Revista Cienﬁca, FCV-LUZ / Vol. XXXV Recibido: 18/11/2025 Aceptado: 07/02/2026 Publicado: 06/03/2026 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico 1 of 10 Revista Cienﬁca, FCV-LUZ / Vol. XXXVI UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Eﬃcacy of Punica granatum L. and Matricaria chamomilla L. herbal extracts on wound healing Eﬁcacia de los extractos herbales de Punica granatum L. y Matricaria chamomilla L. en la cicatrización de heridas Eren Polat 1,a, * , Asiye Ozlem Ates 2,b , Ahmet Selim Ozkan 2,c , Emre Kaya 3,d , Ozgur Ozoner 4,e , Merve Pekince-Ozoner 5,f , Nazım Tanrikulu 6,g , Hakan Serin 7,h ¹ Firat University, Faculty of Veterinary Medicine, Department of Surgery, Elazig, Türkiye ² Firat University, Faculty of Veterinary Medicine, Elazig, Türkiye ³ Firat University, Faculty of Veterinary Medicine, Department of Biochemistry, Elazig, Türkiye ⁴ Siirt University, Faculty of Veterinary Medicine, Department of Pathology, Siirt, Türkiye ⁵ Siirt University, Faculty of Veterinary Medicine, Department of Histology, Siirt, Türkiye ⁶ Balikesir Metropolitan Municipality Department of Rural Services BAÇEM, Balikesir, Türkiye ⁷ Selçuk University, Faculty of Veterinary Medicine, Department of Biostascs, Konya, Türkiye *Corresponding author: erenpolat@ﬁrat.edu.tr ABSTRACT Dermal injuries, which account for a signiﬁcant poron of healthcare expenditures globally, occur in various forms due to the inﬂuence of various eological factors, primarily trauma. For all these reasons, wound healing is among the most researched topics worldwide. This study aimed to invesgate the eﬀects of ointments obtained from Punica granatum L. (pomegranate) and Matricaria chamomilla L. (chamomile) extracts on secondary wound healing. 42 Sprague Dawley rats were used in the study. In the study, a total of six experimental groups were formed, each consisng of 7 rats: control group, healthy control group, Punica granatum treatment group, Matricaria chamomilla treatment group, Punica granatum and Matricaria chamomilla combinaon group (Punica granatum treatment + Matricaria chamomilla treatment), and the group receiving an applicaon containing olive oil, beeswax, and natural vitamin E. An excisional wound model was created using a biopsy punch on the back of the subjects in all groups except the healthy control group. While ointments prepared from herbal extracts were applied to the rats in the Punica granatum treatment, Punica granatum treatment + Matricaria chamomilla treatment, and Matricaria chamomilla treatment groups, rats in the control group received 0.9 % NaCl, and rats in the olive oil, beeswax, and natural vitamin E group were treated with a mixture of olive oil, beeswax, and vitamin E without any herbal extracts. At the end of the study, it was determined that the Matricaria chamomilla treatment group had the highest mean in terms of wound closure percentage and wound perimeter change percentage, while the Punica granatum treatment group had the lowest mean (P < 0.05). Although the best groups in terms of histopathological scoring were the Punica granatum treatment and Matricaria chamomilla treatment groups, no stascal diﬀerence was observed between the groups (P > 0.05). In terms of oxidave stress parameters (except for superoxide dismutase), the treatment groups were stascally signiﬁcantly closer to the healthy control group than to the control group. Considering all evaluaon criteria, it was concluded that Matricaria chamomilla plant was more eﬀecve on wound healing than Punica granatum plant and was a more suitable opon for clinical use. Key words: Wound healing; Matricaria chamomilla; Punica granatum; rat; excisional wound model. RESUMEN Las lesiones dérmicas representan una parte signiﬁcava del gasto sanitario a nivel mundial y se producen por diversos factores eológicos, principalmente el trauma. Por ello, la cicatrización de heridas es uno de los temas más invesgados a nivel mundial. Este estudio tuvo como objevo invesgar los efectos de ungüentos obtenidos a parr de extractos de Punica granatum L. (granada) y Matricaria chamomilla L. (manzanilla) sobre la cicatrización de heridas por segunda intención. En el estudio se ulizaron 42 ratas Sprague Dawley. Se formaron un total de seis grupos experimentales, cada uno compuesto por 7 ratas: grupo control, grupo control sano, grupo de tratamiento con Punica granatum, grupo de tratamiento con Matricaria chamomilla, grupo de combinación Punica granatum + Matricaria chamomilla (Punica granatum + Matricaria chamomilla) y el grupo que recibió una aplicación que contenía aceite de oliva, cera de abeja y vitamina E natural. Se creó un modelo de herida excisional en la espalda de todas las ratas, excepto en el grupo control sano. Los grupos Punica granatum, Matricaria chamomilla y Punica granatum + Matricaria chamomilla recibieron los ungüentos con extractos herbales; el control recibió solución salina al 0,9 %, y el grupo que recibió la mezcla de aceite de oliva, cera de abejas y vitamina E sin extractos. Al