Comparative study of the anti-inflammatory activity of hydroalcoholic extracts of different parts of the Diospyros mespiliformis plant

 Corresponding author: Issa Samb Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. Comparative study of the anti-inflammatory activity of hydroalcoholic extracts of different parts of the Diospyros mespiliformis plant Ismaïla Bouré Diouf 1, Mareme Thiaw 1, Issa Samb 1, *, Moussa Karé 1 and Mohamed Lamine Gaye 2 1 Department of Chemistry, Organic and Therapeutic Chemistry Team (ECOT), Alioune Diop University (UAD), Bambey, Senegal. 2 Department of Chemistry, Cheikh Anta DIOP University (UCAD), Dakar, Senegal. World Journal of Advanced Research and Reviews, 2025, 26(03), 516-525 Publication history: Received on 27 April 2025; revised on 01 June 2025; accepted on 04 June 2025 Article DOI: https://doi.org/10.30574/wjarr.2025.26.3.2240 Abstract The aim of this study is to verify the anti-inflammatory potential of different parts of the plant Diospyros mespiliformis. This plant is known in traditional medicine to combat various types of ailments. The leaves are used to treat headaches, arthritis, skin infections, gingivitis, toothache and wounds. Infused bark is used to treat stomach aches, coughs, bronchial diseases including tuberculosis, bruises and boils. The roots are used to treat tumors. [1-2] For all these reasons, a comparative study of the anti-inflammatory activity of different parts of the plant was undertaken. This was done by determining the lipoxygenase inhibition capacity of hydroalcoholic extracts (HE) from different parts of the plant. The results showed that leaves were the most active with an IC50 of 0.05281mg/mL, followed by bark with an IC50 of 0.07058 mg/mL and roots with an IC50 of 0.09429 mg/mL. Given that such high activity was obtained with these extracts, their purification could hold great promise in the development of new ways to treat inflammation. Keywords: Diospyros mespiliformis; Anti-inflammatory; IC50; Inflammation 1. Introduction Inflammation is a factor in the onset of many diseases, including diabetes, cancer, infection, obesity, cardiovascular disease and even neurodegenerative diseases such as Alzheimer's. The enzymes involved in inflammatory reactions include lipoxygenases (LOX), which are responsible for the formation of inflammation. [1-4] Among the enzymes involved in inflammatory reactions are lipoxygenases (LOX). These are responsible for the formation of intracellular messengers such as leukotrienes (LT), which play a major role in pain and inflammation regulation pathways. Inhibition of this enzyme therefore appears to be an alternative way of preventing inflammation or suppressing pain. Treatments based on highly effective non-steroidal anti-inflammatory drugs have been developed. However, the side effects - such as gastric bleeding, allergic reactions, kidney and heart problems - that can often result from their use are a persistent dilemma for the medical world. [5] These effects, most often associated with non-target activity on the enzymes involved, still give rise to real concern in the medical community, which is looking for alternatives to alleviate this phenomenon. This situation has prompted reflection on the development of therapeutic compounds capable of specifically inhibiting the enzymes involved in inflammation without leading to an imbalance in the body. Plants such as Diospyros mespiliformis, whose ethnobotanical data reveal pharmacological activities linked to antiinflammatory properties, offer new prospects for the treatment of inflammation. This study aims to discover the therapeutic potential of different parts of the plant that could be selected as potential candidates for the development of drugs to inhibit LOX proteins. World Journal of Advanced Research and Reviews, 2025, 26(03), 516-525 517 1.1. Botanical presentation of the plant Diospyros mespiliformis Hochst Ex A. DC, a member of the Diospyros genus in the Ebenaceae family, is a 10 to 15 m-high tree with a robust, cylindrical trunk and brittle, charcoal-like black bark. It is a species characteristic of savannah woodlands (dry forests) and sometimes wet forests. It is characteristic of heavy, well-drained soils. [6-13] The species is found almost everywhere on the globe. It is found in the flora of several countries, including sub-Saharan Africa and the Gulf of Guinea, southern Africa, central Africa and northern Africa in Egypt. [9, 30] Its presence has been reported in the Near East (Yemen, Israel and Saudi Arabia), in