The Effect of Ethanol Extract of Rose (Rosa damascena) on Intra-abdominal Adhesions After Laparotomy in Rats

Background. Abdominal adhesions are pathological connections in peritoneal surfaces that are created after abdominal surgery. The aim of this study was to evaluate the inhibitory effect of Rosa damascena extract on adhesions, considering the antioxidant properties of rose. Methods. Thirty healthy rats were divided into 3 groups: rats treated by 1% (A) and 5% (B) of R. damascena extract and the control group (C). After administering anesthesia, the abdominal wall was opened and 3 shallow incisions (2 cm) were made on the right wall, and a 2 × 2 piece of peritoneal surface was removed on the left side of the abdominal wall. Then 3 mL of 1% (A) and 5% (B) R. damascena extract was administered into the abdominal cavity. The control group (C) received 3 mL of distilled water. The abdominal cavity was sutured, and a second laparotomy was carried out 14 days later to the created adhesions according to the Canbaz scale, and a histopathologic examination was also performed. All data was analyzed by SPSS volume 16 (Chicago, IL); P < 0.05 was considered statistically significant. Results. The amount of adhesion in group A was significantly lower than that of group C, 1.4 ± 1.265 versus 3 ± 0.816, (P = 0.007). The histological investigation also showed significant differences in the severity of fibrosis (P = 0.029) and inflammation (P = 0.009) between groups A and C; all rats in group B (5%) were found dead. Conclusion. This study indicated the use of R. damascena at a 1% level resulted in a remarkable decrease of intra-abdominal adhesions after laparotomy in rats. Further studies are necessary on this extract and its derivatives for treatment of such diseases in the human model.

Intra-abdominal adhesions are pathological connections or fibrous bands that form between tissues and organs, often as a result of injury during surgery. Adhesions form as a natural part of the body’s healing process, similar to scar formation. The term “adhesion” is used when the scar develops from within 1 tissue across to other tissues, particularly the virtual space such as the peritoneal cavity. Adhesion formation postsurgery typically occurs when 2 injured surfaces are close to one another, causing inflammation and fibrin deposits on the damaged tissues. The fibrin then connects the 2 adjacent structures, so fibrin acts like a glue to seal the injury and builds the fledgling adhesion. Adhesion may develop between solid organs, the intestines, Fallopian tubes, omentum, or the abdominal wall. Different issues can result in intra-abdominal adhesion including peritoneal ischemia, foreign bodies, and infections. These factors cause damage on the surface of organs or the visceral peritoneum resulting in local inflammatory reaction and release of fibrin and its deposition.^1-3 Adhesions can lead to significant postsurgical morbidity, bowel obstruction, infertility, and chronic pelvic pain or chronic abdominal pain and difficulties with subsequent surgeries.⁴ More than 25% of infertility is related to pelvic or abdominal adhesions.⁵ Intra-abdominal adhesions arise after 50% to 97% of abdominal operations⁵ and 60% to 90% of gynecological operations⁶ and are a significant source of postoperative complications. 

In the last century, gynecological surgeries are the main source of intraperitoneal adhesions and related complications; therefore this problem is considered one of the most interesting issues for general and other surgical specialties.^6,7 To prevent the creation of adhesion bands following surgery, many different materials have been studied, such as glucocorticoids, heparin, dextran 70, saline solution, antibiotics, promethazine, antihistamines, inhibitors of prostaglandin synthesis, Ringer’s lactate, calcium channel blockers,⁵ Rofecoxib as cyclohexane-oxygenase inhibitors,⁸ methyl blue,⁹ and octreotide.¹⁰ Many researchers have indicated that antioxidant compounds have a significant impact on reducing abdominal adhesions.¹¹ For example, due to its antioxidant properties, vitamin E can reduce oxidation caused by free radicals, resulting in reduction of adhesions.¹² Statins also demonstrate antioxidant and fibrinolysis activity, resulting in the decrease of abdominal adhesions.¹³ Currently a number of plants with antioxidant properties also have proved to inhibit intra-abdominal adhesions.¹⁴

Rosa damascena known as hip-rose, dog rose, and in Iran “Gole Mohammadi” is one of the most famous species of the Rosaceae family. The flowers are renowned for their fine fragrance and are commercially harvested for rose oil used in perfumery or to make rose water and “rose concrete.” Beside its use as a fragrance, several pharmacological properties, include anti-HIV,¹⁵ antibacterial, antioxidant, antitussive, hypnotic, antidiabetic, and relaxing effects on tracheal chains, have been reported for this plant.^15-17 Studies have demonstrated that rose is a rich source of vitamin C, carboxylic acids, tannins and particularly polyphenols and flavonoids,^18,19 which make it effective against free radical inflammatory cells. Moreover, many other benefits of this plant are reported, such as anticancer and antioxidant properties and preventing mutations.^20,21 In fact, this plant is an important source of healing fractions for the prevention and treatment of diseases caused by free radical activity.²²

