The Antioxidant Potential of Ficus Umbellata Vahl (Moraceae) That Accelerates In Vitro and the In Vivo Anti-Inflammatory Protective Effects

Abstract

Ficus umbellata Vahl (Moraceae), is a plant with health benefits involved in the management of menopause physiological disorders and cancers. This study aimed at investigating the antioxidant and anti-inflammatory activities of aqueous (FU_Aq) and methanolic (FU_MeOH) extracts of Ficus umbellata. Their antioxidant activities were assayed by free radical scavenging using DPPH and ABTS assays, total antioxidant capacity, and ferrous reducing power (FRAP). Further, the effects of FU_Aq and FU_MeOH on murine erythrocyte membrane hemolysis and protein denaturation were investigated. The in vivo anti-inflammatory activity was determined in Wistar rats with carrageenan-induced paw oedema. At tested concentrations, FU_Aq and FU_MeOH demonstrated strong radical scavenging that was dose- and time-dependent, as well as total antioxidant capacity and ferrous ions reducing power. Moreover, they were able to stabilize murine red blood cell membranes against heat induced hemolysis and inhibit the denaturation of egg albumin at concentrations ranging from 0.125–2 mg/mL. Ficus umbellata methanolic extract at doses of 50 and 200 mg/kg endow antiedematous properties with edema inhibition percentages of 71.16 ± 1.72% and 72.98 ± 7.51%, respectively. Our findings shed light on the antioxidant and anti-inflammatory properties of Ficus umbellata that could be used in novel and safe strategies to overwhelm oxidative and inflammatory related diseases.

1. Introduction

Physiological phenomena, such as respiration, metabolism and immune system defense mechanisms, are important for the proper functioning of living organisms through which reactive oxygen species (ROS) are released. Their increased production could lead to disruption of redox signaling, molecular damage, mutations, cell death, and oxidative damage to macromolecules, such as DNA, proteins, and lipids. This damage could result in many diseases, including age-related disorders, cancer, atherosclerosis, and neurodegenerative diseases [1]. Moreover, ROS maintains inflammation by inducing the expression of genes that code for proteins involved in the production of inflammatory mediators [2,3]. Inflammation is a dynamic physiological process of defense, consisting of a set of vascular, cellular, and humoral reactions to eliminate the aggressor agent, cleanse cellular debris, repair damaged tissue, and restore homeostasis. This acute inflammation is manifested by redness, heat, pain, and swelling of the infected area. However, when it persists, it can lead to multiple general biological and clinical effects as altered general condition, combining asthenia, anorexia, fever, sleep disturbance, and cachexia with muscle wasting [4]. In addition, inflammation can be associated with changes in the three-dimensional structure of proteins following exposure to heat or chemicals. This leads to an activation of neutrophils and an overproduction of lysosomal constituents, such as bactericidal enzymes and proteases [5,6].

One of the most appropriate models for studying anti-inflammatory activities in vivo is that of oedema induction with carrageenan. Carrageenan induces acute and local inflammation characterized by an increase in the level of oxidative stress markers (neutrophil-derived free radicals and nitric oxide), proinflammatory mediators (histamine, prostaglandins etc., and proinflammatory cytokines/chemokines) in the body [7]. Overproduction of these mediators is strongly implicated in the pathogenesis of several chronic diseases, including diabetes, hypertension, cardiovascular disease, neurodegenerative diseases, alcoholic liver disease, chronic kidney disease, cancer, and aging [8]. Enzymatic and nonenzymatic antioxidant systems are part of the multiple mechanisms of the human body involved in protection from cellular damage due to reactive oxygen species (ROS) [9]. However, the innate defense may fail in the face of prolonged oxidative stress.

In addition, conventional therapies recommend the uses of analgesic drugs, non-steroidal anti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs (SAIDs), and opioids. However, the side effects caused by long-term use of antioxidants [butylated hydroxyl anisole (BHA) and butylated hydroxyltoluene (BHT)] and synthetic anti-inflammatory drugs (Aspirin, sodium diclofenac, indomethacin, Paracetamol) motivate the scientific community to search for alternative therapeutic solutions [10]. Natural antioxidants are particularly important because they are harmless, low cost and abundant in many plant sources [11]. Thus, medicinal plants are increasingly exploited for this purpose because most of the pharmacological substances and active compounds used to combat the above-mentioned diseases and to produce medicines are of natural origin. They do so through their ability to inhibit the formation of free radicals. Equally, several scientific works have highlighted the anti-inflammatory and antioxidant activities of extracts and pure compounds (phenolics, flavonoids, such as cynaroside) isolated from medicinal plants, such as Libidibia ferrea, Curcuma longa; Spirulina plantensis, Camellia sinensis Elsholtiza bodinieri [12,13,14,15,16,17], and some Ficus species [18,19].