ﬁnal del estudio, el grupo Matricaria chamomilla presentó el porcentaje más alto de cierre de heridas y cambio de perímetro de la herida, mientras que el grupo Punica granatum tuvo los valores más bajos (P < 0,05). En cuanto a la puntuación histopatológica, los grupos Punica granatum y Matricaria chamomilla obtuvieron los mejores resultados, aunque sin diferencias signiﬁcavas entre los grupos (P > 0,05). Los parámetros de estrés oxidavo, excepto la superóxido dismutasa, mostraron que los grupos tratados se acercaban más al control sano que al control. En conclusión, la Matricaria chamomilla demostró ser más efecva que la Punica granatum en la cicatrización de heridas y se considera una opción más adecuada para uso clínico. Palabras clave: Cicatrización de heridas; Matricaria chamomilla; Punica granatum; rata; modelo de herida excisional. https://doi.org/10.52973/rcfcv-e362862

Revista Cienﬁca, FCV-LUZ / Vol. XXXVI UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico INTRODUCTION A wound is a condion in which the integrity of the skin and mucous membranes is impaired due to physical trauma or disease. Physical trauma such as crushing and sprains cause closed wounds where ssue integrity is not completely disrupted, while physical trauma such as cuts, punctures, and laceraons cause open wounds by completely disrupng ssue integrity [1],[2 ,3]. Wound healing is a dynamic and complex biological process that includes inﬂammatory response, ssue formaon and remodeling stages. Pracces for the treatment and care of wounds provide the appropriate environment for these processes to occur within an acceptable meframe [1 , 3 , 4]. The healing eﬀects of herbal extracts on many health problems, especially wounds, have been thoroughly researched in Veterinary and Human Medicine, and their eﬀecveness has been accepted. Among the herbal extracts studied for their eﬀects on wound healing are Punica granatum L. (pomegranate) and Matricaria chamomilla L. (chamomile) [5]. Punica granatum L. herbal extract contains polyphenols (tannins, punicalagin and ellagic acid), phenolic compounds (gallic acid) and triterpenoids (maslinic, ursolic acid and asiac acid). While the gallic acid and tannins it contains have an astringent eﬀect [6], polyphenols are known to smulate dermal ﬁbroblast proliferaon, increase type I procollagen synthesis, and inhibit matrix metalloproteinase-1. Matricaria chamomilla L. plant extract has been reported to have posive eﬀects on wound healing by inhibing histamine release from leukocytes through its ﬂavonoid content (quercen and apigenin); reducing oxidave stress and increasing microcirculaon through its coumarins; and supporng granulaon ssue formaon through its terpenoids, parcularly bisabolol. Although studies invesgang the eﬀects of Punica granatum L. (pomegranate) and Matricaria chamomilla L. on wound healing exist, it is noteworthy that the number of these studies is limited, and some studies, parcularly those reporng increased concentraons of Punica granatum, have slowed the rate of wound healing [5]. In this study, the eﬀecveness of Punica granatum L. (pomegranate) and Matricaria chamomilla L. (chamomile) plants on wound healing was determined and their usability for clinical applicaons was evaluated. The results of the study were evaluated in light of macroscopic ﬁndings such as the wound healing rate coeﬃcient (WHRC), wound closure percentage (WCP), and wound perimeter change percentage (WPCP), as well as laboratory ﬁndings including histopathological and biochemical parameters. A healthy control group (HCG) was included in the study to serve as a reference for the evaluaon of oxidave stress markers and histopathological scores. In addion, a group receiving olive oil + beeswax + vitamin E (OBEG) was incorporated to assess the potenal eﬀects of the excipients and carrier substances used in the preparaon of the ointments and to ensure transparency regarding the formulaon content. MATERIALS AND METHODS Study groups and wound model creaon The study sample consisted of 42 Spraque-Dawley rats (Raus norvegicus) of 10 weeks of age. The rats were randomLy assigned to six groups of seven animals each. The study groups were the control group (CG), the healthy control group (HCG), the Punica granatum group (PG) (Geographical source: Fethiye region of Mugla, Türkiye), the Matricaria chamomilla group (MC) (Geographical source: Bayramic region of Canakkale, Türkiye), the Punica granatum + Matricaria chamomilla group (PG + MC), and the olive oil (Geographical source: Edremit region of Balikesir, Türkiye), + beeswax (Geographical source: Balikesir, Türkiye Botanical origin: Olea europaea, Pinus sp., Quercus sp.) + natural vitamin E group (OBEG). Following a 10-day (d) adaptaon period, experimental wound models were created in rats in the CG, MC, PG, PG + MC, and OBEG groups. Before wound creaon, rats were administered intramuscularly at 10-minute (min) intervals, ﬁrst 8 mg/ kg xylazine hydrochloride, followed by 80 mg/kg ketamine hydrochloride. Aﬅer