Our results showed that wound healing occurred on the fourteenth day in preventive groups treated with spinach aqueous and alcoholic extract for 2 months, and healthy control group, on the twenty-first day in groups treated with spinach aqueous and alcoholic extract for 1 month, and on the thirtieth day in diabetic control group. Furthermore, the results showed that recovery in groups receiving 2 months of aqueous and alcoholic extract of spinach occurred with greater intensity and speed than other groups, particularly the healthy control group, because the complete wound recovery occurred on this day (the third day), and there was no sign of a wound to differentiate from healthy skin of adjacent areas. Indeed, this reflected the positive effect of receiving spinach extract prior to the induction of diabetic ulcer.
Latef et al. found that wounds in diabetic rats treated with trans-retinoic acid were clotted faster than those treated with the carrier. In histological examination, similarly, there was a denser matrix with spindle cells more abundant in healed wound25.
These confirm the results of the present research, owing to, perhaps, the presence of vitamin A in spinach. It has been suggested that vitamin can be useful for the process of healing wounds via stimulating the epithelialization and deposition of collagen by fibroblasts. Vitamin A raises the inflammatory reaction in the wound. It is believed that this increase occurs in inflammatory response due to higher instability of lysosomal membrane, increased input and activation of macrophages, as well as stimulation of collagen synthesis26. Also, vitamin C is one of the compounds found in spinach. It raises the content of collagen, fibroblasts, as well as vascular density in the wounds subjected to gamma radiation. Researchers have found that insufficient vitamin C leads to a very low deposition of collagen, slower angiogenesis, together with significant bleeding, resulting in impaired wound healing27.
Although the above report shows the positive effect of vitamin C on wound healing and those results are consistent with the ones obtained in the current study, inconsistent results suggest that topical application of ascorbic acid does not affect wound healing of Tympanic membrane, leaving no impact on the histological parameters of wound healing28. Vitamin E maintains and stabilizes the membrane mainly by protecting the cell membranes against destruction caused by oxidation29. Vitamin K can have a little direct impact on the healing wound for carboxylation of glutamate in blood clotting factors II, VII, IX, and X. However, the lack of vitamin K may lead to the formation of a hematoma in the wound, thus impairing wound healing and making it susceptible to infection. The greatest effect of vitamin K on healing wounds is associated with its hemostatic capacity29.
Arachidonic and linoleic acids are polyunsaturated fats essential in the diet of prostaglandin synthesis. Free fatty acids are prostaglandins and phospholipid precursors, deficiencies of which can hinder wound healing in humans and animals, mainly since phospholipids are the crucial components of cell basis membrane, whereas prostaglandins have an important role in cellular metabolism and inflammation30.
During the experiments with an animal model, the role of arginine in wound healing become evident. Some mechanisms have been presented to clarify the arginine positive impact on wound healing. The first is that the useful impacts of arginine supplementation on wound healing are alike those associated with growth hormone19. The second proposal is that arginine supplementation may heal the wound by stimulating the responses of lymphocytes T (T cell). Obviously, normal wound healing essentially requires T cells31. The third proposed mechanism considers arginine as a unique platform for nitric oxide production. Nitric oxide is an extremely reactive radical that may have a key role in wound healing. In fact, Arginine can produce proline essential throughout collagen production, while nitric oxide is effective on angiogenesis, apoptosis, proliferation, and differentiation32. Glutamine is one of the main sources of fuel for respiration, supplying nitrogen necessary for the synthesis of amino acids and sugars. Furthermore, it is the precursor of fibroblasts and macrophages in the cell. Also, glutamine is applied as an essential energy source, as well as for lymphocyte proliferation10.
Iron is crucial for hydroxylation of proline and lysine, and hence acute shortage of iron may reduce the production of collagen29.
Any long-term change in proteins, including collagen and nucleic acids, owing to non-enzymatic glycation (i.e., glucose binding with protein amine groups in an amount proportional to the mean glucose concentration) may also contribute to tissue damage. The initial biochemical changes are reversible through appropriate control over blood sugar. As high levels of blood sugar persist, there are irreversible changes, which can eventually lead to the final products of advanced glycation (AGEs)33. Pentosidine is a special marker of glycoxidation. In fact, these end products of advanced glycation alter the physical function of collagen and other proteins. One of the factors delaying wound healing in diabetic patients is the formation of these products and altered functions of proteins, including collagen and their curtailed flexibility. In a research done by Urios in 2007, it was seen that natural flavonoids found in vegetables, such as kaempferol, quercetin, and antioxidants, such as beta-carotene, provide strong inhibitors to prevent the formation of Pentosidine in collagens34.