F. umbellata Vahl (Moraceae) is a plant used in Cameroonian pharmacopoeia in the management of certain physiological disorders related to menopause [20]. Previous studies have revealed that F. umbellata extracts possess antiestrogenic and estrogenic effects in vitro on estrogen receptor (ER) expressing cells and in vivo on estrogen primary targets organs, respectively [21]. In addition, methanolic extracts of F. umbellata showed cytotoxic potential on breast cancer cell lines and an in vivo antitumor activity in ovarian cancer model in Wistar rats [22]. Therefore, the present study was conducted to evaluate the antioxidant and anti-inflammatory activities of Ficus umbellatta aqueous and methanolic extracts.

2. Materials and Methods

2.1. Chemical Reagents and Drugs

Diclofenac sodium and ibuprofen were purchased from Sigma-Aldrich GmbH, Sternheim, Germany. The reagents used for antioxidant assays were purchased from GIBCO (Grand Island, NY, USA).

2.2. Plant Material Harvest and Pre-Treatment

The barks of F. umbellatta Vahl (Moraceae) were harvested at Yaoundé (Centre region, Cameroon). The geographical coordinates indicate 32 N0778863, E0428160_23 m (“GARMIN” GPS). The plant material was then authenticated at the National Herbarium of Cameroon in comparison to the voucher specimen N°99/HNC. The botanical sample was then cut into small pieces and after that air-dried at room temperature and ground.

2.3. Preparation of Extracts

2.3.1. Preparation of Aqueous Extract

One portion of Ficus umbellata powder (2.7 kg) was macerated in distilled water (10 L) for 24 h at room temperature and filtered using Whatman paper N°4. The aqueous filtrate of F. umbellata (FU_Aq) was lyophilized obtaining 229.8 g, corresponding to an 8.5% yield.

2.3.2. Preparation of Methanolic Extract

Ficus umbellata powder (2 kg) was macerated in methanol 99% in proportions 1:4 for 72 h and filtered using Whatman paper N°4. Then, the filtrate was concentrated using rotary evaporator (40 °C, 337 mbar) and dried at room temperature to obtain 162 g of the methanolic extract (FU_MeoH) yielding 8.1%.

2.4. In vitro Antioxidant Assays

Antioxidant activity was determined based on the DPPH, ABTS, TAC (Total Antioxidant Capacity), and FRAP (Ferrous Reducing Antioxidant Power) assays.

2.4.1. DPPH Scavenging Activity

The DPPH scavenging activity of F. umbellata extracts was assessed as described by Alam et al. [23]. Briefly, in a 96-well microplate; 100 µL of sample was introduced and serially diluted from an initial concentration of 15 mg/mL. The 100 µL of freshly prepared DPPH solution was added in each well. The blank sample consisted of 100 µL of methanol and 100 µL of methanolic DPPH solution. The control consisted of the sample dilution solvent and the DPPH solution. All samples were performed three times in darkness at 25 °C, and the absorbance was read at 517 nm in a microplate reader (Tecan Pro 200, Tecan Trading AG, Männedorf, Switzerland) every 15 min for 1 h. Gallic acid was used as positive control and the percentage of the DPPH radical scavenging was determined using the equation given below: (%) = [(A_c − A_t)/A_c] × 100, where A_c is the absorbance of the control and A_t is the absorbance of the sample.

2.4.2. ABTS Scavenging Activity

The scavenging activity of F. umbellata extracts on ABTS radical cation was carried out as described by Re et al. [24] with slight modifications. Briefly, in a 96-well microplate; 100 µL of ABTS solution was added to 100 µL of serial dilution samples from an initial concentration of 1 mg/mL. The samples was then stirred and incubated at room temperature in the dark and optical density was read at 734 nm in a microplate reader (Tecan Pro 200, Tecan Trading AG, Männedorf, Switzerland) every 15 min for 1 h against a blank consisting of 100 µL of ethanol solution and 100 µL of samples. The percentage of the ABTS radical scavenging was determined as above. (%) = [(A_c − A_t)/A_c] × 100, where A_c is the absorbance of the control reaction and A_t is the absorbance in the presence of the sample.

