INTRODUCTION:

Inflammatory diseases are still one of the most important heath problems in the world¹. Inflammation has a considerable impact on mortality and morbidity rates worldwide, contributing to both physical and mental health problems. Chronic diseases are considered to be the biggest threat to human well-being by the World Health Organization (WHO)².

According to Furman et al. (2019), the mortality rate attributable to diseases such as ischaemic heart disease, cerebrovascular accident, cancer, mellitus type 2 diabetes, chronic renal disease, liver failure, autoimmunity, and neurological disorders exceeds 50%,while in Indonesia diseases involving inflammation account for the total number of deaths reached 61 percent ⁴.

Inflammation is a common and natural body reaction that responds to microbial infections or tissue damage. This process involves the migration of numerous cells and substances from the bloodstream to the damaged location, where they work together to eliminate the underlying cause of the disruption⁵.Inflammation is defined by five major indicators, which are sometimes referred to as cardinals characteristics. These symptoms include redness (rubor), growth (tumor), heat (calor), pain (dolor), and reduced function (functio laesa)⁶.

Inflammation occurs due to stimulation from Toll Like Receptors (TLRs) in recognizing pathogenic molecular patterns (PAMPs) and recognizing damage molecular patterns (DAMPs) such as lipopolysaccharide (LPS) that are on the cell surface or in endoplasmic vesicles⁷. After TLR has recognized these foreign pathogens, it will activate macrophages which produce inflammatory mediators including TNF-α, IL-1β, IL-6, nitric oxide, reactive oxygen species, prostaglandin E2⁸. This process causes the release of a precursor, namely arachidonic acid from the phospholipid membrane under the influence of the cyclooxygenase (COX-2) enzyme. Arachidonic acid is then converted into prostaglandin E2 (PGE2) whose role is either directly or via c-AMP. In the inflammatory process, if the level of NF-kb which acts as a pro-inflammatory cytokineis high, the prognosis will worsen⁹.

Inflammation will be characterized by an increase in various cytokines including tumor necrosis factor-α (TNF-α)¹⁰, and interleukin-6 (IL-6)¹¹. TNF-α is a cytokines that affects a variety of cell types. TNF-α plays an important role in establishing a healthy immune response in the context of normal physiology. It is capable of stimulating and activating the immune system, but excessive TNF-α production can be dangerous and can cause disease¹². TNF-α can trigger cellular responses that are achieved from inducing inflammatory gene expression through stimulation of cellular proliferation and differentiation to cellular activation such as apoptosis and necroptosis¹³. Interleukin-6 (IL-6) is a further significant cytokine in an acute inflammatory reaction that has both systemic and local impacts on the body⁵. In different human inflammatory disorders, such as rheumatoid arthritis, IL-6 plays a significant role in inducing inflammation. Specific antibodies targeting this receptor are used as therapeutic agents in the treatment of different types of arthritis¹⁴.Synthetic anti-inflammatory medications now on the market have potentially serious negative effects. Consequently, the creation of strong anti-inflammatory medications with fewer adverse effects from medicinal plant sources is required¹⁵.

Investigating medicinal herbs with anti-inflammatory characteristics is crucial in order to find substitute therapies with fewer adverse effects ¹⁶.Asteraceae family member Carthamus tinctorius Linn, also known as Safflower, is a traditional medicinal plant used by the people of South Sulawesi to cure measles empirically. In order to boost immunity, it is prepared by brewing with hot water¹⁷. The results of research conducted by Kim et al. (2013) Safflowers extract can be a potential anti-inflammatory agent because of its significant effect on inflammatory factors¹⁸. In a 2011 study, Lee et al. discovered that the ethanol extract derived from Safflower flowers had a substantial influence. It significantly increased the expression of Nrf2(Nuclear factor erythroid 2-related factor 2)/HO(Heme oxygenase)-1. Simultaneously, it inhibited the ROS/NF-kB pathway in human vascular endothelial cells generated by TNF-α¹⁹.

Although there has been research conducted on the effectiveness of Safflower, there is still limited information on its anti-inflammatory properties and mechanisms in animal models of inflammation. This study is aimed at providing evidence regarding the effectiveness of Safflower as an anti-inflammatory and its mechanism of action through TNF-α and IL-6 in animal models of inflammation.

MATERIALS AND METHODS:

Chemicals and reagents:

The materials used in this study were Safflower flowers, aluminum foil, 80% ethanol, filter paper (Whatmann®), 0.5% Na-CMC (Food Grade®), distilled water (Onemed®), CFA (Sigma®), EDTA (Merck®), handskun (MaxterTM) and ELISA RATs TNF-α and IL-6 (Bioassay Technology Laboratory®). In addition, rotary vacuum evaporators (Buchi®), cylinders (Pyrex®), Eppendorf tubes, Incubators (Infors HT®), analytical balances (Precisa®) and digital thickness gauges.