Issazadeh and et al. showed that the alcoholic extract of the Iranian spinach variety had antimicrobial properties due to its high content of phenolic compounds, unsaturated fatty acids, amino acids, terpenes, alkaloids, etc. and hence could be used as an antibiotic35. Dehkharghanian also confirmed the presence of polyphenols and flavonoids in aqueous spinach extract36.
Moreover, having compared the amount of phytochemicals and bioactive constituents, including phitobatamin, saponins, phenol, tannins, glycosides, flavonoids, steroids, terpenes and cardenolides, in spinach leaf in aqueous and alcoholic extracts of Spinacia oleracea, Olagoke et al. observed that the aqueous extract of Spinacia oleracea consists of suitable amounts of saponins, flavonoids, terpenes, cardenolides, and phlobatamin, while the alcoholic extract consists of tannins, phenol, glycosides, and steroids22 (Supplementary Table B).
Saponin not only enhances re-epithelialization of the wound but also effectively inhibits inflammatory reactions among the early phase, and promotes matrix synthesis throughout the wound healing process37. flavonoids worked in all phases of wound healing and activated the intracellular signaling pathways essential for healing to happen. These are important events in the clinical management of diabetic wounds, as they can fight inflammation and stimulate tissue regeneration38. Terpenes produce scars with effective tensile strength, function as an adhesive of primary intention, accelerate wound closure, and contribute to collagen deposition39. Also, Cardenolides can boost proliferation of fibroblasts result in enhancement in wound healing process40.
Tannin showed significant wound healing promotion effect, the mechanism may be due to its function in promotion fibroblast proliferation and migration into wounds of NIH3T3 cell, and also a pretention of antibacterial activities41. Studies illustrate that polyphenols from different plant species, take part in different aspects of wound healing, accelerating this process through their antioxidant, anti-inflammatory and antimicrobial properties and their stimulation of angiogenic activities needed for granulation tissue formation and wound re-epithelialization42,43,44. Glycosides have been shown to stimulate fibroblast proliferation and collagen synthesis, increase the production and accumulation of ECM and enhance re-epithelialization by keratinocytes45.
According to such mechanisms and the role of various compounds of spinach in wound healing, it can be argued that this combination can provide a synergistic effect because it recovers faster than the control group. Moreover, receiving the extract 1 month before and one month after induction of disease has a stronger effect on wound healing than receiving it for one month, especially in the group receiving an alcoholic extract of spinach a month before and a month after the induction of disease.
One of the basic characteristics of natural wound healing is the formation of granulation tissue. For example, fibrovascular tissue that contains fibroblasts involves collagen and blood vessels, characterizing the recovery reaction. The vascular component depends on angiogenesis, where the vessels appear days after the wound. The capillary growth into the wound provides a conduit for food and other intermediaries of recovery reaction and also metabolite elimination. Inhibition of angiogenesis can impair wound healing1. Metabolic disorders can aggravate VEGF in the wound periphery. Ischemia and hypoxia characterize tissue damage, which means that after 5 days, the oxygen pressure in the wounds is 6–7 mm Hg. These values range from 45 to 50 mm Hg in normal tissues. Angiogenic can restore tissue perfusion, reestablish microcirculation, and raise oxygen pressure to 30–40 mm Hg46. Therefore, hypoxia increases the expression of VEGF in monocytes, besides other cell types, such as fibroblasts, keratinocytes, myocytes, and endothelial cells47. Hypoxia may enhance tissue expression of VEGF and its receptors that, in turn, helps angiogenic reaction. Hypoxia provides VEGF expression through factor-1 alpha, which is induced by hypoxia48. The VEGF expression curve is parallel with the hypoxia curve, followed by endothelial cells migrating toward areas with the highest oxygen deficiency. The macrophage can maintain the curve since they survive in areas with low oxygen pressure. Indeed, the hypoxic tissue curve is essential for angiogenesis in wound healing, and the elimination of this curve can inhibit capillary growth46.
Our results showed that all study groups except the control had a significant difference statistically with the diabetic group on the third and seventh days. According to the mechanism mentioned above in which VEGF plays a role in wound healing, it can be stated that maximum hypoxia in the affected area in the treatment groups and control were found on the third and seventh days. This is because the VEGF concentration later began to decline, representing a decrease of hypoxia in the affected area through the role of VEGF in stimulating angiogenesis and then compensating the lack of oxygen. As stated earlier, the hypoxic curve is parallel to VEGF one, and when the hypoxia curve declines, its concentration curtails. On the other hand, since the increase in VEGF and angiogenesis occur during the proliferative phase, the results indicates that wound healing in these groups go through the inflammatory phase after three days and step into the next phase of proliferation similar to wound healing in the control group, while a major cause of delay in wound healing in diabetic patients is the prolonged inflammatory phase. This is confirmed by the results of the diabetic group, where the highest level of VEGF is found on the twenty-first day. Another interesting point is the data on group F. As shown in the tables, group F achieves the highest mean on the third and the seventh days, and it manages to make a stark distinction from other groups and even group E on the seventh day. These results indicate that receiving alcoholic extracts of spinach one month before and then one month after the induction of disease and wound leads to maximum effectiveness in increasing VEGF.
