Anti-Inflammatory Effects of Methanol Extract, Hexane, Ethyl Acetate, and Butanol Fraction of Piper crocatum Ruiz & Pav Leaves on Lipopolysaccharide-induced RAW 264.7 Cells

Lipopolysaccharide is a component of the cell wall of gram-negative bacteria, the polyclonal activator of the immune system, stimulates the release of various inflammatory mediators, including nitric oxide (NO), prostaglandin E2, proinflammatory cytokines such as IL-1, IL-6, IL-12, IL-18, TNF-α and TNF-β. Activated macrophages are the main effector cells in the host defense against bacteria through the production of nitric oxide (NO), which is cytotoxic to parasites. Nitric oxide (NO) and reactive oxygen species exert several modulating effects on inflammation and play a key role in regulating the immune response. NO is the main product controlled by nitric oxide synthase (NOS), such as iNOS (inducible nitric oxide synthase). iNOS is induced in macrophages so that its activation leads to organ destruction in several inflammatory and autoimmune diseases. 2,3


INTRODUCTION
Inflammation is a physiological response to stimuli such as infection and tissue injury. This stimulation causes the release of inflammatory mediators such as nitric oxide, histamine, serotonin, bradykinin, and prostaglandins which cause an inflammatory reaction in the form of heat, pain, redness, swelling, and accompanied by impaired function. 1 Lipopolysaccharide is a component of the cell wall of gram-negative bacteria, the polyclonal activator of the immune system, stimulates the release of various inflammatory mediators, including nitric oxide (NO), prostaglandin E2, proinflammatory cytokines such as IL-1, IL-6, IL-12, IL-18, TNF-α and TNF-β. Activated macrophages are the main effector cells in the host defense against bacteria through the production of nitric oxide (NO), which is cytotoxic to parasites. Nitric oxide (NO) and reactive oxygen species exert several modulating effects on inflammation and play a key role in regulating the immune response. NO is the main product controlled by nitric oxide synthase (NOS), such as iNOS (inducible nitric oxide synthase). iNOS is induced in macrophages so that its activation leads to organ destruction in several inflammatory and autoimmune diseases. 2

Extraction and Fractionation
Piper crocatum Ruiz and Pav dry powder (600 g) were macerated with methanol (MeOH) (3x2L) solvent for 48 hours. The macerate was evaporated in vacuo, and 93 g of thick extract was obtained. Eightythree grams of the viscous extract was fractionated with n-hexane, ethyl acetate and n-butanol as solvents, and each solvent was evaporated in vacuo with a rotary evaporator to obtain the thick fraction of n-hexane (22 g), ethyl acetate fraction (20 g) and viscous fraction n-butanol (8 g and BFPC) with concentrations of 100, 50, 25 g/mL and positive control (dexamethasone) with the concentration of 5, 2.5 g/mL, negative control, and normal control. Then it was stimulated using LPS (1 g/ mL) and then incubated again for 24 hours in a 5% CO2 incubator at 37oC. The amount of nitrite in the culture medium was measured as an indicator of NO production. The amount of nitrite was calculated using NO assay kit reagent (Catalog No: E-BC-K035-M). Then, 100 L of the culture supernatant was added to 100 L of NO reagent and then incubated for 10 minutes in a dark room. The absorbance was measured at 550 nm on a microplate reader. The nitrite concentration was calculated using the sodium nitrite standard curve. 14

Cell Viability
Cell viability test of RAW 264.7 on the administration of MEPC, EAFPC, HFPC and BFPC from red betel leaf showed a decrease in the number of live cells with increasing concentration. The concentration of the test substance is : 200; 100; 50; 25 and 12.5 g/mL. Dexamethasone as a positive control with a concentration of 20; 10; 5 and 2.5 g/mL.

Production of Nitric Oxide (NO)
The NO production test was carried out using the Nitric Oxide (NO) Colorimetric Assay Kit. Tests were carried out on MEPC, HFPC, EAFPC, and BFPC from red betel leaf at concentrations of 100, 50 and 25 g/mL, dexamethasone as a positive control at concentrations of 5 and 2.5 g/mL, negative control (LPS-induced cells), and standard control. (cells without LPS induction).
Graph of NO levels calculated from the n-hexane fraction, ethyl acetate fraction, butanol fraction, red betel leaf methanol extract and dexamethasone, normal and LPS-induced cells in RAW cells 264.7.

DISCUSSION
Cell viability test was carried out using the MTT method (3, 4, 5-dimethyltiazo l-2-1-il)-2,5-diphenyl tetrazolium bromide assay to determine the non-toxic concentration of the n-butanol, ethyl acetate, n-hexane and methanol extract of Piper crocatum leaves Ruiz and Pav against 264.7 RAW cells. MTT assay is one of the quantitative tests to determine safe concentrations and is not toxic to cells used in research. The viability test results from MEPC, HFPC, EAFPC, and BFPC from Piper crocatum Ruiz and Pav leaves showed that the concentrations that gave the viability percentage above 80% were 100, 50 and 25 g/mL were used as concentrations for the inhibition test of NO production. Dexamethasone as a positive control, the concentrations were chosen were 5 and 2.5 g/mL because they showed viability above 80%. 15,16 Production of Nitric Oxide (NO) The test of NO production in RAW 264.7 cells was carried out by inducing RAW 264.7 cells with lipopolysaccharide (LPS). Lipopolysaccharide is a substance derived from the outer membrane of gram-negative bacteria, which is an endotoxin that stimulates the release of inflammatory mediators such as NO, prostaglandins, cyclooxygenase. NO is a product produced by macrophages activated by LPS. The resulting NO production is measured as the concentration of nitrite in the culture medium, and NO is very easily oxidized to nitric (NO2) in the solution.
The formed NO2 is added with diazotizing reagent (sulfanilamide) in an acidic medium to form a diazonium salt that reacts with a coupling reagent to form a stable colored azo compound. 17 Cells induced with LPS released higher NO into the medium than cells not treated with LPS. 18 The treated cells showed a decrease in the amount of NO. Treatment of cells with MEPC, HFPC, EAFPC, and BFPC at concentrations of 100, 50 and 25 g/mL showed the ability to reduce NO production.