Animal:

The animals used were adult male mice (Mus musculus) balb/c strain (25-35 g). Mice were placed in a room with a temperature of 25 ± 2^oC with a light/dark cycle of 12 hours. The protocols for treating and using laboratory animals set forth by the Animal Care and Utilization Committee of the Faculty of Veterinary Medicine, Airlangga University, under protocol number 2.KE.089.07.2021 are followed in all animal experiments. A total of 40 mice were acclimatized for 7 days under a controlled environment (25±1°C, Rh 55±5%, and 12:12h light: dark cycle). Mice had a limitless supply to food and waterso they can consume it as needed or as they wish.

Collection and extraction of plants:

Safflower were collected from plantations in Amali District, Bone Regency, South Sulawesi Province, and confirmation/identification of the plant material was determined by the Materia Medica Garden Center Batu with voucher specimen number 074/545 /102.7-A. The extract was produced by maceration using 80% ethanol for 72 hours, with filtration carried out every 24 hours. The ratio of solvent (ethanol 80%) and simplicia (dried flowers of Safflower) is 5:1 (2,500 ml: 500 gr).Then the filtrate was collected and evaporated using a rotary vacuum evaporator at 50^oC. Concentrated extract yields 70.60 g (24.35%).

Phytochemical screening-flavonoid test:

Based on the chemical screening results, 2 mL of 80% ethanol was added, followed by 0.5 g of magnesium powder that looked to dissolve and three drops of concentrated HCl. Mg powder is used as a reducing agent when the reduction process is carried out in an acidic environment with the addition of strong HCl. The reduction of magnesium and concentrated HCl produces a red to bright red color, indicating the presence of flavanoids from the flavanol group.

Safflower's anti-inflammatory effect on the thickness of CFA-induced mice paw edema and measurement of TNF-α and Il-6 levels:

The test subjects used were 40 male balb/c strain mice aged six to seven weeks and weighing 25-35 grams. Mice were injected with 0.01 mL CFA (1 mg/mL in mineral oil) on the right leg plantar posterior part. The mice were randomized into five groups (n = 8) at random, including a negative control group induced by CFA and given 0.2 ml of 0.5% CMC. Group positive control was induced by CFA and given 50 mg of diclofenac sodium.Diclofenac sodium is one of the most often prescribed non-steroidal anti-inflammatory medications (NSAIDs) in the world²⁰. It is usually administered in dosages of 50 to 100 mg. It works by either stabilizing the lysosomal membrane or preventing the release of lysosomal enzymes²¹.Groups P1, P2, and P3 were induced by CFA and given a suspension of Safflower flower ethanol extract at doses of P1:100 mg/kg, P2:200 mg/kg, and P3:400 mg/kg, respectively. The extract is given orally once every day for 5 days.

Before being induced by CFA, male white mice that had been prepped for the study were starved for 18 hours while still being provided water. The initial thickness of the mice's right paw was measured first as the initial thickness (Vo) using a digital micrometer. The thickness gauge works by clamping this tool on the material or part you want to measure so that the results of the thickness measurement can be directly seen on the digital display screen of the thickness gauge.

The anti-inflammatory effect was examined using a CFA-induced mice right paw edema model. Subplantar injection of 0.01 ml of CFA was performed on the eighth day. Mice will exhibit swelling/edema and redness as indicators of inflammation after being triggered. The rationale behind using this approach is that inflammation, or edema, is a sign of inflammation and can be utilized as a gauge for a compound's anti-inflammatory properties²². After CFA induction, at 15 minutes the thickness of the edema on the right sole of the mice was measured again. This is done with the aim of knowing how much influence CFA has in triggering edema. The thickness of the edema was observed (using a digital micrometer) on days 1, 3, and 5 after CFA induction. The research data on plantar thickness values are expressed in mean ± SEM. Data were analyzed using the Post Hoc-LSD test with a 95% degree of confidence.

The TNF-α and IL-6 level were measured, six days after treatment.Animals were sacrificed to collect 0.8 mL blood from cardiac and put it into an EDTA tube. The collected blood was centrifuged at 3000 rpm for 15 minutes. Blood test with ELISA kit TNF-α and ELISA kit IL-6.

