Anti-inflammatory effects of Anredera cordifolia and Piper crocatum extracts on lipopolysaccharide-stimulated macrophage cell line
Abstract
In this study, the anti-inflammatory potential of Anredera cordifolia and Piper crocatum extracts on lipopolysaccharide-induced murine macrophage cell line (RAW 264.7) was observed. Cell viability assay was performed with MTS assay. Parameters measured to determine the anti-inflammatory activity were interleukin-1beta (IL-1beta), tumor necrosis factor (TNF)-alpha, nitric oxide (NO) and IL-6. Both A. cordifolia and P. crocatum at concentration of 50 µg/mL in cell line resulted significant decrease in TNF-alpha level (250.3 and 242.5 pg/mL respectively). A. cordifolia showed significant decrease in IL-1beta level at 50 µg/mL and IL-6 level at 10 µg/mL, whilst P. crocatum showed significant decrease IL-1beta level in three concentrations with lowest level at 50 µg/mL. A. cordifolia showed lowest decrease in NO level at 50 µg/mL but not comparable with normal cells, whilst P. crocatum showed significant decrease in NO level at 50 µg/mL. This research revealed that A. cordifolia and P. crocatum possess the anti-inflammatory potential indicated by the inhibitory activity of the inflammatory mediators including, TNF-alpha, IL-1beta, IL-6, and NO.
Introduction
Inflammation is an important biological response to injury which has been documented in various diseases such as rheumatoid arthritis, inflammatory bowel disease, artherosclerosis, Alzheimer's disease and cancer (Fang et al., 2008). Several responsible markers are present in macrophage during inflammation such as reactive oxygen species (ROS), reactive nitrogen species (RNS), cytokines [Interleukin (IL)-1beta, IL-6, tumor necrosis factor (TNF)-alpha] and nitric oxide (NO), that mediates inflammation and prostaglandin (Jung et al., 2007).
Bacteria lipopolysaccharide (LPS) is known to play its role in increasing the cytokines level as inflammation mediator (Kim et al., 2005). LPS has pro-inflammatory property in its glycolipid that compose Gram-negative bacteria cell wall (Boots et al., 2008). Thus, macrophage and inflammatory mediators induced by LPS are used as targets in anti-inflammatory drug development (Zeilhofer and Brune, 2006; Dewi et al., 2015; Rusmana et al., 2015).
Anredera cordifolia (madeiravine) and Piper crocatum (red betel) are reported to contain bioactive compounds that possess medicinal properties. A. cordifolia possesses antibacterial (Tshikalange et al., 2005), antiobesity and antihypoglycemic (Wang et al., 2011), cytotoxic and antimutagenic (Yen et al., 2001), antidiabetic (Anh and Kim, 2005), and anti-inflammatory activities (MouraLetts et al., 2006).
This research was aimed to observe anti-inflammatory potential of A. cordifolia and P. crocatum extracts on LPS stimulated-murine macrophage cell line (RAW 264.7). The RAW 264.7 cell line is an appropriate model for evaluating and screening of anti-inflammatory agents from plant extract (Dewi et al., 2015; Rusmana et al., 2015; Widowati et al., 2016).
Materials and Methods
Preparation of plant extract
Leaves of A. cordifolia and P. crocatum were collected from the Traditional Medicine Research Center, Bogor, West Java, Indonesia. The plants were identified by the Research Center of Biology, Indonesia Institute of Science, West Java, Indonesia. Extraction of A. cordifolia and P. Crocatum were performed with maceration technique using 96%ethanol. Ethanol filtrate was filtered, and wastes were re-macerated in triplicate. Macerates were concentrated using 50°C evaporator until the pasta form product was obtained. The extracts were stored at -20°C (Widowati et al., 2013a; Widowati et al., 2013b).