anesthesia was achieved, the dorsal regions of the rats were shaved and disinfected, and circular wounds were created using 6 mm diameter biopsy punches (FIG.1). FIGURE 1. Biopsy punch used to create the experimental wound model (a) and formaon of the model (b). Ointment preparaon and applicaons The plants to be used in the study were subjected to solvent extracon in methanol. The methanol was then removed using a rotavapor device (R-300 series, BÜCHI Labortechnik AG, Switzerland) to obtain herbal extracts. Herbal extracts were mixed with olive oil, beeswax, and vitamin E using the bain- marie method to obtain an ointment form. The composion of the ointments included 79 % olive oil, 10 % herbal extract herbal extract (Punica granatum and Matricaria chamomilla), 10 % beeswax, and 1 % vitamin E. Although it varies depending on the speciﬁc formulaons and the type of extract used, the inclusion of herbal extracts in oil-based semi-solid topical formulaons is a frequently reported approach in the literature [7]. The ointments were applied to the PG, MC, and PG + MC groups three mes a day for 10 d, and the wounds were leﬅ open without any dressing. The rats in the OBEG group were administered a mixture of olive oil, beeswax, and vitamin E, without any herbal extract. The wounds of the rats in the CG group were cleaned three mes d with 0.9 % NaCl soluon, whereas the rats in the HCG group received no intervenon. Macroscopic wound evaluaon The area and perimeter of the wounds were measured using Image J soﬅware from photographs taken on d 0, 3, 7, and 10. Using the wound area and perimeter, values such as the wound healing rate coeﬃcient (WHRC), wound closure percentage 2 of 10

Wound healing eﬀects of pomegranate and chamomile / Polat et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico (WCP), and wound perimeter change percentage (WPCP) were obtained and evaluated according to the formulas in TABLE I. The WCP and WHRC formulas used in this study represent simpliﬁed and more understandable adaptaons of the wound healing assessment formulas described by Cukja et al. [8]. The WPCP formula has not been found exactly as such in the literature by us and has not been derived from any source; it was developed for this study as a criterion for evaluang wound closure using simple mathemacal calculaons. TABLE I FORMULAS AND ABBREVIATIONS USED IN THE CALCULATION OF WOUND HEALING RATE COEFFICIENT, WOUND CLOSURE PERCENTAGE, AND WOUND PERIMETER CHANGE PERCENTAGE Formula Explanaon of the abbreviaons in the formula WHRC (A 0 -A t )/t A 0 : Area of wounds on the ﬁrst day of assessment A t : The area of the wound aﬅer a certain number of days t: Number of days passed WCP 100-((A t x 100)/A 0 ) A 0 : Area of wounds on the ﬁrst day of assessment A t : The area of the wound aﬅer a certain number of days WPCP ((P 0 -P t )/ P 0 ) x100 P 0 : The perimeter of the wounds on the inial assessment day P t : Perimeter of the wound on a speciﬁc day WHRC: Wound healing rate coeﬃcient, WCP: Wound closure percentage, WPCP: Wound perimeter change percentage) Histopathological evaluaon At the end of the study (d 10), the rats were euthanized by decapitaon under anesthesia. For histopathological examinaon, skin ssue samples were ﬁxed in 10 % neutral formalin and subjected to roune ssue processing. Subsequently, 5 μm secons were taken from the ssues embedded in paraﬃn blocks using a microtome (HM325, Thermo Scienﬁc, Waltham, MA, USA), stained with hematoxylin and eosin, and prepared for examinaon. The preparaons were examined under a light microscope (Leica Microsystems, Wetzlar, DM 750, Germany), and photographs were taken. Using a modiﬁed version of the method by Greenhalgh et al. [9], 10 randomLy selected areas along the wound line were examined under a 40x objecve, and scored on a scale from 1 to 15 [10]. Areas containing a small number of inﬂammatory cells and granulaon ssue were scored between 1 and 3; areas generally containing a high density of inﬂammatory cells, a small number of ﬁbroblasts, blood vessels, and collagen ﬁbers, and immature connecve ssue were scored between 4 and 6. Areas with a high density of ﬁbroblasts and collagen ﬁbers, thick connecve ssue growth, increased blood vessels, the beginning of epithelial formaon, and a small number of inﬂammatory cells were scored between 7 and 9. Areas consisng of ﬁbroblasts and dense collagen ﬁbers, vascular granulaon ssue, and an epithelial layer parally and/or completely covering it were scored between 10 and 12. Scores between 13 and 15 were given to areas with well- developed epithelial ssue covering a mature connecve ssue layer, and a reduced number of ﬁbroblasts and blood vessels. Determinaon of oxidave stress parameters At the end of the experiment, all blood samples were collected in tubes containing ethylenediaminetetraacec acid (EDTA). Plasma was collected by centrifuging by refrigerated centrifuge (NF NUVE NF800R, Türkiye) blood samples in EDTA tubes at 1630 G for 15 min. Malondialdehyde (MDA), a marker of lipid peroxidaon, was measured in plasma. For the measurement of Glutathione (GSH) and Glutathione Peroxidase (GSH-Px), whole blood was used. Blood samples with EDTA plasma separaon were washed three mes in saline (0.9 % NaCl). Then, erythrocyte Catalase (CAT) and Superoxide Dismutase (SOD) acvies were assessed. All measurements were performed in duplicate. Malondialdehyde levels, which are used to indicate damage to cell membranes and are the end product of lipid peroxidaon, were measured in plasma using the spectrophotometric method (Thermo Scienﬁc Genesys 10S UV-VIS, USA) established. This method is based on the spectrophotometric determinaon of the pink-colored complex formed by the reacon between MDA and thiobarbituric acid (TBA). 