In-vitro anti-inflammatory activity of methanolic extract of Mirablis jalapa and Costus igneus

 Corresponding author: Shrutika V. Bhandare Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution License 4.0. In-vitro anti-inflammatory activity of methanolic extract of Mirablis jalapa and Costus igneus Shrutika V. Bhandare 1, *, Aayesha S. Mujawar 1, Prajakta V. Kamble 1, Vinod S. Pattankude 2 and Dhanraj R. Jadge 2 1 Student of Womens College of Pharmacy, Peth Vadgaon, 416112, Maharashtra, India. 2 Department of Pharmacology, Womens College of Pharmacy, Peth Vadgaon, 416112, Maharashtra, India. World Journal of Advanced Research and Reviews, 2025, 26(03), 456-464 Publication history: Received on 24 April 2025; revised on 31 May 2025; accepted on 03 June 2025 Article DOI: https://doi.org/10.30574/wjarr.2025.26.3.2158 Abstract A protective immunological reaction to irritations, infections, or tissue damage is inflammation. It includes vascular permeability, elevated blood flow, production of signalling chemicals like cytokines and histamine. Enzymes that cause different illnesses can be released when lysosomal membranes lyse during inflammation. By blocking these enzymes or maintaining membrane stability, NSAIDs assist medications like methylprednisolone, diclofenac, aspirin, indomethacin, Diclofenac, Ibuprofen, and Hydrocortisone. Traditional medicine has utilised plants like Costus igneus and Mirabilis jalapa to treat a variety of illnesses. β-sitosterol, β-amyrin are found in Mirabilis jalapa and Costus igneus give it antiinflammatory qualities respectively. Method: The leaves of Mirabilis jalapa and Costus igneus were extracted using methanol, and a phytochemical screening was performed. The human red blood cell (HRBC) membrane stabilization method and heat-induced haemolysis were employed to evaluate the anti-inflammatory properties of Costus igneus and Mirabilis jalapa. The evaluation was conducted at varying concentrations (40, 80, and 100 ug/ml), and the activity was noted in a dose-dependent way. Result and discussion: With the use of HRBC membrane stabilization method, the percentage protection was discovered to be 70%, 78%, and 81%, respectively. Additionally, the heat-induced haemolysis approach yielded 72%, 76%, and 79%, respectively. The HRBC membrane stabilization approach provides 86% protection for the standard aspirin (100 ug/ml), while heat-induced haemolysis provides 82% protection. Conclusion: The bioactive components are associated with their traditional and pharmaceutical application in the management of inflammation, it can be concluded. Both plants have strong anti-inflammatory properties. Keywords: Heat-Induced Hemolysis; HRBC Membrane Stabilization; Costus igneus; Mirabilis jalapa. 1. Introduction The complicated process of inflammation, which is often linked to pain, includes changes in membranes, increased vascular permeability, and increased protein denaturation. Stress is a type of injury that occurs when the cells are injured by bacteria, Chemical substances or physical force. When the body reacts to stress, tissue inflammation occurs.[5]. The body uses inflammation as a defense mechanism in reaction to chemical irritants, allergies, microbial infections, and tissue damage. It is an essential immune response that attempts to get rid of harmful stimuli and initiates the repair process for damaged tissue. [6] World Journal of Advanced Research and Reviews, 2025, 26(03), 456-464 457 Increased blood flow and vascular permeability, as well as the activation of humoral and cellular defense systems, are some of the intricate pathophysiological processes that lead to inflammation. [2] These processes are mediated by a variety of signaling molecules produced by mast cells, brain terminals, endothelial cells, granulocytes, platelets, lymphocytes, and macrophages. Lipids (leukotrienes, prostanoids, and platelet activating factor) are among the mediators. Proteins and peptides (cytokines, growth factors, antibodies, kinines), physiologically active amines (histamine, serotonin), and superoxide anion, hydroperoxide, and hydroxyl radicals are examples