2.4.3. Total Antioxidant Capacity (TAC)

The Phosphomolybdenum method has been used to determine the total antioxidant capacity as reported by Prieto et al. [25]. Briefly, in eppendorf tubes, 100 µL of samples were mixed to 1 mL of a solution consisting of 0.6 M sulfuric acid, 28 mM phosphate sodium, and 4 mM molybdate ammonium. The mixture was incubated for 90 min at 95 °C in a water bath. The operation was done in triplicate and optical density was read at 695 nm in a microplate reader against a blank. The results are expressed as equivalence of ascorbic acid per milligrams of dry weight.

2.4.4. Ferrous Reducing Antioxidant Power (FRAP)

Ficus umbellata extracts reducing power of Fe³⁺ to Fe²⁺ was assessed as described by Benzie and Strain [26]. In microplates, 50 µL of extracts, (concentration of 1 mg/mL) was introduced and then 1950 µL of FRAP solution (40 mM of 2,4,6-tripyridyl-s-triazine in HCl, 20 Mm FeCl₃, and 300 mM acetate buffer at pH 3.6) were added. Blank solutions were made of FRAP solution and solvent. Incubation was done in darkness for 30 min and absorbance was measured at 593 nm. All analyses were done in triplicate. The results were expressed in microgram of test extract as equivalent of ascorbic acid per milligram of dry mass (µg EAA/mg).

2.5. Anti-Inflammatory Assays

The in vitro anti-inflammatory activity of F. umbellata extracts was assessed by evaluating their capability to protect the murine erythrocyte cell membrane against heat [27] and inhibit the denaturation of proteins [28].

2.5.1. Preparation of Red Blood Cell Suspension

Ten milliliters of freshly collected rat blood were introduced in heparinized tubes. After centrifugation (2500× g; 30 min), the solution was washed three times with equal volumes of normal saline. The blood’s final volume was measured and reconstituted with normal saline to achieve a 10% v/v suspension.

2.5.2. Induction of Hemolysis by Heat

The test tubes containing 2 mL of reaction mixture consisted of 1 mL extracts of F. umbellatta at concentrations ranging between 0.125 to 2 mg/mL; 1 mL of phosphate buffer 0.15 M (pH 7.4) and 0.5 mL of 10% erythrocyte suspension were heated (56 °C; 30 min). The ibuprofen was used as a reference drug. The mixture was cooled, centrifuged (2500 g; 20 min), and the absorbance of the supernatants was read at 560 nm. The experiment was performed in triplicate. The percentages of the membrane stabilization activity were determined as below: Inhibition of hemolysis (%) = [(A_control − A_test)/A_control] × 100.

2.5.3. Protein Denaturation Assay

In test tubes were added successively, 1 mL of samples at different concentrations (0.125–2 mg/mL), 0.1 mL of albumin from fresh chicken eggs, and 1.9 mL of phosphate buffered saline (PBS, pH 6.4). Diclofenac sodium at the same concentrations was used as a reference drug. After incubation (37 °C; 20 min) and heating (70 °C; 5 min), the test tubes were cooled and the optical density was read against distilled water, at 660 nm, as blank. The inhibition percentages were calculated as below: % Inhibition = [(A_test − A_control)/A_control] × 100.

2.5.4. κ- Carrageenan Induced Acute Inflammation

The experimental design was composed of four (04) groups of five Wistar rats, each rat weighing between 150 and 200 g. A solution of 0.1 mL of 1% κ-carrageenan suspended in saline was injected in the left hind paw of each rat to induce acute inflammation. The rats were pre-treated with either sterile (0.9% NaCl w/v, s.c) FU50 and FU200 (50 and 200 mg/kg, p.o.) or diclofenac sodium (5 mg/kg, p.o.) as a reference 1 h prior to carrageenan injection. The handling of animals was in accordance with the guidelines and procedures of animal bioethics of the Cameroon Institutional National Ethic Committee (CEE Council 86/609), which adopted all procedures recommended by the European Union on the protection of animals used for scientific purposes. The paw volume of rats was measured by a plethysmometer (Ugo Basile, Varese, Italy) at zero time and then 0.5, 1, 2, 3, 4, and 5 h after injection of carrageenan [29]. The variations of paw volume are expressed as percentages using the formula below: [(V_c − V_t)/V_c] × 100; where V_c and V_t represent the mean increase in paw volume of the control and treated groups, respectively.

