Datagni et al Journal of Drug Delivery & Therapeutics. 2021; 11(5-S):63-70 Available online on 15.10.2021 at http://jddtonline.info Journal of Drug Delivery and Therapeutics Open Access to Pharmaceutical and Medical Research Copyright © 2021 The Author(s): This is an open-access article distributed under the terms of the CC BY-NC 4.0 which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use provided the original author and source are credited Open Research Article Access Full Text Article In vitro anti-inflammatory and antimicrobial activity of Securidaca longepedunculata and Annona senegalensis hydro-alcoholic extract Datagni G’massampou1*, Mouzou Aklesso Pouwelong1, Metowogo Kossi1, Afanyibo Yaovi-Gameli2, Sadji Adodo2, Eklu-Gadegbeku Kwashie1 1 Research Unit in Physiopathology - Bioactive substances and Safety / Laboratory of Physiology-Pharmacology / Faculty of Sciences / University of Lomé, 01BP: 1515, Lomé, Togo 2 National Institute of Hygiene of Togo, 26 Street Nangbéto, B.P 1396, Lomé, Togo. Article Info: _________________________________________ Article History: Received 21 August 2021 Reviewed 29 September 2021 Accepted 05 October 2021 Published 15 October 2021 _________________________________________ Cite this article as: Datagni G, Mouzou AP, Metowogo K, Afanyibo YG, Sadji A, Eklu-Gadegbeku K, In vitro antiinflammatory and antimicrobial activity of Securidaca longepedunculata and Annona senegalensis hydro-alcoholic extract, Journal of Drug Delivery and Therapeutics. 2021; 11(5-S):6370 DOI: http://dx.doi.org/10.22270/jddt.v11i5-S.5090 _________________________________________ *Address for Correspondence: Datagni G’massampou, Research Unit in Physiopathology-Bioactive substances and Safety/ Laboratory of Physiology-Pharmacology/Faculty of Sciences / University of Lomé, 01BP: 1515, Lomé, Togo Abstract ______________________________________________________________________________________________________ Annona senegalensis and Securidaca longepedunculata are two plants traditionnaly used in inflammation and wounds infection treatment after snakebites. This study aims to investigate the in vitro anti-inflammatory and antimicrobial activities of the hydroalcoholic extracts of Annona senegalensis and Securidaca longepedunculata. Antimicrobial activity of the two plant extracts was examined against five bacterial strains with the well diffusion method and the inhibition zones diameters (IZD), minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined using the 96-well microplate dilution method. While antiinflammatory activity was assessed by the albumin denaturation method. The results obtained showed that the hydroalcoholic extract of Annona senegalensis has antimicrobial property against Staphylococcus aureus (IZD=12.22 ± 0.24 mm, MIC=62.5 mg/mL, MBC=125 mg/mL) and against Pseudomonas aeruginosa (IZD=12.06 ± 0.06 mm, MIC=125 mg/mL, MBC=250 mg/mL). Securidaca longepedunculata also showed its antimicrobial activity against Staphylococcus aureus (IZD=12.03 ± 0.03 mm, MIC=125 mg/mL, MBC=250 mg/mL) and Candida albicans (IZD=12.12 ± 0.07 mm, MIC=62.5 mg/mL, MFC=125 mg/mL). In the order hand, Annona senegalensis and Securidaca longepedunculata exhibited concentration-dependent anti-inflammatory activity by reducing significantly (p<0.001) the denaturation of BSA. In addition S. longepedunculata inhibited haemolysis significantly (p<0.001) more than Diclofenac sodium at 200 and 400 µg/mL. Hence, it was concluded that Annona senegalensis and Securidaca longepedunculata possessed anti-inflammatory and antimicrobial properties and can be used in the treatment of inflammation and wounds infection after snakebites. Keywords: Annona antimicrobial, BSA. INTRODUCTION Inflammation and wound infection occuring after snakebite complicate treatment of victims. To treat snake bites in Africa, few victims use modern health facilities 1. The microbes resistance to available antibiotics, the blow of antibiotics and anti-inflammatory drugs available on the market leads to the search for new therapeutic molecules with antimicrobial and anti-inflammatory properties. Medicinal plants are an adequate solution