Statistic analysis:

The data obtained were analyzed statistically using the IBM SPSS Statistics 29 program. The method of analyzing the effect of Safflower ethanol extract and reducing levels of TNF-α and IL-6 in all treatment and comparison groups was analyzed using the One Way ANOVA statistical test, with a confidence level 95% and significance level 5% (α = 0.05). The use of anova is based on the assumption that the data is normally distributed, the variance is homogeneous and the sampling is random and each sample is independent. Then the follow-up analysis method after ANOVA is often called the Post Hoc test²³.The data obtained will also be analyzed using bivariate analysis to analyze the relationship between the variables edema thickness, TNF-α, IL-6. Analysis was carried out using the Spearman correlation test with a significance level of 0.05. Correlation is declared to have a weak relationship if the value of r = 0.00-0.25; moderate relationship if the r value = 0.26-0.50; strong relationship if the r value = 0.51-0.75; and the relationship is very strong/perfect if the r value = 0.76-1.00. If the p-value ≤α (0.05) means that the statistical calculation results are declared meaningful (significant).

RESULTS AND DISCUSSION:

Phytochemical analysis:

The chemical components found in plant extracts are identified by the phytochemical screening procedure. Phytochemical screening use a reagent to identify classes of substances, including terpenoids, flavonoids, alkaloids, tannins, and saponins. Examining the chemical makeup of the product is the first step towards developing new drugs derived from secondary metabolites. By changing to a reddish-crimson tint, the screening result for flavonoids in safflower extract employing the Mg²⁺ + HCl reagent is positive. This may indicate the presence of flavonoid chemicals in the plant extract under investigation.

Flavonoids are a group of naturally occurring chemical compounds found in various plants, known for their diverse pharmacological effects. Previous research has linked flavonoids to a range of health advantages, such as pain-relieving, antioxidant²⁴, anti-bacterial and anti-inflammatory properties²⁵. Hence, the presence of favorable outcomes in the flavonoid screening suggests that the plant extract holds promise for therapeutic purposes, namely in the treatment of inflammatory ailments. The scientific literature has thoroughly investigated the significance of flavonoids in the treatment of inflammatory diseases. Flavonoids have been found to hinder the synthesis of prostaglandins, which play a significant role in the body's inflammatory response²⁶.Additionally, these chemicals have the ability to hinder the activity of enzymes that play a role in the inflammatory pathways, such as cyclooxygenase (COX) and lipoxygenase (LOX)²⁷. Flavonoids can mitigate inflammation and relieve symptoms related to different inflammatory disorders through several mechanisms. Furthermore, studies have demonstrated that flavonoids can inhibit the synthesis of pro-inflammatory cytokines, including IL-6 and TNF-α. Cytokines have a crucial function in promoting the body's inflammatory response. Consequently, inhibiting their production can aid in diminishing the intensity of inflammation²⁸.

The effect of Safflower on Paw Edema, TNF- α, and IL-6 Levels:

Edema is a primary indicator of inflammation, which is the body's physiological reaction to various stimuli like injury, infection, or immune system activities²⁹.The development of edema after CFA induction will reach its peak on day 5 post-CFA inoculation and resolves on day 30 post-injection³⁰. Testing the anti-inflammatory effect obtained quantitative data on the average decrease in the thickness of the edema on the right paw of themice. Average measurements with a digital micrometer thickness gauge can be seen in Figure 1.

Figure 1. The average paw edema thickness of mice

Figure 1 illustrates a progressive rise in edema from days 1 to 3, followed by a decline on day 5. According to a study conducted by Noh et al. in 2022, it has been found that edema will begin immediately after CFA injection and will reach its highest level on day 5. The negative control group (KN) made this observation. On the fifth day, the thickness of mouse paw edema in the KP, P1, P2, and P3 groups decreased. However, there was no statistically significant difference between the positive control group and the treatment group.

Table 1. Post Hoc-LSD Test data for the thickness of mice sole edema

Groups	n	Mean	SD	Min	Max	Anova
Kn	8	5.4262^a	0.39	4.86	5.95
KP	8	4.5338^b	0.54	3.48	5.00	F=9.100
P1	8	4.3938^b	0.43	3.86	5.06	p=0.001
P2	8	4.3625^b	0.15	4.22	4.63
P3	8	4.6438^b	0.44	3.88	5.20

Keterangan:

*a, b Different superscripts in the same column indicate significant differences based on the LSD TEST p≤0,05

