Flavonoids and wound healing -
reported that many species of Dodonaea genus were found to possess flavonoids and terpenoids Beshah et al. According to the findings of Sebelemetja et al. viscosa extract, created polymeric nanoparticle which was evaluated against the oral cavity acid-producing Streptococcus mutans Sebelemetja et al.
Joshi et al. proved the preliminary wound healing studies with ethanol extract of D. viscosa leaves Joshi et al. Nayeem et al.
evaluated the wound healing potential of D. viscosa formulation prepared with methanol and chloroform extract of leaves in experimental animals Nayeem et al.
Literature studies indicate that D. viscosa leaves possess significant wound healing potential. However, the wound healing studies are lacking in the fractions of leaves and the correlation to its phytoconstituents. Our preliminary studies proved the wound healing potential of flavonoid rich fraction of D.
viscosa by in-vitro cell proliferation assay on HACAT cell line Shanthi et al. Although the plant is traditionally claimed to have fast wound-healing effects besides its rich flavonoid content, there has been no scientific data to confirm this claim on flavonoid rich fraction of D.
Hence, the present study aimed to investigate the mechanism of action and the wound healing properties of the flavonoid-rich ethyl acetate fraction of D. viscosa leaves in the forms of new topical formulations by using animal models. FIGURE 2. Wound healing properties of D. viscosa leaves. Stress and obesity are two of the many factors that might contribute to delayed wound healing.
Fresh, healthy leaves of D. vicosa were harvested from the Madurai district, Tamil Nadu, India. The plant material was shade-dried and then ground into a coarse powder.
The mixed extracts were concentrated using a rotavapor to yield the final solvent-free crude extract. To get a flavonoid-rich fraction, the extract was suspended in water and separated with ethyl acetate Seyfi et al. Ethyl acetate fraction 10 mg was refluxed for min with 78 ml of alcohol, water, and hydrochloric acid cocktail The extract was filtered and diluted with ethanol to ml.
An aliquot 5 ml was filtered through a C silica column followed by methanol elution 4 ml. The elution volume was limited to 10 ml before being analyzed using a HPLC Oliveira et al. HPLC analysis was conducted at room temperature in a Shimadzu apparatus outfitted with an SPD-M10A diode array detector and a reverse-phase column Linchosorb RP, 25 cm × 5 mm.
Elution was conducted using a solution of methanol, water, and phosphoric acid For analysis, 10 μL of sample and standards were injected each time.
The peak regions of the HPLC chromatogram from the triple selection were used to compute the flavonoids concentration. Prior to the animal study, the ethyl acetate fraction of D. viscosa was formulated as a D. viscosa ointment DVFO 2. An acute dermal toxicity study was performed according to the Organization for Economic co-operation and Development OECD test guideline OECD, Mortality and aberrant clinical indicators were monitored daily for 14 days until the study was completed.
The body weight was measured before medication administration and then weekly until the investigation was completed. At the end of the experiment, any significant pathological variations were identified.
Groups III and IV were treated with DVFO 2. Before inflicting the experimental wounds, the animals were anesthetized by intraperitoneal i.
The dorsal fur was shaved and a circular stainless-steel stencil with methylene blue was used to outline the projected wound location to be made on the back and the sides of the animals.
Using surgical scissors and forceps, a circular excision mm 2 was formed on the shaved dorsal skin up to a certain depth 1. Day 0 was calculated when a circular excision wound was deemed created. Treatment was administered once daily until full epithelialization was achieved.
One-third of the experimental animals were sacrificed on the seventh post-operative day. Wound granulation tissues excluding any underlying muscle and superfluous tissue were collected.
Following injury on day 15, half of the surviving animals were euthanized, and the retrieved tissue was utilized for histopathological analysis. The remaining animals were monitored until a complete epithelialization occurred Upadhyay et al.
