INTRODUCTION:

Inflammation is the physiological reaction of the body to injuries or harm inflicted upon organs or tissues, which can result from infections, physical injuries, or exposure to chemicals. It is often shown by redness, swelling, discomfort, and heat¹. Inflammation begins when cells are activated and release inflammatory mediators. These mediators are histamine, serotonin, leukotrienes, prostaglandins, and free radicals derived from oxygen and nitrogen^2,3. The proteins, leukocytes, and other fluids within the blood vessels move to the inflammatory tissue⁴. In the wounded region, increased blood flow causes the skin's surface to become red, and pressure on edematous tissue creates discomfort⁵.

Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) are a medication used to relieve pain, fever, inflammation¹, musculoskeletal disorders, osteoarthritis, rheumatoid arthritis, and other comorbid conditions⁶. Long-term usage of NSAIDs may cause skin rashes, gastrointestinal discomfort, anaphylaxis⁷, cardiovascular disorders, and renal impairment⁸. Because the side effects of NSAIDs are highly alarming, an alternative is required, such as using anti-inflammatories from natural products.

Sappan wood (Caesalpinia sappan L.) is a medicinal plant that Indonesians use to make herbal beverages. The utilized part of the sappan is the wood. This plant's wood includes several phenolic compounds, including dibenzoxocins, flavones, homoisoflavonoids, chalcone, xanthone, and brazilin⁹. Brazilin is the primary component of sappan wood, which has anti-oxidants, anti-cancer¹⁰, wound healing¹¹, anti-inflammatory^12,13, and anti-arthritis¹⁴. Moreover, the leaves of sappan wood posseses analgesic and anti-piretic activity¹⁵.

Red ginger rhizome (Zingiber officinale Roxb.) is a herb that has been utilized as a spice and herbal medicine for centuries. It has been claimed that red ginger rhizome has a wide range of biological and pharmacological effects. Red ginger rhizome is used in traditional medicine to cure headaches, indigestion, nausea, vomiting, and cancer. Red ginger rhizome also treats autoimmune illnesses, hypertension, hypercholesterolemia, hyperuricemia, and bacterial infections. In addition, red ginger rhizome also has other bioactivities such as anti-microbial, analgesic, anti-diabetic, anti-inflammatory, anti-oxidant, melanogenesis-inhibiting, anti-cancer, anti-tumor, anti-hyperlipidemic, anti-hypercholesterolemic, anti-hypertensive, anti-alzheimer, androgenic, insecticidal, and immunomodulatory effects. The part of red ginger that is used is the rhizome. The rhizome of red ginger has anti-inflammatory compounds, including 8-gingerol, 10-gingerol, 6-shogaol, 6-gingerol¹⁶.

Phenolic compounds contained in sappan wood and red ginger rhizome are very potential as anti-inflammatory because of their ability to inhibit the enzymes PLA₂ (phospholipase A₂), COX (cyclooxygenase), and LOX (lipoxygenase) so that the release of pro-inflammatory mediators can be reduced¹⁷. An anti-inflammatory activity test was used to determine the anti-inflammatory effect of sappan wood and red ginger rhizome. Due to the lack of relevant in vitro studies, a researcher is interested in applying the RBC membrane stability approach to examine the anti-inflammatory activity of the combination of sappan wood ethanol extract and red ginger rhizome ethanol extract.

MATERIALS AND METHODS:

Materials:

This study is an experimental method carried out at Organic Chemistry and Biochemistry Laboratoriums in Universitas Negeri Surabaya. Dry sappan wood powder, dry red ginger rhizome powder, blood samples, 96% ethanol, ice gel, alsever solution (2% dextrose (Merck, USA), 0.8% sodium citrate (Merck, USA), 0.42% NaCl (Merck), and 0.5% citric acid (Merck, USA)), Na₂HPO₄ (Merck, USA), NAH₂PO₄.2H₂O (Merck, USA) PBS solution pH 7.4, hyposaline solution, isosaline solution, diclofenac sodium, and distilled water were used. The tools needed in this study are analytical balance (OHAUS, USA), three-arm balance (OHAUS, USA), centrifuge tubes, test tubes (IWAKI, Indonesia), test tube racks, micropipette tips, mortar and pestle, 1000mL sample bottles, vacuum rotary evaporator (Buchi Rotavapor^® R-300, Switzerland), pH meter, water bath (D-Lab Tech, China), magnetic stirrer (IKA), refrigerated centrifuge (ThermoScientific Micro Cl-21R, USA), non-refrigerated centrifuge (Hettich Zentrifugen EBA-20, Germany), ultrasonic (ELMA S-80 H, USA), autoclave (Hirayama HVE-50), UV-VIS instrument (Shimadzu UV-1800, Japan), and micropipette (D-Lab, China).

