Biogenic synthesis of nano-sulfur using Punica granatum fruit peel extract with enhanced antimicrobial activities for accelerating wound healing

Microbial wound infections leading to secondary complications in wound healing has resulted in high demand for therapeutic drugs with improved efficacy. Despite achieving enhanced bio-activity and higher bioavailability compared to its bulk form, nano-sulfur (SNP) has been explored to a very limited extent for wound healing applications. In this work, we prepare biogenic SNP (SNP-B) via simple biogenic technique using pomegranate (Punica granatum) peel extract and demonstrate its antimicrobial and wound healing activity. The SNP-B was characterized using powder x-ray diffractometer, FESEM, transmission electron microscopy and Raman spectroscopy. Different wound models (excision, incision, dead space and burn) were used to assess the wound healing potential of SNP-B. The 2% (w/w) SNP-B treated group exhibited enhanced wound contraction rate (excision wound, 99.62 ± 0.59%; burn wound, 99.46 ± 0.59%), breaking strength (393.2 ± 10.87 g cm−2), and granulation tissue weight (166.8 ± 9.45 mg) compared to the control group (excision wound, 84.24 ± 2.78%; burn wound, 90.58 ± 3.2%; breaking strength, 241.3 ± 16.11 g cm−2; granulation tissue weight, 91.17 ± 7.28 mg). The efficacy of 2% (w/w) SNP-B was comparable to that of standard (5% w/w povidone-iodine ointment) in all the wound models analyzed. The SNP-B showed enhanced antibacterial activity with a MIC value of 90, 80, 80, and 60 μg ml−1 for Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis and Staphylococcus aureus, respectively. The results obtained prove the potential of SNP-B as a multifunctional therapeutic agent for topical applications.

IMMUNOMODULATORY Β-GLUCANS ENHANCE THE WOUND HEALING ABILITY OF CHLOROFORM FRACTION OF ACHYRANTHES ASPERA L

Introduction: Wound healing is a multifaceted biological process, and diabetic wounds add more complexity to it. In diabetic wounds, the combination of chloroform fractions of Achyranthes aspera L. (A.aspera) leaves with β-Glucans has not been investigated. The additive effect of these two (A.aspera + β-Glucans) would benefit the inflammatory phase of diabetic wounds, as improper treatment will lead to chronic injuries. Therefore, the goal of this research work was to assess the in-vivo wound healing and anti-inflammatory effects of a combination of chloroform fractions of A.aspera leaves and β-Glucans in a variety of wound models in STZ-induced diabetic rats. Methods: Preliminary phytochemical analyses of A.aspera were conducted to identify various phytoconstituents in the test extract. Acute and sub-acute dermal toxicity tests of A.aspera were carried out on mice and rats, respectively, to see whether there were any abnormalities. Excision and incision wound models, cotton pellet-induced granuloma models, rat paw edema and burn wounds were used to test wound healing and anti-inflammatory actions. To induce diabetes, streptozotocin (STZ) was administered intraperitoneally at a dosage of 65 mg/kg (i.p.). A.aspera (10% w/w) and β-Glucans (2% w/w) ointments were tested separately and in combination for wound healing activities. Silver sulfadiazine (1 % w/w) ointment was used to treat the positive control groups. Excision wound model rats that had been treated with basic ointment were used as negative controls, as were incision wound model rats that had not been treated. A.aspera (400 mg/kg, po) and β-Glucans (30 mg/kg, po) were tested separately and in combination for anti-inflammatory efficacy. Positive control groups were given indomethacin (10 mg/kg, po) for cotton pellet-induced granuloma and rat paw oedema models. Negative controls for both anti-inflammatory activity models were provided 2% Tween 80. The groups were made up of six rats, and the treatments were given topically and orally to assess wound healing and anti-inflammatory effects. The levels of hydroxyproline and hexosamine and the antioxidant enzymes (SOD and CAT) in the granulation tissue were measured in excision wound model. Healed excision wound skin was examined histopathologically. Results: The A.aspera and β-Glucans combination resulted in a significant percentage of wound contraction and a shorter epithelialization time (P<0.01). The combination was found to be the most effective, with the highest percentage of edema reduction (55 %; p<0.01). The combination also exhibited favourable hydroxylamine, hexosamine and anti-oxidant profiles supported by histopathology data. Conclusion: This research showed that the immunomodulatory effect of β-Glucans had significantly enhanced the wound healing, anti-inflammatory, and anti-oxidant potential of A.aspera in diabetic wounds.