0.67 g of TBA was dissolved in 80 mL of 10 % perchloric acid and diluted to 100 mL with dislled water to prepare the TBA soluon. Aﬅer preparing a 10 % trichloroacec acid (TCA) soluon, 3 parts TCA soluon were mixed with 1 part TBA soluon. Before analyzing the samples, a blank tube was prepared to detect any decrease in absorbance that might be caused by substances that could absorb at 532 nm. 0.25 mL of physiological saline was added to the blank tube, and 0.25 mL of plasma was added to the sample tubes. 2.25 mL of color reagent (TBA and 10 % TCA) was added to the test tubes. The tubes were sealed, mixed, and incubated at 100 °C for 20 min. Aﬅer cooling under tap water, the tubes were centrifuged at 850 G for 10 min at + 4 °C, and the resulng supernatant was carefully collected using a pipee and its absorbance was read at 532 nm [11]. Glutathione levels, an important anoxidant, were determined by spectrophotometric detecon of the yellow color resulng from the reacon of 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB) with sulydryl groups in homogenates. A precipitang soluon was prepared by dissolving 1.67 g of glacial metaphosphoric acid, 0.20 g of disodium EDTA, and 30 g of NaCl in 100 mL of dislled water. 42.60 g of Na2HPO4 was weighed and diluted to 1 L with dislled water to prepare 0.3 M Na2HPO4. 20 mg of DTNB was diluted to 100 mL with 1 % Na- citrate soluon to prepare the DTNB soluon (Ellman Reagent). One (1 mL of dislled water was added to the blank tube, and 1 mL of blood samples was added to the sample tubes. 1.5 mL of precipitang soluon was added to all tubes and centrifuged at 850 G for 30 min at + 4 °C. 0.25 mL of the ﬁltrate from the top of the tubes was taken and transferred to clean tubes. 1 mL of 0.3 M Na2HPO4 and 0.125 mL of Elman’s reagent were added. The test tubes were then mixed and read against a blank at a wavelength of 412 nm [12]. 3 of 10

Revista Cienﬁca, FCV-LUZ / Vol. XXXVI UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Catalase acvity, which prevents cellular damage by breaking down hydrogen peroxide (H₂O₂) into water and oxygen, was determined by measuring the rate of this breakdown. A 30 mM H2O2 soluon was prepared by dilung 0.34 mL of 30 % H2O2 to 100 mL with phosphate buﬀer. 8.90 g of Na2HPO4 (2H2O) and 6.81 g of KH2PO4 were weighed and dissolved in 1 L of dislled water. The buﬀer was prepared by adding KH2PO4 to the Na2HPO4 (2H2O) soluon at pH 7.0. Then, 2 mL of sample and 1 mL of phosphate buﬀer were added to a blank tube, and the instrument was zeroed relave to the blank. Aﬅer adding 2 mL of sample and 1 mL of H2O2 soluon to the sample tubes, the diﬀerence in absorbance at 0 and 30 seconds was determined [13]. Glutathione peroxidase acvity was determined based on the enzyme’s ability to convert reduced GSH to oxidized glutathione (GSSG) while reducing peroxides. The resulng GSSG was then reduced back to GSH via glutathione reductase (GR), consuming NADPH. The decrease in NADPH concentraon was monitored spectrophotometrically to calculate GSH-Px acvity. Tris buﬀer was prepared by weighing 121.14 g of Tris, dissolving it in dislled water to adjust the pH to 8.0, then adding 1.68 g of EDTA (5 mM EDTA) and compleng the volume to 1 L. A 0.1 M GSH soluon was prepared by weighing 0.31 g of GSH and dissolving it in 10 mL of dislled water. A GR soluon was prepared by dissolving 10 units of GR in each 1 mL of soluon; 7.22 µl of GR was dissolved in 1 mL of dislled water. A 2 mM NADPH soluon was prepared by dissolving 0.07 g of NADPH in 30 mL of dislled water. 7 mM t-BOOH was prepared by dissolving 10 µL of t-BOOH in 10 mL of dislled water. 