Plant material and extraction
The dried plant material was mixed and macerated with absolute ethanol at a 1:20 ratio (100 g in 1 L solvent) for 7 days. Then the extract was filtrated through Whatman No 1 filter paper and then followed by rotor- evaporated the supernatant by using the BUCHI Switzerland Rotary Evaporator to remove the ethanol and to obtain concentrated, oily extract. The crude extracts were then kept in −20 °C.
MMDP leaves were collected from Probolinggo, East-Java, Indonesia and were identified and authenticated by biologists also the specimen be deposited in an official herbarium that is located at Department of Biology, Universitas Brawijaya (specimen No.0157/Taxonomy). The leaves (1.5 kg) were dried for 5 days, the dried plant material was powdered. The dried powder was subjected to extraction by maceration with 90% ethanol (1:20 ratio, 100 g in 1 L solvent) for 72 h. The maceration process was repeated three times in 24 h cycles. The resulting extraction was filtered through Whatman filter paper and then concentrated at ±60 °C under reduced pressure by using a rotary evaporator to obtain a solid form of the extract. The quercetin content from MMDP extraction was 1.15 μg/g dry weight that quantified by thin layer chromatography (TLC).
Animals & study groups
Female Balb/c mice, aged 8–10 weeks, weight between 20 and 22 g were group-housed in cages with wire-net floors in a room maintained at 24-25 °C and a relative humidity 50–55%. Mice were given normal drinking water ad libitum and fed a standard pellet diet during the experimental period. Mice were housed 5–6 to a cage with free access to food and water on a 12 h light/12 h dark cycle. The experiments were performed in accordance with the guidelines and approval (No.160-KEP-UB) of the Institutional Animal Care and Use Committee of Brawijaya University and followed institutional requirements concerning the care and handling of animals according to Guiding Principles for the Care and Use of Animals for Scientific Purposes in the Institutional Animal Care and Use Committee (IACUC). The mice were randomly divided into five groups, each containing 10 mice. Group1 received 100 μL of 50% ethanol-phosphate-buffered saline (PBS) by rectal administration. Group 2–5 received a single rectal administration of 0.5 mg of trinitrobenzene sulfonic acid (TNBS) in 50% ethanol. MMDP extract was dissolved in 100 uL PBS and group 3–5 orally received daily the MMDP extract either at 150, 300 and 600 mg/kg body weight.
TNBS-induced acute colitis in mice
Colitis was induced in mice by intra-rectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) by using the procedure described by Sang et al. [15]. Briefly, colitis was induced by the intra rectal administration of 0.5 mg TNBS (Sigma Chemical Co., St. Louis, MO, USA) dissolved in 50% ethanol. The volume of TNBS enema was 100 μl. To induce acute colitis, the TNBS was slowly injected into the lumen of the colon via a thin round-tip needle attached to a 1 mL syringe with mice under pentobarbital anesthesia following instillation, the animals were maintained in a head-down position for 2–3 min to prevent instillation leakage [22]. Mice were randomly divided into five groups (Fig. 1). Development of colitis was assessed daily by using an occult blood detection kit (Hemoccult). Mice experienced bloody diarrhea and a significant loss of body weight. The mice were then sacrificed at the end of 7 days. Colon tissue was removed and cleaned, then subjected to ELISA, flowcytometry and histological examination.
Clinical assessment of colitis
Body weight, diarrhea scores, and bleeding scores were assessed daily as previously described [23]. Body weight change was monitored. The body weights and occult blood test results were recorded.
Myeloperoxidase (MPO) activity
The activity of the enzyme MPO was used to assess the infiltration of neutrophils. Briefly, colonic proteins were extracted by lysing cells in 3 mM EDTA, 10% glycerol (pH 7.4), 200 mM NaCl, 1 mM phenyl methyl sulfonyl fluoride (PMSF), and 10 nM Tris. MPO levels were measured in the protein extracts by using a mouse MPO ELISA kit (Elabscience) according to the manufacturer’s instructions.
Histological score of colitis
For histological examination, the colonic tissue was fixed in 10% formalin, dehydrated, paraffin-embedded, processed, sliced into 4-μm-thick sections, and stained with hematoxylin and eosin. The microscopic cross-sections of the colons were histologically investigated. Histological changes were graded semi-quantitatively from 0 to 4 according to previously described criteria as follows: 0) no signs of inflammation. 1) Very low level of leukocyte infiltration. 2) Low level of leukocyte infiltration. 3) High level of leukocyte infiltration, high vascular density and thickening of the colon wall. 4) Severe ulceration, transmural infiltration, loss of goblet cells, high vascular density, and thickening of the colon wall. All slides were evaluated using light microscopy and scored by an independent pathologist blinded to the experimental groups.
Isolation and culture of mesenteric lymph nodes (MLNs) cells
MLN cells were isolated and transferred to ice cold sterile Hank’s balanced salt solution. The nodes were disrupted and passed through a nylon mesh (70 μm pore size). A 96-well plate was pre-coated overnight with anti-CD3 (2 μg/mL) in PBS. Single-cell suspensions of 106 cells/mL were incubated in RPMI 1640 with 10% fetal calf serum and 100 IU/mL penicillin/streptomycin for 72 h in the presence of anti-CD28 antibodies (eBioscience, San Diego, CA, USA). Cell cultures were maintained in a humidified incubator at 37 °C with 5% CO2. Culture supernatant was collected at 72 h and then assayed for cytokines (IL-10 and IL-17) by using ELISA kits, as per the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA).
Flow Cytometry and intracellular staining
All antibodies used for cell labeling were purchased from eBioscience. For intracellular cytokine measurement, MLN cells were stimulated for 5 h with PMA (1 μg/mL, Sigma Aldrich) and ionomycin (50 μg/mL, BD Biosciences) in the presence of monensin (0.1 mg/mL, Sigma Aldrich) and placed in a 37 °C and 5% CO2. MLN cells were washed with PBS and surface-labeled with anti-CD4 –FITC (Biolegend, Uithoorn, Netherlands) and anti CD25-PE (BD Biosciences). MLN cells were fixed and permeabilized (Cytofix/Cytoperm, BD Biosciences) and stained intra cellularly with anti-IL-17-PE (Biolegend, Uithoorn, Netherlands) and anti-FoxP3-PerCP (BD Biosciences). The stained cells were analyzed using FACS Calibur, and the data were analyzed using Cell Quest Pro software.
Statistical analysis
The data were reported as mean ± standard deviation. The statistical significance was evaluated by using one-way analysis of variance (p < 0.05), followed by a post hoc Tukey test.