According to the results, on the fourteenth, twenty-first and thirtieth days, all the treatment groups showed a significant difference with the diabetic control one. However, the noteworthy point was that there was not a statistically significant difference between preventive groups and healthy control group. The mean blood glucose on the thirtieth day in preventive groups dropped from 250 mg/dl as a marker of diabetes in rats to 145 and 115 mg/dl, falling within the non-diabetic range. This proved the hypothesis concerning the effectiveness of aqueous and alcoholic extracts on blood glucose in diabetic rats on the fourteenth day. Two previous studies focused on the impact of aqueous and alcoholic extracts of spinach on blood glucose in diabetic rats. In a research performed by Kumar et al. in 2010, the anti-diabetic effects of alcoholic extract of spinach were revealed when used at 100 mg/kg for fifteen days49. Nevertheless, Gomathi observed the effects of spinach in lowering blood glucose at a dose of 200 mg/kg and 400 mg/kg of aqueous and alcoholic extracts on the twelfth day; this was confirmed by the results of the present study50. Moreover, Gomathi found some improvement in the beta cells of the pancreas in the spinach-receiving groups. Spinach decreases serum glucose through inhibiting intestinal α-glucosidase activity, which reduces the digestion and absorption of disaccharides. On the other hand, differentiation of 3T3-L1 pre adipocytes which is a useful model for studying insulin action was increased in the presence of spinach extract, showing its insulin-like and insulin-sensitizing actions51. Also, spinach thylakoids can increase the secretion of GLP-1, which is an incretin hormone and induces insulin secretion20,52. The effects of spinach and thylakoids on insulin have been very inconsistent in previous studies53,54,55,56.
The decisive role of insulin lies in inhibiting the breakdown of adipose tissue reserves of triglycerides in NEFA. Fatty acids are the main substrates of ketosis in the liver. Hence, insulin functions as a major ketosis regulator by controlling blood levels of fatty acids and other effects. Insulin deficiency or resistance can leave extensive metabolic impacts. As carbohydrates are not applied and may not be deposited in the liver, there are excess blood glucose levels. Meanwhile, the cells are not able to consume glucose, where insulin deficiency transforms glycogen, fat, and protein into glucose. In the absence of glucose, however, insulin is not consumed, thus aggravating the blood sugar levels. Protein synthesis curtails muscle protein transformation into glucose, which leads to muscle weakness and muscle tissue degradation as the breakdown of fat causes weight loss57.
In an investigation to assess the anti-diabetic effect of spinach on alloxan diabetic rats, spinach extract at a dose of 400 mg/kg managed to leave a beneficial effect on weight gain by the twelfth day50. Similarly in the current study, it was expected that receiving spinach extract could, on the fourteenth day, improve weight gain in diabetic rats under treatment; however, the results showed that, on this day only, the preventive groups had a significant difference with the diabetic control group, while it was not the case in one-month groups. On the twenty-first day preventive groups showed a statistically significant difference with the diabetic control group, while there was no such statistically significant difference concerning the healthy control group. Conversely, groups C and D showed a significant difference with the healthy control group. Similarly, group C indicated a statistically significant difference with diabetic control one. The results on the thirtieth day, however, contrasted those above since there was no difference between the treatment groups and those of the diabetic and healthy control. This might have occurred due to the adaptability of the diabetic rats in the control group with the disease rather than the hypothesis of ineffective extract in the treatment groups. As the results for blood sugar suggest, the lowered blood sugar began on the fourteenth day, indicating effective compounds found in spinach, such as vitamins, antioxidants, and flavonoids. Enhanced insulin secretion facilitated the entry of glucose into the cells. This, in turn, prevented damage breakdown of structural proteins to a certain extent and improved weight gain. It was, unlike Gomati’s study in which the effectiveness of spinach on weight gain was observed on the fourteenth day, therefore, revealed that day twenty-one achieved the effect on weight because it was the single day on which groups E, F, and C showed a significant difference with the diabetic control group. The seven-day delay in observing the effect of spinach on weight between the current study and Gumati could be associated with the administered dose of extract, which was 300 mg/kg in the current study and 400 mg/kg in the Gumati study, respectively.