Oxidative stress-induced uncontrolled inflammation is the hallmark of non-healing or chronic wounds. A vast number of chronic wound healing modalities involve the utilization of antioxidants for their free radical scavenging and inflammation curtailing properties. Based on the pharmacological profile of its isomer and the reported antioxidant potential of isovanillin, the present study is aimed to investigate the wound healing and anti-inflammatory activity of isovanillin using in vivo models. Among isomers of vanillin, o-vanillin has shown better pharmacological activity and isovanillin has also been reported for its promising pharmacological role as an antioxidant, antispasmodic and antidiarrheal compound. For evaluation of antioxidant, antimicrobial, wound healing, anti-inflammatory, and anti-angiogenic potential of isovanillin in excision wound model and burn wound models. ELIZA test was used for the estimation of antioxidant and inflammatory parameters. Analgesic activity was assessed by carrageenan induced paw edema, xylene induced ear edema, acetic acid induced writhing test. Antimicrobial activity was evaluated by disc diffusion Antioxidant of isovanillin was assesed with an IC50 value of 30.29 ± 1.86 µg/ml using by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. In wound healing (excision and burn wound) models isovanillin assisted dose-dependent wound contraction and re-epithelialization. There was no significant difference (p > 0.05) when % wound contraction of isovanillin (10% ointment) was compared to the standard treatment group. Isovanillin-treated animals showed complete re-epithelialization, reduced inflammatory cells, and development of hair follicles on histopathological analysis of skin samples, Furthermore elevation of enzymatic antioxidants such as CAT and SOD in liver tissues along with marked (p < 0.05) decrease in IL-6 and TNF-α was observed. To investigate the anti-inflammatory activity two models of acute edema were used. In the carrageenan-induced paw edema model, isovanillin exhibited significant anti-inflammatory activity up to 6 hours, while maximum % inhibition of edema was achieved at the 6th hour (p > 0.05) as compared to indomethacin (10 mg/kg). Anti-inflammatory activity in xylene-induced ear edema shown by isovanillin (10mg/kg) was comparable (p < 0.05) in comparison to dexamethasone. Analgesic activity assessed by acetic acid-induced abdominal writhing in rats showed comparable % inhibition of writhes compared to indomethacin (10 mg/kg). Antimicrobial activity evaluated by disc diffusion showed clear zone of inhibitions by isovanillin against Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and pseudomonas aeruginosa. Based on our investigation, it was concluded that isovanillin possesses antimicrobial, wound healing, and anti-inflammatory activity mediated by upregulation of antioxidant enzymes and downregulation of pro-inflammatory cytokines (IL-6 and TNF-α).
Research Article
Evaluation of the wound healing, anti-inflammatory, and antimicrobial activity of isovanillin by using an animal model
https://doi.org/10.21203/rs.3.rs-2114680/v1
This work is licensed under a CC BY 4.0 License
Version 1
posted
You are reading this latest preprint version
Isovanillin
wound healing
analgesic
antimicrobial
Skin is the body’s outer sheath and functions as a semi-porous obstruction against foreign material. To preserve its physiological functions skin maintenance and mechanical stability are essential as it is prone to various ailments and injuries (Juneja et al. 2020). A wound is any damage or injury that disrupts skin or tissues by bringing changes in their biochemical composition, function, or cellular structure (Masson-Meyers et al. 2020). The phenomenon of wound healing is a restoration activity that is natural but very dynamic and complex requiring synchronicity in several cellular and intercellular signaling mechanisms (Naraginti et al. 2016). The first step starts when vessels contract and platelet come together to form a thrombus which is followed by the migration of several inflammatory cells towards the wound site. Chemotaxis and phagocytosis involving early neutrophils and late macrophages fall in the inflammatory phase that progresses towards the third stage of healing where an epithelial layer is re-established, formation of new vessels occur and integration of collagen takes place. Finally, the fourth step is a phase of maturation where fibroblasts undergo programmed cell death, collagen network fix-up gets completed and vasculature is returned to normal status, and wound contraction takes place (Moeini et al. 2020; Rodrigues et al., 2019). Reactive oxygen species play an indispensable role in the repair process from hemostasis to infection control and TNF-α stimulation, however, unregulated production of ROS builds up oxidative stress and leads the wounds towards chronicity (Cano Sanchez et al. 2018). Inflammation is an advantageous natural response of the body against damaging stimuli, an important stage in injury recuperation but a robust and chaotic inflammatory cascade attributed to the overburden of pro-inflammatory substances such as TNF-α, IL-6 and IL-1β are characteristic of impaired healing mechanisms (Nosenko et al. 2019). Various studies have reported suppression of TNF-α and IL-6 levels as an indicator of reversing the deaccelerated healing mechanisms (de Christo Scherer et al. 2019; Liang et al. 2019; Pourkarim et al. 2022). A vast number of chronic wound healing modalities involve the utilization of antioxidant bioactive compounds (protocatechuic acid, gallic acid, and resveratrol) for their free radical scavenging and inflammation curtailing properties as these compounds can serve as leads for new drugs. (Jia et al. 2019; Son et al. 2018; Yen et al. 2018; Zhao et al. 2020; Zhou et al. 2021; Zhu et al. 2019).