North and South America, and in Madagascar. [6, 10, 12, 18,19, 22, 31] The plant's leaves are used as an astringent, febrifuge, haemostatic, laxative, stimulant and vermifuge. Infusions are used to treat fever, pneumonia, syphilis, leprosy and yaws. The leaves are also used to treat headaches, arthritis and skin infections. [33] 2. Materials and methods 2.1. Harvesting and preservation of plant leaves The raw material used in our study was harvested in Ndiemane, a village located on Senegal's Petite Côte between Mbour and Joal-Fadiouth. After harvesting, the leaves were washed with water and then dried under cover at room temperature in our laboratory. After drying, the samples were crushed and the powder put into glass jars for further processing. 2.2. Extraction of plant leaves This was carried out using the maceration process. This was carried out by cold contacting a 100 g mass of plant material powder with a 100 mL solvent in a 1000 mL flask. The mixture was then left to macerate for 48 hours. The macerate was then collected and filtered on Whatman filter paper using a Büchner filter fitted with a 500 mL volumetric flask. The filtrate obtained was reduced to a quarter of its initial volume on a rotary evaporator, then placed in a refrigerator to dry to obtain the desired crude extract. 2.3. Phytochemical screening tests These were carried out to qualitatively determine the different families of secondary metabolites present in extracts from the plant parts studied. Their identification was based on chemical screening methods written by several authors. [34-44]. Table 1 Screening test results for hydroalcoholic extracts of different plant parts Tests Hydroalcoholic extracts Alkaloids Terpenes Saponins Polyphenols Flavonoids Tannins Coumarins Anthraquinones Quinones Leucoanthocyanins Reducing compounds Carbohydrates Leaves ++ + ++ ++ ++ ++ - ++ ++ ++ ++ ++ Barks ++ + ++ ++ ++ ++ - ++ ++ ++ ++ ++ Roots +++ + +++ +++ ++ ++ ++ ++ + ++ ++ ++ 2.4. Anti-inflammatory activity tests 2.4.1. Preparation Substrate 140 mg linoleic acid is added to 5 mL deoxygenated distilled water by nitrogen bubbling. Next, 18 mL Tween 80 is added to the mixture, which is vortexed for 5 minutes. A 100 µL solution of 2 M NaOH is then added, and the volume adjusted to 50 mL with deaerated distilled water. The linoleic acid solution is then aliquoted and stored in the freezer. World Journal of Advanced Research and Reviews, 2025, 26(03), 516-525 518 Buffer solution The optimum pH of the reaction matrix was obtained using 0.1 M sodium borate buffer adjusted to pH 9.5 with 5 M NaOH. The borate buffer was oxygenated for 30 minutes before use. Enzyme Lipoxygenase solution (EC 1.13.11.12, SIGMA, 50,000 U/mg) was prepared at a concentration of 0.1 mg/mL in distilled water. After shaking, the solution was aliquoted and stored in the freezer until use. As lipoxygenase is heat-sensitive, all experimental steps were performed in an ice bath. Samples A series of dilutions from 0.1 to 0.6 mg/mL was prepared from dry extracts. 2.4.2. Experiments Experimental protocol In a 10 mm cuvette, 900 µL of borate buffer and 100 µL of extract or standard inhibitor (quercetin) at different concentrations are introduced, followed by 10 µL of lipoxygenase solution. The mixture is shaken three times and left at room temperature for 15 minutes. After this incubation, 10 µL of the prepared linoleic acid solution is added. After shaking, absorbance kinetics are measured directly at 234 nm every 5 seconds for 5 minutes. For each assay, a positive control corresponding to 0% inhibition was prepared by mixing 900 µL of borate buffer with 10 µL of lipoxygenase solution, then initiating the reaction by adding the substrate. Percent inhibition was calculated according to equation : %I= (S – E)/S With • S: speed of reaction in the absence of inhibitor (slope of curve) • E: reaction rate in the presence of inhibitor (sample) Standard inhibitor: quercetin Solvent: water Table 2 Percentage inhibition results for the aqueous solution of the inhibitor standard (quercetin) Quercetin Concentration %PI 0,001 10,76 0,01 73,39 0,1 97,58 IC50 value is 0.007 mg/mL World Journal of Advanced Research and Reviews, 2025, 26(03), 516-525 519 Figure 1 Graph showing the percentage of lipoxygenase inhibition as a function of the concentration of the inhibitor standard (quercetin) 3. Results 3.1. Part: Leaves 3.1.1. Extract: Hydroalcoholic (HE) Table 3 Percentage inhibition results for hydroalcoholic extract of plant leaves. Leaves HE Concentrations %PI