The data in Table 1, the results of the LSD test carried out on day 5, provide information on the thickness of the mouse paw edema. The results showed that the KN group was significantly different from the KP group and the treatment group at doses P1:100 mg/kg, P2:200 mg/kg, and P3:400 mg/kg. However, there was no significant difference between the KP group and the P1, P2, and P3 groups. The thickness of the edema on the right leg of the mice from high to low, namely the negative control (KN) was 5.43 mm, then the P3 group had an edema thickness of 4.64 mm, while the control group (KP) had an edema thickness of 4.53 mm. Group P1 has a thickness of 4.39 mm, and group P2 has a thickness of 4.36 mm. The edema thickness values in the negative control group were larger compared to the other groups, primarily due to the lack of anti-inflammatory activity or active components in the CMC Na suspension dose. The positive control group given diclofenac sodiumshowed a lower edema thickness compared to the P3 group and a higher edema thickness compared to the P1 and P2 groups. However, there was no statistically significant difference between the positive control group and the treatment group. Based on the research findings, various dosages of safflower flower extract demonstrated potential in reducing edema thickness. However, the dose of 200 mg/kg exhibited greater potential in lowering edema thickness.The reduction in edema thickness indicates that the anti-inflammatory effect is likely caused by the presence of flavonoid chemicals found in safflower flowers. Wang et al (2014) conducted research that found that kaempferol, a flavonoid found in the ethanol extract of Safflower flowers, can prevent the release of histamine and serotonin in the early phase and inhibit the release of various inflammatory mediators by blocking the action of prostaglandins in the late phase³¹.Anti-inflammatory drugs that work by inhibiting the ability of the hypothalamus to synthesize prostaglandins usually also have antipyretic effects³².

Flavonoids, which are a vast category of phenolic chemicals, possess significant promise as anti-inflammatory agents. They achieve this by effectively suppressing the regulation of enzymes and transcription factors associated with inflammatory mediators in the context of inflammation³³. Flavonoids exhibit anti-inflammatory effects by acting on various pathways, including the inhibition of COX enzymes and lipoxygenase, suppression of neutrophil degranulation, prevention of leukocyte accumulation, and inhibition of histamine, thereby leading to the suppression of eicosanoid and leukotriene production³⁴.Under normal conditions, leukocytes are able to move unrestricted over the whole length of the endothelium. Leukocyte attachment to the endothelium occurs as a consequence of several substances released by the endothelial cells and complement factors in the context of inflammation. Flavonoids can reduce leukocyte numbers and complement activation, resulting in decreased leukocyte adherence to the endothelium and a reduction in the inflammatory response in the affected tissue area³⁵.

TNF-α and IL-6 Levels:

The results of the average TNF-α and IL-6 levels in each group can be seen in Figure 2.

Fig. 2a. Mean level TNF-α

Fig. 2b. Mean level IL-6

The results from Figure 2a shows that the group with the lowest average TNF-α levels was P2, which received a dose of 200 mg/kg extract (126.96 pg/mL ± 11.80). The group P1, which received a dose of 100 mg/kg extract, had a slightly higher average TNF-α level (128.17 pg/mL ± 9.21). The P3 group, which received a dose of 400 mg/kg extract, had an even higher average TNF-α level (148.57 pg/mL ± 9.31). The KP group, which served as the positive control, had an average TNF-α level of 158.05 pg/mL ± 6.41. The KN group, which served as the negative control, had the highest levels of TNF-α at 162.61 pg/mL ± 10.63.

The results of the LSD test in this study regarding TNF-α levels provide insight into how various treatments affected TNF-α levels in the group studied. It is known that TNF-α is a cytokine that plays an important role in the inflammatory process, and its regulation is key in the therapy of various inflammatory and autoimmune conditions. Specifically, there was no significant difference between the negative control group (KN) and the positive control group (KP) (p=0.74), which means that the treatment given to KP, namely diclofenac sodium (NSAID), did not significantly change the levels TNF-α was compared with KN given Na-CMC which does not contain active compounds. This is interesting because diclofenac sodium is generally recognized as an effective anti-inflammatory drug through inhibition of the COX enzyme, which plays a role in prostaglandin synthesis, but does not appear to affect pro-inflammatory cytokines directly^20,36. The difference became real when comparing KP with the Safflower ethanol extract treatment group at doses P1: 100 mg/kg (p=0.035) and P2: 100 mg/kg (p=0.029), indicating that the treatment given to P1 and P2 was effective in reducing levels of TNF-α was significantly compared with the group given diclofenac sodium. This shows the potential anti-inflammatory effect of the extract through another mechanism that is able to reduce TNF-α.The KN group had a significant difference with P1: 100 mg/kg (p=0.016) and P2: 200 mg/kg (p=0.013), which again confirmed the effectiveness of Safflower extract in reducing TNF-α levels compared to the negative control group (KN).

