Anti-inflammatory Activity

3 The evaluation of the anti-inflammatory activity of our ethanol extract was conducted using Winter's method [5]. The extract is administered orally at doses of 150, 300, 400, and 450 mg/kg; the same extract is administered locally (local application) at doses of 5% and 10%, 30 min and one hour, respectively, before the injection of 1% carrageenan into NaCl 0.9% under the plantar fascia of the right hind leg. The circumference of the paste is measured before the injection of carrageenan, and then after each hour from the third hour to the sixth hour after the administration of carrageenan. Two anti-inflammatory references were used, one oral (diclofenac 1%) and the other dermal (indomethacin/pomade).

• S0 = circumference before carrageenan injection ;
• St = circumference at a given time after administration of carrageenan.

Analgesic Activity

4 The analgesic test was evaluated according to the number of abdominal contortions induced by intraperitoneal (I.P) injection of acetic acid (0.7%) (torsion test). Batches of five rats were formed. One and a half hours after oral administration of the extracts, rats received 0.7% acetic acid by the I.P route at a dose of 10 ml/kg. The painful syndrome is characterized by stretching movements of the hind legs and twisting of the dorsal-abdominal musculature. After 5 minutes (latency time) of the acetic acid saline injection, we counted the number of twists for each rat for the next 30 minutes[6].

Cicatrizing activity

5 The experimental protocol used in this study is consistent with standard recommendations for the care of animals used in research and teaching [7]. The various batches received locally either neutral cream (excipient) or extract 5%, or extract 10% and the reference healing sulfadiazine (1%).

• A control lot does not suffer from thermal burns and does not receive any treatment ;
• one lot of positive control in rats receiving sulfadiazine 1% (standard healing) locally ;
• a negative control lot of rats treated with neutral cream (contains no anti-healing molecules) ;
• an experimental lot of five rats each receiving locally the 5% extract mixed with the neutral cream ;
• an experimental lot of five rats each receiving locally 10% extract mixed with neutral cream.

6 The burns were carried out on the back (the dorsal region of the rat) previously shaved, by a metal heated in boiling water (100 °C) until thermal equilibrium (reached at 5 min). After heating, it is removed from the water, quickly wiped off, and then applied without pressure for 20 seconds [8–10]. After the induction of burns, the rats in the treated batches were each given a topical application of the product intended for their batch once a day for 20 days. The photographs taken were processed by the ImageJ^® image processing software that we adapted for our study. This program can provide accurate measurements of microscopic lengths and widths in any unit system. In order to deduce the percentage of wound contraction, the average of the surfaces of the four wounds in the same batch is calculated and compared with the surface of the initial burn, using the following equation [11].

7 The re-epithelialization period is estimated by the number of days required for total wound closure without any sharp residual injury [12].

Statistical Analysis

8 Data are presented as mean ± standard deviation of five repetitions. Statistical analysis was performed using the one-factor ANOVA using GraphPad Prism with 95% confidence limits (P < 0.05).

Anti-inflammatory Activity

9 Inflammation induced by carrageenan injection is a widely used test to evaluate the peripheral anti-inflammatory effect. The results obtained from this test show that the extract of W.f, when administered orally, has an anti-inflammatory effect, with a maximum inhibition of 78.87% ± 7.08 at the 450 mg/kg dose followed by the 400 mg/kg dose that inhibits oedema by 75.14% ± 6.39 while diclofenac and indomethacin have an inhibition of 89.75% ± 3.44 and 89.87% ± 6.20, respectively, (Table 1). This suggests that the ethanolic extract W.f has less anti-inflammatory activity than the reference anti-inflammatory, and the 450 mg/kg dose is the most effective in this test compared to the other doses (Fig. 2)

Table 1

Percentage of inhibition of swelling of rat legs after carrageenan injection

	Oral anti-inflammatory activity test	Dermal anti-inflammatory activity test
Hours	150 mg/kg	300 mg/kg	400 mg/kg	450 mg/kg	Diclofenac	Extract 5%	Extract 10%	Indomethacin
3	10.13 ± 5.06	21.38 ± 2.31	11.94 ± 3.97	18.88 ± 2.66	30.55 ± 5.50	15.55 ± 5.67	25.43 ± 6.03	33.05 ± 4.44
4	10.71 ± 7.12	39.28 ± 1.30	26.07 ± 3.72	29.28 ± 8.27	65.35 ± 6.07	37.53 ± 8.84	60.21 ± 4.68	73.57 ± 9.15
5	13.92 ± 7.46	54.04 ± 1.40	52.85 ± 1.75	69.28 ± 9.29	82.61 ± 1.98	59.52 ± 9.63	75.71 ± 4.29	85.11 ± 11.50
6	24.73 ± 6.84	60.76 ± 1.64	75.14 ± 6.39	78.87 ± 7.08	89.75 ± 3.44	76.14 ± 7.88	96.87 ± 5.85	89.87 ± 6.20