Isovanillin is a phenolic compound (an isomer of vanillin) that is widely used as an intermediate in production of pharmaceuticals such as galanthamine and methyldopa. (Yapar et al. 2021). Isovanillin has been reported to be present in various plants such as Erythrina stricta (gul e nishtar), Coriandrum sativum, Pycnocycla spinose, Mondia whitei, Pimpinella anisum (sweet cumin), Dimocarpus longan, Tachigalia paniculata and Smilax Riparia known for their free radical scavenging and anti-inflammatory properties (Akter et al. 2016; Chen et al. 2012; Cioffi et al. 2002; Koorbanally et al. 2000; Li et al. 2014; Singh et al. 2006). Vanillin is a constitutional isomer of isovanillin that has been extensively studied for its antioxidant, anti-inflammatory, antimicrobial, wound healing, neuroprotective, cardioprotective, and anticancer activity (Al-Baqami et al. 2021; Arya et al. 2021; Bezerra-Filho et al. 2019; Costantini et al. 2021; Lan et al. 2019). Other structurally related compounds such as eugenol have also been reported for their anti-oxidant and wound healing activities. Reported antioxidant activity of isovanillin and difference in pharmacological profile of isomers intrigued to explore wound healing and anti-inflammatory potential of isovanillin by estimating various antioxidant and inflammatory parameters.
2.1 Chemicals and reagents
Isovanillin (glentham life sciences), carrageenan (sigma), acetic acid, xylene, ethanol, Polyfax, silver sulfadiazine, aspirin, dexamethasone, gentamicin sulfate, fluconazole, ketamine, and xylazine injection, ELIZA kits for TNF-α, and IL-6, DPPH (2, 2-diphenyl-1-1picrylhydrazyl), ascorbic acid.
2.2 Evaluation of Antioxidant activity of isovanillin
DPPH (2, 2-diphenyl-1-1picrylhydrazyl) antiradical assay was used to estimate the anti-radical activity of isovanillin by an already established method using ascorbic acid as a reference, and results were presented as IC50 (Asif et al. 2020).
2.3 Preparation of ointment
The ointment base was purchased from a local pharmacy and the incorporation method of ointment preparation was adopted to prepare 5% and 10% isovanillin ointment. For each 10g of ointment, either 0.5g or 1g of isovanillin was added to the base ointment and mixed until a homogenous formulation developed.
2.4 In vivo studies
Albino rats irrespective of their gender, having a weight of 150-200g, were procured from animal use of Faculty of Pharmaceutical Sciences, GCUF, and were provided with standard housing protocols i.e., room temperature 25 ± 2∘C, humidity nearly 50% and 12/12 hours light/dark cycle for acclimatization about a week. Animals were provided with a standard lab diet and water ad libitum. All the experimental protocols using animals were reviewed by the Institutional Review Board of GCUF and were approved by the committee (study number: 19878, reference number: GCUF /ERC/2278).
2.4.1 Evaluation of wound healing activity in excision wound model and burn wound model
To study the wound healing potential of isovanillin two in vivo models were conducted. The assessment of in vivo healing capacity was done using the excision wound model and burn wound model by following a previously documented method with modifications (Yaseen et al. 2020). For excision wound induction 5mm biopsy punch was used while for burn wound induction a preheated rod was used. The experiment protocol was the same in both models however, different positive control was employed in the two models. Burn wounds were treated for 21 days while excision wounds were treated topically for 14 days.
2.4.2 Animal grouping
Group- I (Vehicle control): wounded and received topical base ointment
Group- II (Disease control): wounded but without any treatment
Group- III (Treatment − 1): wounded and received isovanillin 5% as an ointment
Group- IV (Treatment − 2): wounded and received isovanillin 10% as an ointment
Group- V (Positive control): wounded and received a topical preexisted drug
The Vernier Caliper was used to measure the wound size and photographed on respective days (day 3rd, 7th, 10th, 14th, and 21st ). Wounds were photographed and wound size was measured on predetermined days and results were presented as mean ± SEM of percent wound closure. The following formula was used to calculate the percent wound size reduction.