of activated oxygen species. Numerous human illnesses are caused, maintained, exacerbated, and influenced by these mediators. The development of endogenous antiphlogistic and immunomodulatory mechanisms adds to the complexity of these processes, in addition to the vast number of proinflammatory actors. [7] Inflammation symptoms is defensive reaction include Loss of function and redness, discomfort, and edema in the injured area. [5] Histamine, prostroglandins, and kinins are released by wounded tissue cells. Together, these lead to increased capillary permeability and vasodilation, or the widening of blood vessels. Increased blood flow to the damaged area results from this. Through a process called chemotaxis, these compounds also act as molecular messengers that attract certain of the body's defensive cells.[7] By either stabilizing the lysosomal membrane or preventing the Lysosomal membrane stabilization is essential for lowering the inflammatory response because it prevents the release of lysosomal components of active neutrophils, such as bactericidal enzymes and proteases, which cause further tissue inflammation and damage upon extracellular release. [12] Acute and chronic inflammation are two different categories. The body's first reaction to damaging stimuli is acute inflammation, which is brought on by an increase in the flow of plasma and leukocytes from blood into the wounded tissues. Chronic inflammation is characterized by the simultaneous destruction and healing of the tissue caused by the inflammatory process, and it results in a progressive change in the kind of cells present at the site of inflammation. [5] Inflammation can cause lysosomal membrane lysis, which releases the enzyme components that lead to a variety of diseases. [7] Non-steroidal anti-inflammatory drugs (NSAIDs) function by either blocking the release of lysosomal enzymes or stabilizing the lysosomal membranes. Hemolysis, or the lysis of red blood cell membranes, and hemoglobin oxidation can result from exposure to toxic chemicals. The following compounds are harmful: heat, methyl salicylate, phenyl hydrazine, and hypotonic medium.[8] A hypotonic solution causes the RBC membrane to rupture due to an excessive build up of fluid inside the cells. RBC haemolysis occurs at the end. The cell is more susceptible to secondary damage from lipid peroxidation caused by free radicals when the red cell membrane is damaged. Bacterial enzymes and proteases are found in the lysosomes of active neutrophils. Leakage of lysosomal components upon extracellular release causes further tissue damage and inflammation. The integrity of the lysosome membrane is therefore essential for controlling the inflammatory response. This is going to stop its contents from spilling.[8] Human erythrocytes can be utilized to test the extract capacity to stabilize the membrane through heat-induced hemolysis and hypotonic solution-induced hemolysis. [9] The lysis of human red blood cell membranes (HRBCs) caused by hypotonicity can be used as an in-vitro test to determine whether a drug or plant extract has anti-inflammatory properties. [10] [11] One important source of compounds with pharmacological activity is natural products, particularly medicinal plants. Herbs are frequently chosen over synthetic substitutes because of their natural sources, possibility for fewer adverse effects, and more comprehensive therapeutic approach. Instead of only treating symptoms, they are said to be kinder to the body and have the ability to promote recovery. Mirabilis jalapa in Ayurveda also called “gulambasa.” It has been claimed that leaves contain a variety of substances, including β-sitosterol, stigmastrol, ursolic acid, leaves and stems are used as tonics and diuretics. To lessen itching in ailments like urticarial, leaf juice is traditionally administered to wounds and bruises. [2] Constituents isolated from the root and aerial parts of the plant include some rotenoids, an isoquinoline derivative, terpenoids, steroids, phenolic compounds, d-glucoside, ursolic acid, mirabalisoic acid, trigonelline, an antiviral