100 μL of 1 M Tris-HCl buﬀer, 20 μL of 0.1 M GSH, 100 μL of 10 Units of GR, 100 μL of 2 mM NADPH, and 670 μL of dislled water were added to the blank tube, mixed, and pre-incubated at 37 °C for 10 min. The spectrophotometer was zeroed against the prepared blank at a wavelength of 340 nm. 100 μL of 1 M Tris-HCl buﬀer, 20 μL of 0.1 M GSH, 100 μL of 10 Units of GR, 100 μL of 2 mM NADPH, 660 μL of dislled water, and 10 μL of sample were added to the sample tubes. The sample tubes were mixed and pre-incubated at 37 °C for 10 min. 10 μL of 7 mM t-BOOH was added to the tubes. The decrease in opcal density at 0 and 2.5 min at a wavelength of 340 nm was determined [14]. Superoxide dismutase acvity was determined based on the principle of converng superoxide radicals into the less harmful hydrogen peroxide. The removal of superoxide radicals in the presence of SOD inhibited the reducon of nitroblue tetrazolium (NBT). SOD acvity was calculated based on the degree of inhibion in NBT reducon. A reacon soluon was prepared by mixing 0.3 mM xanthine, 0.6 mM Na2EDTA, 0.15 mM NBT, 400 mM Na2CO3, and 1 g/L bovine serum albumin (BSA). Then, soluons of 167 U/L xanthine oxidase, 2 M (NH4)2SO4, and 0.8 mM CuCl2 were prepared. Erythrocyte hemolysate, diluted 1/5, was mixed with an equal volume of chloroform/ethanol (3/5-V/V) and centrifuged at 2980 G for 45 min at + 4 °C. The clear supernatant was separated for SOD acvity determinaon. Then, 0.1 mL supernatant, 2.85 mL reacon soluon, and 0.05 mL xanthine oxidase were added to the sample tubes. In the blank tube, the same amount of deionized water was added in place of the supernatant. All samples were incubated at 25 °C for 20 min. CuCl2 (1 mL) was added to all tubes to stop the reacon. The absorbances of the samples and the blank against the reacon soluon were measured at 560 nm using a spectrophotometer [15]. Stascal analysis The data from the study were analyzed using the SPSS 22.0 soﬅware package. The normality of the data distribuon was assessed based on histogram, coeﬃcient of variaon, skewness, and kurtosis values, Detrended Normal Q-Q Plot graphs, and Shapiro-Wilk test results. In data showing a normal distribuon, the diﬀerence between groups was tested using one-way analysis of variance (One-Way ANOVA). When a signiﬁcant diﬀerence was detected, the Tukey HSD test (if variances were homogeneous) and the Tamhane test (if variances were not homogeneous) were applied to determine between which groups the diﬀerence occurred. For data not following a normal distribuon, the diﬀerence between groups was analyzed using the Kruskal-Wallis test. When a signiﬁcant diﬀerence was found, the Mann-Whitney U test was used for pairwise comparisons. The assumpon of sphericity for macroscopic parameters related to wound healing was tested using Mauchly’s test. Changes on days 0, 3, 7, and 10 were evaluated with Repeated Measures ANOVA and the Bonferroni post hoc test if the assumpon of sphericity was met, and with a Greenhouse-Geisser correcon of the Repeated Measures ANOVA and the Bonferroni test if the assumpon of sphericity was not met. Numerical variables are presented as mean ± standard error, and the stascal signiﬁcance level was accepted as P < 0.05 [16]. RESULTS AND DISCUSSION The dermal injuries, which account for 4 % of global healthcare expenditures, heal through a biological process that involves stages such as hemostasis, inﬂammaon, proliferaon, and remodeling [1],[2 ,[17 , 18 , 19 , 20 , 21]. For faster and healthier wound healing, it is crucial that these stages occur in an appropriate environment [22]. Herbal raw materials can be used today in the treatment of medical condions, including wound healing [3 , 23]. Matricaria chamomilla L. (chamomile) or Matricaria recuta L. (German chamomile) has been used since ancient mes as a medicinal plant for gastrointesnal disorders and skin condions [24]. Scienﬁc evidence explaining or demonstrang the eﬀects of Matricaria chamomilla, a plant frequently used in Germany for the treatment of skin disorders, on wound healing is quite limited [24 , 25 , 26 , 27 , 28]. Punica granatum has been invesgated in various studies for its wound healing potenal, and diﬀerent results have been reported depending on the concentraons used [5]. Nevertheless, extracts obtained from the vegetave (roots, stems, leaves) and generave (ﬂowers, fruits) parts of the plant have been reported to exhibit anoxidant, an- inﬂammatory, and anbacterial acvies due to the presence of various phytochemical compounds [29 , 30], and consequently, they are thought to potenally have beneﬁcial eﬀects on wound healing. 4 of 10

Wound healing eﬀects of pomegranate and chamomile / Polat et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Macroscopic ﬁndings The stascal analysis results of within-group (me- dependent) and between-group (at the same me point) diﬀerences for the WHRC, WCP, and WPCP are presented in TABLE II. TABLE II STATISTICAL ANALYSIS OF WOUND HEALING RATE COEFFİCİENT, WOUND CLOSURE PERCENTAGE, AND WOUND PERIPHERY CHANGE PERCENTAGE VALUES ACCORDING TO GROUPS CG PG MC PG + MC OBEG P WHRC 0-3. days 6.58 ± 1.04 A 3.32 ± 0.79 5.46 ± 1.20 3.68 ± 0.54 4.33 ± 0.86 0.097 3-7. days 2.76 ± 0.41 B 4.21 ± 0.76 3.80 ± 0.62 4.20 ± 0.75 2.95 ± 1.42 0.346 7-10. days 4.21 ± 0.48 AB 6.06 ± 0.50 4.31 ± 0.52 5.28 ± 0.84 5.10 ± 0.42 0.137 P 0.012 0.075 0.487 0.409 0.177 WCP 3. day 42.18 ± 4.83 Aa 19.80 ± 3.88 Ab 35.59 ± 6.97 Aab 24.17 ± 2.67 Aab 30.55 ± 5.43 Aab 0.024 7. day 67.52 ± 3.86 B 55.01 ± 5.53 B 69.62 ± 2.97 B 61.49 ± 6.72 B 60.15 ± 8.11 B 0.192 10. day 96.48 ± 0.65 Cab 93.63 ± 1.96 Ca 98.30 ± 0.45 Cb 97.60 ± 0.54 Cab 97.63 ± 0.51 Cab 0.030 P < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 