The results of the LSD test data measuring TNF-α levels showed that there was no significant difference (p>0.05) between KN and KP (p=0.74). If seen from P1 (p=0.035) and P2 (p=0.029), KP groups is significantly different. This is possible because the positive control group was given diclofenac sodium, a type of non-steroidal anti-inflammatory drug (NSAID) which works by inhibiting the cyclooxygenase (COX) enzyme, which plays a role in the production of prostaglandins^20,36. However, its effect on the cytokine TNF-α may be insignificant or indirect. The cytokine TNF-α has a complex regulatory mechanism and can be influenced by many factors. The KN group also had a significant difference with P1 (p=0.016) and P2 (p=0.013). The P3 treatment group did not have a significant difference from the KN (p=0.32) and KP (p=0.48) groups.

Based on the data in Figure 2b, The lowest average IL-6 levels respectively were the Safflower Ethanol Extract group P2: 200 mg/kg (95.95 pg/mL ± 0.14), the P1 extract group: 100 mg/kg (103.79 pg/mL ± 0.16). P3 extract group: 400 mg/kg (105.18 pg/mL ± 0.12), positive control group (139.97 pg/mL ± 0.11), and finally negative control group (150.06 pg/mL ± 0.28). Based on the data, we can see that all groups given ethanol extract of safflower had lower IL-6 levels than the positive control group, indicating that the extract may have an anti-inflammatory effect that reduces IL-6 levels. Statistical analysis using the LSD (Least Significant Difference) test indicated that there was a significant difference between the negative control group and each treatment group (P1, P2, and P3), with a p value = 0.001 for each comparison. This shows that the reduction in IL-6 levels produced by administering the extract is significant compared to the negative group (KN). The positive control group (KP) had significant differences with each treatment group P1 (p=0.040), P2 (p=0.011), and P3 (p=0.033), which confirmed the effectiveness of the extract in reducing IL-6 levels even compared to Normal conditions will increase IL-6.

In this study, the levels of TNF-α and IL-6 in the treatment group had lower values than the positive group. This is because diclofenac sodium has a mechanism of action by non-selectively inhibiting the action of the cyclooxygenase enzyme (COX-1 and COX-2) which functions to accelerate the synthesis of prostaglandins and thromboxane from arachidonic acid³⁷.Prostanoid production is initiated by COX-1 and contributes to the early stages of inflammation; COX-2, on the other hand, is the primary cause of inflammation and keeps it going. Only about two hours following tissue damage can COX-2 expression be seen, and it will continue to rise while COX-1 expression will continue to fall ³⁸.The anti-inflammatory activity of flavonoids contained in the ethanol extract of Safflower flowers is not only through the COX enzyme inhibition mechanism but also through several other pathways, namely inhibiting lipoxygenae, inhibiting neutrophil degranulation, inhibiting the accumulation of leukocytes, inhibiting the production of histamine resulting in inhibition of eicosanoid and leukotriene biosynthesis³⁴.

The ethanol extract of Safflower effectively decreased NO, IL-6, and IL-1β secretion and suppressed p38, p65, p-JNK phosphorylation through inactivation of the MAPKs/NF-kB signaling pathway³⁹. Lee et al's research in 2021 showed that the ethanol extract of Safflower suppressed inflammation by inhibiting the ROS/NF-kB pathway and regulating the Nrf2/Heme oxygenase(HO)-1 signaling axis and exerting a protective effect against inflammation in TNF-α-induced human vascular endothelial cells¹⁹. HO is the enzyme responsible for breaking down the iron-containing molecule heme into protoporphyrin and iron. It catalyzes the rate-inhibiting steps of the reaction, generating ferrous ions, carbon monoxide (CO), and biliverdin (BV), which is then reduced by biliverdin reductase to form bilirubin.The endoplasmic reticulum (ER) contains heme oxygenase-1 (HMOX1 or HO-1). A single transmembrane domain located at the protein's carbonyl (C-terminal) terminus anchors it in the ER membrane⁴⁰. HO-1 is a target gene for nuclear factor erythroid 2-related factor 2 (Nrf2), which has been shown to protect against various pathologies ¹⁹. The Nrf-2/HO-1 signaling pathway protects the body from oxidative damage. On a transcriptional level, Nrf-2 can regulate the HO-1 gene's expression. In addition, when LPS stimulates RAW 264.7 cells, HO-1 can lessen the production of inflammatory molecules linked to iNOS, including IL-1, TNF-α, and MCP-1. This complementary mechanism protects cells from oxidative damage ⁴¹. This shows that the HO-1 induction effect can reduce levels of IL-6 and TNF-α⁴².Decreased expression of HO-1 will result in greater release of IL-6 from activated macrophages, while overexpression of HO-1 will decrease the synthesis of several pro-inflammatory cytokines, including IL-6 and TNF-α⁴³.