On the 14th day the excision group were decapitated while the burn wound model were decapitated on the 21st day. In a similar way, blood was drawn from the heart, and serum was separated. Similarly, skin from the burned area as well as different organs (liver, kidney, and heart) was stored for studying further parameters
2.43 Estimation of antioxidant enzymes
To Estimate the effect of isovanillin on antioxidant enzymes (catalase and superoxide dismutase) liver tissue homogenates of wounded animals were used. Catalase enzyme activity was determined using hydrogen peroxide as a substrate where catalase enzyme caused decomposition of hydrogen peroxide (H2O2). To estimate superoxide dismutase activity NBT method was adopted (Juneja et al. 2020).
2.44 ELIZA for TNF-α and IL-6
To determine the level of TNF –α and IL-6 in serum samples commercially available kit was used and by following the manufacturer’s specification values were determined as pg/ ml (Kant et al. 2021).
2.45 Histological examination
Skin samples from the wound are was cut and preserved in formalin solution (10%) samples were dehydrated with ethanol and placed in paraffin. H & E staining of tissues was done and observed under the microscope for epidermal changes, inflammatory condition and granulation tissue.referance
2.5 Anti-inflammatory activity
To evaluate the anti-inflammatory activity of isovanillin two studies of acute inflammation, carrageenan-induced hind paw edema and xylene-induced ear edema were conducted by the following already reported methods (Asif et al. 2020). Paw diameter and difference in ear weights were noted and results were expressed as mean ± SEM of percent inhibition of edema calculated by following formula
2.6 Analgesic activity
The effect of isovanillin on hyperalgesia was studied using an acetic acid-induced abdominal writhing model following an established protocol. Twenty-four rats were randomly allocated into four groups (each group having six rats). Rats received different treatments (normal saline 10 mL/kg, isovanillin 5 mg/kg, isovanillin 10 mg/kg, and indomethacin 10 mg/kg respectively) by gastric oral gavage. After 30 minutes of pre-treatment, 10 mL/kg of acetic acid (1%) was injected into each rat via the intraperitoneal route. Then rats were transferred to transparent plastic chambers to observe the writhing caused by acetic acid injection for 5–20 minutes. The number of writhes was counted for each group of rats. Percent inhibition in the abdominal writhing was calculated by the formula referance
2.7 Evaluation of antimicrobial activity
The activity of isovanillin against bacterial and fungal strains was estimated using a disk diffusion test. For that purpose, some microbial strains were used such as Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and pseudomonas aeruginosa, and Aspergillus niger (Balouiri et al. 2016).
2.8 Antiangiogenic activities
The effect of isovanillin on the angiogenesis process was determined by following the chick chorioallantoic membrane method with slight modifications (Lirdprapamongkol et al. 2009). For this purpose, 3–4 days old (fifty chicken eggs) were taken and cleaned with ethanol to remove any debris followed by their placement in an incubator vertically at 37oC and 70–80% humidity. Later, in a laminar flow, signs of any development in the eggs were observed with the help of a candle, and those showing no signs were cast out and the rest were used for the assay. Eggs were divided into three groups where the first and second groups comprised of isovanillin at 100µg mL− 1 and 200µg mL− 1 and the third group was treated with ethanol (control). On the 4th day, a hole was generated aseptically along one side of the egg to draw albumin (2-3mL) for forming an air sac and the hole was covered again with the help of sealing tape. A small window was generated with the help of sterilized forceps or scissors. Sterilized and sampled Whatman’s filter paper discs were inserted in through the window and pictures were taken. Later, the windows were closed with the help of tape and again placed in the incubator for 24 hours. After that time, eggs were taken out of the incubator to assess the effect of various treated discs on angiogenesis. The photographs were taken again and compared with those taken earlier. referance
2.9 Statistical analysis
Results were presented as mean ± SEM. The statistical analysis was determined using either One-way or two-way ANOVA for different assays followed by post hoc Tukey’s test with (p < 0.05) considered significantly different
3.1. Assessment of the anti-radical potential of isovanillin
The free radical scavenging potential of isovanillin was evaluated on the basis of the DPPH assay (Table 1) and the data showed that isovanillin exhibited moderate radical scavenging potential with IC50 values ranging from 30.29 ± 1.86 and expressed in µg mL− 1. Moreover, the IC50 value in the DPPH assay was found to be 3.05 ± 1.15.
| Sample | DPPH |
|---|---|
| Isovanillin | 30.29 ± 1.86 |
| Ascorbic acid | 3.2 ± 1.15 |
3.2. In vivo wound healing potential of isovanillin
3.2.1 Excision wound model
A tremendous increment in the percent contraction in the excised wound was pragmatic in both the tested in vivo models. In the excision model, on the 3rd day of treatment, a non-significant effect of isovanillin at 5% (5.40 ± 0.64%) and base control (5.10 ± 0.50%) was observed in terms of wound reduction (Fig. 1). In contrast, isovanillin at 10% and Povidone-iodine (PI) at 5% demonstrated a significant (p < 0.05) inclination in the percent wound contraction by 9.37 ± 0.41% and 17.83 ± 0.34% respectively in comparison with the base control. However, on the 7th day, a substantial increment in the percent wound contraction was observed in the treatment involving the base control (15.93 ± 0.58%) as compared to the disease control (9.07 ± 1.42%), which indicated the efficacy of the moist environment generated owing to the simple ointment base. Among the isovanillin treated groups, isovanillin at 10% treatment caused a highly significant (p < 0.0001) effect on wound contraction (25.77 ± 1.04%) in comparison with its application at 5%.