WPCP 0-3. days 20.71 ± 4.41 A 11.25 ± 2.11 A 22.16 ± 3.73 A 14.09 ± 2.79 A 18.64 ± 2.36 A 0.114 3-7. days 26.94 ± 4.43 A 26.15 ± 4.89 A 29.90 ± 2.40 A 28.14 ± 5.80 A 23.66 ± 4.01 A 0.897 7-10. days 66.26 ± 3.38 B 63.30 ± 3.50 B 76.80 ± 3.79 B 73.83 ± 3.17 B 73.64 ± 3.53 B 0.053 0-10. days 80.89 ± 1.88 Cab 75.32 ± 3.88 Ca 87.41 ± 2.00 Cb 83.75 ± 2.46 Cab 84.01 ± 2.06 Cab 0.035 P < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 WHRC: Wound Healing Rate Coeﬃcient, WCP: Wound Closure Percentage, WPCP: Wound Periphery Change Percentage. Uppercase leers show the changes over me within each group. Lowercase leers show the diﬀerences between groups at each me point When WHRC data within the same me period were evaluated, no stascally signiﬁcant diﬀerence was found between the groups. Although not stascally signiﬁcant, the WHRC value was found to be higher in the CG and MC groups between d 0-3, and in the PG and PG + MC groups between d 3-7 and 7-10. At the end of the 10th d, it was observed that the mean values of the MC group was stascally signiﬁcantly higher than the other groups in terms of WCP and WPCP (P < 0.05). When the mean WCP and WPCP values of the PG group were compared with those of the other groups, they were found to be stascally signiﬁcantly lower, making it the group with the least improvement in terms of macroscopic ﬁndings. It was determined that the WCP and WPCP averages of the rats in the OBEG group were stascally signiﬁcantly lower than those in the MC group and stascally signiﬁcantly higher than those in the PG group (FIG.2). When the intra-group me-dependent relaonship of WHRC data was evaluated, it was determined that there was no stascally signiﬁcant diﬀerence in any group except the CG group. It was determined that the WHRC averages of the rats in the CG group were stascally signiﬁcantly higher between d 0 and 3 compared to the other d. When the intra-group me-dependent relaonship of WCP data was evaluated, a stascally signiﬁcant diﬀerence was observed in all groups on d 3, 7 and 10. When the intra-group me-dependent relaonship of WPCP data was evaluated, it was determined that there was no stascal diﬀerence between d 0-3 and 3-7 in all groups, but both of these periods were stascally signiﬁcantly lower than d 7-10. 5 of 10

Revista Cienﬁca, FCV-LUZ / Vol. XXXVI UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Day Macroscopic ﬁndings 0 3 7 10 FIGURE 2. Macroscopic view of wounds in rats in all groups on d 0, 3rd, 7th and 10th of the study: CG group (a), PG group (b), MC group (c), PG + MC group (d), OBEG group (e) 6 of 10

Wound healing eﬀects of pomegranate and chamomile / Polat et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Nayak et al. [24], in their study using three diﬀerent wound models (excision, incision, and dead space) and administering Matricaria recuta aqueous extract in the drinking water of rats, reported that the wound closure rate was signiﬁcantly higher compared to the control group. Niknam et al. [5], in their study invesgang the eﬀects of ointments containing diﬀerent concentraons of Punica granatum L. (pomegranate) and Matricaria chamomilla L. (chamomile) extracts, as well as their combinaons, on wound healing, reported that the wound closure rates in all treatment groups were signiﬁcantly higher compared to the placebo group. In the same study, it was reported that increasing the concentraon of the acve component enhanced the wound closure rate of Matricaria chamomilla L., whereas the wound closure rate of Punica granatum L. decreased. Niknam et al. [5] reported that, at d 14, the highest wound closure rate was observed in the group treated with 10 % Matricaria chamomilla, followed by the group treated with a combinaon of pomegranate and chamomile. In this study, the observed decrease in wound closure rate with increasing concentraons of Punica granatum may be a result of dose-dependent biological eﬀects. At low concentraons, the tannins present in Punica granatum may accelerate wound healing through their anoxidant and anmicrobial acvies, whereas at higher concentraons, they may interact with cellular proteins, suppress ﬁbroblast proliferaon, and slow down epithelializaon. Similar to the ﬁndings of Niknam et al. [5], the decrease in wound closure rate observed with the ointment prepared from 10 % Punica granatum L. (pomegranate) extract in this study suggests that increasing the concentraon of this plant may negavely aﬀect wound healing. Jarrahi [31] reported that Matricaria chamomilla accelerates wound healing and shows a promising potenal for the treatment of burn injuries. In their study [31], the wound healing percentage in the group treated with Matricaria chamomilla exceeded 90 % on day 55, whereas in the present study, the WCP and WPCP averages in the MC group of rats exceeded 90 % by day 10. The diﬀerence between these two studies is thought to be aributable to protein denaturaon in burn wounds, which may prolong the healing process. Histopathological ﬁndings The