Anti-Inflammatory Potential of Gandarusa (Gendarussa vulgaris Nees) and Soursoup (Annona muricata L) Extracts in LPS Stimulated-Macrophage Cell (RAW264.7)

Inflammation is one of the important biological responses to injury. Anti-inflammatory is therefore proposed to treat both acute and chronic inflammation. Chemical compounds of various plants are widely used in treatment of inflammation. Objective: This study aims to evaluate anti-inflammatory potential of G. vulgaris extract (GVE) and A. muricata extract (AME) on LPS-stimulated murine macrophage cell line (RAW264.7). Cell viability assay to evaluate nontoxic concentration in cell line was performed with MTS assay. Parameters to determine anti-inflammatory activity between treatment group and non treated cells, were IL-1β, TNF-α, and IL-6 which was measured with Elisa, and NO level which was measured with nitrate/nitrite colorimetric assay. Both GVE and AME of 50 and 10 μg/mL showed high viability (>90%) and it was not significantly different compared to control, makes it suitable for treatment. GVE and AME of 50 μg/mL resulted low TNF-α level in RAW264.7(313.16pg/mL and 264.69 pg/mL respectively), as well as IL-1β level (903.53 pg/mL and 905.00 pg/mL respectively) and IL-6 (175.88 pg/mL and 219.13 pg/mL respectively). Whereas, GVE and AME of 75 μg/mL showed lower NO level (9.76 μM and 9.79 μM respectively) compared to untreated cells. This research revealed that GVE and AME possess the anti-inflammatory potential indicated by inhibition of inflammatory mediators including TNF-α, IL-1β, IL-6 and NO. Anti-Inflammatory Potential of Gandarusa (Gendarussa vulgaris Nees) and Soursoup (Annona muricata L) Extracts in LPS Stimulated-Macrophage Cell (RAW264.7) Dian Ratih Laksmitawati1, Ajeng Prima Prasanti1, Nadia Larasinta1, Gloria Agitha Syauta1, Rivanny Hilda1, Hesty Utami Ramadaniati1, Anisa Widyastuti1, Nadia Karami1, Merry Afni2, Dwi Davidson Rihibiha2, Hanna Sari W. Kusuma2 and Wahyu Widowati3* 1Faculty of Pharmacy, University of Pancasila, Jakarta, Indonesia 2Biomoleculer and Biomedical Research Center, Aretha Medika Utama, Bandung, Indonesia 3Medical Research Center, Faculty of Medicine, Maranatha Christian University, Bandung, Indonesia *Author for correspondence Email: wahyu_w60@yahoo.com JOURNAL OF NATURAL REMEDIES


Introduction
Ipomoea reniformis (IR) also called as merremia emarginata (Burm. f.) is a procumbent herb belonging to the family convolvulaceae. In India, it is commonly known as Undirkana and Mushakparni. The plant is widely distributed in India, Sri Lanka, Philippines, Malaysia, Tropical Africa and mainly grows in rainy and winter season. In India, it is found in Southern part mainly counting Chennai, and some places of Andhra Pradesh [1]. Traditionally, IR has been used to treat diverse clinical conditions ranging from pain; fever to neurological disorders [2]. IR has been claimed to be useful for inflammation, headache, fever, cough, neuralgia, rheumatism and also in liver and kidney diseases [3]. The powder of leaves is used as a snuff during epileptic seizures. Juice acts as purgative and the root is having diuretic, laxative actions and applied in the disease of the eyes and gums [4].