Green Synthesized Silver Nanoparticles Using Tridax Procumbens for Topical Application: Excision Wound Model and Histopathological Studies

The objective of this study was to synthesize silver nanoparticles from the leaves of Tridax procumbens and develop its topical gels using chitosan to investigate the wound healing efficacy concomitant with the histopathological study. Green synthesized silver nanoparticles (AgNPs) were prepared by reacting silver nitrate (0.3 M) with leaf extract and characterized by particle analysis, FTIR, XRD, SEM, BET, and TGA. The results revealed formed AgNPs were nano-sized (138 ± 2.1 nm), monodispersed (PDI: 0.460 ± 0.3), inter-particle repulsion (zeta: −20.4 ± 5.20 mV), stabilized, crystalline and, spherical with size ranging from 80–100 nm as per SEM micro photos. The BET analysis of AgNPs presents the surface area (12.861 m2/g), pore volume (0.037 cc/g), and pore radius (24.50 nm).TGA results show a loss of 13.39% up to 300 °C. The topical formulation was developed by loading AgNPs in chitosan-based gels, evaluated by pH, thermal cycling, centrifugal, and spreadability tests. AgNPs chitosan gels results showed skin compatibility, higher stability, and spreading ability. The maximum antibacterial zone of inhibition was found to be 25 ± 0.98 mm for bacillus subtitles and 30 ± 1.99 mm for Klebsiella pneumoniae, respectively. Nanosilver-containing gel also showed excellent compatibility with erythrocytes. Excision wound model was used to assess the wound healing property of the developed AgNP gels, the results of which indicated a significantly progressive healing process in test-group of animals treated with chitosan-based gels containing AgNPs. A histopathological study further confirmed the almost normal skin structure of treated animal tissue compared to standard and negative control. Thus, green synthesized AgNPs loaded chitosan-based topical gel can potentially be used for wound healing application.

GC-MS Analysis and Wound Healing Efficiency of Ethanolic Leaves Extract of Cassia occidentalis and Pithecellobium dulce in Wistar Albino Rats: Comparative Study