histopathological evaluaon in this study determined that the HCG group showed a stascally signiﬁcant diﬀerence compared to all other groups. Analysis of the mean histopathological scores revealed that the CG group exhibited lower values compared to the PG and MC groups, and higher values relave to the PG + MC group; however, these diﬀerences were not stascally signiﬁcant (TABLE III, FIG. 3). TABLE III STATISTICAL ANALYSIS OF HISTOPATHOLOGICAL SCORES ACCORDING TO GROUPS HCG CG PG MC PG + MC OBEG P Histopathological scores 15.00 ± 0.00 a 10.38 ± 0.31 b 11.33 ± 0.52 b 10.99 ± 0.48 b 10.27 ± 0.70 b 10.72 ± 0.44 b ˂ 0.05 FIGURE 3. Normal histological appearance of the skin, 40x, HxE (a, HCG group); increased connecve ssue in the dermis and mild neovascularizaon, 100x, HxE (b, CG group); acanthosis in the epidermis, 200x, HxE (c, MC group); hair follicle formaon in areas close to the hypodermis, 100x, HxE (d, PG group); single-layer epithelium formaon in the wound area and increased connecve ssue in the dermis, 100x, HxE (e, PG + MC group); healing observed at the wound line, 100x, HxE (f, OBEG group). 7 of 10

Revista Cienﬁca, FCV-LUZ / Vol. XXXVI UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico In the study by do Nascimento et al. [21] invesgang the eﬀects of specially designed bio-interacve membranes containing Punica granatum L. extract on wound healing, it was reported that these membranes were signiﬁcantly more eﬀecve than standard gelan-based membranes in terms of wound shrinkage, granulaon ssue formaon, collagen deposion and organizaon. This suggests that the tannins and phenolic compounds in Punica granatum may increase ﬁbroblast proliferaon through their anoxidant eﬀect. Nayak et al. [24] reported that the wound epithelializaon rate was signiﬁcantly higher in rats given the extract compared to the control group, in their study in which they created three diﬀerent wound models (excision, incision and dead space) in rats and invesgated the eﬀect of adding Matricaria recuta aqueous extract to their drinking water on wound healing. Niknam et al. [5], in their study invesgang the eﬀects of ointments containing diﬀerent concentraons of Punica granatum L. and Matricaria chamomilla L. extracts and their combinaons on wound healing, reported that the epithelial volume, myoﬁbroblast/ﬁbroblast number and collagen ﬁber content in all treatment groups of rats were signiﬁcantly higher than in the placebo group. Although no signiﬁcant diﬀerence in epithelial volume was observed between the treatment groups, higher epithelial volumes were recorded in the groups treated with 5 % pomegranate, 5 % chamomile and 2.5 % pomegranate. In addion, the highest collagen ﬁber content was reported in the group treated with 10 % Matricaria chamomilla. In the study conducted by Lukiswanto et al. [32], the eﬀecveness of Punica granatum on the healing of burn wounds was examined histopathologically; in the group treated with 10 % Punica granatum extract, regular and dense collagen deposion, complete and mature epithelializaon, low inﬂammatory cell inﬁltraon, and appropriate levels of angiogenesis were observed, while in the other treatment groups, these parameters were weaker or immature. In this study histopathological evaluaons revealed no stascally signiﬁcant diﬀerences between the groups. However, wound ssue in rats in the PG and MC groups showed more mature epithelializaon and regular collagen deposion compared to other groups, with scores closer to the HCG group. Although not stascally signiﬁcant, these ﬁndings indicate that both herbal extracts histopathologically support wound healing compared to other groups. Biochemical ﬁndings When oxidave stress parameters were evaluated, it was found that in rats in the CG group, MDA levels were increased, while GSH levels and CAT and GSH-Px acvies were decreased compared to the HCG group (P < 0.05 and P < 0.001). In rats in the PG, MC, and PG+MC groups, MDA levels were decreased, while GSH levels and CAT and GSH-Px acvies were increased. Furthermore, while MDA levels and CAT and GSH-Px acvies in the PG, MC, and PG + MC groups were similar to the averages of the HCG group, GSH levels were similar to HCG levels in all groups except the PG group. No signiﬁcant diﬀerence was observed between the groups in terms of SOD acvity (P > 0.05) (TABLE IV). TABLE VI STATISTICAL ANALYSIS OF OXIDATIVE STRESS PARAMETERS ACCORDING TO GROUPS MDA (nmol/mL) GSH (µmol/mL) CAT (k/g Hb) GSH-Px (U/mg Hb) SOD (U/g Hb) HCG 7.56 ± 0.19 ab 39.20 ± 0.32 a 65.59 ± 1.68 a 141.70 ± 2.73 a 54.03 ± 0.90 CG 10.06 ± 0.22 c 31.18 ± 0.72 c 45.28 ± 1.82 b 111.71 ± 3.36 b 50.84 ± 1.85 PG 8.01 ± 0.28 b 36.90 ± 0.69 b 66.05 ± 1.49 a 141.99 ± 1.67 a 52.82 ± 1.47 MC 7.64 ± 0.21 ab 38.64 ± 0.42 ab 66.11 ± 1.08 a 138.82 ± 1.25 a 52.98 ± 1.76 PG+MC 7.06 ± 0.16 a 40.49 ± 0.53 a 71.27 ± 1.37 a 149.06 ± 2.85 a 53.30 ± 0.53 OBEG 7.68 ± 0.11 ab 39.64 ± 0.22 a 65.73 ± 1.19 a 141.87 ± 2.60 a 52.22 ± 0.50 P < 0.001 < 0.05 < 0.001 < 0.001 - Literature searches revealed no studies