1. Introduction
In recent times, the global use of medicinal plants or herbal medicines as either alternative or supportive medicine to conventional medicine has increased tremendously [
1]. Today, medicinal plants and their bioactive components are widely accepted as an emerging source of alternative therapy for the management and treatment of various human diseases [
2,
3]. It is estimated that over 80% of the world population in one way or the other uses medicinal plants and their phytotherapeutic compounds as alternative primary healthcare due to their bioavailability, accessibility, efficacy, affordability, and low associated side effects [
4,
5]. They are not only used for healthcare remedies but can also serve as novel sources of unlimited raw materials for the discovery of new drugs. In ethnomedicine, many medicinal plants are effectively used for wound healing or wound management because they create a suitable moist environment, demonstrate the shortest healing time, and cause less pain or discomfort to the patients [
1]. Wounds (acute, burns, or chronic wounds) are physical injuries that lead to the disruption of normal structures and functions of the tissues and skin [
6]. They are traumatic and devastating types of injuries and therefore one of the leading global public concerns. Wound healing is a dynamic biochemical and cellular event involving the repair of damaged tissues and skins [
7]. Bioactive components in medicinal plants or herbal medicines have great potential to reduce oxidative stress and inflammatory processes [
8], inhibit infections, and promote blood clotting, as well as accelerate wound healing processes by repairing tissues and regenerating damaged skins [
9]. Many researchers have reported that medicinal plants are successfully used in the management and treatment of different types of wounds [
6,
10,
11,
12], and one such plant is
Cucurbita pepo.
Cucurbita pepo L. is a green vegetable that is globally used as functional food and medicine. It belongs to Cucurbitaceae. It is a rich source of carotenoids, liposoluble vitamins, and polyunsaturated fatty acids [
13]. It has high contents of flavonoids, terpenoids, alkaloids, proteins, lipids, carbohydrates, minerals [
14], and Δ5-, Δ7-, and Δ8-phytosterols [
15], which are likely responsible for its medicinal properties. The extract of different parts of
C. pepo (leaves, seeds, flowers, and fruits) has shown to exhibit various pharmacological activities such as antioxidants, anti-inflammatory [
16], anti-proliferative [
17], anti-depressant, anti-ulcer, inhibition of lipid peroxidation [
18], anti-diabetic [
19], anti-bacterial, anti-viral, anti-hypercholesterolemic, and sedative-hypnotic effects [
20]. In traditional medicine, the leaves are used as an analgesic or blood booster [
21], to remove biliousness, and for treatments of rheumatism, benign prostatic hyperplasia, burns, and wound injuries [
4,
14]. Despite the acclaimed ethnopharmacological use of
C. pepo in the treatment of many diseases, there are few or no scientific data from experimental studies as regards its toxicity profile, wound healing, and anti-inflammatory effects. This present study was conducted to evaluate the toxicity profile, wound healing, and anti-inflammatory properties of phytochemical characterized
C. pepo leaf extract in rats.
4. Discussion
There are several acclaimed therapeutic potentials of C. pepo as alternative medicine in the management and treatment of various diseases such as diabetes, benign prostatic hyperplasia, and rheumatism as well as inflammations, burns, and wound injuries. However, no scientific study has documented the toxicity profile or anti-inflammatory and wound healing potentials of C. pepo. Therefore, it is pertinent to investigate the safety profile, wound healing, and anti-inflammatory properties of phytochemical characterized aqueous leaf extract of C. pepo in rats.
The use of gas chromatography-mass spectrometry (GC-MS) for a very detailed evaluation of bioactive chemicals in plant-based compounds is a well-accepted approach [
45]. The GC-MS analysis of
C. pepo showed the presence of fifteen (15) chemical compounds with 1-heneicosanol (12.32%) and squalene (1.73%) as the most abundant and least abundant, respectively. Six out of the fifteen identified compounds are known to have some pharmacological potential. These include 1-Octen-3-ol, noanal, trans-β-lonone, phytol, trans-farnesol, and squalene. Xiong et al. [
38] reported the antimicrobial properties of 1-Octen-3-ol, while Zhang et al. [
39] and Li et al. [
40] revealed the antifungal properties of noanal. In other studies, trans-β-lonone has been shown to possess anti-inflammatory, cancer-preventing, antibacterial, antifungal, and anti-leishmanial activities [
41], while phytol, trans-farnesol, and squalene have biological activities including anti-inflammatory, antinociceptive, antioxidant, anti-cancer, and antimicrobial properties [
42,
43,
44].
