Anti-inflammatory and antioxidant potential of ethanolic leaf extract of Artabotrys hexapetalus: In vitro and in vivo approach

Introduction

Inflammation is a conserved protective response that eliminates the initial cause of cellular injury and facilitates removal of damaged tissue and subsequent repair. It is elicited by diverse stimuli, including physical injury, ischemia, autoimmune reactions and infectious agents. Clinical manifestations may be transient or persist for days to weeks, as observed in bronchitis, pharyngitis, appendicitis and dermatitis (Laveti et al., 2013) [1] . When inflammatory responses are sustained, however, they become maladaptive and contribute to chronic pathology. Persistent inflammation is implicated in the pathogenesis of multiple chronic diseases, including cancer, diabetes, asthma, myocardial infarction and arthritis (Allegra et al., 2019) [2] . Experimental and clinical studies further demonstrate a close interplay between oxidative stress mediated by free radicals and other reactive species and inflammatory signalling pathways. Accumulation of reactive oxygen and nitrogen species has been associated with disease processes such as tumorigenesis, hepatitis, hepatic injury and immune dysfunction (Sinmisola et al., 2019) [3] . Artabotrys hexapetalus, commonly known as climbing ylang-ylang or harichampa, is a woody evergreen climber of the family Annonaceae. Native to India and other regions of South and Southeast Asia, it is widely cultivated as an ornamental species because of its intensely fragrant yellow-green flowers. The plant has simple opposite leaves and bears bisexual, actinomorphic flowers that occur singly or in small clusters on hooked peduncles. Various parts of the plant have long been used in traditional medicine and several biological activities of its leaves and other parts have been reported (Bailly et al., 2022) [4] . However, to the best of our knowledge, no previous study has examined the antioxidant and antiinflammatory potential of the ethanol leaf extract of A. hexapetalus. Accordingly, the present study aimed to investigate the antioxidant and anti-inflammatory activities of the ethanol extract using in vitro and in vivo approaches.

Materials and Methods

Collection and authentication of A. hexapetalus leaves

The leaves of A. hexapetalus were collected in and around Namakkal District, Tamil Nadu and it was identified, duly authenticated by Department of Pharmacognosy, Siddha Central Research Institute, Arumbakkam, Chennai. The collected leaves were shade dried, powdered and stored for further preparation of ethanolic extract.

Preparation of extract

The powdered leaves of A. hexapetalus were extracted with ethanol by maceration method (Pandey and Tripathi, 2014) [5] .

Phytochemical analysis

Phytochemical screening was carried out by the method described by Odebedy and Sofowara, (1978) [6] .

DPPH radical scavenging activity

The radical scavenging activity of the extract was determined as by Aouachria et al. (2017) [7] . DPPH radical scavenging activity was estimated using the following equation:

The half-maximal inhibitory concentration (IC 50 ) was then obtained from a linear regression plot of percentage inhibition against the extract concentration.

Determination of in vitro anti-inflammatory studies

The in vitro anti-inflammatory activity of the extract was evaluated by membrane-stabilizing activity and inhibition of albumin denaturation. Membrane-stabilizing activity was determined as described by Oyedapo et al. (2010) [8] using 2% (v/v) bovine erythrocyte suspension, with phenylbutazone used as the standard drug. The percentage of membrane stabilization was calculated using the following formula:

The ability of the extract to inhibit albumin denaturation was evaluated according to the method described by Sakat et al. (2010) [9] . The percentage inhibition of albumin denaturation was calculated using the following equation:

Determination of in vivo anti-inflammatory studies

The in vivo anti-inflammatory activity of the extract was evaluated using the carrageenan-induced paw edema model in rats (Kalitha et al., 2018) [10] . Male Wistar rats were divided into three groups: control, test (treated with the extract), and standard (treated with phenylbutazone). Edema was induced by injecting 0.1 mL of 1% carrageenan solution into the right hind paw of each rat. The test extract and standard drug were administered 30 minutes before carrageenan injection. Paw volume was measured at 0, 0.5, 1.0, 2.0, 3.0, and 4.0 h post-injection using a digital plethysmometer. The percentage inhibition of edema was calculated to assess the efficacy of the extract in comparison with the standard drug.

Statistical analysis

All measurements were performed in triplicate and the results are expressed as mean ± Standard error of the mean (SEM) using GraphPad Prism software. Statistical analysis was carried out using one-way analysis of variance (ANOVA), and a p value of less than 0.05 was considered statistically significant.