RAW264.7 Cells Viability Assay
Cell viability was performed with MTS ( 3 -( 4 , 5 -d i m e t h y l t h i a z o l -2 -y l ) -5 -( 3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium) assay (Promega, Madison, WI, USA) is a colorimetric method for determining the number of viable cells in proliferation or cytotoxicity assays.Briefly, 100 μL cells in medium (DMEM supplemented with 10% FBS and 1% penicillin-streptomycin) were plated in 96-well plate (5×10 3 cells per well) and incubated for 24 h at 37°C in a humidified atmosphere and 5% CO 2 . The medium was washed and added with 99 μL of new medium and 1 μL of GVE and AME in various concentrations (0.4, 2, 10, 50, 150, 250, and 500 μg/mL), and DMSO in different plate in triplicate then incubated for 24 h. Untreated cells were served as the control. The 20 μL MTS was added to each well. The plate was incubated in 5% CO 2 at 37°C incubator for 4 h. The absorbance was measured at 490 nm on a microplate reader (MultiSkan Go Thermoscientific). The data is presented as the percentage of viable cells (%) 14,15 . The viability assay was conducted to determine the safe and nontoxic concentration for the next assay.

Pro-Inflammatory Activation of RAW264.7 Cells
The pro-inflammatory activation of cells was performed based on modified method 7,8 . The cells were seeded in 6 well plate in density of 5×10 5 cells per well and incubated for 24 h at 37°C in a humidified atmosphere and 5% CO 2 . The medium (DMEM supplemented with 10% FBS and 1% penicillin-streptomycin) was washed and supplemented with 1600 μL of growth medium and 200 μL of extract in different concentration in 1-2 h prior to LPS treatment. The 200 μL LPS (1 μg/mL) was added into the medium and incubated for 24 h at 37°C in a humidified atmosphere and 5% CO 2 . The growth medium was taken It has been reported that bacterial lipopolysaccharide (LPS) is able to increase cytokines production as inflammation mediator 3,4 . LPS has pro-inflammatory property in its glycolipid which compose gram negative bacterial cell wall 5 . Macrophage and inflammatory mediators activated by LPS are appropriate targets in anti-inflammatory drug development 6,7 .
Natural phytochemicals play a significant role in drug discovery. Plant extracts contain bioactive chemicals and most of them found free from adverse effects 8 . These chemical compounds are widely used in treatment of inflammation 9 . Flavonoids found in plants have a great potential as anti-inflammatory agents. G. vulgaris and A. muricata are common plants to contain such compounds. It has been reported that both plants show significant anti inflammatory activity [10][11][12][13] .
The aim of this research is to evaluate antiinflammatory potential of G.vulgaris extract (GVE) and A. muricata extract (AME) on LPS stimulated-murine macrophage cell line (RAW264.7). The RAW264.7 cell line is an appropriate model for evaluating and screening of anti-inflammatory agents from plant extract 6 .

Plant Extract Preparation
Leaves of G. vulgaris and A. muricata were collected from Traditional Medicine Research Center (Balai Penelitian Tanaman Rempah dan Obat), Bogor, West Java, Indonesia. The plants were identified by herbarium staff, Research Center of Biology, Indonesia Institute of Science, West Java, Indonesia. Simplicia of G. vulgaris and A. muricata of 500 g were extracted with ethanol 96% using maceration technique. Ethanol filtrate was filtered, and wastes were re-macerated in triplicate. Macerates were concentrated using 50°C rotavapor to obtain extract. The extracts were stored at -20°C 14,15 . GVE and AME were used as the experiment.

Cell Culture
The murine macrophage cell line RAW264.7 (ATCC®TIB-71TM) was given by Biomolecular and Biomedical Research Center, Aretha Medika Utama, Bandung. The RAW264.7 cells were grown in Dulbecco's Modified Eagle Medium (DMEM) (Biowest L0104) supplemented for the next assay and centrifuged at 2000×g for 10 min. The supernatant was stored at -79°C for the NO, IL-6, IL-1β and TNF-α concentration and inhibitory activity assay.