In acute and subacute toxicity studies, appropriate animal models such as rats or mice can be commonly used to evaluate the health risk of any plant extracts or chemical substances [
46]. In an acute toxicity study, single-dose oral administration of aqueous leaf extract of
C. pepo up to 5000 mg/kg did not cause any death or toxicity signs. Based on the result, the LD
50 of
C. pepo is higher than 5000 mg/kg, and following OECD [
23] guidelines
C. pepo can be classified as a class 5 substance and therefore considered a safe and non-toxic substance.
In the subacute toxicity study, there was no change in body weight after 14 days of
C. pepo treatment, indicating that it is safe at all dose levels. Adverse effects of medications and chemicals are identified by changes in body weight [
47]. Organ weight changes have long been known as a sensitive sign of chemical-induced organ toxicity, such as organ hypertrophy [
48]. As a result, relative organ weight is an important measure in toxicity investigations. When compared to normal control animals, no significant change (
p > 0.05) in relative organ weight was found after treatment with
C. pepo at all dose levels.
Hepatic and renal function tests are used as fingerprint markers to determine the inherent toxicity and pathological states that are tracked to determine organ toxicity or dysfunction caused by hazardous substances [
49]. Electrolytes, urea, and creatinine can all be used to determine renal toxicity [
50]. Cirrhosis, fibrosis, and liver destruction are all symptoms of hepatotoxicity, which is caused by exposure to toxic substances. Hepatic enzymes such as AST, ALT, and ALP were measured to determine the amount of liver necrosis [
51]. In humans, elevated plasma bilirubin levels suggest haemolytic anaemia [
52]. The
C. pepo administration reduced AST levels but did not affect total protein, ALP, albumin, globulin, or bilirubin levels when compared to the control group. Electrolytes are necessary for the body’s homeostatic balance to be maintained. Sodium keeps the body’s acid–base balance in check and protects it from excessive fluid loss [
53]. Pathological conditions can be caused by changes in plasma electrolyte balance. When compared to the normal control group, there was no significant change (
p > 0.05) in plasma electrolyte levels (Na
+, K
+, Cl
−, and HCO
3−) after treatment with
C. pepo, indicating that there was no harmful effect on electrolyte levels. Furthermore, there was no statistical difference (
p < 0.05) in the values of urea and creatinine after administration of
C. pepo, indicating no detrimental effect on the functionality of the renal system.
Toxic substances primarily influence the haematopoietic system, which is a very sensitive and significant indicator of physiological and pathological conditions in humans and animals [
54]. Toxicants change haematological parameters, resulting in disorders such as anaemia, leukopenia, thrombocytopenia, and bone marrow depression, among others [
52].
C. pepo leaf extract did not have a deleterious effect on WBC, RBC, or platelets, nor did it interfere with the haematopoiesis and leucopoiesis processes according to the findings. When compared to the normal control, no toxic changes in all of the studied haematological parameters were seen after 14 days of treatment with
C. pepo, indicating no haematotoxic effect and safety at all dose levels.
When compared to the control, C. pepo administration reduced atherogenic lipid markers including TC, TAG, and LDL considerably, while HDL and VLDL showed no statistical change (p > 0.05). This suggests that C. pepo may have hypolipidemic properties.
C. pepo administration slightly increased the enzymatic antioxidant parameters GSH, SOD, and CAT with a statistically significant increase (
p < 0.05) in GSH levels in the treated groups compared to the control group. Other studies have shown that intake of toxic chemicals affects antioxidant enzymes and elevates MDA levels [
55]. MDA is a lipid peroxidation biomarker that may be elevated as a result of toxin ingestion or other diseases [
55]. Membrane polyunsaturated fatty acids are reduced by lipid peroxidation, which produces aldehydes including MDA, 4-hydroxynonenal, and acrolein, disrupting and changing the functions of the membrane [
56,
57]. Lipid peroxidation has been considered an indication of oxidative stress [
58,
59]. It alters the membrane’s structure by causing changes in fluidity and permeability [
60]. Reduced MDA levels and higher GSH, SOD, and CAT levels seen in this study have been reported to protect against oxidative stress [
61]. The results supported that
C.