Cell culture
The murine macrophage cell line RAW 264.7 (ATCC®TIB-71TM) was given by Biomolecular and Biomedical Research Center, Aretha Medika Utama. The cell lines were grown in Dulbecco's Modified Eagle Medium (DMEM) (Biowest L0104) supplemented with 10% fetal bovine serum (FBS) (Biowest S181H), 5 uL penicillin-streptomycin (iLabware l0018-100), and maintained at 37°C in humidified atmosphere and 5% CO2 until the cells were confluent. The cells were then washed, harvested using trypsin-EDTA (Biowest L0931-500) (Dewi et al., 2015; Rusmana et al., 2015; Widowati et al., 2016).
Viability assay
Cell viability was evaluated by MTS assay. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay (Promega, USA). Briefly, 100 μL cells in medium (DMEM supplemented with 10% FBS and 1% penicillin-streptomycin) were plated (5 x 103 cells per well) and incubated for 24 hours at 37°C in a humidified atmosphere and 5% CO2. The medium was discarded and then added with 90 uL new medium and 10 uL of A. cordifolia and P. crocatum extracts in DMSO at final concentrations (0.4, 2, 10, 50, 150, 250, and 500 ug/mL) in different plate in triplicate and incubated for 24 hours. Untreated cells were served as the control. The 20 uL MTS was added to each well. The plate was incubated in 5% CO2 at 37°C incubator for 4 hours. The absorbance was measured at 490 nm on a microplate reader (MultiSkan Go Thermoscientific). The data is presented as the percentage of viable cells (%). The viability assay was performed to determine the safe and nontoxic concentration for the next assay (Dewi et al., 2015; Rusmana et al., 2015; Widowati et al., 2016; Laksmitawati et al., 2016).
Pro-inflammatory activation of cells
The pro-inflammatory activation of cells was performed based on Khan et al. (1995) modified method. The cells were seeded in 6-well plates in density of 5 x 105 cells per well and incubated for 24 hours at 37°C in a humidified atmosphere and 5% CO2. The medium (DMEM supplemented with 10% FBS and 1% penicillin-streptomycin) then washed and supplemented with 1600 uL growth medium and 200 uL A. cordifolia and P. crocatum extracts in different concentrations (10, 50 and 75 ug/mL) in 1-2 hours prior to the LPS treatment. The 200 uL LPS (1 ug/mL) was added into the medium and incubated for 24 hours at 37°C in a humidified atmosphere and 5% CO2. The growth medium was taken for the next assay and centrifuged at 2,000 x g for 10 min. The supernatant was stored at -79°C for the NO, IL-6, IL-1beta and TNF-alpha concentration and inhibitory activity assay (Dewi et al., 2015; Rusmana et al., 2015; Widowati et al., 2016; Laksmitawati et al., 2016).
Measurement of TNF-alpha, IL-beta and IL-6, concentration and inhibitory activity
Biolegend ELISA kit was used to measure TNF-alpha (430901), IL-6 (431301), and IL-beta (432601) concentration. Briefly, antibody solution was added into each well of 96-well plates, and then incubated in 4°C overnight. After washing the plate, cell-free supernatant treated with A. cordifolia and P. crocatum extracts on cell lines, were added and then shaked for 2 hours. Antibody solution was added and incubated for 1 hour in orbital shaker. Avidin-HRP solution and TMB substrate solution was added to each well. TMB will be oxidated by peroxidase enzymes as indicated by blue color. Concentrations of TNF-alpha, IL-beta, and IL-6 were determined by comparing the OD of the samples to the standard curve. LPS-stimulated cells without A. cordifolia and P. crocatum extracts, were served as positive control. The normal cell was used as negative control (Dewi et al., 2015; Rusmana et al., 2015; Widowati et al., 2016; Laksmitawati et al., 2016).
Measurement of nitrite associated with NO concentration and inhibitory activity assay
The determination of nitrite associated with NO production was performed based on Abnova Kit (KA 1342) protocol. After pre-incubation of cell lines with LPS and A. cordifolia and P. crocatum extracts for 24 hours, the quantity of nitrite accumulated in the cell free supernatant was measured as an indicator of NO production. A 200 uL assay buffer was added in the blank well and 100 uL of standard solution with 100 uL assay buffer was added into the standard well. Briefly, 100 uL of cell medium was mixed with 100 uL 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-stimulated cells without extract was used as positive control. The normal cell was used as negative control (Dewi et al., 2015; Rusmana et al., 2015; Widowati et al., 2016; Laksmitawati et al., 2016).