Measurement of TNF-α, IL-β, and IL-6, Concentration and Inhibitory Activity
Biolegend ELISA kit was used to measure quantification of TNF-α (430901), IL-6 (431301) and IL-β (432601). Briefly, antibody solution was added into each well of 96 well plate, and then incubated in 4 0 C overnight. Cellfree supernatant after treated with GVE and AME in different final concentration (10, 50 and 75 μg/mL), were added and then shaked for 2 h. Antibody solution was added and incubated for 1 h in orbital shaker. Avidin-HRP solution and TMB substrate solution was added to each well. TMB will be oxidated by peroxidase enzymes that indicated performed blue colour. Concentration of cytokines were determined by comparing the OD of the samples to the standard curve. LPS-stimulated cells without GVE and AME, were served as positive control.
The normal cell was used as negative control 6,7 .

Measurement of NO Concentration and Inhibitory Activity
The determination of nitrite associated with NO production was performed based on Abnova Kit (No cat. KA 1342) protocol. After pre-incubation of RAW264.7 cells with LPS and GVE and AME for 24 h, the quantity of nitrite accumulated in the cell-free supernatant was measured as an indicator of NO production. Two hundred μL assay buffer was added in the blank well and 100 μL of standard solution with 100 μL assay buffer was added into the standard well. Briefly, 100 μL of cell medium was mixed with 100 μL assay buffer. The mixture was incubated at room temperature for 10 min. The absorbance at 540 nm was measured in a microplate reader (MultiSkan Go Thermoscientific). The quantity of nitrite was determined from sodium nitrite standard curve. The LPS-induced cell without extract was used as positive control. The normal cell was used as negative control 6,7 .

Statistical Analysis
All data was derived from three independent experiments. Statistical analysis was conducted using SPSS software (version 20.0). Value were presented as Mean± Standard Deviation. Significant differences between the groups were determined using the Analysis of variance (ANOVA) followed by Duncan post hoc Test.

Effect of G.vulgaris and A. muricata Extracts on Viability of RAW264.7 Cell Line
The RAW264.7 cell viability assay was the preliminary study to test the effect of GVE and AME toward RAW264.7 cell viability. Viability was measured by MTS assay based on the conversion of yellow tetrazolium salt to form a purple formazan product. Percentage of cells viability was determined by comparing cells viability value of treatments to the control. As shown in Table 1, cell treated with GVE and AME at concentration of 150, 250, and 500 µg/mL resulted low viability which indicates toxicity to RAW264.7, whereas concentration of 0.4, 0.2, 10 and 50 µg/mL of both GVE and AME showed high viability (>90%). Viable cells obtained at concentration of 10 and 50 µg/ mL in both GVE and AME, appeared to reach normal level (control), makes such concentrations suitable for treatment of RAW264.7 cells. Therefore, further analysis of GVE and AME uses concentration in range of 10 and 150 µg/mL.

Effect of G.vulgaris and A. muricata Extracts on TNF-α Level in LPS-Induced RAW264.7 Cell Line
TNF-α is a multi functional cytokine which can exert regulatory, inflammatory and cytotoxic effects on a wide range of lymphoid and non-lymphoid cells and tumor cells. GVE and AME showed the inhibitory activity against TNF-α production based on the lower concentration of TNF-α compared to the positive control (LPS-stimulated cells free supernatant without extract). As shown in Table 2, GVE and AME decreased TNF-α level compared to positive control. Treatment of AME at concentration of 50 µg/ mL in RAW264.7 resulted lowest TNF-α level 264.69 pg/mL among other treatments. Level of TNF-α in LPS-induced RAW264.7 treated with AME of 50 µg/mL, was significantly different compared to TNF-α level in positive control (Table 2). These results indicate AME of 50 µg/mL decreased TNF-α level to play its role as antiinflammatory. Whereas GVE at concentration of 50 µg/ mL also generated relatively low TNF-α level (313.16 pg/ mL). Both GVE and AME of 50 µg/mL showed significant difference compared to positive control, and resulted good inhibitory activity of TNF-α over positive control (36.46 and 46.82% respectively). AME 50 Ug/mL was the best activity to lower TNF-A level and comparable with negative control.