Due to the antioxidant and anti-inflammatory properties of flavonoid compounds in R. damascena, and to their increasing role towards improving adhesion, these components play an important role in inhibiting complete lysis of fibrin. It also plays a role in preventing collagen production and accumulation, all of which are caused by the adverse effects of inflammatory factors and free radicals. Therefore these antioxidant and anti-inflammatory properties can prevent intra-abdominal adhesions after abdominal surgery and cesarean.¹⁴

The aim of this study was to determine the effect of R. damascena extract on adhesions after laparotomy and to measure phenolic compounds and antioxidant capacity of extract to determine its effective fractions. 

This study was approved by the Ethics Committee of Medical Plants Research Center, Shahrekord University of Medical Sciences, Shahrekord, Iran. The authors attempted to respect all ethical principles of working on laboratory animals to impose the lowest stress on them under observation of a veterinary specialist doctor.

Extract preparation. The rose oil was extracted using the maceration process. For this purpose, 2 L ethanol (70%) was added to the flask containing 100 g of dried powder of fresh R. damascena petals. The solution was left at laboratory temperature (25°C). After 48 hours, the extract was screened through filter paper, and the pulp was squeezed to discharge any remaining solvent. This step was repeated twice; then the extract was concentrated using a vacuum distillation until the volume was reduced to 20 mL. The concentrated extract was dried in an oven at 50°C then scraped with a spatula and rubbed with a mortar.¹⁴ Concentrations of the extract at 1% and 5% were prepared using distilled water. The solutions were sterilized by passing through a 0.2 µm filter. 

Measurement of phenolic compounds. Gallic acid with the Folin-Ciocalteu assay was used to determine the phenol content,²³  with some modifications. Different concentrations of standard gallic acid (12.5 ppm, 25 ppm, 50 ppm, 62.5 ppm, 100 ppm, and 125 ppm in methanol 60%) were prepared. Then, 0.1 mL from each sample was transferred into a test tube and 0.5 mL, and 10% Folin-Ciocalteu assay was added as the reactive agent. The solutions were left for 8 minutes at room temperature and then 0.4 mL of sodium carbonate (7.5%) was added. The tubes were maintained for 30 minutes at laboratory temperature (25°C) and then assayed in 3 intervals by a spectrophotometer (Spectroquest UV-Vis, Unico, Dayton, NJ) at 765 nm wavelength. To measure the overall phenol in the extracts, 0.01 g to 0.02 g of the extracts were dissolved in 60% methanol, reaching 10 mL; then, using the Folin-Ciocalteu method, the overall level of phenol was measured. However, instead of using the standard solution, 0.1 mL extract solution was added. Finally, the overall phenol level of the extract was obtained in milligrams/grams from the optical density read in gallic acid equivalent.¹⁴

Measurement of flavonoid compounds. Rutin is a common dietary flavonoid. In this study, total rutin flavonoids were evaluated using chloride aluminum colorimetry and typical methods for rutin extraction,²⁴ with a slight modification. First, different concentrations of standard rutin (25 ppm, 50 ppm, 100 ppm, 250 ppm, and 500 ppm) were prepared in 60% methanol. Then, from each solution, 1 mL was transferred to test tubes and mixed with 1 mL of 2% chloride aluminum. Afterwards, 6 mL of potassium acetate (5%) was added, and the optical density level was read after 40 minutes at 415 nm wavelength. The concentration levels of the standard solutions were assayed in 3 intervals. To measure the overall level of flavonoids in the extracts, 0.01 g to 0.02 g of the extracts were dissolved in 60% methanol, reaching 10 mL. Then using chloride aluminum colorimetry, the total level of flavonoids was measured. However, instead of using the standard solution, 1 mL of the extract was added. The total flavonoid level was calculated in mg/1 g extract, equivalent to rutin.