Statistical analysis
All data were derived from three independent experiments. Statistical analysis was conducted using SPSS software (version 20.0). Data 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.
Results
Effect on viability of RAW 264.7
The cell line viability assay was the preliminary study to test cytotoxicity of A. cordifolia and P. crocatum extracts toward cell line. Viability was measured by MTS assay indicated by the conversion of yellow tetrazolium salt to form a purple formazan product. Percentage of viable cells was determined by comparing cells viability value of treatments to the control.
A. cordifolia and P. crocatum extracts at concentrations of 150, 250 and 500 µg/mL were toxic to cell line indicated by low viability (Table I). Whereas concentration of 0.4, 2, 10 and 50 µg/mL of both A. cordifolia and P. crocatum extracts showed high viability (>90%). Viable cells obtained at concentration of 10 and 50 µg/mL in both A. cordifolia and P. crocatum extracts, that appeared to reach normal level, makes such concentrations suitable for further analysis. Concentration of 50 µg/mL showed viability that close to normal (~100%), and starting at 150 µg/mL showed toxicity (<100%). Thus, range between 50-150 µg/mL was also chosen as safe concentration. Based on linier regression (data are not shown), concentration of 75 µg/mL in both A. cordifolia and P. crocatum extracts, showed high viability (>90%). Therefore, concentration of A. cordifolia and P. crocatum extracts used were 10, 50 and 75 µg/mL.
Table I: Effects of A. cordifolia and P. crocatum on viability of RAW 264.7 cell line
| Concentration (µg/mL) | Cell viability | |
|---|---|---|
| A. cordifolia | P. crocatum | |
| Control | 100.0 ± 4.8d | 100.0 ± 3.2d |
| 0.4 | 123.6 ± 3.9f | 129.6 ± 5.9f |
| 2 | 129.6 ± 5.9f | 126.5 ± 2.5f |
| 10 | 119.4 ± 0.5f | 113.9 ± 12.5e |
| 50 | 112.4 ± 1.3e | 112.0 ± 3.3e |
| 150 | 82.2 ± 1.1c | 65.3 ± 1.5c |
| 250 | 36.9 ± 3.4b | 16.6 ± 0.5b |
| 500 | 2.9 ± 1.1a | 1.6 ± 1.3a |
| Data are presented as mean ± SD of three replications; Superscript letter ( a-f ), in each column indicates significance different among concentration based on Duncan post hoc test with p<0.05 is considered as significantly different | ||
Effect on TNF-alpha level in LPS-induced cell line
A. cordifolia and P. crocatum extracts showed the inhibitory activity against TNF-alpha production based on the lower concentration of TNF-alpha compared to the positive control (LPS-stimulated cells free supernatant without extract).
Both A. cordifolia and P. crocatum extracts at concentration of 50 µg/mL in cell line resulted significant decrease TNF-alpha level (250.3 and 242.5 pg/mL). Results of both A. cordifolia and P. crocatum extracts at 50 µg/mL were comparable with negative control (235.3 pg/mL), which indicates both treatments possess good anti-inflammatory.
Effect on IL-1beta level in LPS-induced cell line
Inhibition the production of IL-1 is an important approach in finding the anti-inflammatory agent. A. cordifolia and P. crocatum extracts showed the inhibitory potential against IL-1beta production (Table II).
A. cordifolia and P. crocatum extracts decreased the IL-1beta level in LPS-induced cell line, which was significantly different compared to positive control (Table II). A. cordifolia showed lowest IL-1beta level (909.2 pg/mL) at 50 µg/mL, which was comparable to normal cell (890.2 pg/mL). Treatment with P. crocatum in three concentrations showed decreasing IL-1beta level which was comparable to normal cells. P. crocatum showed its lowest level at 873.4 pg/mL.