Preparation of Sappan Wood and Red Ginger Rhizome:

The plant samples used were sappan wood and red ginger rhizomes. Sappan wood was purchased at Rengganis Herbal Shop in Gresik, East Java, while red ginger rhizome was purchased from Wonokromo Market in Surabaya. Sappan wood is obtained in a dry state. Red ginger rhizomes were cleaned under running water, cut into thin slices, and dried. After drying, the sample is ground to a fine powder. The fine powder is then used for the extraction process.

Extraction of Sappan Wood and Red Ginger Rhizome:

Extraction was carried out for three days using the maceration technique, and every 24 hours replaced with a new solvent. Every 500 grams of powdered sappan wood and red ginger rhizome was macerated in 1000 mL of 96% ethanol. Then, the macerate was concentrated with a vacuum rotary evaporator at 40 °C until a concentrated extract was obtained. The extracts will hereinafter be denoted as SWE (sappan wood ethanol extract) and RGRE (red ginger rhizome extract). Unused extracts were stored in a sealed container and refrigerated.

Preparation of Alsever's Solution:

The formulation of Alsever's solution followed by Vakkalagadda and Lankalapalli¹⁸. Alsever solution contained of dextrose (2%), sodium citrate (0.8%), citric acid (0.05%), and NaCl (0.42%) in 100 mL distilled water. The solution was sterilized using an autoclave¹⁸.

Preparation of PBS solution with pH 7.4 (10 mM):

Preparation of the PBS solution according to Shinde et al.¹⁹. To achieve a concentration of 10 mM, 1.15 grams of Na₂HPO₄, 0.26 grams of NaH₂PO₄.2H₂O, and 9 grams of NaCl were dissolved in distilled water up to 1000 mL. The solution was utoclaved for sterilization²⁰.

Preparation of Isosaline Solution:

0.625 grams of NaCl were dissolved in 250 mL of PBS solution and then sterilized using an autoclave²⁰.

Preparation of Positive Control:

Diclofenac sodium was finely ground using a mortar and pestle. Next, 0.025 grams was weighed and dissolved into 25 mL of isosaline solution. Then, stir it with a magnetic stirrer for 1 hour and 12 minutes at 500 rpm and 37 °C. After stirring, sonication was performed at 50 °C for 10 minutes. Then, the diclofenac sodium solution was diluted at 10, 20, 30, 40, and 50 ppm.

Preparation of Formula of Test (SWE:RGRE):

Stock solutions was prepared based on Table 3 then diluted into 400, 200, 100, 50, and 25 ppm.

Preparation of RBC Suspension:

Blood samples were taken from male mice (Mus musculus) DDY (Deutschland Denken Yonken) strains aged 3-4 months weighing above 30 grams obtained from the Farma Veterinary Center Surabaya. Blood collection using the cardiac puncture method.

The preparation of RBCs according to Nagaharika et al.²¹ with slight modifications. Mice blood was centrifuged for 10 minutes at 3000 rpm. The supernatant was collected using a sterile pipette, and the erythrocytes were washed with isotonic solution (NaCl 0.85% w/v) until the supernatant was clear. Next, a 10% RBC suspension was made with isosaline²¹.

Test for Anti-Inflammatory Activity:

Test for anti-inflammatory activity a was performed according to Menon & Latha²² with slight modifications. Tested sample volume was 4.5 mL, contained of 1 mL PBS pH 7.4, 0.5 mL of 10% RBC suspension, 1 mL of each sample, and 2 mL of hyposaline (0.25% w/v NaCl). In the drug control solution, the suspension of RBCs was replaced with an isotonic solution. In the negative control, the sample was substituted with an isotonic solution. Next, incubate at 37 for 30 minutes in a water bath, then centrifuge at 5000 rpm for 10 minutes. A sterile pipette was used to remove the supernatant. After that, the absorbance was read at 560 nm²³. Calculation of membrane stability percentage according to Oyedapoo²⁰:

(1)

when, A = absorbance of tested sample; B = absorbance of drug control; C = absorbance of negative control

The negative control shows 100% lysis or 0% stability^20,22. The calculated percentage of membrane stability was then transformed into a linear regression curve to derive an equation for calculating the IC₅₀.