Measurement of flavonol compounds. Total flavonols were also measured using chloride aluminum colorimetry and rutin extraction procedures; however the optical density level reading was obtained after 2.5 hours at 440 nm wavelength.²⁵ 

Measurement of antioxidant activity. A β-carotene model was employed to measure the antioxidant activity of the extract.²⁶ The authors combined 0.5 mL chloroform, 5 mL β-carotene (0.2 mg), 20 mL linoleic acid (20 mg), and 0.2 mL TWEEN 40 before incubating it at 50°C for 10 minutes to remove the solvent. The control solution was diluted with distilled water and 4 mL aliquots of this solution were then combined with the sample as follows: The control solution was prepared including 0.2 mL of ethanol and 0.2 mL of the sample was prepared with 0.15 mL ethanol and 0.05 mL of the R. damascena extract. The optical density of the control solution was recorded at t₀ and t₉₀ at 470 nm, similar to the standard group. Samples were incubated in a bain-marie at 50°C. The antioxidant activity was measured on the basis of the ability of the samples to prevent the washing of β-carotene. The antioxidant activity was calculated through AA = 100 [1-(A_o- A_t )/(A^o_o – A^o_t )]; where, A_o is the optical density at t_o, A_t is the optical density of the sample at t₉₀, and A^o_o and A^o_t are optical density values in the control samples at t_o and t₉₀, respectively.

Selection and maintenance of animals. For this study, 30 healthy male Wistar rats, aged about 3 months (200 g to 250 g), were used. The rats were randomly divided into 3 groups of 10 rats in each group. Rats in groups A and B were treated with 1% and 5% R. damascena extract, respectively. Rats in group C (control) received only distilled water. None of the rats had a history of surgery or other medical interventions. They were kept at the standard conditions, fed standard pellets (Razi Co, Karaj, Iran); their water was also kept at 23ºC -25ºC, with 12:12-hours of light/dark photoperiod.

Induction of adhesion lesions. Adhesion lesions were induced under anesthesia. Surgery for all subjects was carried out under the same standard conditions by 1 person. Both groups of rats were anesthetized by intramuscular administration of a mixture of 20 mg/kg ketamine 10% (Alfasan Co, Netherlands) and 2 mg/kg xylazine 2% (Alfasan Co, Netherlands). While anesthetized, each rat was laid supine on a surgical table and the abdominal skin was shaved and disinfected with 10% betadine. Under sterile conditions, a 3 cm incision was made on the midline of the abdomen, the abdominal wall was opened and 3 shallow, longitudinal, and transverse incisions (2 cm in length) were made with a No. 24 scalpel on the right wall of the abdomen. A 2 × 2 piece of peritoneal surface was removed from the left side of the abdominal wall with surgical scissors. Then, to prevent the formation of peritoneal adhesions due to the presence of surgical suture material, 4 sutures at 1 cm intervals were placed using absorbable catgut. Fascia and skin were closed with 4 sutures at 1 cm intervals using a nonabsorbable silk. Finally, the incised area of the skin was again disinfected and the rats were left at a suitable temperature (23ºC-25ºC) to become conscious. External sutures were removed on day 10 of treatment, under general anesthesia.²⁷

It should be noted that all surgeries and inducing intra-abdominal lesions were carried out in accordance with the approval of the Morals and Ethics Committee, Shahrekord University of Medical Sciences (Shahrekord, Chaharmahal and Bakhtiari, Iran).

The treatment period was 14 days. The first day of the treatment period was considered the day surgery was performed. Immediately after making the lesions, 3 mL of R. damascena extract at concentrations of 1% (A) and 5% (B) was administered into the abdominal cavity of the rats; then the cavity was sutured. Control group (C) received only 3 mL of distilled water; it is proven that water has no impact on abdominal adhesions.²⁷

Macroscopic examinations. A second laparotomy was performed 14 days after making the lesions. For this, the abdomen of each rat was opened, and scoring was performed on the adhesions by an observer blinded to the study groups. Total adhesion scores were calculated for each rat separately, according to the scoring method developed by Ahmet Canbaz²⁷ (Table 1). 

Histopathological evaluation. Fourteen days after surgery, a sample of adhesion tissue from each animal was taken, fixed in 10% neutral buffered formalin and processed into paraffin and wax. Then, transverse incisions (5 µm thick) were made using a microtome fixed blade. All incisions of samples were stained by hematoxylin and eosin. A blinded pathologist performed histopathological evaluations using a LB-256 Binocular Biological Microscope (Labomed, Los Angeles, CA). Adhesion scoring was performed separately, based on the severity of fibrosis and inflammation²⁸ (Table 2). 

Statistical analysis. Samples were numbered, and the data were recorded separately. To document and show comparisons of obtained data, photographs were taken of the abdominal adhesions. Data analysis was performed through SPSS volume 16 (Chicago, IL) using Kruskal-Wallis and Mann-Whitney tests; P < 0.05 was considered statistically significant.