to these problems. Victims often use medicinal plants. Annona senegalensis (A. senegalensis) and S. longepedunculata are medicinal plants used in envenomation management in Togo 2. A. senegalensis is a small tree that is widely distributed in Africa 3. It is usually 2 to 6 meters tall and has an aromatic ISSN: 2250-1177 [63] senegalensis, Securidaca longepedunculata, anti-inflammatory, flower that is used to flavour foods. Its ripe fruit has a yellow colour and a pleasant smell. It’s fruit is edible 4. Previous studies have shown that this plant has anti venom 4, 5, anti diarrhea 6, anti cancer 7, Spermatogenic 8, anti convulsion 9 properties. A methanol root extract has analgesic and antiinflammatory effects 10. Ethanol and methanol extracts from roots, leaves and bark have antimicrobial effects 11. Securidaca longepeduncula is also called «snake tree». It is a 7 m or 10 m tall shrub with a smooth bark and grey branches. Its pink to purple flowers make it easy to recognize during its flowering season 12. Previous studies have shown that this plant has: antioxidant 13, antiplasmodiale 14, antiparasitic 15 properties. The methanol extract, petroleum ether fraction and methanol fraction have CODEN (USA): JDDTAO Datagni et al Journal of Drug Delivery & Therapeutics. 2021; 11(5-S):63-70 anti-inflammatory effects 16. The methanol extract and chloroform fraction have antibacterial properties 17. In vitro antimicrobial property of A. senegalensis and S. longepedunculata extracts The control of inflammation and wound infection caused by snakebites is important in envenomation management. Antiinflammatory Medicinal plants are used to treat several adverse effects of synthetic anti-inflammatory drugs. Antimicrobial plants are the source of new molecules that can counteract microbial resistance. The aim of this study is to evaluate in vitro anti-inflammatory and antimicobial properties of S. longepedunculata and A. senegalensis using their hydroalcoholic extracts. Microbial strains Microbial suspensions preparation MATERIAL AND METHODS Plants materials Roots of A. senegalensis were collected from Gblainvié in Zio prefecture (Togo), located at 30 km north of Lomé. The roots of S. longepedunculata were collected from Anié locality in the prefecture of Anié (Togo). This locality is located 187,7 Km north of Lomé. Both plants have been identified in Botany and Plant Ecology Laboratory of Faculty of Science (University of Lome), where voucher specimen was deposited in the herbarium under the number TOGO 15673 (A. senegalensis) and TOGO 15676 (S. longepedunculata). Roots of the plants were cleaned out with water, cut into small pieces, dried at the Animal Physiology laboratory at 22°C and then reduced into powder using THOMAS-Wiley, LABORATORY MILL, Model 4 mill. Preparation of the hydro-alcoholic extracts 400g of powder from each plant were extracted in 4L of an ethanol/water mixture (50:50, V/V) for 72 hours under intermittent manual agitation. The crude extract was filtered on Whatman paper and evaporated in vacuum at 45°C using a Rotavapor (Heidolph2, Germany). The extracts were in the form of crystals and have been stored in the refrigerator at 4°C. Phytochemical Study of A. senegalensis and S. longepedunculata Phytochemical screening The phytochemical analysis was performed for detection of phyto-constituents present in the extracts using standard procedure by 18. Determination of total phenols and tannins Total phenols were measured in the extracts by the FolinCiocalteu method and for the determination of tannins; a second dosage of the phenols was performed after fixing tannins by PVP (Polyvinyl pyrrolidone). Total tannins content was determined by absorbance difference between the first and second assay according to the method of Maksimovic et al., 19. Determination of total flavonoids Flavonoids content was determined according to the method used by Mimica-dukic 20. Briefly, to 2 ml of extract / rutin at different concentrations (5-100 µg/ml), 2 ml of aluminum chloride (20 mg/ml) and 6 ml of sodium acetate (50 mg/ml) were added. Absorbance was read at 440 nm after 2.5 hours of incubation. ISSN: 