Similarly, on the 10th day, all the treatment groups exhibited excellent results in wound contractions however, maximum reduction (80.57 ± 0.94%) was observed by the application of PI at 5% followed by isovanillin at 10% (76.71 ± 0.66%), isovanillin at 5% (71.34 ± 1.03%), base control (66.90 ± 0.79%) and disease control (32.76 ± 1.29%) treatments. On the 14th day, maximum percent wound size reduction (99.29 ± 0.28%) was observed by the PI at 5% which was, however, not significant with the isovanillin at 10% (96.77 ± 0.36%) but significantly different with respect to isovanillin at 5% (86.67 ± 0.40%), base control (81.63 ± 1.00%) and disease control (71.38 ± 1.06%).
3.2.2 Effect of isovanillin on the antioxidant production and inflammatory mediators
A significant (p < 0.05) increment in the CAT and SOD activities of the liver tissues after the isovanillin treatment was observed (Fig. 2) in the excision wound model and results were expressed in U mg− 1 protein. Similarly, the isovanillin treatment also resulted in a significant reduction in the serum concentrations of IL-6 and TNF-α in comparison with the control (p < 0.05), and the results were expressed in pg mL− 1.
3.2.3. Histopathological analysis of tissues in excision wounds
The histopathological data obtained, indicated the completion of re-epithelialization (a hallmark of the healed surface) under isovanillin treatment whereas, in the disease control group, no epithelialization was recorded. In addition, in the base control group, the broken epithelium was observed which an indication of delayed healing was. In the control group, neovascularization was observed but it was absent in the treatment groups which depicted that the healing in the treatment groups occurred some days before the final day of the model (when animals were decapitated). This was further evidenced by the presence of mature follicles which explained the success of restoration activity (Fig. 3).
3.2.4. Burn wound healing model
The application of isovanillin ointment resulted in a remarkable effect on the percent contraction in the burn wound rats (Table 2). On day 0, there existed a non-significant difference (p > 0.05) in the wound size as all the rats were being burnt using the same tool. However, on the 3rd day, negative values regarding the wound contractions were obtained which indicated the development of inflammation in the wounded area where isovanillin at 10% exhibited significant activity in comparison with the control and non-significant effect was evident under isovanillin treatment @ 5%. On the 7th day, isovanillin application at 10% demonstrated significantly good results in terms of percent wound reduction than the control and lower dose of isovanillin (5%) whereas, maximum efficiency was visible in the treatment group involving the application of standard drug (silver sulfadiazine). Observations were taken from days 10–21 explained that all the treatments resulted in a significant reduction in the wound size where the maximum decline was observed in the standard drug (98.34 ± 0.95%) ointment followed by isovanillin at 10% (96.84 ± 1.13%), isovanillin at 5% (84.13 ± 1.24%) and control groups (71.39 ± 2.13%) at day 21st (Fig. 4).
3.2.5 Isovanillin on antioxidant enzymes and inflammatory mediators (Burn wound model)
Application of isovanillin led to a significant (P < 0.05) increment in the activities of SOD and CAT in liver tissues as compared to the control treatment and the results obtained, were expressed in U mg− 1. Similarly, a significant (P < 0.05) decrement in the serum concentrations of IL-6 and TNF-α was observed after isovanillin treatment and the values were expressed in pg mL− 1 (Fig. 5).
3.2.6 Tissue histopathology of the burn wound
The histopathological examination explained that the broken, interrupted epithelialization, formation of new blood vessels, and presence of inflammatory cells were indicative of the early stages of the healing process as evident in animals decapitated on the 14th day. On the 21st day, complete epithelialization was observed in the isovanillin-treated group. On the other hand, the epithelial layer was observed to be developed in the base control group but the hair follicles were very mild. The presence of neovascularization in the control group and its absence in the treatment groups suggested the start of the healing process in the treatment groups’ days before the completion of the model (decapitation day). The observations were further supported by the presence of mature follicles and fibrotic tissues as a sign of restoration activity (Fig. 6).