Statistical Analysis:

The results were reported as the mean IC₅₀ value. Statistical analysis was performed using IBM SPSS Statistics 25. The IC₅₀ differences were examined using parametric statistical methods with the One Way Anova test followed by the Games-Howell test. Results were considered significantly different when p < 0.05.

Ethical Clearance:

This study followed the Health Research Ethics Code, Health Research Ethics Committee, School of Medicine, Universitas Airlangga Number 147/EC/KEPK/FKUA/2022.

RESULT:

Results of Sappan Wood and Red Ginger Rhizome Extraction:

Extraction is performed to remove a soluble component using an appropriate solvent. Maceration was used in this research. The maceration method is used because the method is cheap, simple, and done without heating so that natural ingredients are not damaged²⁴.

The solvent used in maceration is 96% ethanol because ethanol is an organic solvent that can dissolve flavonoid and phenolic compounds in the sample²⁵. After maceration, the macerate was filtered and concentrated using a vacuum rotary evaporator to produce a concentrated extract. The extract was subsequently weighed, and the yield percentage was calculated. The yield percentages for sappan wood and red ginger rhizome were 8.708% and 7.031%, respectively.

Results of Anti-inflammatory Activity:

DISCUSSION:

The anti-inflammatory activity of SWE, RGRE, F1, F2, and F3 is seen from the percentage of RBC membrane stability induced by hypotonic solution. It should be noted that when the IC₅₀ value decreases, anti-inflammatory activity increases. The concentration is related to the RBC membrane stability²⁶. Haemoglobin absorbance decreases as concentration increases. But, stability increases, and percent stability increases (Table 1, Table 2, and Figure 1). The decrease in haemoglobin absorbance value means that RBCs are more stable and less lysed.

Figure 1: Effect of test samples and diclofenac sodium on RBC membrane stability. SWE, sappan wood ethanol extract; RGRE, red ginger rhizome ethanol extract; F1, formula 1; F2, formula 2; F3, formula 3

Table 2: Mean difference of IC₅₀ (ppm) between diclofenac sodium and samples

Test sample difference	Mean differences	p
Diclofenac sodium - SWE	152.91	0.000009*
Diclofenac sodium - RGRE	7.89667	0.999
Diclofenac sodium - F1	98.65333	0.001*
Diclofenac sodium - F2	105.59667	0.0009*
Diclofenac sodium - F3	96.16333	0.007*

Notes: SWE, sappan wood ethanol extract; RGRE, red ginger rhizome ethanol extract; F1, formula 1; F2, formula 2; F3, formula 3. Post-hoc Games-Howell test *significantly different (p < 0.05).

Table 3: Formulation of SWE, RGRE, F1, F2, and F3

Sample Name	SWE (mL)	RGRE (mL)	Ratio
SWE	10	0	1:0
RGRE	0	10	0:1
F1	5	5	1:1
F2	6.67	3.33	2:1
F3	3.33	6.67	1:2

Note: SWE, sappan wood ethanol extract; RGRE, red ginger rhizome ethanol extract; F1, formula 1; F2, formula 2; F3, formula 3.

According to Table 1, the percentage of RBC membrane stability for all test samples is directly related to the concentration, so the greater the concentration, the greater the membrane stability. At higher concentrations, the quantity of polyphenolic compounds in SWE and RGRE increases. As a result, numerous compounds accumulate on the RBC membrane. These compounds bind to the lipid bilayer of the RBC membrane at its hydrophilic head, forming a protective layer that prevents hypotonic solutions from diffusing into the RBC membrane^27,28. IC₅₀ value is related to the potential of a drug to produce a specific effect²⁹. The smaller the IC₅₀ value, the stronger its activity³⁰. If the IC₅₀ value of an extract is lower than that of the standard drug, it indicates the extract's potential as an anti-inflammatory agent³¹. SWE gave an IC₅₀ value 47.63 ppm, RGRE = 181.26 ppm, F1 = 101.93 ppm, F2 = 94.98 ppm, and F3 = 104.22 ppm. All test samples gave IC₅₀ values lower than positive control (IC₅₀ = 200.55 ppm), indicating that SWE, RGRE, F1, F2, and F3 have anti-inflammatory activity.