2250-1177 The pathogenic bacterial strains assayed in this study were Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 700603, including the yeast Candida albicans ATCC 35659. These strains come from bacteriology laboratory strain bank of the National Institute of Hygiene (Togo). [64] Grown 24 hours old Microorganisms were used. To obtain these young colonies of microorganisms, the selected germs were isolated on Muller-Hinton agar (MHA) for bacteria and on Sabouraud Chloramphenicol agar (SCA) for Candida albicans. Incubation was done at 37°C for bacteria and 25°C for Candida albicans for 18 to 24 hours. Microbial suspensions of densities of 0.5 MC Farland diluted 10-1 were prepared in normal physiological saline with young colonies of microorganisms. The S. longepedunculata and A. senegalensis extracts solutions were prepared at a concentration of 250 mg/mL in distilled water and then sterilized on millipore membrane of 0.45 μm porosity and 47 mm in diameter 21. Presumptive Test It is a presumptive test that has made it possible to identify the active extracts starting from a high concentration. The antibiotic susceptibility testing was performed by the agar well diffusion method with some changes 21. The high concentration of extract in this study is 250 mg/mL. The microbial suspensions used were equal to 0.5 Mac Farland (≈108 CFU/mL). The inoculum was introduced on culture medium prepared under standard conditions. These were MHA for bacteria and SCA for Candida. The quality of these medium was evaluated by sterility and fertility tests before use. After inoculation of the medium, wells of 6 mm in diameter were made using a sterile hollow punch concentric ally in the agar. Gentamicin solution’s 30 μg/mL (for Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae) and Nystatin solution’s 250 mg/mL for Candida albicans were used as reference drugs. For negative controls, sterile distilled water was used in place of the extract. After 30 minutes of pre-diffusion at laboratory temperature, the Petri dishes were incubated for 24 h at 35˚C for the bacteria and 25˚C for the yeasts. The microbial growth inhibition zone diameter’s was measured using an electronic reading chart. Extracts having an inhibition diameter ≥ 12 mm (including disc) were used for the determination of MIC and MBC/MFC. The tests were repeated in triplicate 22. Minimum Inhibitory Concentrations (MIC) and Bactericidal/Fungicidal (MBC/MFC) determination Extracts that showed a growth inhibition diameter of 12 mm according to our presumptive test were used for the MIC and MBC/MFC determination. This test was performed using 96-well microplate dilution method 23, 24, 25. From a stock extract solution of 250 mg/mL. A successive dilutions series of the two plants extracts (250, 125, 62.5, 31.25, 15.625, 7.8125 and 3.90625 mg/mL) were placed in Mueller Hinton Broth (MHB). The wells were inoculated with a microbial suspension at 6x105 CFU/mL. Quality control was performed with MHB (not inoculated). CODEN (USA): JDDTAO Datagni et al Journal of Drug Delivery & Therapeutics. 2021; 11(5-S):63-70 Another control was performed with MHB seeds to facilitated reading. The tests were performed in a sterile environment. The preparations were covered with parafilm and incubated at the appropriate temperature for 24 h. After incubation, the wells were observed with the naked eye. Turbidity presence corresponded to microbial culture presence. The MIC of the extract on tested strain corresponds to extract lowest concentration for which no culture was observed. Then, 100 μL was taken from wells that did not show visible microbial growth to the naked eye and plated on MHA for bacteria and SCA for Candida albicans. Incubation was carried out at the appropriate temperature for 24 h. The tests were performed in triplicate. The lowest concentration for which no colonies were found was considered the MBC or MFC of the extract on the strain tested. The MBC/MIC ratio was used to determine the antibiotic activity of the extract on the microbial strain. Assessment of the in vitro anti inflammatory activity of A. senegalensis and S. longepedunculata extracts