that directly evaluated the eﬀects of Punica granatum L. and Matricaria chamomilla plants on wound healing through serum oxidave stress parameters. However, in a study by Niknam et al. [5], the anoxidant acvies of methanol fracons of chamomile and pomegranate ﬂowers were invesgated, and it was reported that both plants have strong anoxidant capacity, but pomegranate showed a higher acvity compared to chamomile. In the present study, when serum oxidave stress parameters of rats were measured, a high anoxidant eﬀect was observed in both pomegranate and chamomile groups. Interesngly, in the groups where pomegranate, which has the highest anoxidant capacity in the study by Niknam et al. [5], was applied, MDA levels were found to be highest, except for the control group. This may be due to the local applicaon of the ointment prepared from Punica granatum and the relavely delayed wound healing in these animals compared to other groups. CONCLUSION In this study observed that the 10 % chamomile ointment accelerated wound healing, whereas pomegranate slowed it down. However, when evaluated in conjuncon with the exisng literature, it is believed that an ointment prepared and applied at lower concentraons of pomegranate enhances wound healing, with its anoxidant capacity playing a parcularly signiﬁcant role in this process. Considering all the data, this study concluded that Matricaria chamomilla was more eﬀecve in wound healing than the other groups and more suitable for clinical use. 8 of 10

Wound healing eﬀects of pomegranate and chamomile / Polat et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Ethical statement This study was approved by the Local Ethics Commiee on Animal Experiments at Firat University (dated 05.04.2024, Session Number 23589). Conﬂicts of Interest The authors of this arcle declare that there are no conﬂicts of interest. Author Contribuons Conceptualizaon of the study: EP and AOA; Preparaon of herbal extracts and formulaon of ointments: NT; Project implementaon: EP, AOA, and ASO; Histopathological analyses: OO and MPO; Biochemical analyses: EK; Stascal analysis: EP, HS; Manuscript wring: EP and AOA. Financial Support This project was ﬁnancially supported by the Scienﬁc and Technological Research Council of Türkiye (TÜBİTAK) within the scope of the 2209-A Project Support Program. BIBLIOGRAPHIC REFERENCES [1] Bakr G. Wound repair and experimental wound models. 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Revista Cienﬁca, FCV-LUZ / Vol. XXXVI UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico [23] Kazemian H, Ghafourian S, Sadeghifard N, Houshmandfar R, Badakhsh B, Taji A, Shavalipour A, Mohebi R, Ebrahim- Saraie HS, Houri H, Heidari H. In vivo Anbacterial and Wound Healing Acvies of Roman Chamomile (Chamaemelum Nobile). Infect. Disord. Drug Targets. [Internet]. 2018; 18(1):41-45. doi: hps://doi.org/ gc77d4 [24] Nayak SB, Raju S, Rao VC. Wound Healing Acvity of Matricaria recuta L. Extract. J. Wound Care. [Internet]. 2007; 16(7):298-302. doi: hps://doi.org/qsq2 [25] Drummond EM, Harbourne N, Marete E, Jacquier J, O’Riordan D, Gibney ER. An In Vivo Study Examining the An-İnﬂammatory Eﬀects of Chamomile, Meadowsweet, and Willow Bark in a Novel Funconal Beverage. J. Diet. Suppl. [Internet]. 2013; 10(4):370-380. doi: hps://doi. org/qsq4 [26] Duman F, Ocsoy I, Kup FO. Chamomile Flower Extract- Directed CuO Nanoparcle Formaon for Its Anoxidant and DNA Cleavage Properes. Mater. Sci. Eng. C. [Internet]. 2016; 60:333-338. doi: hps://doi.org/f78hgs [27] Kolodziejczyk-Czepas J, Bijak M, Saluk J, Ponczek MB, Zbikowska HM, Nowak P, Tsirigos-Maniecka M, Pawlaczyk I. Radical Scavenging and Anoxidant Eﬀects of Matricaria chamomilla Polyphenolic–Polysaccharide Conjugates. Int. J. Biol. Macromol. [Internet]. 2015; 72:1152-1158. doi: hps://doi.org/f7ﬅc7 [28] Srivastava JK, Pandey M, Gupta S. Chamomile, a Novel and Selecve COX-2 İnhibitor With An-Inﬂammatory Acvity. Life Sci. [Internet]. 2009; 85(19-20):663-669. doi: hps://doi.org/kds3 [29] Rummun N, Somanah J, Ramsaha S, Bahorun T, Neergheen-Bhujun VS. Bioacvity of Nonedible Parts of Punica granatum L.: A Potenal Source of Funconal Ingredients. Int. J. Food Sci. [Internet]. 2013; 2013:602312. doi: hps://doi.org/gb64kz [30] Rahimi HR, Arastoo M, Ostad SN. A comprehensive review of Punica granatum (pomegranate) properes in toxicological, pharmacological, cellular, and molecular biology researches. Iran J. Pharm. Res. [Internet]. 2012 [cited 13 Jul 2025]; 11(2):385-400. Available in: hps:// goo.su/wafAr [31] Jarrahi M. An Experimental Study of the Eﬀects of Matricaria chamomilla Extract on Cutaneous Burn Wound Healing in Albino Rats. Nat. Prod. Res. [Internet]. 2008; 22(5):422-427. doi: hps://doi.org/d8nrph [32] Lukiswanto BS, Miran A, Sudjarwo SA, Primarizky H, Yuniar WM. Evaluaon of Wound Healing Potenal of Pomegranate (Punica granatum) Whole Fruit Extract on Skin Burn Wound in Rats (Raus norvegicus). J. Adv. Vet. Anim. Res. [Internet]. 2019; 6(2):202-207. doi: hps:// doi.org/gk7sv4 10 of 10