Correlation of Edema, TNF-α, and IL-6

Table 2. Correlation of Edema, TNF-α, and IL-6

Variabel	r	p
Edema - IL-6	0,321	0,044
TNF-α - IL-6	0,366	0,020
Edema - TNF-α	0,102	0,531

This study aims to determine the relationship between signs of inflammation in the form of edema thickness in the legs of mice on the fifth day and levels of TNF-α and IL-6 to see the response of ethanol extract of Safflower flowers to mice induced by CFA. In this study, the correlation between edema and IL-6 had a correlation coefficient r of 0.321, indicating a moderate positive relationship between edema thickness and IL-6 levels. The p value of 0.044 indicates that this relationship is statistically significant. This means that in the present study, increased edema thickness tended to be associated with increased IL-6 levels, indicating that IL-6 may play a role in the inflammatory process that causes edema. The correlation test between TNF-α and IL-6 with a correlation coefficient r of 0.366 shows that there is a moderate positive relationship between TNF-α and IL-6 levels. The p value of 0.020 indicates that this relationship is also statistically significant. This shows that increased levels of TNF-α are associated with increased levels of IL-6, indicating an interplay between these two proinflammatory cytokines in the context of inflammation. The correlation test between edema and TNF-α with a correlation coefficient r of 0.102 shows a weak and positive relationship between edema thickness and TNF-α levels. The p value of 0.531 indicates that this relationship is not statistically significant, which means there is no strong evidence in this study that TNF-α plays a direct role in edema formation, or that this relationship may be influenced by other factors not measured in this study.

Overall, these results indicate that IL-6 has a stronger and statistically significant association with edema thickness than TNF-α. The positive relationship between TNF-α and IL-6 also confirms their role in the inflammatory response, although their relationship to edema directly may be different and complex. These results are important for understanding the mechanisms of inflammation and designing possible therapeutic strategies to overcome inflammation by targeting specific cytokines.

CONCLUSION:

The ethanol extract of Safflower has potential anti-inflammatory activity by reducing levels of TNF-α, IL-6 and reducing edema of the right paw of mice CFA-induced.

CONFESSION:

The authors thank the Ministry of Research, Technology and Higher Education of the Republic of Indonesia and the Faculty of Medicine, Universitas Airlangga.

CONFLICT OF INTEREST:

Nothing has been announced.

REFERENCES:

1. Manvar MN, Desai T, Desai TR. In-vitro Anti-inflammatory and Anti-Arthritic Activities of Fruits of Vernonia anthelmintica Willd. (Asteraceae). Pharm Res. 2014; 4: 186-188.

2. Pahwa R, Goyal A, Jialal I. Chronic Inflammation. 2022nd ed. StatPearls Publishing, Treasure Island (FL); 2021. Accessed June 8, 2022. https://www.ncbi.nlm.nih.gov/books/NBK493173/#_article-19530_s2_

3. Furman D, Campisi J, Verdin E, et al. Chronic inflammation in the etiology of disease across the life span. Nat Med. 2019; 25(12): 1822-1832. doi:10.1038/s41591-019-0675-0

4. KemenkesRI-P2PTM. Rencana Aksi Kegiatan Pengendalian Penyakit Tidak Menular. Published online . 2017.

5. Kumar V, Abbas AK, Aster JC, Perkins JA. Robbins Basic Pathology. 2018.

6. Hannoodee S, Nasuruddin DN. Acute Inflammatory Response. StatPearls Publishing, Treasure Island (FL); 2022. Accessed June 8, 2022. https://www.ncbi.nlm.nih.gov/books/NBK493173/#_article-19530_s2_

7. Cronkite DA, Strutt TM. The regulation of inflammation by innate and adaptive lymphocytes. J Immunol Res. 2018; 2018. doi:10.1155/2018/1467538

8. Mustika A, Fatimah N, Sari GM. The self-nanoemulsifying drug delivery system of Petiveria alliacea extract reduced the homeostatic model assessment-insulin resistance value, interleukin-6, and tumor necrosis factor-α level in diabetic rat models. Vet World. 2021; 14(12): 3229-3234. doi:10.14202/vetworld.2021.3229-3234

9. Sudoyo AW, Setiyohadi B, Alwi I, K. Simadibrata M. Buku Ajar Ilmu Penyakit Dalam. In: Interna Publishing. Jilid 1.; 2009: 1423+45.