2.6. Statistical Analysis

The data were expressed as mean ± standard deviation. The effects of FU_Aq and FU_MeOH were assessed using One-way and Two-way analysis of variance (ANOVA). The differences between group pairs were performed using the post hoc test of Tukey and Bonferroni. All analyses were performed with GraphPad Prism 5.0 (San Diego, CA, USA). p < 0.05 was considered statistically significant.

3. Results

3.1. Antioxidant Activities

3.1.1. DPPH Scavenging Activity

F. umbellata extracts exhibited a DPPH scavenging activity in a concentration-dependent manner.

Concerning FUaq, it appears that the percentages of inhibition increase significantly up to 7.5 mg/mL and then drop to 15 mg/mL. FU_aq thus exerts a dose-dependent antiradical activity at a given time with a peak of activity at the concentration of 7.5 mg/mL, while FU_MeOH exhibited a dose-dependent DPPH antiradical activity at a given time with a peak activity observed at 3.75 mg/mL (

Table 1. DPPH scavenging activity of F. umbellatta extracts.

% Scavenging
	Time	15 min		30 min		45 min		60 min
Conc (mg/mL)		FUMeoH	FUAq	FUMeoH	FUAq	FUMeoH	FUAq	FUMeoH	FUAq
15		96.20 ± 0.84	92.96 ± 2.52	96.16 ± 0.88	90.83 ± 2.96	95.91 ± 0.88	91.67 ± 2.51	95.62 ± 0.92	91.33 ± 3.01
7.5		95.89 ± 0.37 a	140.21 ± 4.61 b	95.87 ± 0.402 a	141.72 ± 7.04 b	95.65 ± 0.36 a	143.33 ± 7.17 b	95.40 ± 0.38 a	146.65 ± 7.24 b
3.75		94.66 ± 0.42 c	117.49 ± 2.59 d	95.61 ± 0.29 c	119.46 ± 2.55 d	95.57 ± 0.21 c	121.14 ± 2.53 d	95.40 ± 0.27 c	124.22 ± 2.41 d
1.87		73.10 ± 1.09 e	101.98 ± 3.15 f	76.79 ± 1.14 e	110.18 ± 4.91 f	78.30 ± 1.28 e	111.35 ± 6.03 f	78.88 ± 1.3 e	114.80 ± 6.25 f
0.937		57.46 ± 2.85 g	68.12 ± 1.04 h	59.43 ± 3.08 g	72.22 ± 1.88 h	59.27 ± 3.29 g	77.74 ± 1.79 h	55.35 ± 3.5 g	79.87 ± 3.15 h
0.468		49.67 ± 0.82	41. 30 ±1.19	50.51± 0.88	44.34 ± 0.99	49.13 ± 0.96	44.48 ± 0.31	47.17 ± 1.61	46.25 ± 1.08
0.234		44.64 ± 1.8 i	23.52 ± 0.09 j	44.45 ± 1.93 i	22.64 ± 0.23 j	42.40 ± 2.06 i	21.22 ± 0.17 j	39.49 ± 2.16 i	18.35 ± 0.19 j
0.117		37.13 ± 0.48 k	15.77 ± 1.22 l	36.16 ± 0.43 k	14.26 ± 1.28 l	33.12 ± 0.45 k	10.53 ± 1.25 l	29.3 ± 0.51 k	5.74 ± 1.35 l

FU_MeoH: Ficus umbellata methanolic extract; FU_Aq: Ficus umbellata aqueous extract. Values are average of three independent experiments and represent mean ± standard deviation. Values with different letters for a given time and concentration of extracts are significantly different from each other. Significance at p < 0.05.

). These results are confirmed by the IC₅₀ values, which vary from 0.60 to 0.76 mg/mL for FU_MeOH and from 0.82 to 0.76 mg/mL for FU_aq. However, these activities are significantly lower than that of the gallic acid standard, with IC₅₀ values ranging from 1.67 to 1.74 µg/mL.

3.1.2. ABTS Scavenging Activity

Similarly, FU_aq and FU_MeOH exhibited dose and time-dependent ABTS antiradical activities at the tested concentrations (0.0019 to 0.125 mg/mL). The highest activities were observed at 0.125 mg/mL, regardless of incubation time, with approximately 100% of scavenging (

Table 2. ABTS scavenging activity of Ficus umbellata extracts.