Membrane stabilisation assay The method described by Javed et al., 26 and Joshi et al., 27 was used to perform this test. Wistar rats were anesthetized using light diethyl ether and blood was collected from the retro-orbital sinus in heparinised tubes. The collected blood was centrifuged at 1500 rpm for 10 min and washed three times with the same volume of normal saline. The reconstitution of red blood cells (RBC) with saline solution was 10% v/v suspension. To 1 mL of the RBC suspension were added 1 mL of plant extract or the reference drug Diclofenac sodium at different concentrations (50-1600 µg/mL) and 2 mL of hyposaline solution (0.36%). After 30 min of incubation at 37 °C, centrifugation (3000 rpm) was performed for 20 min. The assay was performed in three replicates for each concentration and the membrane stabilisation percentage reflecting anti-inflammatory activity was determined after reading the absorbance at 560 nm. The inhibition percentage was calculated by following formula. Anti-inflammatory activity (%) = (A0 - At / A0) x 100 A0 was the control absorbance (without extract) and At was the absorbance of extract or drug presence. Inhibition of bovine protein serum albumin (BSA) denaturation incubation, and at 70°C during 5 min. After cooling, 2.5 mL PBS (pH 6.3) was added to each sample. The test was carried out in three replicates for each concentration and the protein denaturing inhibition percentage that reflects antiinflammatory activity was determined after reading the absorbance at 660 nm. The inhibition percentage was calculated by following formula. Anti-inflammatory activity (%) = (A0 - At / A0) x 100 A0 was the control absorbance (without extract) and At was the absorbance of extract or drug presence. Data Analysis Data were expressed as Mean ± SD (standard deviation) for presumptive test and Mean ± SEM (standard error of the Mean) for anti-inflammatory tests using the GraphPad Prism 7 software. Statistical differences between groups were determined by ANOVA followed by Dunnett test and considered significant for p < 0.05. RESULTS Phytochemical Study of A. senegalensis and S. longepedunculata Phytochemical screening Phytochemical analysis of hydroalcoholic extracts A. senegalensis and S. longepedunculata revealed the presence of flavonoids, tannins, carbohydrates, alkaloids, phenols, and saponosides. The results of phytochemical screening are resumed in table 1. Table 1 : Phytochemical screening Metabolites A. senegalensis S. longepedunculata Alkaloids + + Tannins + + Flavonoids + + carbohydrates + + Phenols + + Saponosides + + + : presence The anti-inflammatory effect of extracts was investigated using Saleem et al., method 28. To 0.45 mL of bovine serum albumin (BSA) solution (5% w/v), 0.05 mL of extract at different concentration or reference drugs (Diclofenac sodium) were added (1600; 800; 400; 200; 10 and 50 µg/ml). Incubation took place twice : at 37°C during 20 min Determination of total flavonoids, phenols and tannins Table 2 shows that A. senegalensis and S. longepedunculata contain phenolic compounds. Table 2 : Total phenols, flavonoids and tannins content Extracts Total flavonoids Total phenols Total tannins (mgRE/g) (mgGAE/g) (mgGAE/g) A. senegalensis 15.62 ± 0.71 31.77±2.24 5.15±2.24 S. longepedunculata 29.62±0.95 11.09±0.62 1.07±0.62 Total phenols and tannins are expressed in mg Gallic Acid Equivalent/g extract. Flavonoids are expressed in mg Rutin Equivalent/g extract. ISSN: 2250-1177 [65] CODEN (USA): JDDTAO Datagni et al Journal of Drug Delivery & Therapeutics. 