10. Dhalendra G, Satapathy T, Roy A. Animal Models for Inflammation: A Review. Asian J Pharm Res. 2013; 3(4): 207-212.

11. Mustika A, Fatimah N, Safitri I, Susanti N, Noor NS. Clinacanthus nutans L Extracts Reduce the Serum Tumor Necrosis Factor-α, Malondialdehyde, and Interleukin-6 Levels and Improve the Langerhans Islet Area in Diabetic Rat Models. Clin Med Insights Endocrinol Diabetes. 2023; 16: 1-7. doi:10.1177/11795514231196462

12. Jang DI, Lee AH, Shin HY, et al. The role of tumor necrosis factor alpha (Tnf-α) in autoimmune disease and current tnf-α inhibitors in therapeutics. Int J Mol Sci. 2021; 22(5): 1-16. doi:10.3390/ijms22052719

13. Wajant H, Siegmund D. TNFR1 and TNFR2 in the control of the life and death balance of macrophages. Front Cell Dev Biol. 2019; 7(May): 1-14. doi:10.3389/fcell.2019.00091

14. Abbas AK, Lichtman AH, Pillai S. Basic Immunology Functions and Disorders of the Immune System Fifth Edition. 2016. doi:10.1016/B978-0-323-39082-8.01001-2

15. Anbarasi A, Vidhya R, A, R. Evaluation of In Vitro Anti-Inflammatory Activity of Tephrosia purpurea (Seed). Asian J Pharm Res. 2015; 5(2): 83. doi:10.5958/2231-5691.2015.00012.x

16. Shinde PP, Khule SD, Sonawane S, Shelke S. Analgesic activity and anti-inflammatory activity of methanolic extract of plant Sida cordata in carrageenan-induced paw edema in rats. Asian J Pharm Res. 2021; 2021: 143-146. doi:10.52711/2231-5691.2021.00027

17. Hamsidi R, Widyawaruyanti A, Hafid AF, et al. In vitro antimalarial activity of chloroform, n-butanol, and ethyl acetate fractions of ethanol extracts of carthamus tinctorius Linn. Flowers. Asian J Pharm Clin Res. 2018; 11(2): 121-123. doi:10.22159/ajpcr.2018.v11i2.15856

18. Kim DH, Hwang EY, Son JH. Anti-Inflammatory Activity of Carthamus tinctorious Seed Extracts in Raw 264.7 cells. J Life Sci. 2013; 23(1): 55-62. doi:10.5352/jls.2013.23.1.55

19. Lee YJ, Lee YP, Seo CS, et al. The modulation of Nrf-2/HO-1 signaling axis by carthamus tinctorius L. Alleviates vascular inflammation in human umbilical vein endothelial cells. Plants. 2021; 10(12): 1-15. doi:10.3390/plants10122795

20. Kaplan AA, Yurt KK, Deniz ÖG, Altun G. Peripheral nerve and diclofenac sodium: Molecular and clinical approaches. J Chem Neuroanat. 2018; 87(July 2017): 2-11. doi:10.1016/j.jchemneu.2017.08.006

21. Gorla US, Savithri M, Rao GSNK, Niharika Y, Sathya PDS, Harika V. Evaluation of anti-inflammatory activity of Hydroalcoholic extract of Ananas cosmosus fruit peel by HRBC membrane stabilisation. . Asian J Pharm Res. 2018; 8(1): 33. doi:10.5958/2231-5691.2018.00006.0

22. Safitri RA, Rahayu MP, Widodo PG. Uji Aktivitas Antiinflamasi Ekstrak Batang Karamunting (Rhodomyrtus tomentosa) Terhadap Tikus Jantan Galur Wistar Test of Antiinflamatory Activity Extract of Karamunting ( Rhodomyrtus tomentosa ) Stem to Male Rat Wistar Strain. J Surya Med. 2023; 9 (1): 330 – 334. https://doi.org/10.33084/jsm.v9i1.5202

23. Riadi S, Roswandi I, Kurnia TE, Ff IR. Analisa Pengaruh Penambahan Polydon Terhadap Ketahanan Fisik Dan Perbedaan Kualitas Supplier. J Teknol UMJ. 2021; 13(2): 179-192.

24. Meena AK, Singh U, Yadav AK, Singh B, Rao MM. Pharmacological and Phytochemical Evidences for the Plants of Wedelia Genus Vitex - A Review. Int J Pharm Clin Res. 2011; 2(1): 1-9.

25. Priyanka N, Brahmeswari P, Babu GR, Sowjanya M, Ramayyappa M. Evaluation of Anti-bacterial and Anti-inflammatory Activities of Ethanolic Extract of Hibiscus hirtus Linn. Leaves. Asian J Pharm Res. 2022: 5-10. doi:10.52711/2231-5691.2022.00002

26. Jaseela K, Kumar A, Babu DV, Swamy VBN. Studies on Anti-depressant and Anti-inflammatory Activities of Curcuma aromatica Rhizome in Experimental Animal Models. Asian J Pharm Res. 2016; 6(2): 79. doi:10.5958/2231-5691.2016.00014.9

27. Al-Khayri JM, Sahana GR, Nagella P, Joseph B V., Alessa FM, Al-Mssallem MQ. Flavonoids as Potential Anti-Inflammatory Molecules: A Review. Molecules. 2022; 27(9). doi:10.3390/molecules27092901

28. Mayangsari E, Mustika A, Nurdiana N, Ardhayudicva S. Potency antiinflammatory of ethanol extract gel of Kepok banana peel (Musa balbisiana). Med J Malaysia. 2023; 78(4): 488-490.