For every third-day post-wounding, the perimeter of the excision wound was traced on a transparent paper until the wound closed. The wound area was measured by retracing the injury on millimeter scale graph paper and the percentage of wound contraction was estimated using the following formula:.
The wound was monitored for the presence of complete epithelialization. Basically, it was measured from wounding day baseline until the eschar separated itself from the raw wound Nayak et al. The rats were anesthetized before and throughout the wound infliction. Groups III and IV were given DVFO at 2.
Using a sterile scalpel, a 6 cm long and 2. Following the incision, the skin was held together and sewn at 1 cm intervals of interrupted sutures using a surgical thread and a curved needle no. The wounds were left naked, and the formulations were topically administered to the wound daily.
On day 8, the sutures were removed, and the formulation continued to be applied. The extracted skin tissues were dried until they reach a constant weight in a hot air oven 60°C—70°C. Subsequently, the dry tissues were hydrolyzed by 6 N HCl at °C for 4 h in sealed glass vials.
After neutralizing the acidic tissue hydrolysate to pH 7. The reaction was stopped by adding 0. A UV visible spectrophotometer was used to detect the absorbance at nm.
The standard calibration curve for pure 4-hydroxy-L-proline was plotted and utilized to estimate the sample concentrations in the test samples Murthy et al. The hydrolyzed fraction 0. Subsequently, 0.
The step was followed by heating in a boiling water bath for 20 min before cooling. Then, 1. The reaction was left for 30 min before measurement of color intensity at nm against a blank. On the 7th and 15th days of the study, cross-sectional full-thickness skin specimens from each group were obtained to examine for histopathological changes.
The regenerated tissues were examined qualitatively for keratinization, epithelization, inflammation, fibroblastic proliferation, collagen depositions and neovascularization under a light microscope.
The protein expression of collagen type 3 COL3A1 , basic fibroblast growth factor bFGF and vascular endothelial growth factor VEGF in granulation tissue from seven-day-old wounds was determined by western blotting Feng et al.
Protein concentration was estimated by using a Bradford reagent. The proteins were trans-blotted poly vinylidene fluoride PVDF membrane and incubated with COL3A1, bFGF, VEGF and β-actin primary antibodies overnight at 4°C, at room temperature with the corresponding secondary antibodies 1: for 1—2 h.
The desired proteins were detected by a Western Max-HRP-Chromogenic detection kit and 5-Bromochloro-3 ' -indolyl phosphate p-toluidine salt-Nitro Blue Tetrazolium BCIP-NBT solution using β-actin as the internal control. Statistical evaluations were performed with an IBM SPSS version 23 and with Microsoft excel Results of parametric tests were expressed in terms of mean ± SD.
Kruskal Wallis test, followed by the Mann-Whitney U -test, was used in the multivariate analyses. The ethanol extract of D. viscosa was fractionated with ethyl acetate solvent to yield an ethyl acetate fraction The fraction was greenish brown semi-solid mass which was soluble in water.
The flavonoids in the ethyl acetate fraction were estimated by HPLC using quercetin and kaempferol as standards. The flavonoids were identified by comparing the retention time in HPLC chromatograms of the fraction with the standards run in Similar condition.
The retention time was The amounts of flavonoids were calculated from the peak area of the HPLC chromatogram of ethyl acetate fraction Figure 3.
Quercetin and kaempferol were determined to be 6. FIGURE 3. HPLC analysis of ethyl acetate fraction of A Quercetin, B Kaempferol and C Ethyl acetate fraction of D. No evidence of toxicity with respect to clinical signs, remarkable bodyweight observations, or mortality, were recorded in all the experimental animals.
The percentage wound contraction findings based on the excision wound model experiment following topical administration of ethyl acetate fraction ointment at different concentrations 2.
The control group exhibited a contraction of Nevertheless, the 2. A full-thickness skin defect was induced by skin excision and the various groups were treated with base, povidone-iodine ointment and DVFO ointment at different concentrations until complete epithelialization.