% Scavenging
Conc (mg/mL)	15 min		30 min		45 min		60 min
	FUMeoh	FUAq	FUMeoH	FUAq	FUMeoH	FUAq	FUMeoH	FUAq
0.125	101.98 ± 0.177	101.63± 2.30	102.04 ± 0.19	105.28 ± 28	102.11 ± 0.18	105.69 ± 0.55	103.5 ± 0.29	105.22 ± 5.38
0.062	95.27 ± 0.64	97.07 ± 1.12	96.98 ± 0.68	98.24 ± 1.06	98.18 ± 0.77	99.37 ± 1.08	98.28 ± 1.26	99.98 ± 1.53
0.0312	81.95± 1.91	84.068 ± 0.92	82.69 ± 2.23	84.19 ± 1.05	83.43 ± 2.71	84.26 ± 1.23	73.22 ± 5.41	73.83 ± 1.85
0.0156	72.84 ± 0.95 a	78.73 ± 0.155 b	71.67 ± 0.96 a	78.20 ± 0.2 b	71.25 ± 1.2 a	77.21 ± 0.39 b	50.95 ± 2.27	59.33 ± 0.52
0.0078	68.36 ± 0.48 c	73.48 ± 0.90 d	67.22 ± 1.12 c	73.23 ± 0.68 d	66.71 ± 1.43 c	72.58 ± 0.48 d	43.41 ± 2.92	53.05 ± 0.93
0.0039	65.02 ± 0.36 e	71.64 ± 0.08 f	62.75 ± 0.43 e	70.66 ± 0.07 f	61.56 ± 0.39 e	69.65 ± 0.06 f	33.55 ± 0.76 a	46.94 ± 0.15 b
0.0019	65.48 ± 1.66	66.83 ± 0.51	63.60 ± 2.27	65.69 ± 0.57	62.69 ± 2.72	64.73 ± 0.74	35.86 ± 5.32	38.78 ± 1.35

FU_MeoH: Ficus umbellata methanolic extract; FU_Aq: Ficus umbellata aqueous extract. Values are average of three independent experiments and represent mean ± standard deviation. Values with different letters for a given time and concentration of extracts are significantly different from each other. Significance at p < 0.05.

). However, the results are not time-dependent at higher concentrations. These activities are also shown by the IC₅₀ values, which vary from 1.40 to 0.62 mg/Trolox dry weight (Dw) but remain significantly lower than those of gallic acid whose IC₅₀ = 32.33 µM/Trolox Dw.

3.1.3. Total Antioxidant Capacity (TAC)

In this study, the aqueous and methanolic extracts of F. umbellatta showed total antioxidant activity with 389.50 ± 2.66 and 734.98 ± 4.51 µg EAA/mg DW, respectively (

Table 3. Total antioxidant capacity of F. umbellata extracts.

Extracts	µg Eq AA/mg DW
FUMeOH	734.98 ± 4.51 a
FUAq	389.50 ± 2.66 b

FUMeoH: Ficus umbellata methanolic extract; FUAq: Ficus umbellata aqueous extract; µg Eq AA/mg DW: microgram equivalent of ascorbic acid per milligram of dry weight. Values are average of three independent experiments and represent mean ± standard deviation. The superscript letter indicates significance at p < 0.05.

).

3.1.4. Ferrous Reducing Antioxidant Power (FRAP)

Regarding the ferrous reducing antioxidant power, both extracts of F. umbellata, exhibited the ability to reduce ferric ions with 166.99 ± 14.50 and 223.72 ± 8.63 µg EAA/mg DW for FU_aq and FU_MeOH, respectively (

Table 4. Ferrous Reducing Antioxidant Power of F. umbellata extracts.

Extracts	µg Eq AA/mg DW
FUMeOH	223.72 ± 8.63 a
FUAq	166.99± 14.5 b

FUMeoH: Ficus umbellata methanolic extract; FUAq: Ficus umbellata aqueous extract; µg Eq AA/mg DW: microgram equivalent of ascorbic acid per milligram of dry weight. Values are average of three independent experiments and represent mean ± standard deviation. The superscript letter indicates significance at p < 0.05.

).

3.2. In Vitro Anti-Inflammatory Activities

3.2.1. Erythrocyte Membrane Stabilization

The effects of F. umbellatta extracts on the hemolysis of murine red blood cells are shown in

Table 5. Inhibitory effects of ibuprofen and F. umbellatta extracts on hemolysis of erythrocytes membrane.