29. Goli V, Vijay Bhaskar K, Prasad Macharla S, Suvarna Devi P, Ramchander T. www.asianpharmaonline.org Effects of Anti-Inflammatory Activity of Mimosa pudica. Asian J Pharm Res. 2011; 1(3): 69-71.

30. Noh ASM, Tan DC, Khir NAM, Shafin N, Ismail CAN. A Review on Complete Freund’s Adjuvant-Induced Arthritic Rat Model: Factors Leading to its Success. IIUM Med J Malaysia. 2022; 21(4): 3-12. doi:10.31436/imjm.v21i4.2026

31. Wang Y, Chen P, Tang C, Wang Y, Li Y, Zhang H. Antinociceptive and anti-inflammatory activities of extract and two isolated flavonoids of Carthamus tinctorius L. J Ethnopharmacol. 2014; 151(2): 944-950. doi:10.1016/j.jep.2013.12.003

32. Karthik KNS, Kumar KR. Evaluation of Analgesic, Anti-Inflammatory of stem bark ethanolic Extract of Michelia champaca Linn. Asian J Pharm Res. 2017; 7(2): 94. doi:10.5958/2231-5691.2017.00016.8

33. Sanjaya GRW, Linawati NM, Arijana IGKN, Wahyuniari IAI, Wiryawan IGNS. Flavonoid dalam Penyembuhan Luka Bakar pada Kulit. J Sains dan Kesehat. 2023; 5(2): 243-249. doi:10.25026/jsk.v5i2.1247

34. Hayatillah R, Hapsari WK. Anti - Inflamasi Tanaman Kedondong (Spondias Dulcis G. Forst.): Review. 2023;2(4):63-69.

35. Pradini NKV, Hardiana I, Raningsih NM. UJI Ekstrak Etanol Bunga Kenanga (Cananga odorata) Terhadap Penurunan Edema Pada Mencit Putih Jantan (Mus musculus). J Farm Kryonaut. 2023; 2(2): 123-130. https://doi.org/10.59969/jfk

36. Alfaro RA, Davis DD. Diclofenac. 2023; 5: 1-6.

37. Humaira AF, Cahyani DF, Setyowati KA, Fitri MK, Hafizah Q, Hasina R. Uji Efektivitas Antiinflamasi Produk Jamu Serbuk Ikan Gabus Secara In Vitro. J Kedokt Unram. 2023; 1(1): 1-4.

38. Qorib MF, Khairul Rizki Purba A, D’Arqom A. Dinamika Ekspresi Cox1 dan Cox2 Sebagai Landasan Tatalaksana Nyeri dan Inflamasi. J Kedokt Unram. 2022; 2022(4): 1233-1239.

39. Kim SY, Hong M, Deepa P, et al. Carthamus tinctorius Suppresses LPS-Induced Anti-Inflammatory Responses by Inhibiting the MAPKs/NF-κB Signaling Pathway in HaCaT Cells. Sci Pharm. 2023; 91(1): 14. doi:10.3390/scipharm91010014

40. Consoli V, Sorrenti V, Grosso S, Vanella L. Heme oxygenase‐1 signaling and redox homeostasis in physiopathological conditions. Biomolecules. 2021; 11(4): 1-23. doi:10.3390/biom11040589

41. Sun LP, Shi FF, Zhang WW, Zhang ZH, Wang K. Antioxidant and anti-inflammatory activities of safflower (Carthamus tinctorius L.) honey extract. Foods. 2020; 9(8): 1-16. doi:10.3390/foods9081039

42. Kim DH, Burgess AP, Li M, et al. Heme oxygenase-mediated increases in adiponectin decrease fat content and inflammatory cytokines tumor necrosis factor-α and interleukin-6 in Zucker rats and reduce adipogenesis in human mesenchymal stem cells. J Pharmacol Exp Ther. 2008; 325(3): 833-840. doi:10.1124/jpet.107.135285

43. Durante W. Protective role of heme oxygenase-1 against inflammation in atherosclerosis. Front Biosci. 2018; 16(6): 2372-2388. doi:10.2741/3860

Received on 19.12.2023 Modified on 11.03.2024

Research J. Pharm. and Tech 2024; 17(11):5567-5574.

DOI: 10.52711/0974-360X.2024.00850