As shown in Table 1, the IC₅₀ value of SWE is lower than RGRE. This difference can be attributed to variations in the content of polyphenolic compounds within these two extracts. Polyphenolic compounds play a crucial role in stabilizing the membranes of RBCs²⁷. Specifically, the SWE exhibits a higher total polyphenolic content (27.65 mg GAE/g), when compared with RGRE (21.90 mg GAE/g)³². Furthermore the polyphenol content in the SWE is approximately 10.67%, whereas in RGRE is 2.53%³³.

This research is inline with previous studies. Nirmal et al.⁹found that the ethanol extract of sappan wood possesses anti-inflammatory properties. At a 0.1 g/mL concentration, the extract had a 61.9% inhibitory effect on albumin denaturation. Meanwhile, Mueller et al.³⁴ reported that sappanwood can reduce the secretion of proinflammatory cytokines (IL-6 and TNF- α) by 80% at a concentration of 50 g/mL. Mueller et al.³⁴also evaluated the anti-inflammatory activity from five compounds in sappanwood (episappanol, protosappanin, brazilin, a mixture of protosappanin B and isoprotosappanin B, and sappanol). All compounds significantly suppressed the proinflammatory cytokines (IL-6 and TNF-α). Another study by Min et al.³⁵ examined the anti-inflammatory efficacy of sappanwood-isolated compounds. Protosappanin A and 3-deoxysappanchalcone showed anti-inflammatory activity by decreasing NO expression in LPS-induced macrophage RAW 264.7 cells.

The difference between the IC₅₀ values of each test sample was highly significant (p < 0.001). Further analysis by comparing the IC₅₀ value of sodium diclofenac and the IC₅₀ value of samples is presented in Table 2. The difference in IC₅₀ of SWE, F1, F2, and F3 is significant because it gives a p < 0.05. Meanwhile, there was no significant difference in the IC₅₀ value of RGRE (p > 0.05). It indicates that sappan wood extracts F1, F2, and F3 have more anti-inflammatory activity than sodium diclofenac. SWE has the highest difference in the IC₅₀ mean average, so that it will provide good anti-inflammatory activity compared to diclofenac sodium.

The method was used for anti-inflammatory assay is RBC membrane stability method. This method used because of the similarity between RBC membranes and lysosomal membranes³⁶. During inflammation, lysosomal enzymes and hydrolytic components are released, causing damage to organelles and surrounding tissues^37,38. Like the lysosomal membrane, when the RBC membrane contacts with hypotonic solution, the membrane will lyse, accompanied by the release of haemoglobin³^,³⁹. Released haemoglobin can be measured using a UV-Vis spectrophotometer at 560 nm^40,41.

The intracellular calcium concentration is related to the deformability and volume of erythrocyte cells. Adding plant extracts increases cell volume surface area ratio due to the interaction between compounds in plant extracts and membrane proteins. Calcium entrance into RBCs can be changed by the protective impact of sappan wood ethanol extract and red ginger rhizome ethanol extract⁴². An explanation related to the stability of the RBC membrane when induced by hypotonic solution is an increase in the cell surface area/volume ratio caused by membrane expansion, cell shrinkage, or both. The increase in the surface area/volume ratio of RBCs can make RBCs less osmotically fragile⁴³.

Membrane-stabilizing compounds protect cell membranes against harmful substances and their ability to interfere with phospholipase release⁴⁴. One of the compounds that have this activity is the phenolic compound. Phenolic compounds work similarly to NSAIDs, inhibiting other pro-inflammatory mediators besides COX by inhibiting gene activity or expression. In addition, phenolic compounds as anti-inflammatory work by inhibiting eicosanoid synthesis, inhibiting immune cell activation, nitric oxide synthase, and COX-2 enzyme synthesis⁴⁵.