Table II: Effects of A. cordifolia and P. crocatum on TNF-alpha, IL-1beta, IL-6 and NO levels in RAW 264.7 cell line
| Treatment | TNF-alpha (pg/mL) |
IL-1beta (pg/mL) |
IL-6 (pg/mL) |
NO (pg/mL) |
|---|---|---|---|---|
| Negative control | 235.3 ± 16.3a | 890.2 ± 24.2a | 167.6 ± 40.6a | 6.0 ± 1.5a |
| Positive control | 491.5 ± 28.1d | 1110.5 ± 18.8c | 607.0 ± 46.2d | 33.4 ± 1.0f |
| A. cordifolia extract | ||||
| 75 µg/mL | 328.6 ± 14.0b | 987.2 ± 14.0b | 330.4 ± 12.8c | 33.2 ± 1.6f |
| 50 µg/mL | 250.3 ± 22.7a | 909.2 ± 19.6a | 234.7 ± 13.8b | 22.8 ± 2.1d |
| 10 µg/mL | 355.3 ± 24.5b | 928.2 ± 29.9b | 217.8 ± 14.7ab | 25.7 ± 0.6e |
| P. crocatum extract | ||||
| 75 µg/mL | 334.8 ± 23.2b | 896.4 ± 88.9a | 333.4 ± 37.5c | 9.9 ± 0.1b |
| 50 µg/mL | 242.5 ± 25.6a | 873.4 ± 11.1a | 196.0 ± 10.3ab | 6.3 ± 1.5a |
| 10 µg/mL | 411.7 ± 44.0c | 911.5 ± 11.2a | 189.0 ± 5.2ab | 13.0 ± 1.1c |
| Data are presented as mean ± SD of three replications; Superscript letter (a-d) in each column indicates significance different among treatments based on Duncan pos hoc test with p<0.05 is considered as significantly different | ||||
Effect on IL-6 level in LPS-induced RAW 264.7
Results showed LPS induced inflammation and increased IL-6 level in cell line which was indicated by high level of IL-6 in positive control (607.0 pg/mL) and significantly different compared to negative control (167.6 pg/mL). A. cordifolia at 10 µg/mL showed significant decreased IL-6 level (217.8 pg/mL), as well as P. crocatum at 10 and 50 µg/mL (189.0 and 196.0 pg/mL respectively). The results were also confirmed by the percentage inhibiton of A. cordifolia and P. crocatum extracts which peaked at 10 µg/mL. Both extracts were comparable to normal cell.
Effect on NO level in LPS-induced cell line
The positive control shows the highest concentration of nitrite concentration compared to the negative control and extract-treated cells (Table II). The percent of inhibition was determined by the value of positive control nitrite concentration minus the nitrite concentration of treatment divided to the nitrite concentration of positive control. Both A. cordifolia and P. crocatum extracts resulted lower NO than positive control (Table II). A. cordifolia showed the lowest NO level at concentration of 50.0 µg/mL (22.8 pg/mL), yet it was significantly different compared to normal cell (6.0 pg/mL). Whereas P. crocatum showed lowest NO level at 50.0 µg/mL (6.3 pg/mL), which was comparable with normal cell.
Discussion
In this study, A. cordifolia and P. crocatum extracts showed no toxicity to cell line at concentration in range between 10-150 µg/mL. Non toxicity 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 (Dewi et al., 2015; Rusmana et al., 2015; Widowati et al., 2016; Laksmitawati et al., 2016).
LPS successfully induced inflammation of macrophage cell line (RAW 264.7). Cytokines production as inflammation mediator are enhanced by LPS (Kim et al., 2005; Mahajna et al., 2014; Rusmana et al., 2015). LPS is one of components in outer membrane of Gram-negative bacteria as endotoxin that induces production of pro-inflammatory mediators such as NO, IL-1, IL-6, TNF-alpha, interleukins, prostanoids and leukotrienes (Mahajna et al., 2014; Dewi et al., 2015; Rusmana et al., 2015; Widowati et al., 2016; Laksmitawati et al., 2016).