Effect of G.vulgaris and A. muricata Extracts on IL-1β level in LPS-Induced RAW264.7 Cell Line
IL-1 which refers to two proteins (IL-1α and IL-1β), is a potent immuno-modulator which mediates a wide range of immune and inflammatory responses including activation of B and T cells 16 . Inhibiting the production of IL-1 is important in finding the anti-inflammatory agent. GVE and AME showed the inhibitory potential against IL-1β production ( Table 3). Effect of GVE and AME level on IL-1β level in LPS-induced RAW264.7 is presented in Table 3.
As shown in Table 3, GVE and AME at concentration of 10, 50 and 75 µg/mL decreased IL-1β level in LPSinduced RAW264.7, which was significant compared to positive control and comparable with negative control. GVE at concentration of 50 µg/mL resulted highest decreased IL-1β level of 903.53 pg/mL.

Effect of G.vulgaris and A. muricata Extracts on IL-6 Level in LPS-Induced RAW264.7 Cell Line
IL-6 is one of the cytokines that possess biological activities due to acute inflammation 17 . IL-6 along  with TNF-α and IL-1 is elevated in septic or aseptic inflammation, makes it appropriate target in prevention and treatment of inflammatory disease 18 . In this study, GVE and AME decreased IL-6 compared to positive control, as shown in Table 4. The results showed that LPS induced inflammation and increased IL-6 level in RAW264.7 which was indicated by high level of IL-6 in positive control (599.83 pg/mL) and significantly different compared to negative control. Levels of IL-6 in treatment of GVE and AME were lower and significantly different compared to positive control. These results indicate both GVE and AME are able to decrease IL-6 in inflammation-induced cell. GVE and AME at concentration of 50 µg/mL showed significant decrease in IL-6 level (175.88 pg/mL and 219.13 pg/mL respectively), and significantly different than positive control.

Effect of G.vulgaris and A. muricata Extracts on NO Level in LPS-Induced RAW264.7 Cell Line
The positive control of this test showed the highest concentration of NO concentration compared to the negative control and extract treated cells ( Table 5). The percent of inhibitory activity was determined by the value of positive control nitrite concentration minus the nitrite concentration of treatment divided to the nitrite concentration of positive control. Although NO level of treatment group was higher than negative control, both GVE and AME significantly resulted lower NO than positive control (Table 4), which indicated GVE and AME reduce NO level in inflammation-induced cell. GVE and AME at concentration of 75 µg/mL showed lower NO level (9.76 µM and 9.79 µM respectively). Decrease in NO level by GVE and AME showed both treatments supress inflammation properly, makes it promising in reducing NO to play its role in inflammation 18 .