Standard deviations 0,01 2,68 0 0,02 7,11 0,006666667 0,04 27,95 0,004355556 0,08 86,60 0,006466667 IC50 value is 0.05281 mg/mL Figure 2 Graph showing the percentage inhibition of lipoxygenase as a function of the concentration of the hydroalcoholic extract of plant leaves World Journal of Advanced Research and Reviews, 2025, 26(03), 516-525 520 3.1.2. Extract: Aqueous (H) Table 4 Percentage inhibition results for aqueous plant leaf extract Leaves H Concentrations %PI Standard deviations 0,01 1,02 0,00175 0,02 1,46 0,0035 0,04 3,52 0,0336 0,08 25,52 0,023311111 IC50 value is 0.15394 mg/mL Figure 3 Graph showing the percentage of lipoxygenase inhibition as a function of the concentration of the aqueous extract of plant leaves 3.2. Part: ROOTS Table 5 Percentage inhibition results for hydroalcoholic extract of plant roots Roots H Concentrations %PI Standard deviations 0,01 0,00 0 0,02 1,57 0 0,04 25,72 0,012644444 0,08 38,96 0,029777778 IC50 value is 0.09429 mg/mL World Journal of Advanced Research and Reviews, 2025, 26(03), 516-525 521 Figure 4 Graph of percentage lipoxygenase inhibition as a function of plant root hydroalcoholic extract concentration 3.3. Part: BARKS Table 6 Percentage inhibition results for hydroalcoholic extract of plant bark Barks H Concentrations %PI Standard deviations 0,01 0,00% 0 0,02 6,54% 0,0586 0,04 9,23% 0,012444444 0,08 63,74% 0,023266667 IC50 value is 0.07058 mg/mL Figure 5 Graph of percentage lipoxygenase inhibition as a function of plant root hydroalcoholic extract concentration In order to compare the lipoxygenase activities of the different hydroalcoholic extracts of the plant parts, a comparative graph of their activities was drawn. World Journal of Advanced Research and Reviews, 2025, 26(03), 516-525 522 Figure 6 Comparative graph of lipoxygenase inhibition percentages as a function of the concentration of hydroalcoholic extracts from different parts of the plant 4. Discussions The results show that our different extracts had higher IC50 values than the standard inhibition reference, quercetin IC50= 0.007mg/mL) used during the study. Between extracts from different parts of the plant, the hydroalcoholic leaf extract gave the best enzyme inhibition, with an IC50 value of 0.05281 mg/mL. This was followed by the bark hydroalcoholic extract, with an IC50 value of 0.07058 mg/mL. Roots came third, with an IC50 value of 0.09429 mg/mL. To justify the choice of solvent used in the study, the inhibition capacity of the aqueous extract of the most active part, namely the leaves, was checked in parallel and gave an IC50 of 0.15394 mg/mL. This value reinforces the idea that hydroalcoholic extracts are superior to aqueous extracts of the same plant part for lipoxygenase inhibition. An extract is considered highly active and promising when its IC50 is less than or equal to 23μg/mL. It is considered moderate or good when its IC50 is between 23 and 53 μg/mL, lesser or weak between 53 and 83μg/mL and insignificant when it is greater than 83μg/mL. [44-54] According to this classification, leaf extract with an IC50 value of 0.05281 mg/mL (52.81 μg/mL) exhibits good lipoxygenase inhibitory activity. The hydroalcoholic extract of roots with an IC50 of 0.07058 mg/mL (70.58 μg/mL) shows less or poor activity. Roots with an IC50 of 0.09429 mg/mL (94.29 μg/mL) have a very low to non-significant inhibitory activity. Anti-inflammatory properties are often attributed to the presence or detection of high levels of phenolic compounds in plant extracts. [2, 5] Chemical screening tests carried out on various extracts revealed the high presence of compounds from this family in our extracts. The presence of these phenolic compounds could be at the origin of its proven antiinflammatory properties. 5. Conclusion This study provides an overview of the inhibitory effect of extracts from different parts of the plant on LOX. Among the extracts tested, the leaf extract with an IC50 value of 0.05281 mg/mL showed the best activity, followed by the hydroalcoholic bark extract with a lower or low activity IC50 of 0.07058 mg/mL. The root extract, with an IC50 of 0.09429 mg/mL, was considered very weak or even insignificant. The hydroalcoholic extract remains a mixture of several molecular families, so it is interesting for the future to continue purification and compound isolation studies on the leaf hydroalcoholic extract. Compliance with ethical standards Disclosure of conflict of interest No conflict of interest to be disclosed. 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