Percentage of inhibition of swelling of rat legs after carrageenan injection

Fig. 2

Effect of W.f ethanolic extract administered orally at different doses and diclofenac (1%) on carrageenan-induced edema in rats. The values are expressed as an average ± SEM; P < 0.05 is considered significant in relation to the control

Effect of W.f ethanolic extract administered orally at different doses and diclofenac (1%) on carrageenan-induced edema in rats. The values are expressed as an average ± SEM; P < 0.05 is considered significant in relation to the control

10 The ethanolic extract of W.f administered locally dermally, at doses of 5% and 10%, has a superior anti-inflammatory effect compared to indomethacin; the 10% cream reduces it more significantly (Fig. 3). The swelling of the legs injected by carrageenan, with a maximum inhibition, reaches 96.87% ± 5.85; below this concentration and with a cream of 5%, the anti-inflammatory activity is less pronounced and the maximum inhibition reaches only 76.14% ± 7.88. At the same time, the drug (indomethacin ointment) used as a maximum positive control of inhibition reaches 89.87% ± 6.20.

Fig. 3

Effect of W.f ethanolic extract administered dermally to 5% and 10% creams and indomethacin on carrageenan-induced oedema in rats. The values are expressed as an average ± SEM; P < 0.05 is considered significant in relation to the control

Effect of W.f ethanolic extract administered dermally to 5% and 10% creams and indomethacin on carrageenan-induced oedema in rats. The values are expressed as an average ± SEM; P < 0.05 is considered significant in relation to the control

11 This anti-inflammatory activity of the ethanolic extract, administered either orally or locally, can be explained on the one hand by the difference in chemical composition and on the other hand by the likely existence of polar phenolic compounds. In addition, phenolic compounds, represented mainly by flavonoids and tannins, are currently of great scientific interest, as they are considered to be powerful antioxidants, antibacterial, antifungal, and anti-inflammatory [13–15]. In addition, this plant has a wealth of phytochemical compounds (like tannins, mucilage, alkaloids, coumarins, free quinone, etc.) [4]. The 10% cream showed significant inhibition of induced rat leg edema following carrageenan injection with maximum efficacy at 5 and 6 hours. Indeed, the injection of carrageenan causes the release of several chemical mediators that are responsible for the inflammatory process. This inflammatory response is biphasic, with the initial phase lasting about one hour due to the release of histamine and serotonin, while bradykinin is released in the second phase (1.5–3 hours), and prostaglandin biosynthesis occurs beyond the third hour [16]. These mediators increase the permeability of the capillaries in the region. As a result, the exudate escapes from the bloodstream into the interstitial space. This exudate is the cause of localized edema, which in turn compresses nerve endings and thus determines a sensation of pain [17,18]. Taking these indications into account, it was proposed that the observed effect may be due to the ability of the W.f extract to inhibit prostaglandin synthesis by the cyclooxygenase route.

Analgesic Activity

12 The evaluation of the analgesic activity of the studied extract of W.f was carried out using the acetic acid method. Only the 450 mg/kg dose, considered to be an effective anti-inflammatory, was studied for its possible analgesic activity. The results of this test are shown in figure 4, and it is noted that the abdominal contractions of the rats treated with the extract are significantly lower than those of the control group that received only a physiological solution of NaCl with 41.20 ± 2.30 and 82.20 ± 5.04, respectively. Rats treated with the reference analgesic have a slightly greater effect than rats treated with the extract with 53.40 ± 4.94 abdominal contractions.

Fig. 4

Analgesic activity of the test extract compared to tramadol and the control group N = 5;***: P < 0.001

Analgesic activity of the test extract compared to tramadol and the control group N = 5;***: P < 0.001

13 Contractions induced by intraperitoneal injection of acetic acid is a method used to study the peripheral analgesic effect of a substance. The pain caused by the injection of the latter is due to the release of serotonin, histamine, bradykinin, and prostaglandins (PGEβα, PGFβα). These chemical mediators stimulate peripheral nociceptive neurons and induce increased vascular permeability [16,19]. The root extract of the plant studied inhibits abdominal torsion in a significantly dose-dependent manner. This analgesic effect may be related to the inhibition of the release of chemical mediators of pain.