protein, alanine, alpha-amyrins, arabinose, beta-amyrin, campesterol, daucosterol, and dopamine. [4] World Journal of Advanced Research and Reviews, 2025, 26(03), 456-464 458 Figure 1 Mechanism of Inflammation Proteous substances have been reported to have anti-tumor and plant virus inhibitory effect, while leaves have been utilized to treat and reduce inflammation. These studies have resulted in the isolation of soluble starch, pigments of centrospermae, sterols and fatty acids, sugars, amino acids, and triterpenes. [13] The plant’s aerial components, which include four steroids, B-sitosterol acetate, ursolic acid 4, and one diterpene, trans-phytol triterpenes, are discussed in this communication. [2] Costus igneus called “Fiery Costus” and “Spiral Flag” are other names for the medicinal plant Costus igneus. [2] Native to South and Central America. Commonly referred to as the “insulin plant”. This plant is relatively new to India, having originated in South Central America.[13] In Southern India, the entire Costus igneus plant is utilized for its anti-diabetic properties, which also shield the body from illness, preserve mental health, and increase life expectancy. Preliminary phytochemical screening revealed that this plant extract included proteins, carbohydrates, steroids, alkaloids, tannins, glycosides, saponins, fixed oils, and flavonoids. [13] Based on previous studies, there is a strong belief that eating the leaves of this plant can help diabetics control their blood glucose levels. It was discovered that people who consumed the plant’s leaves had lower blood glucose levels. It has been demonstrated that the administration of Costus igneus stem extract in both aqueous and ethanolic form inhibits the production of urinary stones in rats with experimentally induced urolithiasis by ethylene glycol. The components of the Costus igneus plant preparation indicate that it may reduce oxidative stress. [14] The goal of this work was to identify the mechanism by which β-amyrin, which was extracted from Costus igneus leaves,[2] β-sitosterol which extracted from Mirabalis jalapa had an anti-inflammatory effect [1]. Phenol, cardiac glycosides, triterpene, tannin, carbohydrates, World Journal of Advanced Research and Reviews, 2025, 26(03), 456-464 459 alkaloids, terpenoid, and protein presence techniques were among the main chemicals that were examined. Major chemicals found in Costus igneus, along with their pharmacological and therapeutic properties. Pharmacological property include anti-diabetic, anti-proliferative, anti-microbial, anti-urolithiatic, anti-inflammatory, anti-oxidant, neuroprotective, hypolipidemic, and other effects. [4] 2. Experimental work Collection and authentication of plant: Both plants were gathered during the July-August monsoon season from local area (garden) of Ichalkaranji, Kolhapur, Maharashtra. To preserve the phytochemical content, the plant materials were shade-dried at room temperature (around 25 °C) after being cleaned with distilled water, and authenticated by Dattajirao Kadam Arts, Science and Commerce college Ichalkaranji, Kolhapur, Maharashtra, India. Voucher specimens were prepared and archived for reference. • Mirabilis jalapa: Known as Gulambasa in Ayurveda is known for its diverse bioactive compounds like betasitosterol, ursolic acid, and anthraquinone. Traditionally, its leaf juice is used for wounds, itching, and bruises. [3][2] • Costus igneus: Referred to as the “insulin plant,” is prized for its anti-diabetic, anti-inflammatory, and antioxidant properties. Rich in phytochemicals like flavonoids, alkaloids, and glycosides, its leaves help lower blood glucose and cholesterol levels. [4][13] Figure 2 Leaves of Mirabilis jalapa Figure 3 Leaves Costus igneus Table 1 Taxonomy of Mirabilis jalapa Botanical Name Mirabilis jalapa Kingdom Plantae Family Nyctaginaceae Genus Mirabilis Species jalapa Table 2 Taxonomy of Costus igneus Botanical Name Costus igneus Kingdom Plantae Family Costaceae Genus Costus Species igneus World Journal of Advanced Research and Reviews, 2025, 26(03), 456-464 460 • Biological and pharmacological properties Mirabalis jalapa: Diuretic, carminative, conjunctivitis, abdominal colic, purgative, anti-bacterial, anti-fungal, anti-inflammatory, anti-spasmodic, anti-viral, and antigonorrheal • Biological and pharmacological of properties Of Costus igneus: anti-inflammatory, anti-diabetic, antimicrobial, anti-oxidant, anti-Urolithiatic, neuroprotective, hypolipidemic, and anti-proliferative [4] [13] 3. Material and methods 3.1. Extraction of Mirabilis jalapa and Costus igneus The coarsely powerdered leaves of Costus igneus were extract with methanol by using maceration process of extraction. About 50 gram powdered leaves extracted with 250 ml of methanol through a week of cold maceration at room temperature.[15] The coarsely powerdered leaves of Mirabilis jalapa were extracted with methanol by using maceration process of extraction. 200 mL of methanol were used to extract roughly fifty grams of powdered leaves over the course of 48 hours at room temperature. [16] Extract of both supernatant evaporated after they had been filtered through Whatman No. 1 filter paper. The extract amounts for Costus igneus and Mirabilis jalapa were 1.96 g and 1.26 g, respectively. 3.2. Procedure for analysis of anti-inflammatory activity: • Preparation of Red Blood Cells (RBCs) (10% v/v) • Method for analysis anti-inflammatory activity o Human red blood cell Stabilization method o Heat induced hemolysis [20] Preparation of Red Blood Cells (RBCs) (10% v/v): Human donors who were willing and in good health provided the red blood cells. (Never took NSAIDS before to expressing). The EDTA tube was filled with three mL of the red blood cells, which were then centrifuged at 3000 rpm for fifteen minutes at room temperature. After removing the supernatant and placing the residue in the centrifugation tube, sufficient isosaline solution was added, and centrifugation was repeated. Three iterations of the procedure were carried out until the isosaline solution’s colour became evident. To make a 10% red blood cell suspension, one drop of red blood cells was combined with nine drops of isosaline. [19] Method for analysis anti-inflammatory activity: HRBC membrane Stabilization method: Different concentration of test solution (40,80,100ug/ml)consisted of 1 mL sample solution; 2 mL hyposaline;1 mL of sodium phosphate buffer and 0.5 mL (10% v/v) suspension of RBCs in isosaline. The test control solution consisted of 2 mL hyposaline; 1 mL of sodium phosphate buffer, 1 mL of isosaline and 0.5 mL (10% v/ v) of RBCs suspension in isosaline. The standard solution consisted of 2 mL hyposaline; 1 mL of sodium phosphate buffer, 1 mL aspirin (100μg/mL) and 0.5 mL (10% v/v) suspension of RBCs in isosaline. After 30 minutes of incubation at 37°C, each assay mixture was centrifuged at 3000 rpm. Liquid supernatant was decanted, a colorimeter set at 570 nm was used to measure the absorbance. The formula is used to determine the percentage of protection and hemolysis. [7] [18] [20] % Hemolysis = (Abs of Test / Abs of Control) x 100 % inhibition = (Abs control – Abs sample) x 100 /Abs control Abs control =Absorbance of control Abs sample = Absorbance of sample Figure 4 Effect of extracts different concentration for HRBC membrane stabilization method World Journal of Advanced Research and Reviews, 2025, 26(03), 456-464 461 Heat induced hemolysis: Two mL of hyposaline, one mL of sodium phosphate buffer, half a mL of 10% v/v RBC suspension, and one mL of sample were used to make the test solution. 1 mL standard solution, 2 mL hyposaline, 1 mL sodium phosphate buffer, and 0.5 mL (10% v/v) suspension of RBCs in isosaline. 0.5 mL (10% v/v) RBC suspension in isosaline, 1 mL of sodium phosphate buffer, 1 mL of isosaline, and 2 mL of hyposaline made up the control solution. After that, the solution was incubated in a water bath at 56°C for 30 minutes. After that, the fluid was centrifuged at 3000 rpm for 15 minutes. The absorbance of the solution was measured using a colorimeter set at 570 nm. A formula was used to calculate the protection and hemolysis percentages.