Aim: To identify and compare the bioactive compounds in the ethanolic leaves extracts of Cassia occidentalis and Pithecellobium dulce and to evaluate the wound healing efficiency in Wistar Albino rats. Study Design: The leaves ethanolic extracts was analysed by GC-MS and the extract was prepared in the form of a cream by ethanolic leaves extracts of C. occidentalis and P. dulce at 5% (w/v), 10% (w/v), and also in combination, a simple ointment base was developed with a composition of (1:1) Topical application of 5% (w/v) and 10% (w/v) was utilised in excision wound models. For excision wound models, the treatment duration was ten days. The day on which the wound was inflicted was designated as day '0'. Wound healing Activity: Excision wound Model: The animals were randomly separated into eight groups of six rats each: Group I: Control.; Group II: Standard group, treated with Framycetin sulfate cream (Soframycin, Aventis);. Group III: Treated with ethanolic extract of C. occidentalis (ELCO) (5% w/v); Group IV: Treated with ethanolic extract of C. occidentalis (ELCO) (10% w/v);Group V: Treated with ethanolic extract of P. dulce (ELPD)(5% w/v); Group VI: Treated with ethanolic extract of P. dulce (ELPD) (10 % w/v);Group VII: Treated with ethanolic extract of C. occidentalis and P. dulce (ELCO & ELPD 1:1) (5% w/v); Group VIII: Treated with ethanolic extract of C. occidentalis and P. dulce (ELCO & ELPD 1:1) (10 % w/v) till complete epithelization. Next dead space wound model and histology was studied. Place and Duration of Study: The GC-MS was carried out at Lab in Chennai. The extraction procedures were done at Department of Microbiology, Acharya Nagarjuna University, Guntur and treatment of wound healing activities were conducted at Ratnam Institute of Pharmacy in Nellore, Andhra Pradesh, India, and housed in the Department of Pharmacology between October to January 2016. Methodology: To study bioactive compounds, GC-MS was adopted, for wound healing activity: Excision wound Model, Dead space wound model and histology procedures was applied. Results: In the current study, ethanol leaves extract (EL) of Cassia occidentalis and Pithecellobium dulce were compared using GC-MS and their wound healing efficacy in wistar rats was examined. The GC-MS analysis of EL from both plants revealed 14-16 distinct bioactive phytochemical components with varying molecular weights and retention duration (RT). Excision and dead space wound models were utilised to assess the wound healing activities of EL extracts on rats. Wound concentration, full epithelialzation time, granulation, tissue weight, and hydroxyproline content were used to measure healing. In the excision wound model, the standard group (Framycetin sulphate cream) and group-VII (10% w/v; 1:1) combination EL treatment exhibited 98.5 ± 0.54 % and 98.4 ± 0.46 % wound healing activity, respectively. When compared to the control, the granulation tissue weight and hydroxyproline content in the dead space wound rose considerably. Histological examination revealed fewer inflammatory cells and more collagen, indicating a role in accelerating wound healing activity. Conclusion: The results of our investigation indicate unequivocally that ethanolic leaf extracts of these plant species are effective at encouraging wound healing. The 10% (ELCO+ELPD) tropical treatment drastically reduced the wound as compared to standard and also increased granulation and hydroxyproline content. However, it requires more clinical examination before being considered for wound therapy.

Wound-Healing Potential of Cucurbita moschata Duchesne Fruit Peel Extract in a Rat Model of Excision Wound Repair

Background and Aim. Pumpkin (Cucurbita moschata Duchesne) is a medicinal plant with different pharmacological effects such as antioxidant, antidiabetic, hepatoprotective, and anticancer effects. In the present study, we aimed to investigate wound-healing activity of pumpkin fruit peel in a rat model of excision wound repair. Materials and Methods. Hydroalcoholic extractions of pumpkin fruit peel were obtained and used to prepare two different cold cream-based formulations, namely, 10% and 20% pumpkin peel extracts (PPEs). These formulations, phenytoin cream, and cold cream were topically used once daily for 14 days to compare their wound-healing effects in a rat model of excision wound repair. Wound sizes were monitored at different intervals. Skin tissue samples were subject to H&E staining for histopathological analysis. Blood samples were also taken on day 14 to measure serum levels of nitrite. Results. Both 10% and 20% PPE formulations resulted in a significant reduction of wound sizes compared to positive and negative controls. Wound closure rate was estimated to be higher in 20% PPE-treated rats. According to histopathological analysis, treatment with 20% PPE improved parameters associated with efficient wound repair, including better regeneration of epidemic layer, higher density of dermis collagen fibers, and lower presence of inflammatory cells. Also, both formulations lowered serum concentrations of nitrite. Conclusion. Given the obtained data from our study, the hydroalcoholic extract of Cucurbita moschata Duchesne fruit peel is proposed to be effective in accelerating the process of excision wound repair partly due to its antioxidant effect in terms of decreasing nitrite concentration.