	% Inhibition of Hemolysis
Concentration (µg/mL)	Ibuprofene	FUMeoH	FUAq
0.125	47.83 ± 2.46 a	34.17 ± 2.17 b	33.04 ± 3.92 b
0.25	59.27 ± 1.39 c	33.008 ± 1.57 d	38.31 ± 1.48 d
0.5	75.13 ± 0.314 e	30.026 ± 2.06 f	37.08 ± 2.31 g
1	92.048 ± 0.71 h	37.85 ± 1.7 i	31.44 ± 0.71 j
2	93.52 ± 0.84 k	30.91 ± 2.39 l	23.69 ± 0.13 m

FUMeoH: Ficus umbellata methanolic extract; FUAq: Ficus umbellata aqueous extract; data are presented as the means of three independent experiments and represent mean ± SD of hemolysis inhibition percentage. Values with different letters for a given concentration are significantly different from each other. significance at p < 0.05.

. At the tested concentrations (0.125–2 mg/mL), Ficus umbellatta extracts have stabilized the murine red blood cell membranes within the range of 33.04 ± 3.92% to 23.69 ± 0.13% and 30.91 ± 2.39% to 34.17 ± 2.17% for FUaq and FU_MeOH, respectively. These activities were less important compared to ibuprofen used as standard with percentages of 47.83 ± 2.46% to 93.52 ± 0.84 for concentrations ranging from 0.125 to 2 mg/mL.

3.2.2. Inhibition of Albumin Denaturation

Table 6. Inhibitory effects of sodium diclofenac and F. umbellatta extracts on albumin denaturation.

	% Inhibition of Denaturation
Concentration	Sodium Diclofenac	FUMeOH	FUAq
0.125	72.14 ± 1.37 a	60.18 ± 1.54 b	59.85 ± 2.25 b
0.25	70.52 ± 1.48 c	60.79 ± 0.93 d	60.57 ± 1.93 d
0.5	74.11 ± 1.73 e	60.99 ± 1.95 f	62.81 ± 2.35 f
1	73.73 ± 1.35 g	61.09 ± 0.52 h	59.99 ± 2.52 h
2	74.17 ± 1.32 i	64.39 ± 2.86 j	62.27 ± 1.68 j

FUMeoH: Ficus umbellata methanolic extract; FUAq: Ficus umbellata aqueous extract. Data are presented as the means ± SD of denaturation inhibition percentage. Values with different letters are significantly different from each other at a given concentration. Significance at p < 0.05. Values are average of three independent experiments and represent mean ± SD. The different superscript letters mean significance at p < 0.05.

/Figure 1 revealed the effects of F. umbellatta extracts on albumin-induced denaturation. At the tested concentrations, FUaq and FU_MeOH exhibited an inhibitory capacity of protein denaturation ranging from 59.85 ± 2.25% to 62.27 ± 1.68% and from 60.18 ± 1.54% to 64.39 ± 2.86%, respectively. However, no significance was observed between both extracts, and their effects were significantly lower compared to sodium diclofenac (72.14 ± 1.37% at 0.125 mg/mL) used as a reference drug.

3.3. In Vivo Anti-Inflammatory Activities

In this study, the injection of carrageenan solution induced edema in hind paw leading to the vascular phase of inflammation. The pre-treatment of rats with diclofenac sodium (5 mg/kg) and methanolic extract of F. umbellatta at doses 50 and 200 mg/kg significantly prevented the development of paw oedema over time, respectively, at 2, 3, 4, and 3 h after carrageenan administration (

Table 7. Effects of sodium diclofenac and F. umbellatta extracts on carrageenan induced acute inflammation.

	% Inhibition of Paw Edema
Time (h)	Diclo5	FU 50	FU 200
0.5	28.68 ± 2.31	27.56 ± 2.16	33.75 ± 1.74
1	46.41 ± 2.85	47.88 ± 2.69	51.88 ± 0.94
2	56.93 ± 2.98	48.75 ± 2.52 *	56.13 ±1.09
3	66.31 ± 2.29	51.51 ± 2.92 ***	55.06 ± 1.51 **
4	70.77 ± 1.00	58.81 ± 2.51 **	68.02 ± 1.64
5	72.98 ± 7.51	66.24 ± 1.28	71.16 ± 1.72

Data are presented as the means ± SD of edema inhibition percentage. * p < 0.05; ** p < 0.01; *** p < 0.001 compared to the positive control. Diclo5: positive control (animals treated with diclofenac sodium at 5 mg/kg + 0.9% NaCl p.o.); FU50: animals treated with the methanolic extract of F. umbellatta at 50 mg/kg + 0.9% NaCl p.o.; FU200: animals treated with the methanolic extract of F. umbellatta at 200 mg/kg + 0.9% NaCl p.o.