CONCLUSION:

SWE (IC₅₀ = 47.63 ppm), RGRE (IC₅₀ = 181.26 ppm), F1 (IC₅₀ = 101.93 ppm), F2 (IC₅₀ = 94.98 ppm), and F3 (IC₅₀ = 104.22 ppm) showed anti-inflammatory activity, because the IC₅₀ values lower than diclofenac sodium (IC₅₀ = 200.55 ppm). For single extracts, SWE is better as an anti-inflammatory. Meanwhile, in the formulations, F2 excels as an anti-inflammatory agent.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors thanks to the Directorate of Research, Technology, and Community Service, Directorate General of Higher Education, Research, and Technology, Ministry of Education and Culture for providing fiscal funding in 2022 through the Rector's Decree Number 313/UN38 /HK/PP/2022 on March 16, 2022. This research can be carried out with the support of laboratories and other facilities at the Department of Chemistry, Universitas Negeri Surabaya.

REFERENCES:

1. Su S, Hua Y, Wang Y, Gu W, Zhou W, Duan JA, et al. Evaluation of the anti-inflammatory and analgesic properties of individual and combined extracts from Commiphora myrrha, and Boswellia carterii. J Ethnopharmacol. 2012; 139(2):649–56. doi: 10.1016/j.jep.2011.12.013.

2. Abdulkhaleq LA, Assi MA, Abdullah R, Zamri-Saad M, Taufiq-Yap YH, Hezmee MNM. The crucial roles of inflammatory mediators in inflammation: A review. Vet World. 2018; 11(5):627–35.

3. Anosike CA, Obidoa O, Ezeanyika LU. Membrane stabilization as a mechanism of the anti-inflammatory activity of methanol extract of garden egg (Solanum aethiopicum). DARU, J Pharm Sci. 2012; 20(1):1.

4. Benly. Role of Histamine in Acute Inflammation. J Pharm Sci Res. 2015; 7(6):373–6.

5. Y MI, Saehu MS, Bina P, Kendari H. Efek Antiinflamasi Fraksi Dari Ekstrak Etanol Batang Galing ( Cayratia trifolia L . Domin ) Secara In Vitro. J Farm Sains dan Prakt. 2021; 7(3):289–97.

6. Ghosh R, Alajbegovic A, Gomes A V. NSAIDs and cardiovascular diseases: Role of reactive oxygen species. Oxid Med Cell Longev. 2015; 2015(Table 1).

7. Kowalski ML, Makowska JS. Seven steps to the diagnosis of NSAIDs hypersensitivity: How to apply a new classification in real practice? Allergy, Asthma Immunol Res. 2015; 7(4):312–20.

8. McDowell K, Clements JN. How can NSAIDs harm cardiovascular and renal function? J Am Acad Physician Assist. 2014; 27(4):12–5.

9. Nirmal NP, Panichayupakaranant P. Antioxidant, antibacterial, and anti-inflammatory activities of standardized brazilin-rich Caesalpinia sappan extract. Pharm Biol. 2015; 53(9):1339–43.

10. Meiyanto E, Lestari B, Sugiyanto RN, Jenie RI, Utomo RY, Sasmito E, et al. Caesalpinia sappan L. heartwood ethanolic extract exerts genotoxic inhibitory and cytotoxic effects. Orient Pharm Exp Med. 2019; 19(1):27–36. doi: 10.1007/s13596-018-0351-9

11. Nirmal NP, Prasad RGS V, Keokitichai S. Wound healing activity of standardized brazilin rich extract from Caesalpinia sappan heartwood. J Chem Pharm Res. 2014; 6(10):195–201.

12. Mueller M, Weinmann D, Toegel S, Holzer W, Unger FM, Viernstein H. Compounds from Caesalpinia sappan with anti-inflammatory properties in macrophages and chondrocytes. Food Funct. 2016; 7(3):1671–9.

13. Mekala K, Radha R. A review on sappan wood-a therapeutic dye yielding tree. Res J Pharmacogn Phytochem. 2015; 7(4):227.

14. Jung EG, Han K Il, Hwang SG, Kwon HJ, Patnaik BB, Kim YH, et al. Brazilin isolated from Caesalpinia sappan L. inhibits rheumatoid arthritis activity in a type-II collagen induced arthritis mouse model. BMC Complement Altern Med. 2015; 15(1):1–11. doi: 10.1186/s12906-015-0648-x

15. Saptarini NM, Deswati DA. Analgesic and antipyretic activities of ethanolic extract of sappan wood (Caesalpinia sappan l.) leaves. Res J Pharm Technol. 2021; 14(10):5213–6.