Wound healing, as indicated by the formation of granular tissue, occurred on day 18 in the 2. Hydroxyproline levels in granuloma tissue treated with D. viscosa ethyl acetate fraction is represented in Figure 4. In the present study, the topical application of DVFO 2.
FIGURE 4. Effect of DVFO on hydroxyproline content. Values are expressed as Mean ± SD. Statistical analysis was done by Kruskal—Wallis followed by Mann-Whitney U test.
Povidone iodine-treated groups. Hexosamine is one of the components of glycosaminoglycans. FIGURE 5. Effect of DVFO on hexosamine content. Tensile strength is a crucial characteristic in an incision wound model Figure 6. The minimum tensile strength of the wound in the control group was In comparison the mean strength of the animals from the DVFO group increased considerably in a dose-dependent manner.
The mean tensile strength of a wound treated with 2. FIGURE 6. Effect of DVFO on Tensile strength. On day 7, many new capillaries and fibroblasts were seen in the granulation tissue in the DVFO- and standard treated groups, which were more prevalent than in the control group Figure 7 indicating that DVFO stimulates neovascularization while also increasing fibroblast proliferation and migration.
FIGURE 7. Control group [ A on 7 th Day and B on 15 th Day], Standard treatment group [ C on 7 th Day and D on 15 th Day], 2. On day 15, wound healing processes were well-organized following treatment with either DVFO or povidone-iodine. Additionally, all treated tissues lacked fibrinoid necrosis.
The histology of an excision biopsy of a skin wound on day 15 revealed healed skin structures with normal epithelialization, adnexal restoration, and extensive fibrosis within the dermis in the DVFO and standard treated groups.
In contrast, the control groups lag behind the treated groups in forming ground substances in the granulation tissue, as observed in the tissue sections. The DVFO- and standard treatment groups had full epithelialization, while the control group had partial epithelialization Figure 7.
In fact, in the control group, collagen bundles were loosely packed, and wounds were only mildly cellular with fibroblasts Figure 8. FIGURE 8. A Normal skin, B Wound control group, C Standard treatment group, D 2. Western blotting was used to examine COL3A, VEGF and bFGF protein expression levels Figure 9.
The protein expression of COL3A, VEGF and bFGF in the standard group was considerably more significant than in the control group. Overall, our findings suggest that DVFO promotes wound healing by increasing the protein expression levels of COL3A, VEGF and bFGF. FIGURE 9. Western blot analysis of important growth factors involved in the regulation of wound healing and regeneration.
The skin tissue of animals treated with DVFO showed increased expression of FGF, VEGF and COL3A as compared to control. Values are expressed as Mean ± SEM. Our work provides the scientific data to validate the traditional claims that D.
viscosa leaves have wound-healing effects, possibly by increasing the protein expression levels of COL3A, VEGF and bFGF. Wound healing is considered a complicated and dynamic process that typically encompasses several phases signifying healing stages and necessitates the involvement of many cell types in various cellular activities Lopez-Jornet et al.
Hemostasis is the first stage of wound healing, followed by inflammation, proliferative and maturation phases.
Many growth factors, cytokines, and chemokines are involved in the signalling network that controls and executes this intricate process.
The TGF-beta, FGF, VEGF, Granulocyte Macrophage Colony Stimulating Factor GM-CSF , PDGF, Connective Tissue Growth Factor CTGF , Interleukin IL family, and TNF-α families are of particular significance Barrientos et al. Failure to progress through normal stages of phases of wound healing can ultimately result in delayed wound healing.
Several factors contributing to the delayed healing are diabetes, metabolic disorders, infection, venous disease, deficiencies, and age. Most existing medications used to treat wounds are costly, and some of them cause allergic responses and drug resistance Prasad and Dorle, Plants or their phytocompounds may impact some of these wound healing stages by speeding up the healing process.