Cicatrizing Activity

14 Part of the objectives of this work was to determine the healing activity of the ethanolic extract of W.f. To do this, two types of ointments were formulated with the tested extracts: the first based on the 10% extract and the second based on the 5% extract. The difference in wound surface sizes between the batches of W.f extract at 5% and 10% on the one hand and those of the negative control batches and sulfadiazine is represented in figure 5.

Fig. 5

The reduction of wound surfaces according to the days after treatment with extracts and controls. Values are expressed as a mean ± SEM; P < 0.05 is considered significant in comparison to the control

The reduction of wound surfaces according to the days after treatment with extracts and controls. Values are expressed as a mean ± SEM; P < 0.05 is considered significant in comparison to the control

15 The evolution of the size of the wounds treated with the 10% and 5% extract compared to the untreated one shows that in the first days after burns, rats from the two lots treated with the 5% and 10% extract have a significant reduction in the size of the wounds induced by the cutaneous burn compared with untreated rats (Table 2). After 6 days of the first treatment, the lot of the 10% extract recorded a surface area 0.41 ± 0.16 cm² smaller than that of the 5% extract (0.49 ± 0.13 cm²) corresponds, respectively, to shrinkage percentages of 93.20% ± 3.36% and 84.50% ± 3.84, against 48.47% ± 2.15 of the control lot and 81.88% ± 2.24 of the reference drug. To date 12, the wounds of the lot of the extract 10% have totally healing, while those of the lots of the extract 5% and control still have average surfaces of 0.3 cm² and 0.38 cm² corresponding, respectively, to retraction percentages of 98.09% ± 1.28 and 95.79% ± 1.02.

Table 2

Percentage (%) of area wound reduction of extracts and controls by day

Days	Extract 5%	Extract 10%	Negative control	Medication
1	20.21 ± 3.53	21.04 ± 3.32	3.63 ± 1.37	43.29 ± 4.09
3	41.6 ± 5.64	57.77 ± 10.1	24.73 ± 5.02	56.37 ± 2.60
6	84.5 ± 3.84	93.20 ± 3.36	48.47 ± 2.15	81.88 ± 2.24
9	96.82 ± 1.78	100 ± 0.0	65.48 ± 4.02	93.69 ± 0.68
12	98.09 ± 1.28	100 ± 0.0	75.56 ± 3.29	95.79 ± 1.02
15	100 ± 0.0	100 ± 0.0	91.36 ± 2.39	100 ± 0.0
17	100 ± 0.0	100 ± 0.0	96.91 ± 2.07	100 ± 0.0
18	100 ± 0.0	100 ± 0.0	100 ± 0.0	100 ± 0.0

Percentage (%) of area wound reduction of extracts and controls by day

16 Comparison of the healing effect of Withania extract at a dose of 10% compared to the standard (Sulfadiazine) shows that wounds treated with sulfadiazine recorded their first size reductions with a retraction percentage on day 3 of 56.37% (Fig. 6), so this percentage remains lower than those recorded in rats treated with the 10% extract during the same period of medication and is around 57.77%. During the 12 days of the experiment, the rats in the control batch recorded areas and retraction percentages lower than those of the treated batch, until the last day when the latter's pleasures healed completely, while those in the control batch still have an average area of 0.38 cm² correspond to a retraction percentage of 75.56%. The phytochemical screening we have done has allowed us to characterize this plant which contains flavonoids, coumarins, oses, holosides, and mucilages [4]. According to the results obtained, it can be said that the ointment of the base of the Withania frutescens extract has a dose-dependent healing activity, since the surface reductions and retraction percentages recorded in wounds treated with the 10% extract exceed those treated with the 5% extract. Like any natural product, the healing effect of the extract is due to the various phytochemical components of its composition. According to Bahramsoltani et al. [20], natural healing products manifest their effects through one of the following mechanisms: antimicrobial, anti-inflammatory, antioxidant, stimulation of collagen synthesis, and cell proliferation.

Fig. 6

Effects of the concentration of extract in the cream preparing comparing with the reference medicament

Effects of the concentration of extract in the cream preparing comparing with the reference medicament