3.3. Anti-inflammatory activities
3.3.1. Carrageenan-induced hind paw edema model
A significant (p < 0.0001) inhibition of edema was observed after each hour (measured from 1-6th hour) in isovanillin and indomethacin-treated groups. In the initial hours (1 and 2), there was no significant difference between the two doses of isovanillin however, during the 4th and 6th hour, a significantly increased inhibitory effect was pragmatic as compared to the control treatment group where the high dose (10 mg kg− 1) proved to be more effective than the low dose (5 mg kg− 1). Moreover, during the first two hours, a non-significant edema inhibitory effect was observed between indomethacin (10 mg kg− 1) and isovanillin (5 and 10 mg kg− 1). Furthermore, up to the 6th hour, the effect of indomethacin (66.15 ± 1.62%) was relatively more significant than isovanillin at 5 mg kg− 1 (48.84 ± 1.04%) whereas the effect was still non-significant than isovanillin treatment at 10 mg kg− 1 (63.03 ± 0.97%) (Fig. 7).
3.3.2. Xylene-induced ear edema
Our findings regarding the effect of isovanillin in suppressing the ear edema (Fig. 8) depicted that all the treatments performed significantly (p < 0.0001) better as compared to the control where the maximum reduction was observed in the standard drug i.e., dexamethasone at 1 mg kg− 1 (71.11 ± 0.73%) followed by isovanillin at 10 mg kg− 1 (68.73 ± 0.84%) and at 5 mg kg− 1 (60.79 ± 0.82%). The significant difference between the two doses of isovanillin indicated that the anti-inflammatory effect was highly dose-dependent.
3.4. Analgesic activity
The number of writhes after injecting acetic acid into each group was noted where the highest number of writhes was observed in the control group (78.83 ± 1.14%). Whereas, significant reductions in the percentage of writhing were observed in the isovanillin and indomethacin treatments with maximum reduction (51.33 ± 1.28%) were evident in the indomethacin at 10 mg kg− 1 treatment followed by isovanillin at 10 mg kg− 1 (40.39 ± 1.02%) and at 5 mg kg− 1 (34.18 ± 1.26%). So, the final results concluded that the suppressing effect of indomethacin was significantly better than both the doses of isovanillin (Fig. 9).
3.5. Antimicrobial activities
The antimicrobial effects of isovanillin (Fig. 10) on the basis of measurement regarding the zone of inhibition indicated that the application of isovanillin at 400µg was relatively more significant than its application at 200µg against all the bacterial strains however, isovanillin (200 and 400µg) treatments did not exhibit any distinct effect against the fungus (Aspergillus niger). In contrast, the reference drug used against bacterial strains (gentamicin sulfate) and fungal strains (fluconazole) significantly inhibited their respective microbial activities. Among bacterial strains, maximum inhibition of gentamicin sulfate was observed against Staphylococcus epidermidis (27.03mm) whereas, fluconazole decreased the zone of inhibition of Aspergillus niger (18.97mm) respectively.
3.6. Anti-angiogenic activities
Results regarding the effect of isovanillin on angiogenesis (Fig. 11) demonstrated that a normal vasculature development was observed in the treatment where the isovanillin at 100µg disc was placed. The isovanillin did not cause any damage to the vasculature development in the chick chorioallantoic membrane.
A wound is any damage or injury that disrupts skin or tissues by bringing changes in their biochemical composition, function, or cellular structure (Masson-Meyers et al. 2020). The step includes vessel contraction and thrombosis formation by platelets the establishment of the epithelial layer, formation of new vessels, integration of collagen, and wound contraction (Moeini et al. 2020). Oxidative stress-induced uncontrolled inflammation is the hallmark of non-healing or chronic wounds which. A vast number of chronic wound healing modalities involve the utilization of antioxidants for their free radical scavenging and inflammation curtailing properties (Rodrigues et al. 2019). DPPH (2, 2-diphenyl-1-1picrylhydrazyl) anti-radical assay was used to estimate the anti-radical activity of isovanillin by an already established method using ascorbic acid as a reference, and results were presented as IC50 (Asif et al. 2020)
Vanillin which is a constitutional isomer of isovanillin has been extensively studied for its antioxidant, anti-inflammatory, antimicrobial, wound healing, neuroprotective, cardioprotective, and anticancer activity (Al-Baqami et al. 2021; Arya et al. 2021). Among isomers of vanillin, o-vanillin has shown better pharmacological activity and isovanillin has also been reported for its promising pharmacological role as an antioxidant, antispasmodic and antidiarrheal compound. Based on the pharmacological profile of its isomer and the reported antioxidant activity of isovanillin, the present study aimed to investigate the wound healing and anti-inflammatory activity of isovanillin using in vivo models. For evaluation of antioxidant, antimicrobial, wound healing, anti-inflammatory, and anti-angiogenic potential of isovanillin already reported in vitro and in-vivo models were used. ELIZA test was used for the estimation of antioxidant and inflammatory parameters (Bezerra-Filho et al. 2019; Costantini et al. 2021; Lan et al. 2019).