Discussion
The result of present study showed both GVE and AME extract showed no toxicity to RAW264.7 at concentration of 0.4, 2, 10, and 50 μg/mL. Non toxicity of substrate performed with MTS assay, was recorded by over 90% of viable cells.Viability test is crucial in pharmacology to determine adverse effect of bioactive substance in living organism prior to clinical use of drug or chemical compounds [19][20][21] .
In this study, LPS was used to induce inflammation in RAW264.7 cell line. It has been reported that bacterial LPS is able to increase cytokines production as inflammation mediator 3,4 . LPS compose outer membrane of gram negative bacteria as endotoxin that induces production of proinflammatory mediators such as NO, IL-1, IL-6, TNF-α, interleukins, prostanoids and leukotrienes 3,4 . LPS induces inflammation via Toll-like receptor 4 (TLR4) binding.TLR4 is a transmembrane protein that recognizes lipopolisaccharide specifically. TLR4 signaling pathway may activates Nuclear Factor Kappa B (NF-κB) and Activation protein 1 (AP-1) which later induces the secretion of proinflammatory mediators such as NO, TNFα, IL-1 and IL-12 22,23 .
Anti-inflammatory activities of GVE and AME were observed through markers such as IL-1β, TNF-α, NO and IL-6 inhibitory activity assays in LPS-induced macrophage cell line (RAW264.7). Both GVE and AME extract of 50 µg/mL resulted low TNF-α level in LPSinduced RAW264.7, with lowest level generated from AME. These results indicate both GVA and AME of 50 µg/mL play its role as anti-inflammatory, yet it did not exceed normal level. The TNF-α is an important cytokine involved in inflammatory response via activation of NF-κB, cytokine and adhesion molecule inducer 24,25 . The TNF-α is an important target of anti-inflammatory agent screening 5 . In presence of anti-inflammatory, TNF-α that exists in cascades is blocked 26 . Endogenous pyrogens consisting of TNF-α along with IL-1β and IL-6, cause fever during inflammation, following up-regulated inflammatory responses that later triggers production of acute phase reactants 27 . GVE and AME at concentration of 50 μg/mL reduced IL-1β level in RAW264.7. IL-1β is prototypic proinflammatory cytokine that exert pleiotrophic effects on a variety of cells and play key roles in acute and chronic inflammatory as well as autoimmune disorders. IL-1β is produced mainly by blood monocytes. IL-1β, TNF-α and IL-6, simultaneously promote fever during inflammation due to up-regulated inflammatory responses that later triggers production of acute phase reactants 5 .
In this study, GVE and AME at concentration of 50 μg/mL reduced IL-6 level in RAW264.7, with lowest IL-6 level was obtained in treatment of GVE. The IL-6 production has been detected in many cell types. Macrophages and monocytes are the primary source of cytokine during acute inflammation. IL-6 is pleiotropic cytokine to modulate inflammatory response 26,27 . IL-6 along with TNF-α and IL-1 is elevated in condition of septic or aseptic inflammation, makes it effective in prevention and treatment of inflammatory disease 18 .
The result of present study showed GVE and AME at concentration of 50 μg/mL reduced NO level in RAW264.7 cell, with lowest NO was obtained in treatment of GVE. Additional inflammatory pathways promoted by TNF-α, results nitric oxide (NO) 26,27 . NO inhibitory activity is frequently used as appropriate target in antiinflammatory agent screening. NO is responsible in host immune defense, vascular regulation, neurotransmission and other system in normal condition. Excess inducible NO Synthase (iNOS) is especially asssociated with various human diseases including inflammation 15,17 .
It has been reported that active compounds from plants play important role in prevention and treatment of various diseases 9,28 . Anti-inflamatory activity of leaf extract of G. vulgaris has been documented in previous studies. Phytochemical analysis of G. vulgaris extract revealed the presence of flavonoids glycosides, saponins, steroids, tannins and polyphenols. Anti-inflammatory effects are present due to inhibition of mediators in inflammation by glycosides or steroids 9 . According to Jothimanivannan et al. (2010), flavonoid content also play key role in anti-inflammatory acitivity of GVE 10 . Kim et al. (2004) reported flavonoid contained in plants possess cellular mechanism in anti-inflammatory activity by inhibiting eicosanoid that produces phospholipase A2, cyclooxigenase and lypoxigenase. Inhibition of these enzymes will reduce prostanoid and leucotrien level 11 . AME is effective for both acute and chronic inflammation. It significantly decreases both TNF-α and IL-1β levels in CFA-induced arthritis model 29 . Phytochemical test conducted on ethanolic extract of A. muricata indicates presence of alkaloids, saponins, flavonoids, tannins, triterpenes and steroid. Flavonoids have a great potential as anti-inflammatory agents. Flavonoids and tannins have been reported to inhibit prostaglandin synthesis [29][30][31][32] . Recent study showed certain flavonoids such as flavon, posses anti-inflammatory properties that regulates pro-inflammatory genes cyclooxigenase-2(COX-2), nitrite oxide synthase (NOS), and cytokines 11 . Other substances present in extract such as tannins, may give the synergistic effect to the flavonoids.

Conclusion
This research revealed that ethanol extracts of G.vulgaris and A. muricata possess the anti-inflammatory potential indicated by inhibition of inflammatory mediators including IL-1β, TNF-α, NO and IL-6.