2021; 11(5-S):63-70 = 12.06 ± 0.06 mm. A. senegalensis root extract has not active on : Klebsiella pneumonia (K. pneumonia), Escherichia coli (E. coli) and Candida albicans (C. albicans).In addition, S. longepedunculata extract was active on S. aureus with IZD = 12.03 ± 0.03 mm and C. albicans with IZD = 12.12 ± 0.07 mm. S. longepedunculata root extract has not active on K. pneumoniae, E. coli and P. aeruginosa. (Table 3) In vitro antimicrobial property of A. senegalensis and S. longepedunculata extracts Antimicrobial presumptive test The results showed that A. senegalensis extract was active on Staphylococcus aureus (S. aureus) with IZD = 12.22 ± 0.24 mm and on Pseudomonas aeruginosa (P. aeruginosa) with IZD Table 3 : inhibition zone diameter obtain in presumptive test IZD (mm) A. Senegalensis P. aeruginosa ATCC 27853 12.06 ± 0.06 GN S. longepedunculata 250 mg/mL 30 μg/mL NA 14.80 ± 0.79 K. pneumonia ATCC 700603 NA NA 11.84 ± 0.34 S. aureus ATCC 29213 12.22 ± 0.24 12.03 ± 0.03 27.30 ± 0.81 E. coli ATCC 25922 NA NA 20.65 ± 0.06 C. albicans ATCC 35659 NA 12.12 ± 0.07 250 mg/mL NY 250 mg/mL 29.09 ± 0.04 IZD = inhibition zone diameter, GN = Gentamicin, NY = Nystatin, NA = not active A. senegalensis extract was active on S. aureus with MIC = 62.5 mg/mL and MBC = 125 mg/mL. He is also active on P. aeruginosa with MIC = 125 mg/mL and MBC = 250 mg/mL. The MBC/MIC ratio is 2 for the two bacteria on which A. senegalensis extract was active. S. longepedunculata extract was active on C. albicans with MIC = 62.5 mg/mL and MBC = 125 mg/mL. He is also active on S. aureus with MIC = 125 mg/mL and MBC = 250 mg/mL. The MFC/MIC or the MBC/MIC ratio is 2 for the two bacteria on which S. longepedunculata extract was active. (Table 4) Determination of Minimum Inhibitory Concentrations (MIC) and Bactericidal/Fungicidal (MBC/MFC) MICs and MBCs/MFCs were determined for germs that were susceptible to extra ts with inhibition diameters ≥ 12 mm. The bacteriostatic and bactericidal effects of the extracts on the germs were determined by the ratio of MBC/MIC or MFC/MIC ≤ 1 (Bactericidal); MBC/MIC or MFC/MIC ≥ 2 (Bacteriostatic). Table 4 : MIC and MBC of microorganisms A. senegalensis Germs CMI (mg/ml) CMB (mg/ml) S. longepedunculata MBC/CMI CMI MBC/CMI MFC/CMI (mg/ml) CMB (mg/ml) MFC/CMI P. aeruginosa 125 250 2 0 0 0 K. pneumonia 0 0 0 0 0 0 62.5 125 2 125 250 2 E. coli 0 0 0 0 0 0 C. albicans 0 0 0 62.5 125 2 S. aureus Assessment of the in vitro anti inflammatory activity of A. senegalensis and S. longepedunculata extracts significantly (p<0.001) inhibited haemolysis compared to diclofenac. Membrane stabilisation assay These results provide evidence for the membrane stabilizing effect of both extracts as an additional mechanism for their anti-inflammatory activity (Figure 1). The analysis showed concentration-dependent protection of the cell membrane by the hydroalcoholic extracts of A. senegalensis and S. longepedunculata. For example, at the dose of 50 µg/mL, A. senegalensis and S. longepedunculata ISSN: 2250-1177 [66] CODEN (USA): JDDTAO Datagni et al Journal of Drug Delivery & Therapeutics. 2021; 11(5-S):63-70 100 d ic lo fe n a c * i n h ib it io n % 80 *** * A . s e n e g a le n s is S . lo n g e p e d u n c u la ta 60 *** *** 40 * 20 00 0 16 80 0 0 40 10 20 0 *** *** 50 0 C o n c e n tra tio n (µ g /m L ) Figure 1: Effet of A. senegalensis and S. longepedunculata hydroalcoholic extracts on red blood cell membrane stabilisation ***p < 0,001 ; *p < 0,05 (compared to diclofenac). % inhibition = (A0 - At / A0) x 100 .A0 was the control absorbance (without extract) and At was the absorbance of extract or drug presence. Inhibition of bovine protein serum albumin (BSA) denaturation The results in figure 4 showed that At 50 µg/mL, hydroalcoholic extracts of A. senegalensis and S. longepedunculata significantly (p<0.05) inhibited protein denaturation compared to Diclofenac. In addition, at the dose of 400 µg/mL, the inhibition of BSA denaturation by the two plants were more significant (p<0.001) than Diclofenac (Figure 2). 150 d ic lo fe n a c i n h ib it io n % A . s e n e g a le n s is S . lo n g e p e d u n c u la ta 100 * *** *** 50 *** * 00 16 0 80 0 40 0 20 10 50 0 * 0 C o n c e n tra tio n (µ g /m L ) Figure 2 : Effet of A. senegalensis and S. longepedunculata hydro-alcoholic extract on BSA denaturation ***p < 0,001 ; *p < 0,05 (compared to diclofenac). % inhibition = (A0 - At / A0) x 100 .A0 was the control absorbance (without extract) and At was the absorbance of extract or drug presence. DISCUSSION The search for natural anti-inflammatory and antimicrobial agents with fewer side effects has crucially increased nowadays. This study was then investigated in order to evaluate the antiinflammatory and antimicrobial activities of hydroalcoholic extracts of A. senegalensis and S. longepedunculata. ISSN: 2250-1177 [67] The antimicrobial assay was performed in other to detect the sensitivity of five germs (Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 700603; Candida albicans ATCC 35659) in the presence of A. senegalensis and S. longepedunculata hydro-alcoholic extracts. Results revealed that 40% of the germs tested were CODEN (USA): JDDTAO Datagni et al Journal of Drug Delivery & Therapeutics. 