To study the wound healing potential of isovanillin two in vivo models were conducted. The assessment of in vivo healing capacity was done using the excision wound model and burn wound model by following a previously documented method with modifications (Yaseen et al., 2020). For excision wound induction 5mm biopsy punch was used while for burn wound induction a preheated rod was used. The experiment protocol was the same in both models however, different positive control was employed in the two models. Burn wounds were treated for 21 days while excision wounds were treated topically for 14 days. In wound healing (excision and burn wound) models isovanillin assisted dose-dependent wound contraction and re-epithelialization. There was no significant difference (p > 0.05) when % wound contraction of isovanillin (10% ointment) was compared to the standard treatment group. Isovanillin-treated animals showed complete re-epithelialization, reduced inflammatory cells, and development of hair follicles on histopathological analysis of skin samples, furthermore elevation of enzymatic antioxidants such as CAT and SOD in liver tissues along with a marked (p < 0.05) decrease in IL-6 and TNF-α was observed.
To evaluate the anti-inflammatory activity of isovanillin two studies of acute inflammation, carrageenan-induced hind paw edema and xylene-induced ear edema were conducted by following already reported methods (Asif et al. 2020). Paw diameter and difference in ear weights were noted and results were expressed as mean ± SEM of percent inhibition of edema calculated by following formula. In the carrageenan-induced paw edema model, isovanillin exhibited significant anti-inflammatory activity up to 6 hours, while maximum % inhibition of edema was achieved at the 6th hour (p > 0.05) as compared to indomethacin (10 mg/kg). Anti-inflammatory activity in xylene-induced ear edema shown by isovanillin (10 mg/kg) was comparable (p < 0.05) in comparison to dexamethasone.
The effect of isovanillin on hyperalgesia was studied using an acetic acid-induced abdominal writhing model following an established protocol. A number of writhes were counted for each group of rats. Percent inhibition in the abdominal writhing was calculated by formula. The study design in this model was the same as the excision model except the current model lacked the diseased control group and the rat skin was burned rather than excised. Moreover, in this study, the 21-day model was employed instead of 14 days. For causing the burn wound, a metal rod was heated using a burner and was placed on the shaved rat skin for nearly 30 seconds. The process was repeated to obtain third-degree burns and photographs of the wounds were taken on day 0. Later, each group received the above-mentioned treatments until the 21 days when they were decapitated.
Analgesic activity assessed by acetic acid-induced abdominal writhing in rats showed comparable % inhibition of writhes compared to indomethacin (10 mg/kg). In vitro testing method was employed to check the anti-microbial effects of the isovanillin against different bacterial strains such as Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli as well as fungal strain such as Aspergillus niger. Antimicrobial activity evaluated by disc diffusion showed a clear zone of inhibitions by isovanillin against Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and pseudomonas aeruginosa (Balouiri et al. 2016). Based on our investigation, it was concluded that isovanillin possesses antimicrobial, wound healing, and anti-inflammatory activity by upregulation of antioxidant enzymes and downregulation of pro-inflammatory cytokines (IL-6 and TNF-α). Isovanilli8n can prove as an effective treatment for wound healing in different medical procedures.
This study signifies the anti-oxidant, analgesic, anti-inflammatory activities in wound healing (excision and burn wound) models isovanillin assisted dose-dependent wound contraction and re-epithelialization. Isovanillin-treated animals showed complete re-epithelialization, reduced inflammatory cells, and development of hair follicles on histopathological analysis of skin samples, elevation of enzymatic antioxidants such as CAT and SOD in liver tissues along with marked decrease in IL-6 and TNF-α was observed. Thus highlighting isovanilin potential as wound healing agent which was event by its wound assay again excision and burn. In the carrageenan-induced paw edema model, isovanillin exhibited significant anti-inflammatory activity up to 6 hours, while maximum % inhibition of edema was achieved at the 6th as compared to indomethacin at 10 mg/kg. Analgesic activity assessed by acetic acid-induced abdominal writhing in rats showed inhibition of writhes compared to indomethacin at 10mg/kg. So, it was concluded that isovanillin possesses antimicrobial, wound healing, and anti-inflammatory activity by upregulation of antioxidant enzymes and downregulation of pro-inflammatory cytokines (IL-6 and TNF-α).
- Akter, K., Barnes, E. C., Loa-Kum-Cheung, W. L., Yin, P., Kichu, M., Brophy, J. J., . . . Jamie, J. F. (2016). Antimicrobial and antioxidant activity and chemical characterisation of Erythrina stricta Roxb.(Fabaceae). Journal of ethnopharmacology, 185, 171-181.