Herbal remedies have generated much interest for the treatment of wounds as they are affordable and safe. Maver et al. Thus, the potential for obtaining affordable medicines from traditional plant-based medicine has been investigated Phan et al. Flavonoid rich fraction of many medicinal plants such as Tephrosia purpurea L.
In line with this, earlier reports indicated that the plant D. viscosa possesses rich polyphenolic components, mainly flavonoids which are well known for their significant wound healing properties. The contribution of various flavonoids in wound healing is enormous.
In this context, the plant D. viscosa has been selected for the current investigation. Moreover, several experimental studies have explored the wound healing potential of the whole extract, such as a group of researchers have disclosed the preliminary wound healing studies with ethanol extract of D.
Similarly, another study has unveiled the wound healing potential of D. All the previous studies have used the whole crude extract of D. viscosa , and no one has explored the wound healing potential of the fractions of leaves together with mechanism of action. In the previous in-vitro wound healing studies, the authors compared the potential of ethanol extract and flavonoid rich fraction Ethyl acetate fraction of D.
viscosa by in-vitro cell proliferation assay on HaCaT cell line Shanthi et al. Hence, the potential of the flavonoid rich fraction of D. viscosa as topical formulation was evaluated for wound healing efficacy. Our investigation on the acute dermal toxicity investigation indicated that DVFO was safe.
Our study, which employed two distinct models to test the wound healing impact of D. viscosa ethyl acetate fraction on various stages of wound healing, indicated that similar to conventional treatment, DVFO decreased wound size considerably as compared to the control.
Accelerated wound contraction is associated with fibroblast activation, which is mediated by specialized myofibroblasts of granulated tissues Moulin et al. Contraction minimizes the amount of extracellular matrix required to repair the damage and thereby shortens the healing process.
In addition, by reducing the distance that migratory keratinocytes must cover, contraction promotes re-epithelization Strodtbeck, Furthermore, if the medicine is more effective, the wound will heal quickly Prasad and Dorle, Therefore, the significant effect of DVFO on the wound healing process in rats may be attributed to an increase in fibroblast activities, required for proper wound closure.
The positive effect of DVFO treatment was further ascertained with a histopathological examination, which confirmed normal skin histology with the reappearance of intact epidermis and dermis, fibroblast and collagen deposition compared to the control group that had disrupted tissues in the dermal wound area.
The DVFO-group exhibited faster epithelialization, while the control group exhibited a partial epithelialization. Granulation tissue development over the wound section hastens re-epithelialization due to the arrangement of epithelial cells surrounding the newly formed tissue to establish a barrier between the wound area and the external environment Esimone et al.
The wound re-epithelialization cycle is mainly associated with a faster wound healing process that can be indicated by hydroxyproline level as well.
Hydroxyproline concentration is a critical component of the collagen fiber triple helix Shoulders and Raines.
An increase in hydroxyproline concentration suggests a decrease in cellular proliferation and collagen formation. In addition, hexosamine levels increase in the early phases of wound healing, indicating that fibroblasts are actively synthesizing ground material mucopolysaccharides on which collagen may be deposited Ross and Benditt, In the excision wound model, topical administration of DVFO 2.
Tensile strength is an essential indicator of the quality and amount of epithelialized collagen as well as the strength and degree of wound healing Raghuwanshi et al. In terms of wound healing tissue tensile strength, DVFO has an excellent wound healing property.
The improved tensile strength of the treated incision wounds may be related to increased collagen content and fiber stability.
Collagen, a component of developing cells, is synthesized by tissue going for repair. Collagen molecules are generated and laid down at the wound site, where they crosslink to create fibers. Wound strength is gained by both collagen remodeling and the creation of stable intra- and intermolecular crosslinks in the fibers.
Consequently, wound healing effects of DVFO 2. On day 15 after the injury, histopathological sections of both DVFO and positive control revealed newly formed collagen aligned with just a few cells.
Additionally, wound healing and cell differentiation were also seen. The control wound tissue had low inflammatory cells and high fibroblasts producing the epidermal layer, with the new collagen seemingly disordered.