Results

Qualitative phytochemical analysis

The preliminary qualitative phytochemical analysis of A. hexapetalus leaf ethanolic extract revealed the presence of alkaloids, carbohydrates, flavonoids, phenols, saponins, tannins and terpenoids, whereas glycosides was not detected. The results are presented in Table 1.

DPPH radical scavenging activity

As shown in Table 2, the IC 50 values for A. hexapetalus leaf extract and ascorbic acid (standard) were 161.31 and 106.15 µg/mL, respectively. The plant extract exhibited concentration-dependent DPPH radical scavenging activity, with the highest activity observed at 500 µg/mL.

Membrane stability

For A. hexapetalus leaf extract, the ability to stabilize red blood cell membranes against heat and hypotonicity-induced hemolysis was evaluated to investigate its underlying antiinflammatory mechanism. As shown in Table 3, the extract protected RBCs against hemolysis in a concentrationdependent manner. The highest inhibition was observed at 500 µg/mL. Phenylbutazone was used as the standard drug.

Inhibition of albumin denaturation

The anti-inflammatory activity of A. hexapetalus leaf extract was evaluated by its ability to inhibit albumin denaturation, with phenylbutazone as the standard. As shown in Table 4, the extract exhibited its highest inhibitory activity at 400 µg/mL.

In vivo anti-inflammatory activity

Table 5 illustrates the anti-inflammatory effect of A. hexapetalus leaf extract on carrageenan-induced paw edema in rats. The negative control group (carrageenan only) showed the highest paw volume, confirming successful induction of inflammation. The standard drug, phenylbutazone, produced a significant reduction in paw edema, validating its known anti-inflammatory effect. A. hexapetalus leaf extract produced a comparable reduction in paw volume to phenylbutazone, indicating similar efficacy.

Discussion

In the present study, we evaluated the antioxidant and antiinflammatory effects of A. hexapetalus leaf extract. Phytochemical analysis indicated that the extract contained phenolic compounds. Kousalya and Doss (2020) [11] demonstrated that A. hexapetalus aqueous leaves extract showed the presence of various phytochemicals like alkaloids, flavonoids, tannins, phenols, glycosides, diterpenes, saponins, carbohydrates, proteins and amino acids. These compounds demonstrated radical-scavenging activity, protected RBC membranes against heat and hypotonicity-induced hemolysis and inhibited albumin denaturation. As primary natural antioxidants, phenolic compounds likely account for the extract's total phenolic content, its free-radical scavenging and antioxidant activities and its observed anti-inflammatory effects. DPPH is a stable nitrogen-centered free radical that is violet and turns yellow upon reduction by hydrogen or electron-donating antioxidants. Substances with this activity are termed antioxidants or free-radical scavengers (Pavithra and Vadivukkarasi, 2015) [12] . In the present study, A. hexapetalus leaf extract scavenged DPPH radicals in a dependent manner, with scavenging activity increasing as extract concentration increased. Thus, A. hexapetalus extract may be an important source of free-radical scavengers and could support treatment of oxidative stress-related ailments. These results agree with Krishna Satya et al. (2017) [13] , who reported potent DPPH activity and high total phenolic and flavonoid contents in A. hexapetalus leaf extract. The extract's flavonoids and phenols likely contribute to the observed radical-scavenging activity and overall antioxidant potential. RBC membrane stabilization is used to assess in vitro anti-inflammatory activity because the RBC membrane resembles the lysosomal membrane. A. hexapetalus leaf extract inhibited heat and hypotonicity-induced hemolysis in a dose-dependent manner, indicating RBC membrane stabilization. This activity may result from phytoconstituents interacting with membrane components and preventing release of phospholipase A2, thereby reducing phospholipid hydrolysis and formation of inflammatory mediators. These findings support membrane stabilization as one mechanism underlying the extract's antiinflammatory effect and suggest the extract helps preserve lysosomal and RBC membrane integrity during inflammation. Lysosomal enzymes released into the cell can exacerbate inflammation and tissue damage by preventing their release, the extract may limit such damage. The extract's maximum percentage inhibition of hemolysis was comparable to that of phenylbutazone, corroborating the observations of Bajaj and Wakode (2017) [14] . Protein denaturation is associated with the inflammatory process. In the present study, A. hexapetalus leaf extract effectively prevented albumin denaturation in a concentrationdependent manner, indicating the presence of secondary metabolites, such as phenolic compounds, that preserve protein structure and protect against denaturation (Kousalya and Doss, 2020) [11] . The carrageenan-induced paw edema model is a standard laboratory test used to study acute inflammation and screen the anti-inflammatory properties of drugs and plant extracts. In the present study, the ethanolic leaf extract of A. hexapetalus showed significant antiinflammatory activity in carrageenan-induced acute paw edema in albino rats (Suman et al., 2015) [15] . Alkaloids and polyphenols present in A. hexapetalus likely contribute to these anti-inflammatory effects. Means bearing different superscripts within a column (between treatment groups) differ significantly