2021; 11(5-S):63-70 susceptible to hydro-alcoholic extracts of A. senegalensis and S. longepedunculata. A. senegalensis extract was found to be active on Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) with inhibition zone diameters of 12.22 mm and 12.06 mm respectively. For the two bacteria on which A. senegalensis extract was active, the MIC = 62.5 mg/mL for S. aureus and MIC = 125 mg/mL for P. aeruginosa. The MBC extract for these two bacteria is 125 mg/mL for S. aureus and 250 mg/mL for P. aeruginosa. However, this extract was not active on E. coli, K. pneumoniae and C. albicans Our results corroborate with the work Swhich showed that Kaurenoic Acid isolated from A. senegalensis root bark inhibited S. aureus and P. aeruginosa but has no effect on Klebsiella pneumoniae and Escherichia coli 29. This also corroborates with those of More et al., which showed that A. senegalensis methanol extract is not active on Candida albicans 30. Our results are different from the work of Awa et al 31 in which methanol extract from A. senegalensis stem bark acted on E. coli with inhibition zone diameter equals 12 mm. Lino and Deogracious 32 showed that the acqueous extract inhibited S. aureus with a diameter of 18 mm. Similarly, the raw flavonoids isolated from stem bark of A. senegalensis inhibited E. coli an inhibition zone diameter equal to 18 mm 33. The MIC and MBC obtained with methanol and ethanol extracts of this plant are different from the values obtained with hydro-alcoholic extract in this study. Thus, A. senegalensis methanol extract was active on S. aureus with MIC=250 µg/ml and MBC>1000 µg/ml 11. The ethanol extract from A. senegalensis root bark acted on S. aureus with a MIC=500 µg/ml 34. In addition, S. longepedunculata extract was active on S. aureus and C. albicans respectively with 12.03 mm and 12.12 mm inhibition diameters. For the two microbes on which A. senegalensis extract was active, the MIC are 125 mg/mL for S. aureus and 62.5 mg/mL for C. albicans. S. longepedunculata extract has an MBC=250 mg/mL for S. aureus and an MFC=125 mg/mL for C. albicans. However S. longepedunculata is not found to be active on E. coli, P. aeruginosa and K. pneumoniae. Our results are same with those obtained by 35 which showed that S. longepedunculata roots ethyl acetate fraction and n-butanol fraction have inhibitory effects on S. aureus and C. albicans. The two fractions had an inhibition zone diameter of 12 mm each on both germs. Acqueous extract yielded 13 mm inhibition zone diameter with S. aureus and MIC=1000 mg/mL 32. The lack of sensitivity of the microorganisms used in this work to the extracts of A. senegalensis and S. longepedunculata could be explained by: an inaccessibility of the molecules contained in these two extracts to the microbial cell due to the impermeability of the membrane of the microbes to the molecules contained in these extracts; or an affinity of the molecules contained in these two extracts for the bacterial target or an expulsion of the antibiotic molecules contained in these extracts by chromosomal efflux pumps. The diversity observed in the sensitivity of K. pneumoniae to the extracts could be explained first by the capsule surrounding the bacteria presence as a natural protection [36]. Difference between our results and those obtained with methanol and ethanol extracts is explained by the fact that hydro-alcoholic extract (50:50) would not have been able to extract sufficient the active ingredients extracted acting microbe with methanol and ethanol. These active ingredients would increase the bactericidal potency of the two extracts when methanol and ethanol are used