Potential of A. cordifolia and P. crocatum extracts were observed through inflammatory markers such as TNF-alpha, IL-1beta, IL-6, and NO inhibitory activity assays in LPS-induced macrophage cell line (RAW 264.7). Both A. cordifolia and P. crocatum extracts of 50 µg/mL decreased TNF-alpha level in LPS-induced RAW 264.7, which was comparable to normal cell. The TNF-alpha is an important cytokine involved in inflammatory response via activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), cytokine and adhesion molecule inducer (Libby, 2002; de Cassia da Silveira E Sa et al., 2014). TNF-alpha is therefore used as an important target of anti-inflammatory agent screening (Boots et al., 2008; Dewi et al., 2015; Laksmitawati et al., 2016). TNF-alpha that exists in cascades, is blocked in presence of anti-inflammatory (Dinarello, 2010). Endogenous pyrogens consisting of TNF-alpha along with IL-1beta and IL-6, cause fever during inflammation following up-regulated inflammatory responses that later triggers production of acute phase reactants (Damte et al., 2011).
A. cordifolia showed lowest IL-1beta level at 50.0 µg/mL, which was comparable to normal cell. Whereas P. crocatum in three concentrations showed decreasing IL-1beta level which was comparable to normal cells. IL-1beta is a key role in acute and chronic inflammatory and autoimmune disorders. IL-1beta is prototypic pro-inflammatory cytokine that employs pleiotrophic effects on a variety of cells. IL-1beta is produced mainly by blood monocytes (Damte et al., 2011).
In this study, A. cordifolia and P. crocatum extracts at concentration of 10 ug/mL reduced IL-6 level in cell line that comparable to normal cell, with lowest IL-6 was obtained in treatment of P. crocatum. IL-6 is pleiotropic cytokine to modulate inflammatory response (Kostek et al., 2012). IL-6 is present in many cell types. IL-6 along with TNF-alpha and IL-1, is elevated in septic or aseptic inflammation, makes it appropriate target in prevention and treatment of inflammatory disease.
A. crocatum showed lowest NO level in cell line that almost exceed normal level, that indicates good anti-inflammatory activity. NO originate from additional inflammatory pathways promoted by TNF-alpha (de Cassia da Silveira E Sa et al., 2014). NO inhibitory activity is often used as appropriate target in anti-inflammatory 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 (Kang et al., 2011).
Active compounds from plants is documented to play important role in prevention and treatment of various diseases (Leontowicz et al., 2006). A. cordifolia has been reported in previous studies to posses medicinal properties (Moura-Letts et al., 2006; Sukandar et al., 2011; Yuziani et al., 2014; Wahjuni et al., 2014). Phytochemical analysis of A. cordifolia indicate that the leaves contain considerable amounts of saponins, alkaloids, and flavonoids (Astuti et al, 2011). It has been reported that flavonoid inhibits inflammatory mediators including TNF-alpha, IL-1beta and IL-6, IL-8, and COX-2 (Tunon et al., 2009; Serafini et al., 2010).
The result of present study showed anti-inflammatory properties of P. crocatum. There are only few studies regarding antimedicinal properties of P. crocatum. Wicaksono et al.(2009) reported P. crocatum methanol extract inhibits the growth of human breasts cancer (T47D) cells via inhibition of p44/p42 phosphorylation. Recent study shows P. crocatum leaves extract to act as anti-inflammation in Wistar rats with atherosclerosis through decrease of TNF-alpha and IL-6 levels (Wahjuni et al., 2016). Anti-inflammatory properties of P. crocatum might be correlated with compounds contained in the plants. Chromatographic analysis of P. crocatum shows flavonoids, alkaloids, polyphenolic compounds, tannins and essential oils. These compounds are known to have efficacy as antibacterial, anti-inflammatory, and anti-pyretic (Tunon et al., 2009; Serafini et al., 2010).
Conclusion
Extracts of A. cordifolia and P. crocatum possess anti-inflammatory potential indicated by inhibition of inflammatory mediators including TNF-alpha, IL-1beta, IL-6, and NO on LPS-induced macrophage cells.
Acknowledgement
This study was funded and supported by the Biomolecular and Biomedical Research Center, Aretha Medika Utama, Bandung, Indonesia for laboratory facilities and research methodology. We are thankful to Seila Arumwardana, Merry Afni, Hanna Sari W. Kusuma from Biomolecular and Biomedical Research Center, Aretha Medika Utama, Bandung, Indonesia for their valuable assistance.