Conclusion

The ethanolic leaf extract of A. hexapetalus demonstrated significant antioxidant and anti-inflammatory activities in both in vitro and in vivo models. Its effects on DPPH scavenging, membrane stabilization, albumin denaturation and carrageenan-induced paw edema support its traditional

Acknowledgement

International Journal of Advanced Biochemistry Research https://www.biochemjournal.com use and suggest potential for development as a natural antiinflammatory therapeutic.

References

1. Laveti D, Kumar M, Hemalatha R, Sistla R, Naidu VG, Talla V, et al. Anti-inflammatory treatments for chronic diseases: a review. Inflamm Allergy Drug Targets. 2013;12(5):349-361. https://doi.org/10.2174/18715281113129990057
2. Allegra M. Antioxidant and anti-inflammatory properties of plants extract. Antioxidants. 2019;8(11):549. https://doi.org/10.3390/antiox8110549
3. Sinmisola A, Oluwasesan BM, Chukwuemeka AP. Blighia sapida K.D. Koenig: A review on its phytochemistry, pharmacological and nutritional properties. J Ethnopharmacol. 2019;235:446-459. https://doi.org/10.1016/j.jep.2019.01.017
4. Bailly C, Hénichart JP. Advocacy for the medicinal plant Artabotrys hexapetalus (Yingzhao) and antimalarial yingzhaosu endoperoxides. Molecules. 2022;27(19):6192. https://doi.org/10.3390/molecules27196192
5. Pandey A, Tripathi S. Concept of standardization, extraction and pre phytochemical screening strategies for herbal drug. J Pharmacogn Phytochem. 2014;2(5):115-119.
6. Odebiyi OO, Sofowora EA. Phytochemical screening of Nigerian medicinal plants. Lloydia. 1978;41(3):234246.
7. Aouachria S, Boumerfeg S, Benslama A, Benbacha F, Guemmez T, Khennouf S, et al. Acute, sub-acute toxicity and antioxidant activities (in vitro and in vivo) of Reichardia picroides crude extract. J Ethnopharmacol. 2017;208:105-116. https://doi.org/10.1016/j.jep.2017.07.009
8. Oyedapo OO, 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(4):46-51.
9. Sakat S, Juvekar AR, Gambhire MN. In vitro antioxidant and anti-inflammatory activity of methanol extract of Oxalis corniculata Linn. Int J Pharm Pharm Sci. 2010;2(1):146-155.
10. Kalita S, Kumar G, Karmakar D, Kalita J, Tripathy S, Pal R. Evaluation of anti-inflammatory activity of Moringa oleifera leaf extract in experimental models. Biomed Pharmacother. 2018;108:838-846. https://doi.org/10.1016/j.biopha.2018.09.088
11. Kousalya P, Doss VA. Artabotrys hexapetalus (L.f.) Bhandari: Ethnomedicinal uses, pharmacological properties and bioactive compounds-review. J Huazhong Univ Sci Technolog. 2020;50(7):1-11.
12. Pavithra K, Vadivukkarasi S. Evaluation of free radical scavenging activity of various extracts of leaves from Kedrostis foetidissima (Jacq.) Cogn. Food Sci Hum Wellness. 2015;4(1):42-46. https://doi.org/10.1016/j.fshw.2015.02.001
13. Krishna Satya A, Sowjanya KM, Swathi J, Narendra K. Estimation of in vitro antioxidant activity and total phenolic, flavonoid content of selected medicinal plants. Int J Pharm Sci Rev Res. 2017;43(2):142-147.
14. Bajaj S, Wakode S. Effect of extraction solvent on total phenol content, total flavonoid content, antioxidant and anti-inflammatory activity of Artabotrys hexapetalus. Int J Biol Pharm Allied Sci. 2017;6(8):1562-1577.
15. Suman S, Raghavendra HG, Suresh Babu M. Antiinflammatory activity of ethanolic leaf extract of Artabotrys hexapetalus. Indo Am J Pharm Res. 2015;5(1):285-294.