16. Zhang S, Kou X, Zhao H, Mak KK, Balijepalli MK, Pichika MR. Zingiber officinale var. rubrum: Red Ginger’s medicinal uses. Molecules. 2022; 27(3).

17. Maleki SJ, Crespo JF, Cabanillas B. Anti-inflammatory effects of flavonoids. Food Chem. 2019; 299(July):125124. doi: 10.1016/j.foodchem.2019.125124

18. Vakkalagadda RK, Lankalapalli S. In vitro Anti-cancer, Anti-inflammatory and Anti-arthritic activity of Ethanolic extract of Ochna obtusata leaves. Res J Pharm Technol. 2022; 15(9):3999–4004.

19. Shinde UA, Phadke AS, Nair AM, Mungantiwar AA, Dikshit VJ, Saraf MN. Membrane stabilizing activity - A possible mechanism of action for the anti-inflammatory activity of Cedrus deodara wood oil. Fitoterapia. 1999; 70(3):251–7.

20. Oyedapo O, Akinpelu BA, Akinwunmi KF, Adeyinka MO, Sipeolu FO. Red blood cell membrane stabilizing potentials of extracts of Lantana camara and its fractions. Int J Plant Physiol Biochem. 2010; 2(October):46–51.

21. Nagaharika Y, kalyani V, Rasheed S, Ramadosskarthikeyan. Anti-inflammatory activity of leaves of Jatropha gossypifolia L. by hrbc membrane stabilization method. J Acute Dis. 2013; 2(2):156–8. doi: 10.1016/s2221-6189(13)60118-3

22. Menon DB, Latha K. Phytochemical screening and in vitro anti-inflammatory activity of the stem of Coleus forskohlii. Pharmacogn J. 2011; 3(23):75–9. doi: 10.5530/pj.2011.23.11

23. Vijayraja D, Jeyaprakash K. Preliminary phytochemical analysis, in vitro antioxidant and anti-inflammatory activity of Turbinaria ornata (Turner) J. Agardh. Res J Pharm Technol. 2017; 10(7):2243–8.

24. Puspitasari AD, Proyogo LS. Perbandingan Metode Ekstraksi Maserasi Dan Sokletasi Terhadap Kadar Fenolik Total Ekstrak Etanol Daun Kersen (Muntingia calabura). J Ilm Cendekia Eksakta. 2017; 1(2):1–8.

25. Hakim AR, Saputri R. Narrative review: Optimasi etanol sebagai pelarut senyawa flavonoid dan fenolik. J Surya Med. 2020; 6(1):177–80.

26. Francis P, Suseem SR. Phytochemical analysis and anti-inflammatory screening of Strychnos colubrina Linn. Res J Pharm Technol. 2016; 9(2):165–9.

27. Kaźmierczak T, Bonarska-Kujawa D, Męczarska K, Cyboran-Mikołajczyk S, Oszmiański J, Kapusta R. Analysis of the Polyphenolic Composition of Vaccinium L . Extracts and Their Protective Effect on Red Blood Cell Membranes. Membranes (Basel). 2023; 13(6):589.

28. Wu H, Bak KH, Goran G V., Tatiyaborworntham N. Inhibitory mechanisms of polyphenols on heme protein-mediated lipid oxidation in muscle food: New insights and advances. Crit Rev Food Sci Nutr. 2022; 0(0):1–19. doi: 10.1080/10408398.2022.2146654

29. Berrouet C, Dorilas N, Rejniak KA, Tuncer N. Comparison of drug inhibitory effects (IC 50) in monolayer and spheroid cultures. Bull Math Biol. 2020; 82(6).

30. Maryam S. Kadar antioksidan dan IC50 tempe kacang merah (Phaseulus vulgaris L) yang difermentasi dengan lama fermentasi berbeda. Pros Semin Nas Fmipa Undiksha V. 2015;347–52.

31. Hashmi Z, Sarkar D, Mishra S, Mehra V. An in-vitro assessment of anti-inflammatory, antioxidant, and anti-hyperglycemic activities of traditional edible plants - Murraya koenigii, Mentha spicata, and Coriandrum sativum. J Biomed Ther Sci. 2022; 9(1):1–10.

32. Amalia RT, Tukiran, Sabila FI, Suyatno. Phytochemical Screening and Total Phenolic Compounds of Red Ginger (Zingiber officinale) and Secang Wood (Caesalpinia sappan) As Preliminary Test of Antiarthritis. Chim Nat Acta. 2021; 9(1):14–9.

