SUPPLEMENTARY MATERIAL

Transcription

1 SUPPLEMENTARY MATERIAL Immune modulation properties of herbal plant leaves: Phyllanthus niruri aqueous extract on Immune Cells of Tuberculosis patient - in vitro study Denise Utami Putri 1, Ning Rintiswati 2, Marsetyawan HNE Soesatyo 3* and Sofia Mubarika Haryana 3 1 Faculty of Medicine, Graduate Program of Medical and Health Science, Universitas Gadjah Mada, Yogyakarta, Indonesia 2 Faculty of Medicine, Department of Microbiology, Universitas Gadjah Mada, Yogyakarta, Indonesia 3 Faculty of Medicine, Department of Histology and Cell Biology, Universitas Gadjah Mada, Yogyakarta, Indonesia Correspondence: *) Marsetyawan HNE Soesatyo Department of Histology and Cell Biology, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia. Tel : marshnes@ugm.ac.id Abstract Disease progression in Tuberculosis (TB) is depended on host s immune system. Phyllanthus niruri, a traditional herb, has long been used to boost immune system in Indonesian society. This study aimed to observe the potential role of P. niruri in inducing immune cells activity in TB patients by in vitro approach. Peripheral blood mononuclear cells (PBMCs) and macrophages were collected from active pulmonary TB patients. After stimulation with graded doses of P. niruri aqueous extract, cell proliferation, phagocytic activity, and nitric oxide (NO) release were analysed. P. niruri aqueous extract induced proliferation of PBMCs, increased NO release, and improved macrophages phagocytic activity. These effects were observed in a dose-dependent

2 manner. This may lead to further research for the potential role of P. niruri as immunomodulatory adjuvant therapy for TB patients. Keywords: Tuberculosis, Phyllanthus niruri, immune modulation, phagocytic activity, herbal plant

3 SUPPLEMENTARY MATERIALS 3. Experimental Sample Recruitment The study was approved by Ethical Committee Faculty of Medicine, Universitas Gadjah Mada (Ref No. KE/FK/121/EC/2015). All subjects provided written informed consent for participation in the study. Subjects were patients in Provincial Lung Clinic (BP4) Respira, Bantul and Primary Health Care centers in Yogyakarta, Indonesia. Inclusion criteria for patients were age years, newly diagnosed Tuberculosis (TB) as determined by a positive acid-fast bacilli (AFB) sputum smear, and 14 days of treatment with anti-tuberculous therapy (ATT). Exclusion criteria included >14 days of ATT, current pregnancy or lactation, corticosteroid use in the past 30 days, current use of cytotoxic or immunosuppressive drugs, known malignancy, untreated diabetes mellitus (DM), and presence of HIV coinfection. 20ml of peripheral blood samples were obtained by venipuncture from all 12 TB patients in heparinized tube, and immediately sent for assay. Extract Preparation Dried leaves of Phyllanthus niruri were obtained and specified by botanical expert at herbal manufacturing company, CV Merapi Farma, Yogyakarta, Indonesia between January and February The dried leaves was made into aqueous extract by maceration technique at Integrated Research and Testing Laboratory of Universitas Gadjah Mada which has been standardized and accredited nationally. For the study, extract in paste form was dissolved with DMSO solution as stock, and graded concentrations were made by diluting stock with culture medium into 25, 50, 100, 200 and 400 μg/ml. Analysis of Anti-oxidant Activity The aqueous extract sample was sent to Integrated Research and Testing Laboratory UGM for analysis of 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging activity, and its 50%

4 inhibitory concentration (IC 50 ) was measured. The study was done in triplicates using spectrophotometry UV-Vis method as per UGM Laboratory protocol. Peripheral Blood Mononuclear Cell (PBMC) and Macrophage Culture Subjects were divided into 2 groups randomly, 4 patients at the first group, and 8 patients at the second group. From the first group patients, mononuclear cells from peripheral blood were isolated and cultured for cell viability and proliferation assay, and from the second group patients, peripheral macrophage were grown for phagocytic activity and nitric oxide (NO) release assay. Among the first group, 3 patients were male and 1 was female, and mean age was 36 years old. PBMC was isolated from peripheral venous blood and treated by Ficoll-gradient density separation. After centrifugation, PBMC layer containing lymphocyte and monocyte were taken out using sterile pipette and resuspended in RPMI 1640 (Sigma) culture medium with addition of 10% FBS, 2% PenStrep, and 0,5% Fungizone. Number of PBMC was calculated using tryphane blue dye with 1:20 dilution. The cells were seeded into 96-well culture plate, and into 24-well culture plate with coverslips at a density of 1x10 5 cells/well, and incubated at 37 o C under 5% CO 2. Eight patients were initially recruited for the second group study, however, during the study, cells from 2 samples were contaminated and underwent technical error, therefore in the final result, only samples from 6 patients were included in analysis. Among these 6 patients, 4 were male and 2 were female, comprising of 66.67% and 33.33% male and female, respectively, with mean age of 32 years old. Peripheral Blood Mononuclear Cell (PBMC) Proliferation Assay Cell proliferation and viability assay were evaluated on human PBMC by principle of reduction of tetrazolium MTT (3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolium bromide) (Sigma) by reductase system. After incubation for 48 hours, mononuclear cells seeded in 96-well culture plate were supplemented with 25, 50, 100, 200 and 400 μg/ml P. niruri aqueous extract in duplicate. The RPMI medium was used as the no-drug control. After additional incubation for 24 hours at 37 o C under 5% CO 2, a solution of MTT (5mg/ml, 10µl per well) was added and further incubated at 37 o C in 5% CO 2 for 2 hours to allow formazan crystal formation in viable

