THE RED FRUITS (Pandanus conoideus) EXTRACT IMMUNOMODULATION ON THE MAMMARY TUMOR BEARING MICE T LYM

International Journal of Current Medical Science and Dental Research (IJCMSDR) Volume 1 Issue 1 ǁ May-June 2019 ǁ PP 01-05 ISSN: 2581-866X || www.ijcmsdr.com

THE RED FRUITS (Pandanus conoideus) EXTRACT IMMUNOMODULATION ON THE MAMMARY TUMOR BEARING MICE T LYMPHOCYTES AND MACROPHAGES Yulhasri1, Kusmardi Kusmardi2*, Aryo Tedjo3, Eva Zakiyah4, Suprianto5, Novianto5 1

Department of Biochemistry and Molecular Biology, Faculty of Medicine, Universitas Indonesia 2 Department of Anatomical Pathology, Faculty of Medicine, Universitas Indonesia 3 Department of Medical Chemistry, Faculty of Medicine, Universitas Indonesia 4 Laboratory of Yasmin, Cipto Mangunkusumo Hospital, Jakarta 5 Faculty of Pharmacy, Prof. Dr. HAMKA Muhammadiyah University, Jakarta

ABSTRACT: Objective: To investigate the effect of the red fruits (Pandanus conoideus) oil extract (RFOE) on activities of T lymphocytes (TL) derived from spleen and peritoneal macrophages (PM) of mammary tumor bearing mice in vitro. Materials and Methods: The RFOE was obtained by ethanol extraction consists of fraction 1-5 and used at 2 ppm concentrations. TL and PM were isolated, stimulated to proliferate while 24 hours of in vitro cultures. TL and PM activity were studied with the fraction 1 to 5, β- carotene, tocopherol, linoleic acid, RFO and negative control. Results and Conclusion: our study showed that fraction 2 and 3 of RFOE, β-carotene and tocopherol have effect in enhancing the CD8, CD 25 expression on T lymphocytes cultures; and fraction 1 on the CD 54 and CD64 expression of PM cultures.

KEY WORDS: T Lymphocytes, macrophages, red fruit, mammary tumor, Pandanus conoideus I. INTRODUCTION Herbal preparations are effectively and extensively used for their medicinal properties, and have become [1,2] increasingly popular worldwide.

Herbal medicines generally have fewer side effects than synthetic [3] compounds, and their effectiveness can be improved by modern pharmacological methods. Several plants used [4-5] in the traditional system of medicines, have been shown to modulate immune response.

Pandanus conoideus, commonly known as red Papua fruit, belongs to family Pandanaceae and is a well-known as traditional medicinal herbs from Papua. Various workers have studied the effector mechanism of Pandanus conoideus [4,5] (PC) extract using different models.

Extract of this fruits is reported to possess anti-inflammatory, [4,5] antipyretic, antioxidant and antiulcerogenic properties.

However, no studies have been reported on the effect of extract Pandanus conoideus on the activities of T lymphocytes (TL) derived from spleen and peritoneal macrophages (PM) of mammary tumor bearing mice in vitro.

II.

METHODS

Preparation of extract: The Extraction of the red fruits (Pandanus conoideus) oil (RFO) was performed using 0,5 grams of RFO, was added by 6 ml etanols and Butylated Hydroxy Toluene (BHT) with comparison 1:1, shaked 10 seconds, incubated in 85 oC temperature in water bath for 6 minutes. Samples were shake 3 minutes, vortex for 10 seconds. Saponification are conducted by enhance KOH. The supernatan were adding Petroleum Ether (PE): Diethyl Ether (DE) = 2:1, continued by chromatography analysis.

Carotenoid identification with thin layer chromatography (TLC): Carotenoid that has been rather seen condensed then carried over TLC plate. Eluent PE:DE: Acetone (40:10:10) (v/v) was transferred into chamber, incubated 10 minutes. After seen some layers at plate, lifted and dried, Rf are calculated, conducted preparative method by isolating every layer, carried over centrifuge tube by enhance PE:DE: Acetone (8 ml:2 ml: 2 ml), [6] centrifugation at 1500 rpm for 10 minutes. Supernatant are dried, the weight was deliberated. .

Isolation of TL of mammary tumor bearing mice: The spleen should be place in a sterile 60 mm culture dish containing 5 ml of serum free medium. The spleen is gripped at one end with sterile fine forceps and, using

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THE RED FRUITS (Pandanus conoideus) EXTRACT… sterile broad forceps, is squeezed progressively along its length, starting at the end held by the fine forceps. The empty “skin” of the spleen should be removed from the dish to waste. This is a clean and effective way of obtaining spleen lymphocytes which can be mastered with practice. The cell suspension should be pipeted up and down using a 5 ml pipet to disaggregate any large clumps, and subsequently placed into a sterile tube and allowed to settle for 2-3 min. The resultant supernatant should be taken into a fresh tube and centrifuged once for 7 min at 20 oC. The lymphocytes should be centrifuged once more after resuspension in serum-free medium and counted. An optional step which uses ammonium chloride to lyze spleen erythrocytes can be added after the first centrifugation step of spleen cells. When the supernatant has been decanted, the pellet is resuspended in the small amount of remaining medium and then 5 ml ammonium chloride buffer is added. After 5 minutes at room temperature, the supernatant should turn a clear red following lysis of the erythrocytes. The volume should be made up to 10 with serum-free medium before centrifuging the mixture at 200 g for 5 min.