). No significant differences have been observed between Ficus umbellata extract’s activities and Diclo 5 during experimentation at t = 5 h. These results suggested antiedematous effects of methanolic extracts of F. umbellatta at different stages of acute inflammation.

4. Discussion

The current article focused on the antiradical, antioxidative and anti-inflammatory activities of F. umbellata. DPPH (2,2-diphenyl-1-picrylhydrazyl) is a stable free radical whose electrons can delocalize and form free radicals that do not dimerize from one another; leading to its visible discoloration from purple to yellow and absorption at 517 nm. So, DPPH assay is one of the most adequate methods to assess antioxidant activities of various natural substances in vitro because of its fastness, reliability, and reproducibility [30,31,32]. The DPPH reactivity in the presence of F. umbellata extracts carried out in this study consisted of tracking the inactivation of the DPPH radical by the different extracts over time by recording the absorbance every 15 min for 60 min. The DPPH and ABTS radical scavenging abilities are mainly based on the electron transfer ability of Ficus umbellata’s antioxidant components. Several works have highlighted the DPPH and ABTS antiradical activities of ethanolic and methanolic aqueous extracts of leaves, fruits, barks, and roots of medicinal plants, such as Cassia species, Olive tree, Citrus species, Camellia sinensis (L.) O. Kuntz, Ficus bengalensis L., Ficus racemosa L. and Ficus carica [18,19,33,34,35]. Methanolic extract have exhibited stronger scavenging activities than aqueous extract at the tested concentrations. This could be due to its richness in phenol compounds more specifically flavonoids and flavonols, as shown by [22]. This variability in phenol compound content and antiradical activities in both extracts is due to the polarity of the different compounds toward water and methanol [36]. In addition, it is well known that phenol compound content is closely related to free radical scavenging activities [33,37,38].

The total antioxidant capacity assay of Ficus umbellatta extracts revealed that FU_MeOH exhibited higher capacity than FU_aq, confirming that aqueous and alcoholic extracts of some Ficus varieties have been shown to possess high total antioxidant activity [37,39,40]. The high total antioxidant capacity of FU_MeOH is correlated to its high content in secondary metabolites, such as flavonoids, flavonols, and alkaloids [20]. These are able to donate electrons to transform reactive species into non-reactive and more stable compounds [41].

Ferrous ions are involved in many physiological processes leading to the production and accumulation of free radicals in the body causing much damage. Therefore, the ferrous reducing antioxidant power of an extract or a pure compound could be of a great importance to alleviate neurodegenerative disorders, metabolic diseases, anemias, cardiovascular diseases, and cancers [42]. Nowadays, it has been clearly shown that extracts from Ficus species are also endowed with ferrous reducing antioxidant power [19,39]. Murugan et al. [43] revealed the strong ferrous reducing antioxidant power of extracts from Osbeckia parvifolia. These results suggest that some bioactive compounds from F. umbellata extracts as phenol compounds are able to reduce ferrous metal ions, which could be connected to the type of functional groups from their structure. Polyphenolic compounds, such as flavanols and flavonoids, have been recognized as strong ferrous reducing antioxidant power [35,38]. Previous studies which aimed at evaluating the antioxidant potential of species from Ficus genus have demonstrated strong antioxidant power comparable to controls with either scavenging activities or reducing potential [39,44]. EC₅₀ values of 9.06 ± 2.21 μg/mL and 369.19 ± 12.04 μg/mL were obtained from Ficus sur fruits and leaves, respectively [44]. With the DPPH assay, EC₅₀ ranging between 210.3 ± 3.22 and 358.3 ± 2.05 µg/mL were obtained from six Ficus species [45]. This difference in activity with regard to the present study could be due to the difference in the part/organs of the plant been used and the geographical region where the plants were collected; both factors influence the type and relative abundance of secondary metabolites present.