References
Anh MJ, Kim JW. Identification and qualification of steroidal saponins in polygonatum species by HPLC/ESI/MS. Arch Pharm Res. 2005; 8: 592-97.
Astuti SM, Sakinah M, Andayani R, Risch A. Determination of saponin compound from Anredera cordifolia (Ten) steenis plant (Binahong) to potential treatment for several diseases. J Agric Sci. 2011; 3: 224-32.
Boots AW, Wilms LC, Swennen EL, Kleinjans JC, Bast A, Haenen GR. In vitro and ex vivo anti-inflammatory activity of quercetin in healthy volunteers. Nutrition 2008; 24: 703-10.
Damte DM, Reza MA, Lee SJ, Jo WS, Park SC. Anti-inflammatory activity of dichloromethane extract of Auricularia auricula-judae in RAW 264.7 cells. Toxicol Res. 2011; 27: 11-14.
de Cassia da Silveira E Sa R, Andrade LN, dos Reis Barreto de Oliveira R, de Sousa DP. A review on anti-inflammatory activity of phenylpropanoids found in essential oils. Molecules 2014; 19, 1459-80.
Dewi K, Widyarto B, Erawijantari PP, Widowati W. In vitro study of Myristica fragrans seed (Nutmeg) ethanolic extract and quercetin compound as anti-inflammatory agent. Int J Res Med Sci. 2015; 3: 2303-10.
Dinarello CA. Anti-inflammatory agents: Present and future. Cell 2010; 140: 935-50.
Fang SC, Hsu CL, Yen GC. Anti-inflammatory effects of phenolic compounds isolated from the fruits of Artocarpus heterophyllus. J Agric Food Chem. 2008; 56: 4463-68.
Jung CH, Jung H, Shin YC, Park JH, Jun CY, Kim HM, Yim HS, Shin MG, Bae HS, Kim SH, Ko SG. Eleutherococcus senticosus extract attenuates LPS-induced iNOS expression through the inhibition of Akt and JNK pathways in murine macrophage. J Ethnopharmacol. 2007; 113: 183-87.
Kang CH, Choi YH, Choi IW, Lee JD, Kim GY. Inhibition of lipopolysaccharide-induced iNOS, COX-2 and TNF-a expression by aqueous extract of orixa japonica in RAW 264.7 cells via supression of NF-kB activity. Trop J Pharmaceut Res. 2011; 10: 161-68.
Khan TZ. Wagener JS, Bost T, Martinez J, Accurso FJ, Riches DW. Early pulmonary inflammation in infants with cystic fibrosis. Am J Respir Crit Care Med. 1995; 151: 1075-182.
Kim AR, Cho JY, Zou Y, Choi JS, Chung HY. Flavonoids differentially modulate nitric oxide production pathways in lipopolysaccharide-activated RAW 264.7 cells. Arch Pharm Res. 2005; 28: 297-304.
Kostek MC, Nagaraju K, Pistilli E, Sali A, Lai SH, Gordon B, Chen YW. IL-6 signaling blockade increases inflammation but does not affect muscle function in the mdx mouse. BMC Musculoskeletal Disord. 2012; 13: 106-17.
Leontowicz H, Leontowicz M, Drzewiecki J, Haruenkit R, Poovarodom S, Park Y-S, Jung S-T, Kang S-G, Trakhtenberg S, Gorinstein S. Bioactive properties of snake fruit (Salacca edulis Reinw) and mangosteen (Garcinia mangostana) and their influence on plasma lipid profile and antioxidant activity in rats fed cholesterol. Eur Food Res Technol. 2006; 223: 697-703.
Libby P. Inflammation in atherosclerosis. Nature 2002; 420: 868-74.
Mahajna S, Azab M, Zaid H, Farich BA, Battah FFA, Mashner S, Saad B. In vitro evaluations of cytotoxicity and anti-inflammatory effects of Peganum harmala seed extracts in THP-1-derived macrophages. Eur J Med Plants. 2014; 5: 165-75.