Efficacy of Aloe vera Gel on the Excision Wound Healing in Sprague dawley Rats

Wound is a pathological processes in all living things. Wound includes the overlapping mechanisms. The failure in wound healing causes infection. This study aimed to analyze the efficacy of Aloe vera gel in excisionwound on the rat’s back. This study used 18 male, Sprague Dawley rats, and they were induced excision wound on the back. The rats were separated into 3 groups, K1 = control, K2 = treated with betadin gel, K3 = treated with Aloe vera gel. The therapy was given twice a day for 7 days. The wound measurement was observed in day 3 and 7. The data were analyzed using SPSS. The result showed that Aloe vera gel potentially promote wound healing through the decrease microscopical condition of wound, re-epithelialization, and deposition of collagen better than in group K1 and K2 (p≤0.05). In conclusion, Aloe vera gel promote excision wound healing in Sprague Dawley rats.

Evaluation of in vivo Wound Healing Activity of Ursolic Acid Rich Chloroform Extract of Hedyotis herbacea Linn Ointment

Aim: This study evaluates the effect of in vivo wound healing potential of Ursolic acid rich chloroform extract of Hedyotis herbacea Linn ointment using incision and excision wound model. Study Design: Wound healing potentials of Hedyotis herbacea were analysed by Incision and excision wound model. Place of Study: Nandha College of Pharmacy, Erode, Tamilnadu. Methodology: Hedyotis herbacea was subjected to extraction (cold maceration), with solvents of increasing polarity. All the extracts were estimated for the presence of phytoconstituents by HPTLC. As the study has been focused on the phytoconstituent based biological activity, the Ursolic acid rich chloroform extract was chosen for the study of wound healing activity. The Chloroform extract of Hedyotis herbacea was incorporated into ointment base, to prepare the ointment. The ointments prepared with Chloroform extract (2.5% and 5%) of Hedyotis herbacea (CEHH) were subjected for evaluation of excision and incision wound model. Wistar albino rats were divided into four groups each consisting of six animals; group I (left untreated) considered as untreated control, group II treated with 5% w/w povidone-iodine ointment served as standard, group III treated with CEHH 2.5% w/w ointment, and group IV treated with CEHH 5% w/w ointment were considered as test groups. All the treatments were given once daily. Wound healing effect was assessed by the rate of wound contraction, level of total protein, Hydroxy proline, Hexosamine and Hexuronic acid and histopathology studies in an excision wound model. Tensile strength was also measured in both excision and incision wound. Results: Wound healing activity of CEHH 5% w/w ointment treated group was greater than CEHH 2.5% w/w and untreated groups in both excision and incision wound model. The high rate of wound contraction (*P< 0.001), high tensile strength (*P< 0.001), and elevated total protein, Hydroxyproline, Hexosamine, and Hexuronic acid content were observed in animals treated with CEHH ointments when compared to the untreated control group of animals. Histopathological studies of the CEHH ointments treated groups also revealed the effectiveness in wound healing. Conclusion: These results justified the claimed traditional use of the Hedyotis herbacea as wound healing plant.

Wound Healing Activities of Hydromethanolic Crude Extract and Solvent Fractions of Bersama abyssinica Leaves in Mice