Generally, during biological phenomena in human beings, such as metabolism and respiration, free radicals and reactive oxygen species are yielded. However, their excessive production causes oxidation of macromolecules (proteins, lipids, proteins), and DNA damage, which are strongly involved in the pathogenesis of most chronic diseases, such as atherosclerosis, neurodegenerative diseases, angiocardiopathy, and cancers [46,47]. Natural antioxidants can help to fight against these deleterious effects by preventing and avoiding oxidation of biomolecules and regulating the immune system. The increased production of ROS triggers a cascade of reactions leading to the onset of an inflammatory response that can be chronic and lead to pathologies mentioned above, hence it is necessary to assess the anti-inflammatory activities of aqueous and methanolic extracts of F. umbellata.

The exposure of cells to such chemicals as methyl salicylate, phenyl hydrazine, hypotonic solutions, or heat, provokes hemoglobin oxidation and hemolysis [48]. On the other hand, free radicals, such as lipid peroxides and superoxides are responsible for cell membrane destabilization. Flavonoids are polyphenolic compounds that are effective scavengers of these free radicals. Thus, we can postulate that these polyphenolic compounds found in Ficus umbellata extracts could be responsible for the membrane stabilizing effect observed in this study. This protective effect against heat-induced hemolysis of F. umbellatta extracts noticed in this study suggests their ability to protect the plasma membrane against lysis and then of lysosomal membranes because of their structural similitudes. The mechanism involved for the maintenance of cell life is the inhibition of releasing serum proteins because they can activate neutrophils and extend the inflammatory response for a long time [49].

F. umbellatta extracts have shown the antidenaturing activity of egg albumin at concentrations ranging from 0.125 to 2 mg/mL. These results corroborate those of Dharmadeva et al. [50] who extracted Ficus racemosa L. from bark at concentrations ranging from 0.01 μg/mL to 0.1 μg/mL. Murugan et al. [43] have shown that the methanol extracts of O. parvifolia at 1 mg/mL concentration significantly protected the albumin denaturation and lysis of erythrocyte membrane induced by hypotonic solution, which is comparable to the standard Diclofenac sodium. In fact, protein denaturation occurred very often in an uncontrolled or prolonged activation of inflammation leading to dangerous alterations, such as production of autoantigens associated with type III hypersensitivity reactions and autoimmune diseases, such as rheumatoid arthritis. The denaturation results from the disruption of electrostatic, hydrogen, hydrophobic, and disulfide bonds that maintain the three-dimensional structure of proteins [51]. The inhibitory activity of F. umbellatta extracts are probably due to their chemical composition because phenol compounds could interact with two sites present in some proteins, such as ovalbumin of tyrosine-, threonine- and lysine-rich bonds. Therefore, F. umbellatta extracts could protect protein denaturation by inhibiting the release of autoantigens in living organisms.

Chemical external stimuli, such as carrageenan, leads to a two-phase inflammatory reaction, since carrageenan is a phlogogenic substance. The initial or early phase lasting 90 min after injection of carrageenan is characterized by the release of serotonin, histamine, and bradykinin. After injection of carrageenan, the late phase can last up to 5 h and it is characterized by infiltration of neutrophil and prostaglandin release, mediated by cyclooxygenases. These mediators increase capillary permeability and thus forms an exudate responsible for the oedema that also compresses the nerves and causes the sensation of pain [52]. In this case, FU200 exhibited the strongest antiedematous activity 5 h after injection of carrageenan. Similar observations were made by Mehta et al., 2013 after administration of ethanolic extracts form aerial parts of Cassia species in carrageenan induced paw oedema in Wistar albino rats. Different extracts of Ficus species have also demonstrated high antiedematous activity in rodents [40]. In addition, Almeida et al. [12] have reported the in vivo anti-inflammatory activities of several extracts related to Libidibia ferrea organs’ medicinal plant. Thus, the antiedematous effects of methanolic extracts from F. umbellata could be helpful to attenuate the delirious effects of an acute inflammation.

5. Conclusions

In summary, this study provides experimental evidence that aqueous and methanolic extracts of F. umbellata endow free radical scavenging, total antioxidant capacity, and ferrous reducing power, in a dose-dependent manner. They also exhibit protective effects against hemolysis of murine erythrocyte membranes induced by heat and denaturation of proteins at concentrations ranging from 0.125 to 2 mg/mL. Moreover, methanolic extract of F. umbellata at 200 mg/kg has presented a strong in vivo anti-inflammatory response in a carrageenan induced paw oedema model. Further studies on other models are necessary to validate methanolic extracts of F. umbellata as a potential antioxidative and anti-inflammatory agent.