Hydroethanolic Leaf Extract of Cordia vignei Hutch and Dalziel Inhibits Carrageenan-Induced Foot Oedema in Chicks, Prostaglandin E2-Induced Paw Oedema in Mice, and Bradykinin-Induced Paw Oedema in Mice

Background Cordia vignei Hutch and Dalziel (Fam. Boraginaceae) is a woody plant found in west tropical Africa. The aim of this research is to find out if the leaf extract of this plant prevents oedema in animal models. Methods (a) Inflammation was induced in the animals by injecting 100 µl of 2% lambda carrageenan into the subplantar tissue of the right footpads of 7-day-old chicks 1 h before or after oral administration of 30–300 mgkg−1 CVE. Oedema was measured for 5 h using the water displacement method. (b) Oedema was induced in ICR mice by subplantar injection of prostaglandin E2 (PGE2) (50 µl of 1 nM) 30 minutes before or after CVE administration. Oedema was measured for 3 h. (c) Oedema was induced in ICR mice by subplantar injection of bradykinin (BK) (10 nmol/paw) 30 min before or after administration of extract. Results We found that CVE significantly (P < 0.05) prevented inflammation that was induced by injecting carrageenan into the footpads of the chicks. Also, we observed that CVE prevented inflammation produced by injecting PGE2 into the subplantar tissue of mice. Finally, we also report that CVE prevented inflammation produced by injecting BK into the subplantar tissues of mice. All these effects were observed in both preventive and curative protocols. Conclusion We conclude that Cordia vignei leaf extract has potential anti-inflammatory activity.


Introduction
Infammation is a process by which the body's immune system reacts to the destruction of tissues caused by chemical, physical, or microbial factors. It is the normal physiological attempt of the body to repair injuries and defend against foreign invasions [1]. Infammatory response involves recruitment of cells including neutrophils and tissue macrophages that release mediators to enhance vasodilation, vascular permeability, and exudation of fuid into the site of injury or infection and these are responsible for the swelling [2,3].
Presently, NSAIDs are the main therapies for infammatory conditions [4]. However, due to the side efects of these conventional drugs, natural products that have potential anti-infammatory activity are being explored globally as alternatives or supplements to standard treatment [5]. One of these useful plants in traditional medicine in Ghana is Cordia vignei. It is used to treat diseases such as sore, colitis, and prostate cancer [6][7][8][9]. Tere is limited data to support this claim. Our purpose of this research is to test the antiinfammatory activity of hydroethanolic extract of Cordia vignei leaf on carrageenan-induced oedema in chicks, PGE 2induced paw oedema, and BK-induced paw oedema in mice.

Carrageenan-Induced Foot
Oedema. Seventy (70) healthy chicks were weighed and put into 14 Groups (n � 5). Chicks in Groups I-VII were used for prophylactic study, and chicks in groups VIII-XIV were used for curative study. Te initial foot volume of each chick was measured using the water displacement method [17]. All the chicks in both prophylactic and curative studies were given injection of 100 µl of 2% (w/v) lambda carrageenan into the right footpad to induce oedema.
Te foot volume of the chick was measured at every 1 h for 5 h.
Te increase in foot volume was calculated as shown in the following equation: where Fvt is the foot volume of the chick at various times measured after induction and Fvo is the foot volume before induction.

Prostaglandin E 2 -Induced Paw Oedema in Mice.
Tirty (30) healthy ICR mice were put into six groups (n � 5), and the initial right hind paw thickness of each mice was measured with a digital caliper (VC1346i, MP Lab Equip, USA). Mice in groups I and II received saline (10 mlkg −1 p.o.), mice in group III orally received 10 mg/kg diclofenac, and mice in groups IV, V, and VI orally received 30, 100, and 300 mgkg −1 CVE, respectively. In the prophylactic study, PGE 2 (50 µl of 1 nM) was injected 30 min post-CVE administration. In the curative test, mice were given oral treatments of diclofenac or CVE 30 min after the PGE 2 challenge. Infammation was measured at 30 min intervals for three hours.

Bradykinin-Induced Paw Oedema.
Mice were put into 6 groups of 5 and were given a subcutaneous injection of 5 mg/ kg of captopril to inhibit kininase II activity. Mice received a subplantar injection of bradykinin (10 nmol/paw) 30 min after oral administration of saline (1 mlkg −1 ) or extract (30-100 mgkg −1 ). In the therapeutic protocol, mice were given CVE (30, 100, and 300 mgkg −1 ) orally thirty minutes after a bradykinin challenge. Infammation was measured for three hours at thirty minutes intervals using a digital caliper.

Statistical Analysis.
Te results were presented as the mean ± SEM. GraphPad Prism for Windows version 6.01 (GraphPad Software Inc., San Diego, CA, USA) was used for all statistics. Raw values were normalized as percentages. Data with two independent variables were analyzed using a two-way ANOVA followed by Bonferroni's post hoc test. Data with one independent variable was analyzed using Student's t-test. Table 1.
In the curative protocol, both Diclofenac and CVE inhibited oedema over the course of the study   (Figure 2(d)).