Background. Bersama abyssinica leaves are traditionally used for management of wounds in several communities of Ethiopia, despite no scientifically approved studies done on wound healing. Our study planned to work out the wound healing effects of B. abyssinica leaves extract in mice. Methods. B. abyssinica leaves were extracted with 80% hydromethanol and fractioned with chloroform, hexane, and water. The acute dermal toxicity of the crude extract was evaluated in mice. The crude extract formulated at 5% and 10% w/w ointment was investigated in excision, incision, and burn wound models and solvent fractions in the excision model using simple ointment (negative control) and nitrofurazone 0.2% w/v (positive control). We evaluated histopathological analysis, wound contraction rate, complete epithelialization period, and skin durability. One-way ANOVA followed by the post hoc Tukey HSD test with IBM SPSS software version 23.0 was used for data analysis, and p < 0.05 was considered statistically significant. Results. Hydromethanolic crude extract produced 5% (99.5%) and 10% (100%) wound contraction on the 16th day of the treatment and 5% (18.8) and 10% (28.2) percent reduction in the epithelization period on the excision wound healing model. Hydromethanolic crude extract produced 5% (47.5) and 10% (61.17) percent durability on the incision wound healing model. Hydromethanolic crude extract produced 5% (99.82%) and 10% (100%) wound contraction on the 20th day of treatment and 5% (13%) and 10% (21.7%) reduction in the epithelization period on the burn wound healing model. The chloroform fraction produced 5% (90.17%) and 10% (91.01%), hexane fraction produced 5% (85.81%) and 10% (86.78%), and aqueous fraction produced 5% (99.17%) and 10% (99.38%) wound contraction on the 14th day of the treatment and 5% (18.8) and 10% (28.2) percent reduction in the epithelization period on the excision wound healing model. Both hydromethanolic crude extract and solvent fractions at 5% and 10% (w/w) were significant ( p < 0.001) compared with negative control. Conclusion. The results of this study showed that both 5% w/w and 10% w/w of 80% hydromethanolic crude extract and solvent fractions of B. abyssinica leaves have wound healing effects.

Effects of Methanolic Extract Based-Gel From Saudi Pomegranate Peels With Enhanced Healing Potential on Excision Wounds in Diabetic Rats

Introduction: Current study was designed to evaluate the wound healing activity of a Saudi pomegranate peel extract on excision wound healing in experimentally induced diabetes in rats.Methodology: Animals were divided into three groups: diabetic excision wound with no treatment, diabetic excision wound with gel alone and diabetic excision wound with Saudi pomegranate peel extract in gel. Animals were monitored for clinical signs, weekly body weight, morbidity and mortality during entire study period. The efficacy parameters evaluated were percent wound contraction, Hydroxyproline content, estimation of Transforming Growth Factor ß1 (TGF-ß1), Vascular Endothelial Growth Factor (VEGF), and Epidermal Growth Factor (EGF) in wound lysates by ELISA, mRNA expression of TGF-ß1, VEGF, and EGF in wound lysates by qPCR, Estimation of nitric oxide (NO) and NO synthase (NOS) in Wound Lysates and histopathology of skin for reepithelization, neovascularization, and inflammation.Results: The Saudi pomegranate peel extract in gel (5.0 g extract per 100 g gel) showed significant wound healing activity when compared to the vehicle control [p &lt; 0.05] following 21 days of treatment. Animals in the control and treatment groups were apparently normal through the study with no significant differences in body weights between groups. Expression of mRNA of TGFβ1, EGF and VEGF in wounds was the highest on day 14 post treatment 4.3, 3.5 and 0.9 fold higher respectively in the treatment group when compared to vehicle control, and on day 21, the values were 0.12, 0.3 and 0.83, respectively. No statistically significant differences were observed in TGF-ß1 levels in wounds on days 4, 7, 14 and 21 post treatment when compared to the vehicle control (p &gt; 0.05). Significantly higher levels of VEGF were observed in treatment group on day 7 and 21 when compared to vehicle control (p &lt; 0.05). Significantly higher levels of EGF were observed in treatment group on day 7 and 21 when compared to vehicle control (p &lt; 0.05). Mean hydroxyproline levels were higher in treatment group on days 4 and 7 when compared to vehicle control. NO levels in treatment group were significantly lower on days 7, 14 and 21 when compared to vehicle control (p &lt; 0.05). NOS activity in treatment group were significantly lower on days 4 and 7 when compared to vehicle control (p &lt; 0.05). Histopathological changes in skin wound in the treatment group were consistent with wound healing when compared to the vehicle group.Conclusion: This study’s findings suggest that topical application of SPPE gel effectively enhanced wound healing in experimentally induced diabetic conditions.