pepo possesses antioxidant properties. Nkosi et al. [
62] reported the antioxidative activity of
C. pepo against carbon tetrachloride-induced hepatotoxicity in rats fed with a low protein diet.
In humans and animals, exposure to various hazardous chemicals raises oxidant levels and induces oxidative DNA damage, which leads to cellular damage and tissue necrosis [
63]. A histopathological examination can assist us to determine whether organs have been damaged at the cellular level as a result of a harmful chemical action. The liver and kidneys of rats given varying doses of
C. pepo showed no signs of pathological damage. These findings backed up the results of biochemical tests, as well as the safety of
C. pepo leaf extract in rats.
The wound healing process aids in the recovery of injured tissue layers and cellular structure. The wound area shrinks as the fibroblast stage begins [
64]. Contraction, granulation, epithelization, and collagenation are all part of this complicated process [
65,
66]. Microbial infections, the presence of free radicals, and metabolic disruptions can all cause the multifactorial process of wound healing to be delayed or interrupted [
67]. Wound closure, wound contraction, and functional barrier reorganization are significant markers in the wound healing process [
68]. Again, because the type of injury has an impact on wound healing, healing phases of wound models, including excision and incision wound models and dead space, are all affected differently [
69]. The fundamental goal of wound management is to heal the damage as quickly as possible while causing the least amount of pain and discomfort to the patient. In the current study, the effect of
C. pepo leaf extract on tissue regeneration was investigated utilizing experimental rat models. Rats were continuously observed and treated topically with prepared ointment from
C. pepo leaf extract as well as a reference drug after various types of wounds were created. When compared to the control group animals, the
C. pepo leaf treatment groups and the reference drug-treated group (povidone-iodine) in the excision and incision wounds showed significant (
p < 0.05) reductions in the wound area, rapid epithelization, and rapid rate of wound closure.
It has been reported that, at wound sites, high amounts of reactive oxygen species such as oxygen singlet, hydroxyl radicals, superoxide anion, and hydrogen peroxide stimulate collagen breakdown and hence extracellular matrix degradation (ECM). When the ECM is damaged, processes such as angiogenesis and re-epithelization that are necessary for wound healing are reduced [
70,
71]. The photograph section of the post-excision wound contraction is shown in
Figure 9. From the result, there was a great tissue restoration in the groups that were treated with
C. pepo, and this wound restoration manifested as the day progressed, significantly showed on day 4, and completely healed on day 16. Interestingly, the improvement in the excision wound healing compared favourably to the group that was treated with 5%
w/
w PI (standard drug). On day 16, the extract-treated group revealed a stronger healing effect compared to the untreated group. Our study showed that
C. pepo increased % WC and tensile strength and reduced epithelialization days which are evidence of potent wound healing capacity. A phytochemical enriched antioxidant such as squalene may boost the production of antioxidants such as superoxide dismutase and catalase in excised wounds and lower the activity of malondialdehyde [
42,
72]. The findings from the antioxidant activities as well as the bioactive constituents of
C. pepo leaf extract may have synergistically aided in the elicited wound healing potential of the resource plant.
This study also investigated the anti-inflammatory activity of
C. pepo extract in egg albumin-induced paw inflammation in rats with results indicating significant inhibition of paw inflammations in all test rats pre-treated with the extract before induction. Paw circumferences of these treated rats were also found to be significantly lower than those of the control rats and compared favourably with those of rats pretreated with aspirin, a standard anti-inflammatory agent. The entire results, therefore, suggest that
C. pepo extract may contain substances with anti-inflammatory activities and may have achieved this effect by truncating the established physiologic inflammatory pathway of egg albumin-induced inflammation. Usually, egg albumin induces edema due to its ability to trigger the release of histamine and serotonin and eventual prostaglandin release, leading to inflammation [
73]. Another inflammatory pathway is activating the release of cyclo-oxygenase (COX) which also leads to prostaglandins release and eventual onset of pain, swelling (inflammation) and fever [
37]. The extract at all administered doses progressively reduced the increased paw circumference (oedema) induced by egg albumin in the test rats and may have achieved this effect by inhibiting/reducing the release of histamine and serotonin due to egg albumin injection and by inhibiting COX activity, having produced activity like that of aspirin, a non-steroidal anti-inflammatory agent known for its anti-inflammatory activity via COX activity inhibition [
37].