- Al-Baqami, N., & Hamza, R. (2021). Synergistic antioxidant capacities of vanillin and chitosan nanoparticles against reactive oxygen species, hepatotoxicity, and genotoxicity induced by aging in male Wistar rats. Human & Experimental Toxicology, 40(1), 183-202.
- Arya, S. S., Rookes, J. E., Cahill, D. M., & Lenka, S. K. (2021). Vanillin: a review on the therapeutic prospects of a popular flavouring molecule. Advances in traditional medicine, 21(3), 1-17.
- Asif, M., Saadullah, M., Yaseen, H. S., Saleem, M., Yousaf, H. M., Khan, I. U., . . . Shams, M. U. (2020). Evaluation of in vivo anti-inflammatory and anti-angiogenic attributes of methanolic extract of Launaea spinosa. Inflammopharmacology, 28(4), 993-1008.
- Balouiri, M., Sadiki, M., & Ibnsouda, S. K. (2016). Methods for in vitro evaluating antimicrobial activity: A review. Journal of pharmaceutical analysis, 6(2), 71-79.
- Bezerra-Filho, C. S., Barboza, J. N., Souza, M. T., Sabry, P., Ismail, N. S., & de Sousa, D. P. (2019). Therapeutic potential of vanillin and its main metabolites to regulate the inflammatory response and oxidative stress. Mini Reviews in Medicinal Chemistry, 19(20), 1681-1693.
- Cano Sanchez, M., Lancel, S., Boulanger, E., & Neviere, R. (2018). Targeting oxidative stress and mitochondrial dysfunction in the treatment of impaired wound healing: a systematic review. Antioxidants, 7(8), 98.
- Chen, W., Tang, S., Qin, N., Zhai, H., & Duan, H. (2012). Antioxidant constituents from Smilax riparia. Zhongguo Zhong yao za zhi= Zhongguo zhongyao zazhi= China journal of Chinese materia medica, 37(6), 806-810.
- Cioffi, G., D'Auria, M., Braca, A., Mendez, J., Castillo, A., Morelli, I., . . . De Tommasi, N. (2002). Antioxidant and Free-Radical Scavenging Activity of Constituents of the Leaves of Tachigalia p aniculata. Journal of Natural Products, 65(11), 1526-1529.
- Costantini, E., Sinjari, B., Falasca, K., Reale, M., Caputi, S., Jagarlapodii, S., & Murmura, G. (2021). Assessment of the Vanillin Anti-Inflammatory and Regenerative Potentials in Inflamed Primary Human Gingival Fibroblast. Mediators of Inflammation, 2021.
- de Christo Scherer, M. M., Marques, F. M., Figueira, M. M., Peisino, M. C. O., Schmitt, E. F. P., Kondratyuk, T. P., . . . Fronza, M. (2019). Wound healing activity of terpinolene and α-phellandrene by attenuating inflammation and oxidative stress in vitro. Journal of tissue viability, 28(2), 94-99.
- Jia, R., Li, Y., Cao, L., Du, J., Zheng, T., Qian, H., . . . Yin, G. (2019). Antioxidative, anti-inflammatory and hepatoprotective effects of resveratrol on oxidative stress-induced liver damage in tilapia (Oreochromis niloticus). Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 215, 56-66.
- Juneja, K., Mishra, R., Chauhan, S., Gupta, S., Roy, P., & Sircar, D. (2020). Metabolite profiling and wound-healing activity of Boerhavia diffusa leaf extracts using in vitro and in vivo models. Journal of traditional and complementary medicine, 10(1), 52-59.
- Kant, V., Jangir, B. L., Sharma, M., Kumar, V., & Joshi, V. G. (2021). Topical application of quercetin improves wound repair and regeneration in diabetic rats. Immunopharmacology and Immunotoxicology, 43(5), 536-553.
- Koorbanally, N. A., Mulholland, D. A., & Crouch, N. R. (2000). Isolation of isovanillin from aromatic roots of the medicinal African liane, Mondia whitei. Journal of herbs, spices & medicinal plants, 7(3), 37-43.
- Lan, X.-B., Wang, Q., Yang, J.-M., Ma, L., Zhang, W.-J., Zheng, P., . . . Yu, J.-Q. (2019). Neuroprotective effect of Vanillin on hypoxic-ischemic brain damage in neonatal rats. Biomedicine & Pharmacotherapy, 118, 109196.
- Li, Z. H., Wang, Q. M., & Pan, Y. M. (2014). Study on the antioxidant compounds extracted from longan (Dimocarpus longan Lour.) shell. Asian Journal of Chemistry, 26(15), 4602.
- Liang, M., Chen, Z., Wang, F., Liu, L., Wei, R., & Zhang, M. (2019). Preparation of self‐regulating/anti‐adhesive hydrogels and their ability to promote healing in burn wounds. Journal of Biomedical Materials Research Part B: Applied Biomaterials, 107(5), 1471-1482.