The histopathology of wound granulation tissue demonstrated a faster wound closure rate and increased hydroxyproline content in the DVFO-treated group, which corroborated with prior findings on wound healing processes in dogs El-Tookhy et al. The histopathological findings backed with the biochemical results showed a considerable increase in collagen and fibroblastic deposition in DVFO and standard drug treated rats compared to control animals.
The wound healing proliferation phase includes the formation and vascularization of granulation tissue, collagen production and subsequent maturation. Collagen type III is present in remodeling wound tissues and is generated by proliferating juvenile fibroblasts in response to growth factors such as basic fibroblast growth factor bFGF and transforming growth factor-β TGF-β Ganeshkumar et al.
The angiogenic molecule bFGF increases neovascularization and blood flow to granulation tissue. Increased oxygen supply promotes collagen fiber development in granulation tissue Kondo and Ishida, Further studies indicated that VEGF increases endothelial cell hydrogen sulfide generation and release, resulting in endothelial cell proliferation, migration and permeability, micro vessel formation and wound healing.
Furthermore, VEGF stimulates wound epithelialization and collagen deposition Bao et al. The growth factors bFGF and VEGF have been known to play an important role in angiogenesis.
Angiogenesis, or the formation of new blood vessels, is involved in various physiological and pathological processes, including ovulation, embryogenesis, wound healing, inflammation, malignant tumor development, retinopathies, rheumatoid arthritis, and angiogenesis-dependent disorders Bitto et al.
In the DVFO treated group, both FGF and VEGF were up-regulated. Increased expression of growth factors was identified in the DVFO-treated group in the current investigation, showing that ethyl acetate fraction increases angiogenesis and collagen formation and mediates the wound healing process.
Wounds are considered to be a serious problem that affects the healthcare sector in many countries, primarily due to diabetes and obesity. Wounds become worse because of unhealthy lifestyles and habits. Wound healing is a complicated physiological process that is essential for restoring the epithelial barrier after an injury.
Numerous studies have reported that flavonoids possess wound-healing properties due to their well-acclaimed anti-inflammatory, angiogenesis, re-epithelialization, and antioxidant effects.
Hence, we have compiled existing evidence on the manipulation of flavonoids towards achieving skin wound healing, together with current limitations and future perspectives in support of these polyphenolic compounds as safe wound-healing agents, in this review.
Song HS, Park TW, Sohn UD, Shin YK, Choi BC, Kim CJ et al The effect of caffeic acid on wound healing in skin-incised mice. Korean J Physiol Pharmacol — Younan GJ, Heit YI, Dastouri P, Kekhia H, Xing W, Gurish MF, Orgill DP Mast cells are required in the proliferation and remodeling phases of microdeformational wound therapy.
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Unit of Geriatry, Department of Medicine, University Laboratory of Medical Research, University of Verona, Policlinico GB Rossi piazzale AL Scuro 10, , Verona, Italy.
You can also search for this author in PubMed Google Scholar. Correspondence to Salvatore Chirumbolo. Reprints and permissions. Chirumbolo, S.
Flavonoids in propolis acting on mast cell-mediated wound healing. Inflammopharmacol 20 , 99— Download citation. Received : 05 January Accepted : 06 February Published : 17 February Issue Date : April Anyone you share the following link with will be able to read this content:.
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Access this article Log in via an institution. References Alexander A, Walker CL The role of LKB1 and AMPK in cellular responses to stress and damage. x Article PubMed Google Scholar Frankland Sawaya ACH, Barbosa da Silva Cunha I, Marcucci MC Analytical methods applied to diverse types of Brazilian propolis.
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x Article PubMed CAS Google Scholar Song HS, Park TW, Sohn UD, Shin YK, Choi BC, Kim CJ et al The effect of caffeic acid on wound healing in skin-incised mice. Conflict of interest The author declares that he has no conflict of interest.
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