5 cells. Medium was removed and 150μl DMSO was added to dissolve the purple formazan crystals formed in the viable cells. The plate was shaken for 30 minutes, and optical density (OD) determined at 550nm using spectrophotometer. Color intensity was proportional with the number of viable cells. Each result of the duplicate was expressed as a percentage of the mean duplicate of the nodrug control cells and mean was calculated for each measurement. Data was shown to be inhomogeny, thus non-parametric Kruskal-Wallis test was used to analyze the data (p=0.006). Assay of Macrophage Phagocytic activity Macrophages were cultured into 24-well culture plate with cover slips for 7 days until maturation. Cells were stimulated with graded doses of P. niruri aqueous extract (50, 100, 200, and 400 ug/ml), RPMI culture medium was used as the no-drug control. After additional incubation for 4 hours at 37 o C under 5% CO 2, latex beads were added into each well and further incubated for 1 hours. Supernatant were collected for nitric oxide release analysis, and stored in - 80 o C prior to assay. Cover slips were washed 3 times with PBS, and fixed with absolute methanol. 20% Giemsa dye was used for staining. Phagocytosis of latex beads was observed under light microscope and calculated in 4 different areas of cover slip as shown below: Phagocytic activity was expressed as phagocytic index (PI), calculated using the following formula: PI = (% phagocytic cells containing 1 bead) (multiplicity of infection) Data was distributed both normally and homogeny, and oneway ANOVA test was used to compare each mean of % phagocytic cells and multiplicity of infection (MOI, equals to mean number of beads divides by phagocytic cell containing beads). Statistical analysis showed significant difference for both mean number of phagocytic cells and MOI (p<0.001) (Supplementary material Table S1 and S2, respectively).

6 Nitric Oxide (NO) Release Assay The effect of P. niruri stimulation on nitric oxide (NO) release by macrophage induced with latex beads was determined by an assay of nitrite levels in the supernatants using modified Griess method by Green (Green et al., 1982). Chromogenic (Griess) reagent was made by adding 0.1% N-(1-napthyl)ethylenediamine dihydrochloride (NED) (Sigma) dissolved in sterile water, and 1% Sulfanilamide (Sigma) dissolved in 5% phosphoric acid, and mixed in equal volume. For standard nitric, NaNO 2 was dissolved in sterile water (2mM stock), and graded dilution was made in µm by adding culture medium. 100µl Griess reagent was put into 96-well plate with flat bottom and 100µl supernatant from macrophage culture or graded doses of nitric standard was added into the wells. Control medium only was used as blank. After 15 minutes reaction at room temperature, the OD of the pink color was measured at 550 nm in spectrophotometer. Standard curve was made based on simple linear regression from standard NaNO 2 reading. NO concentration was quantified based on the standard curve in µm units. References GREEN, L. C., WAGNER, D. A., GLOGOWSKI, J., SKIPPER, P. L., WISHNOK, J. S. & TANNENBAUM, S. R Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem, 126,

7 Table S1. Number of phagocytic cells (p<0.001) P. niruri dose: 0ug/mL (%) 50ug/mL (%) 100ug/mL (%) 200ug/mL (%) 400ug/mL (%) Mean ± SD 57.5± ± ± ± ±6.98

8 Table S2. Multiplicity of infection (p<0.001) P. niruri dose: 0ug/mL 50ug/mL 100ug/mL 200ug/mL 400ug/mL Mean ± SD 2.53± ± ± ± ±0.82

9 Figure S1. Percentage of peripheral blood mononuclear cell viability (p=0.006)

10 Figure S2. Phagocytic index (PI)

11 Figure S3. Macrophages stained with Giemsa, observed under light microscope in 400x magnifications. (A): Control macrophages without addition of P. niruri, (B), (C), and (D): with addition of 100, 200, and 400 µg/ml P. niruri, respectively. Cells were stimulated with latex beads. Arrows: Latex beads ingested by macrophage.

12 Figure S4. Nitric oxide regression curve

13 Figure S5. Activity of P. niruri as antioxidant and in increasing NO release