- Masson‐Meyers, D. S., Andrade, T. A., Caetano, G. F., Guimaraes, F. R., Leite, M. N., Leite, S. N., & Frade, M. A. C. (2020). Experimental models and methods for cutaneous wound healing assessment. International journal of experimental pathology, 101(1-2), 21-37.
- Moeini, A., Pedram, P., Makvandi, P., Malinconico, M., & d'Ayala, G. G. (2020). Wound healing and antimicrobial effect of active secondary metabolites in chitosan-based wound dressings: A review. Carbohydrate polymers, 233, 115839.
- Naraginti, S., Kumari, P. L., Das, R. K., Sivakumar, A., Patil, S. H., & Andhalkar, V. V. (2016). Amelioration of excision wounds by topical application of green synthesized, formulated silver and gold nanoparticles in albino Wistar rats. Materials Science and Engineering: C, 62, 293-300.
- Nosenko, M., Ambaryan, S., & Drutskaya, M. (2019). Proinflammatory cytokines and skin wound healing in mice. Molecular Biology, 53(5), 653-664.
- Pourkarim, R., Farahpour, M. R., & Rezaei, S. A. (2022). Comparison effects of platelet-rich plasma on healing of infected and non-infected excision wounds by the modulation of the expression of inflammatory mediators: experimental research. European Journal of Trauma and Emergency Surgery, 1-9.
- Rodrigues, M., Kosaric, N., Bonham, C. A., & Gurtner, G. C. (2019). Wound healing: a cellular perspective. Physiological reviews, 99(1), 665-706.
- Sadraei, H., Ghanadian, M., Asghari, G., & Azali, N. (2014). Antidiarrheal activities of isovanillin, iso-acetovanillon and Pycnocycla spinosa Decne ex. Boiss extract in mice. Research in pharmaceutical sciences, 9(2), 83.
- Sadraei, H., Ghanadian, M., Asghari, G., & Madadi, E. (2014). Antispasmodic activity of isovanillin and isoacetovanillon in comparison with Pycnocycla spinosa Decne. exBoiss extract on rat ileum. Research in pharmaceutical sciences, 9(3), 187.
- Singh, G., Maurya, S., De Lampasona, M., & Catalan, C. A. (2006). Studies on essential oils, Part 41. Chemical composition, antifungal, antioxidant and sprout suppressant activities of coriander (Coriandrum sativum) essential oil and its oleoresin. Flavour and fragrance journal, 21(3), 472-479.
- Son, J. H., Kim, S.-Y., Jang, H. H., Lee, S. N., & Ahn, K. J. (2018). Protective effect of protocatechuic acid against inflammatory stress induced in human dermal fibroblasts. Biomedical Dermatology, 2(1), 1-5.
- Yapar, G., Duran, H. E., Lolak, N., Akocak, S., Türkeş, C., Durgun, M., . . . Beydemir, Ş. (2021). Biological effects of bis-hydrazone compounds bearing isovanillin moiety on the aldose reductase. Bioorganic Chemistry, 117, 105473.
- Yaseen, H. S., Asif, M., Saadullah, M., Asghar, S., Shams, M. U., Bazmi, R. R., . . . Yaseen, M. (2020). Methanolic extract of Ephedra ciliata promotes wound healing and arrests inflammatory cascade in vivo through downregulation of TNF-α. Inflammopharmacology, 28(6), 1691-1704.
- Yen, Y. H., Pu, C. M., Liu, C. W., Chen, Y. C., Chen, Y. C., Liang, C. J., . . . Chen, Y. L. (2018). Curcumin accelerates cutaneous wound healing via multiple biological actions: The involvement of TNF‐α, MMP‐9, α‐SMA, and collagen. International wound journal, 15(4), 605-617.
- Zhang, S., Wang, H., Dai, J., Niu, Y., Yin, Q., & Zhou, L. (2021). Solubility determination, model evaluation and solution thermodynamics of isovanillin in 15 pure solvents and 4 binary solvents. Journal of Molecular Liquids, 116847.
- Zhao, C.-C., Zhu, L., Wu, Z., Yang, R., Xu, N., & Liang, L. (2020). Resveratrol-loaded peptide-hydrogels inhibit scar formation in wound healing through suppressing inflammation. Regenerative Biomaterials, 7(1), 99-107.
- Zhou, X., Ruan, Q., Ye, Z., Chu, Z., Xi, M., Li, M., . . . Xie, W. (2021). Resveratrol accelerates wound healing by attenuating oxidative stress-induced impairment of cell proliferation and migration. Burns, 47(1), 133-139.
- Zhu, L., Gu, P., & Shen, H. (2019). Gallic acid improved inflammation via NF-κB pathway in TNBS-induced ulcerative colitis. International immunopharmacology, 67, 129-137.