[7][18][20] % Hemolysis = (Abs of Test / Abs of Control) x 100 % inhibition = (Abs control – Abs sample) x 100 /Abs control Abs control =Absorbance of control Abs sample = Absorbance of sample Figure 5 Effect of extracts different concentration for heat induced haemolysis method 3.3. Statical analysis The statistical analysis utilizing one-way ANOVA for both the heat-induced hemolysis and HRBC membrane stabilization procedures revealed no significant differences between the groups. The HRBC method had an F-value of 11.781 and a p-value of 0.0280, whereas the heat-induced hemolysis method had an F-value of 11.3649 and a p-value of 0.0265. These results showed that the methanol extracts of Mirabilis jalapa and Costus igneus exhibited anti-inflammatory activity similar to the reference drug (p<0.5), supporting the extracts' consistency and effectiveness in both in vitro models. 4. Result and discussion Table 3 Phytochemical test for Mirabalis jalapa Sr. No. Test Procedure Inference 01 Salkowaski test Filtrate + few drops of Conc. H₂SO₄. + 02 Copper acetate test Extract + distilled water+ 3-4 drops of copper acetate solution + 03 Libermann burchard test Extract+ 2ml acetic acid solution+ 1-2 drops of Conc. H₂SO₄ along the side of test tube. + World Journal of Advanced Research and Reviews, 2025, 26(03), 456-464 462 Table 4 Phytochemical test for Costus igneus Sr. No. Test Procedure Inference 01 Salkowaski test Filtrate + few drops of Conc. H₂SO₄. + 02 Copper acetate test Extract + distilled water+ 3-4 drops of copper acetate solution + 03 Libermann burchard test Extract+ 2ml acetic acid solution+ 1-2 drops of Conc.H₂SO₄ along the side of test tube. + Table 5 Human red blood cells stabilization method Sr. No. Treatment Concentration (µg/mL) % Haemolysis % Protection 01 Control - 100 00 02 Test 40 30 70 03 Test 80 21 78 04 Test 100 18 81 05 Standard 100 14 86 Figure 6 Comparison of % haemolysis and protection of HRBC membrane stabilization method Human red blood cells stabilization method: The methanolic extract of the leaves of Mirabilis jalapa and Costus igneus was tested for its in vitro anti-inflammatory qualities using the HRBC membrane stabilization technique. The strongest anti-inflammatory activity and 81% HRBC protection in hypotonic solution were demonstrated by 100 µg/ml out of all doses. The results showed 86% protection display the benchmark. Table 6 Heat induced hemolysis method Sr. No. Treatment Concentration (µg/mL) % Haemolysis % Protection 01 Control - 100 00 02 Test 40 27 72 03 Test 80 23 76 04 Test 100 20 79 05 Standard 100 18 82 World Journal of Advanced Research and Reviews, 2025, 26(03), 456-464 463 Figure 7 Comparison of % haemolysis and protection of Heat induced haemolysis method Heat induced hemolysis method: The mechanism of the anti-inflammatory action of the methanolic extract of Costus igneus and Mirabilis jalapa at varying doses was investigated using heat-induced hemolysis. It proves that all concentrations effectively stopped heat-induced hemolysis. The greatest percentage protection was 79% at concentrations of 100 µg/ml, while the lowest percentage was 72% at values of 40 µg/ml. The present study confirm that phytoconstuituents derived from Mirabilis jalapa and Costus igneus has antiinflammatory activity. In the HRBC technique, the extract at 100 µg/ml showed 81% protection, close to the standard aspirin (86%), suggesting strong membrane stabilization, which may prevent lysosomal enzyme release and reduce inflammation. In the heat-induced hemolysis method, the extracts also showed effective protection (72–79%) across tested concentrations. One-way ANOVA analysis confirms the statistical analysis for the HRBC membrane stabilization and heat-induced haemolysis method. Both shows that There are no any notable variations between the groups (p<0.5) and confirm that Mirabalis jalapa and Costus igneus have anti-inflammatory property. 5. Conclusion Both Costus igneus and Mirabilis jalapa are excellent medicinal herbs that have been utilised for millennia in Ayurvedic medicine. They found ß-sitosterol, ß-amyrin are among the bioactive substances found in Mirabalis jalapa leaves and Costus igneus respectively. 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