as maceration solvents. ISSN: 2250-1177 [68] Inflammation is a physiological process that defends body against aggression that results in tissue alteration. Inflammation primary function is to eliminate the aggressor and allow tissue repair. Short-term inflammation known as acute inflammation is a beneficial phenomenon of the body that allows it to regain its physiological integrity. Whereas the negative inflammation aspect occurs when it lasts and becomes a chronic inflammation. In this case, inflammatory reaction should be controlled by the drugs 37. In this study, the effect of S. longepedunculata and A. senegalensis extracts on membrane stabilization was indexed via the ability to protect RBCs from heat-induced haemolysis. The red blood cell membrane stabilization can be extrapolated to the lysosomal membrane because the two membranes are analogous. The haemolytic effect of a hypotonic solution is expressing by excessive accumulation of fluid inside the cell that caused membrane rupture 38. Results showed that S. longepedunculata and A. senegalensis protected red blood cell membrane against haemolysis. This effect occured in dose dependent manner. However, S. longepedunculata showed a higher protective capacity than the reference drug used for the 200 µg/mL dose. This could be explained by the control of surface/volume ratio of cells or by strengthening of the red blood cells membrane by the plants. Protein denaturation is a process by which proteins lose their structure due to the presence of other compounds, external stress, or heat, thus leading them to lose their biological functionality. Therefore, denaturation of tissue proteins is recognized as a marker of inflammation. In our study, Both extracts inhibited BSA protein denaturation in concentration depent manner. It is possible that bioactive compounds in the extracts protect lysosomal membranes against injury by interfering with activation of phospholipases. The extracts may block an exagerated release of pro-inflammatory molecules, including histamine, serotonin, tachykinine, bradykinine and complement proteins 39. Our results corroborate with other studies that had demonstrated the anti-inflammatory effect of A. senegalensis 40, 41 and S. longepedunculata42,43. To better understand the effect of the two plants, their preliminary phytochemical study was carried out. With phytochemical screening, the presence of alkaloids, tannins, flavonoids, carbohydrates, saponosides and phenols were revealed in the two extracts. This is in accordance to the works of 44 and 45. Quantitative tests confirmed the presence of these phenolic compounds. The phenolic compounds present the plants could explain their antimicrobial and antiinflammatory effects because many studies demonstrated the antibacterial properties of tannins 46 and flavonoids 47; anti-inflammatory properties of phenols such as coumarin 48, flavonoids 49 and tannins 50. In fact, these compounds present in the extract having functional groups serve as electron donors by breaking the free radical chain. Flavonoids also have the property of stabilizing the structure of biological membranes. Phenolic compounds also are able to inhibit either the production or the action of proinflammatory mediators, resulting in anti-inflammatory capacity. CONCLUSION In summary, A. senegalensis and S. longepedunculata have remarquable antimicrobial and anti-inflammatory properties. These properties could be related to the phytochemical compounds present in the hydroalcoholoic CODEN (USA): JDDTAO Datagni et al Journal of Drug Delivery & Therapeutics. 2021; 11(5-S):63-70 extracts of A. senegalensis and S. longepedunculata. Taken together, these two medicinal plants can be used in the patients suffering of inflammation and microbial infection of wounds by snake bites. However, further studies are required to understand the exact mechanism of action of various constituents present in the two plants. ACKNOWLEDGMENTS The authors are grateful to the bacteriology laboratory of National Institute of Hygiene (INH) of Lome for assistance in antimicrobial test. 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