Rifa'i et al.,

  2008). By utilizing IL-2 CD8 have a higher

• affinity than the affinity of CD4 D4 (Rifa'i et al.,
• 2008). IL-2 produced from CD D4 addition is
• used to regulate itself and also us used by CD8 as
• 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

  Pourreza. 2005. Humoral immunity of broilers is affected by oil extracted propolis (OEP) in the diet, International Journal of Poultry Science, 4(6), 414-417.

  5. Lee, Y.H., Y. Ishida, M. Rifa’i, Z. Shi, K.

  10. Taheri, H.R., H.R. Rahmani, and J.

  bioregulator, Universitas Brawijaya Press, Malang.

  9. Rifa’i, M. 2011. Imunologi dan

  International Immunology, 20 (7), 937- 947.

  CD8

  8. Rifa’i, M., Z. Shi., S.Y. Zhang., Y.H. Lee., H. Shiku., K. Isobe dan H. Suzuki. 2008.

  Journal of Experimental Medicine, 200 (9), 1123-1134.

  7. Rifa’i, M., Y. Kawamoto, I. Nakashima, dan H. Suzuki. 2004. Essential role of CD8

  Ethnopharmacology , 79, 331–334.

  6. Murad, J.M., Calvi, S.A., Soares, A.M., Bankova, V., Sforcin, J.M. 2002. Effects of propolis from brazil and bulgaria on fungicidal activity of macrophages against paracoccidioides brasiliensis, Journal of

  Isboe, dan H. Suzuki. 2008. Essential role of CD8

  4. Krell, R. 1996. Propolis value added products from beekeeping fao agricultural service bulletin, Food and Agricultural Organization of The United Nation , 124, P. 157-194.

  Medical Science.

  tumor immunity by agaricus, propolis and paffia in mice. Suzuka Inc, University of

  3. Gu, Y. 2005. Antioxidant activity and anti-

• CD122
• regulatory T cells in the recovery from experimental autoimmune encephalomyelitis, The Journal of Immunology ,180 (2), 825-832.
• and CD8
• , but there is difference in function of efektor (Abbas, 2005).
• CD122
• regulatory t cells in the maintenance of t cells homeostasis, The
• CD62Lˉ. Ethanol extract of propolis can decrease the proliferation of T cells memory CD8
• CD12
• Regulatory T cells recognize activated T cells via conventional MHC class 1-αβTCR interaction become IL-10 producing active regulatory cells,
• 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.

  2. Cheeke, P.R. 2010. Actual and potential aplication of yucca schidigere and quillaja saponaria saponin in human and animal nutrition, J. Anim Sci, 77:1-10.

  Cellsular and molecular immunology fifth edition, Elsevier Inc, USA.

  REFERENCES 1. Abbas, A.K and Lichtman, A.H. 2005.

  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.

  ACKNOWLEDGEMENTS

  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

  CONCLUSIONS

  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

  a stimulant for cells proliferate.T cells CD8

• cells will lyse other cells infected by a similar antigen (Rifa'i, 2004).