The cell suspension should be added to the syringe and allowed to run into the nylon wool by briefly releasing the clamp. A volume of medium equal to thet of the cell suspension is applied, and again run into the nylon wool. Athen the assembly is incubated in a 37 oC incubator for 45-60 min. Elution of unbound TL is achieved by addaing more medium to the top of column. The eluted TL should be washed twich before use by [7-11] centrifugation at 200 g for 5 min.

Culture of PM: PM were culture a day before were washed twice in RPMI medium and incubated in a CO2 5% [10] incubator, 37 oC. .

6

Culture of TLs and PMs with extract and positive control: 500 μl of 2 x 10 cells/ml were cultured in 24 well microtiter plates. After 24 hours incubation, TLs and PMs were treated with 2 ppm of fraction 1 to 5 of RFOE, positive control (β- carotene, tocoferol, linoleic acid, RFO) and medium as negative control. All the above cell treatments were performed in triplicate. After incubation at 37ºC in 5% carbon dioxide for 48 h, cells were harvested for estimation of their activities by flow cytometry. [11] Viability assay: Viability of TL and PM was determined by the standard trypan blue dye exclusion test.

6 Approximately, 2 x 10 cells/ml of TL and PM were treated with two-fold diluted concentration of PC extracts, the positive control and the negative control with medium alone, for 2 days in a humidified incubator at 37°C with 5% CO. The total number of viable and non-viable cells was counted under a microscope with the help of a [12-14] haemocytometer, following staining by trypan blue. The percentage of viable cells was calculated. .

Flow cytometric analysis: Flow cytometric analysis was performed using a BD FACS Calibur. .

[13-14]

Statistical analysis: Each experiment with PBLs was run in triplicate, and the results are expressed as their mean± SEM. The data were analyzed using one-way analysis of variance, followed by Duncan multiple comparisons test. P<0.05 was considered significant.

III.

RESULTS

A. The characteristics of RFOE: The Rf value were obtained from TLC that produce 5 factions (Figure 1) were 0,6034, 0,6781, 0,7241, 0,8045 and 0,9770. From Figure 1 and Rf value were showed that faction 3, 4 and 5 very bunch up, whereas faction 2 have span of that very wide, whereas faction 1 is the most come near solvent.

Figure 1. The result of TLC of RFOE that showed 5 factions (fraction 1-5).

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THE RED FRUITS (Pandanus conoideus) EXTRACT… B. The influence of RFOE on TL.

Figure 2. The influence of 5 fractions of RFOE, some positive and negative controls on percentage of TL. ( 1. negative control. 2-6. Fraction 1-5 respectively, 7. Tocopherol. 8. Linoleic acid, 9. β-carotene standard, 10.

DMSO, 11. Crude extract of red fruits). From figure 2 and the result of Duncan’s test were indicated that RFOE, faction 2 of RFOE, tocopherol and ß-caroten have higher effect on enhancing the TL proportion of mammary tumor bearing mice. It also were showed that other fractions and negative control have lower effect on TL proportion.

In line with its influence toward the TL as totalize, RFOE also has effect on enhanching the percentages of CD3+CD4+ (T helper lymphocytes), data are not presented. Whereas its influence to percentage of CD3+CD8+ T supresor/cytotoxic) showed no effect, neither positive control nor negative control.

C. The expression of RIL-2Ra (CD25) on TL.

Figure 3. The influence of 5 fraction of RFOE, some positive and negative controls on the expression RIL-2Ra (CD25) on TL. (1. negative control. 2-6. Fraction 1-5 respectively, 7. Tocopherol. 8. Linoleic acid, 9. β-carotene standard, 10. DMSO, 11. Crude extract of red fruits). From figure 3 was indicated that faction 2, ß-carotene and

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THE RED FRUITS (Pandanus conoideus) EXTRACT… tocopherol have higher effect on enhanching the expression RIL-2Ra (CD25) on TL than other fraction and negative control.

D. The influence of RFOE on PM.

Figure 4. The influence of 5 fraction of RFOE, some positive and negative controls on the percentages of CD64 on the PM cultures. From figure 4 and the result of Duncan’s test were indicated that fraction 1 of RFOE and ß-carotene have higher effect on enhancing the PM proportion (CD64) of mammary tumor bearing mice.

The expression of ICAM-1 and RIL-2Rα PM.: The result of Duncan’s test (data were not presented) were indicated that RFOE, faction 2 of RFOE, tocopherol and ß-carotene have higher effect on enhancing the expression of ICAM-1 (CD54+) of mammary tumor bearing mice. It also were showed that the percentage of PM that express RIL-2Rα at PM cultures were enhanced by ß-carotene, tocopherol and faction 2 of RFOE.

IV.

CONCLUSION

In general, faction 2 of RFOE have equivalent ability with ß-carotene and tocopherol to immunomodulate TL and PM.

ACKNOWLEDGEMENT We highly appreciate University Indonesia for funding this research.

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