Moura-Letts G, Villegas LF, Marçalo A, Vaisberg AJ, Hammond GB. In vivo wound-healing activity of oleanolic acid derived from the acid hydrolysis of Anredera diffusa. J Nat Prod. 2006; 69: 978-89.
Rusmana D, Elizabeth M, Widowati W, Fauziah N, Maesaroh M. Inhibition of inflammatory agent production by ethanol extract and eugenol of Syzygium aromaticum (L.) flower bud (clove) in LPS-stimulated Raw 264.7 cells. Res J Med Plant. 2015; 9: 264-74.
Serafini M, Peluso I, Raguzzini A. Flavonoids as anti-inflammatory agents. Proc Nutr Soc. 2010; 69: 273-78.
Sukandar EY, Fidrianny I, Adiwibowo IF. Efficacy of ethanol extract of Anredera cordifolia (Ten) steen is leaves on improving kidney failure in rats. Int J Pharmacol. 2011; 7: 850-55.
Tshikalange TE, Meyer JJ, Hussein AA. Antimicrobial activity, toxicity, and the isolation of bioactive compounds from plants used in sexually transmitted diseased. J Ethnopharmacol. 2005; 96: 515-19.
Tunon MJ, Garcia-mediavilla, MV, Sanchez-Campos S, Gonzales-Gallego J. Potential of flavonoids as anti-inflammatory agents: Modulation of pro-inflammatory gene expressions and signal transduction pathways. Curr Drug Metab. 2009; 10: 256-71.
Wahjuni S. Anti-hypercholesterolemia of Anredera cordifolia in hypercholesterolemia rat Wistar through decrease of malondialdehyde and 8-hydroxy-diguanosine. Indones J Biomed Sci. 2014; 8: 4-7.
Wahjuni S, Wita IW, Mantik AIN. Anti-inflammatory effect of red Piper crocatum leaves extract decrease TNF-alpha and IL-6 levels in Wistar rat with atherosclerosis. Bali Med J. 2016; 5: 51-56.
Wang L, Bang CY, Choung SY. Anti-obesity and hypolipidemic effects of Boussin-gaultia gracilis Miers var pseudo-baselloides Bailey in obese rats. J Med Food. 2011; 14: 17-25.
Wicaksono BD, Handoko YA, Arung ET, Kusuma IW, Yulia D, Pancaputra AN, Sandra F. Antiproliferative effect of the methanol extract of Piper crocatum ruiz & pav leaves on human breast (T47D) cells in vitro. Tropical J Pharm Res. 2009; 8: 345-52.
Widowati W, Mozef T, Risdian C, Yellianty Y. Anti-cancer and free radical scavenging potency of Catharanthus roseus, Dendrophthoe petandra, Piper betle, and Curcuma mangga extracts in breast cancer cell lines. Oxidants Antioxid Med Sci. 2013a; 2: 137-42.
Widowati W, Wijaya L, Wargasetia TL, Bachtiar I, Yelliantty Y, Laksmitawati DR. Antioxidant, anti-cancer and apoptosis-inducing effects of Piper extracts in HeLa cells. J Exp Integr Med. 2013b; 3: 225-30.
Yen GC, Chen HY, Peng HH. Evaluation of the citotoxicity, mutagenicity, and antimutagenicity of emerging edible plants. Food Chem Toxicol. 2001; 39: 1045-53.
Yoon WJ, Ham YM, Kim S-S, Yoo BS, Moon JY, Baik JS, Lee NH, Hyun C-G. Suppression of pro-inflammatory cytokines, iNOS and COX-2 expression by brown algae Sargassum micracanthum in RAW 264.7 macrophages. Eur Asia J Bio Sci. 2009; 3: 130-43.
Yuziani Y, Harahap U, Karsono K. Evaluation of analgesic activities of ethanolic extract of Anredera cordifolia (Ten) steenis leaf. Int J PharmTech Res. 2014; 6: 1608-10.
Zeilhofer HU, Brune K. Analgetic strategies beyond the inhibition of cyclooxygenases. Trends Pharmacol Sci. 2006; 27: 467-74.