33. Putri DK, Tukiran, Suyatno, Sabila FI. Antioxidant Activity from The Combination Ethanol Extract Secang Wood ( Caesalpinia sappan L .) And Red Ginger Rhizome ( Zingiber officinale Roxb .). Int Jt Conf Sci Eng 2021 (IJCSE 2021). 2021; 209(Ijcse):143–7.

34. Mueller M, Weinmann D, Toegel S, Holzer W, Unger FM, Viernstein H. Compounds from Caesalpinia sappan with anti-inflammatory properties in macrophages and chondrocytes. Food Funct. 2016; 7(3):1671–9.

35. Min BS, Cuong TD, Hung TM, Min BK, Shin BS, Woo MH. Compounds from the heartwood of Caesalpinia sappan and their anti-inflammatory activity. Bioorganic Med Chem Lett. 2012; 22(24):7436–9. doi: 10.1016/j.bmcl.2012.10.055

36. Sravani D, Aarathi K, Sampath Kumar NS, Krupanidhi S, Vijaya Ramu D, Venkateswarlu TC. In vitro anti-inflammatory activity of Mangifera indica and Manilkara zapota leaf extract. Res J Pharm Technol. 2015; 8(11):1477–80.

37. Manvar MN, Desai TR. In-vitro Anti-inflammatory and Anti-Arthritic Activities of Fruits of Vernonia anthelmintica Willd. (Asteraceae). Asian J Pharm Res. 2014; 4(4):186–8.

38. Anbarasi A, Vidhya R. Evaluation of In Vitro Anti-Inflammatory Activity of Tephrosia purpurea (Seed). Asian J Pharm Res. 2015; 5(2):83.

39. Thawkar B, Kale M, Oswal M, Maniyar K, Kadam K, Kamat S. To study anti-inflammatory activity of 70% methanolic extract of Triumfetta rhomboidea: In vitro study. Res J Pharm Technol. 2016; 9(3):241–4.

40. Siswanty PW, Wibowo MA, Harlia. Aktivitas Toksisitas Antioksidan Dan Antiinflamasi Secara In Vitro Dari Ekstrak Metanol Daun Mangga Bacang (Mangifera foetida L). J Untan. 2017; 6(1):42–9.

41. Teja K, Satyanarayana T, Saraswathi B, Goutham B, Mamatha K, Samyuktha P, et al. Phytochemical and In vitro Anti-inflammatory Activity on Abrus precatorius . Asian J Res Pharm Sci. 2019; 9(1):50.

42. Paramita S, Ismail S, Marliana E, Moerad EB. Anti-inflammatory activities of curcuma aeruginosa with membrane stabilization and carrageenan-induced paw oedema test. EurAsian J Biosci. 2019; 13(2):2389–94.

43. Abe H, Katada K, Orita M, Nishikibe M. Effects of Calcium Antagonists on the Erythrocyte Membrane. J Pharm Pharmacol. 1991; 43(1):22–6.

44. Kumar A, Ghosh S, Vaishali. An experimental evaluation of Ageratum conyzoides on membrane stabilization and protein denaturation during acute inflammation and arthritis. Biomed Pharmacol J. 2011; 4(2):313–7.

45. Ambriz-Pérez DL, Leyva-López N, Gutierrez-Grijalva EP, Heredia JB. Phenolic compounds: Natural alternative in inflammation treatment. A Review. 2. 2016; 2(1).

Received on 25.02.2023 Modified on 20.08.2023

Research J. Pharm. and Tech. 2024; 17(3):1250-1255.

DOI: 10.52711/0974-360X.2024.00195

Concentration (ppm)	RBC membrane stability (%)					Concentration (ppm)	RBC membrane stability (%)
Concentration (ppm)	SWE	RGRE	F1	F2	F3	Concentration (ppm)	Diclofenac sodium
25	38.43	14.20	4.23	21.21	17.26	10	10.04
50	64.20	16.40	23.00	30.71	25.07	20	12.02
100	97.03	22.30	39.62	48.68	44.04	30	14.43
200	213.98	51.10	105.87	98.01	87.49	40	16.12
400	498.70	111.07	253.25	179.87	177.73	50	18.48
IC₅₀	47.63	181.26	101.93	94.98	104.22	IC₅₀	200.55