Relative Potency of CVE.
Te potency of the extract was compared to that of diclofenac by estimating the ED 50 values in each protocol ( Figure 3). In the prophylactic study, the ED 50 was 4.490 ± 1.48 and 22.78 ± 1.9 for diclofenac and CVE, respectively (Figure 3(a)). In the curative study, the ED 50 was 5.60 ± 1.94 and 12.66 ± 2.51, respectively, for diclofenac and CVE (Figure 3(b)). In both protocols, diclofenac and CVE were efective in inhibiting oedema; however, diclofenac was found to be more potent than CVE.

Bradykinin-Induced Paw
Oedema. Subplantar injection of 1 µg bradykinin (10 nmol/paw) evoked acute infammation characterized by oedema of the injected paw. Te maximal oedema response of the control mice occurred on the 90 th min with increase in paw thickness to 70.57 ± 12.47% of the initial (Figure 5(a)). In contrast, pretreatment of mice with CVE signifcantly prevented infammation induced by bradykinin. Te percentage increases in paw thickness of the CVE-treated mice at the 90 th min were as low as 18 (Figure 5(b)).

Discussion
Carrageenan-induced oedema as described by Winter [18] is a widely used acute infammatory model for screening novel compounds for anti-infammatory activity. Carrageenan injection produced a rapidly developed biphasic acute infammatory response characterized by swelling, heat, and pain [19]. In phase one, which occurs between 0 and 2 h, infammatory mediators such as serotonin, nitric oxide, and histamine are released during the frst 1 h, and then bradykinin release follows. Treatment of phase 1 with NSAIDs does not result in inhibition [20,21]. Te phase two which occurs between 2 and 5 h is mediated by the release of prostaglandin, and unlike phase 1, treatment of phase 2 with NSAIDs leads to signifcant improvement [22][23][24]. Diclofenac which was used as a reference drug in this study is known to inhibit cyclooxygenase 2 which releases prostaglandin from arachidonic acids in infammatory cells [25,26]. Similarly, CVE signifcantly prevented infammation in both prophylactic and curative protocols. It could be possible that the anti-infammatory activity of CVE is due to the inhibition of some of the above compounds especially prostaglandins which mediate infammation.
Prostaglandin E 2 (PGE 2 ) is the most ubiquitous prostaglandin in the biological system. It is principally involved in infammatory processes that lead to swelling, redness, heat, and pain [27]. Intradermal administration of PGE 2 causes vasodilation which enhances accumulation of cells and efector molecules that result in oedema as observed in this study. Infammatory response in phase one is due to direct activation of intradermal PGE 2 receptors whereas the sustained response was due to subsequent synthesis of prostaglandins from tissue macrophages through the arachidonic pathway [27]. Unlike other proinfammatory mediators, this synthetic pathway of PGE 2 is very sensitive to treatment with NSAIDs. Inhibition of PGE 2 by NSAIDs, analgesic drugs, and steroid hormones produces marked clinical improvement in infammatory, pyretic, and pain conditions. Bradykinin causes vasodilatation and increases vascular permeability through the B2 receptor. Te infammatory efect of bradykinin is due to its ability to elicit the release of vasodilators such as prostacyclin (PGI 2 ) and 4 Evidence-Based Complementary and Alternative Medicine nitric oxide (NO) [28]. Terefore, inhibition of the B2 receptor by a synthetic or organic compound could possibly prevent infammation.
Anti-infammatory efect of diclofenac in prophylactic and curative protocols is mainly by the inhibition of COX 2, as mentioned above. Inhibition of PGE 2 -induced oedema by Evidence-Based Complementary and Alternative Medicine 7 CVE could possibly be due to direct blockade of PGE 2 receptors or inhibition of further synthesis and/or release of PGE 2 from macrophages [29,30]. Anti-infammatory efect of CVE in this study could be due to the various phytochemical ingredients found in the extract. For instance, favonoids are widely known to exhibit anti-infammatory, antioxidant, and free radical scavenging efects [31]. Alkaloids, terpenoids, tannins, and saponins are also known to possess health benefts including antiinfammatory, anti-cancer, antiviral, antibiotic, antioxidants, and free radical scavenging efects [32,33].
It could be inferred from this study that CVE has antiinfammatory activity. However, further investigation is required to ascertain its mechanism of action.

Conclusion
We conclude that hydroethanolic extract of Cordia vignei leaf inhibits carrageenan-induced foot oedema in chicks and prostaglandin E 2 -induced paw oedema in mice.

Data Availability
Te data supporting the conclusions of this article are available at Kwame Nkrumah University of Science and Technology Library repository and could be accessed through the corresponding author upon reasonable request.

Ethical Approval
Te ethical approval for the study was granted by the Department of Pharmacology Ethical Committee

Conflicts of Interest
Te authors declare that there are no conficts of interest.