The total phenolic content of R. damascena was 109.1 mg/g gallic acid equivalent. Total flavonoid content was 48.5 mg/g rutin equivalent/g, and the total flavonol content was 37.5 mg/g rutin equivalent (based on dry extract). The antioxidant activity of the R. damascena extract was 50% of β-carotene.

Up to 1 day after treatment, all rats in group B (5% extract) were found dead. In group A (1% extract), there were no symptoms of abdominal ascites or viscous liquid and mortality until the end of study; at the end of the study all rats were euthanized. The second laparotomy surgery, done for evaluation in both surviving groups, showed adhesions in the inner peritoneal lining. The amount of adhesions was considerably lower in group A (1% extract) compared to group C (control); for group A, the frequency of adhesion with zero grad (no adhesion bands) was 2 cases. In group C, all samples had band adhesions. It is worth noting that in group C adhesions reached to the intra-abdominal parts, such as the intestines (Figure 1); this was not seen in group A (Figure 2). In group A (1% extract), adhesion grade 1 was most frequently observed with 5 cases (50%); in group C, adhesion grade 3 was most frequently observed with 4 cases (40%) (P = 0.007) (Table 3). 

Table 4 presents the results of the mean degree of adhesion in groups A and C, based on macroscopic and histological studies (fibrosis and inflammation severity). In a statistical comparison of the degree of adhesion between the 2 groups, macroscopic investigation of the adhesion in group A was significantly lower than that of the control group (P = 0.007). 

Histopathologic investigation also showed significant difference in the severity of fibrosis (P = 0.029) and inflammation (P = 0.009) between these groups. Figure 3 shows tissue samples prepared from adhesion bands of the control group that represents the severity of fibrosis and inflammation in this group. 

Although the pathophysiology of intra-abdominal adhesions is widely understood, there is no absolute solution to this problem yet. Treatment of adhesions after they have formed is costly and associated with high patient morbidity and mortality⁴; therefore, emphasis should be placed on preventing adhesions from forming.^4-7 Natural antioxidants such as medicinal plants have been among the agents investigated for the prevention of intra-abdominal adhesions.¹⁴ In this study, the inhibitory effects of hydroalcoholic extract of R. damascena was evaluated in intra-abdominal adhesions in Wistar rats. Results indicated the use of 1% extract was effective against intra-abdominal adhesions.

In group B (5% extract), all rats were found dead up to 1 day posttreatment. The reason is unclear, but it has been shown that antioxidants in certain circumstances may act as pro-oxidants^29,30 and, based on their concentration, can result in oxidative stress, which can lead to tissue damage.^31,32 Further studies will be necessary to prove these rats died from toxicity caused by the extract. Latifi et al³³ found in a study on the anti-inflammatory effects of hydroalcoholic extract of R. damascena in rats, that administration of the extract led to improved wound healing in the gut; however, intraperitoneal injection in high doses (> 500 mg/kg) caused lethal effects in some rats.³³ Low doses (< 500 mg/kg) in this study were considered nontoxic.³³ In a literature survey, other studies^34-36 revealed that the hypnotic, antiseizure, and laxative effects of R. damascena extract have been investigated following intraperitoneal injection.^34-36 However, in these studies, 500 mg/kg to 1000 mg/kg of extract were given as single doses; the animals did not have any significant problems with these doses so the researchers confirmed its safety in low doses. Further study is needed to determine the exact lethal dose and confirm its lingering toxicity in long-term use.

Intra-abdominal adhesions are a common problem following abdominal surgery, causing fatal complications such as ileus, intestinal obstruction, and infertility.³⁷ For example, in a study of 2,295 patients with small bowel obstruction, more than 64% of complications were due to intra-abdominal adhesions and 86% had undergone previous abdominal surgeries.³⁸ 

However, adhesions form as part of the natural healing process after surgery due to the presence of agents that can lead to oxygen depletion and ischemia.³⁹ These agents are oxygen-free radicals produced in the early phases of ischemia, which due to their rapid reaction with oxygen, remarkably in turn, decrease the amount of oxygen. The main source of production of superoxide and other free radicals may be the mitochondrial cytochrome oxidase or xanthine oxidase of endothelial cells which release following tissue damage.⁴⁰ Both hydrogen peroxide and superoxide anion are toxic for cells including endothelial cells, platelets, and fibroblasts, because they can cause extracellular cytolysis.⁴¹ Cytolysis and lipid peroxidation of cell membranes can result in increased vascular permeability as well as exudate formation that initiates the adhesion process.⁴² So far, none of the performed historical interventions have been able to completely prevent the formation of intra-abdominal fibrous bands. It seems that R. damascena, as a good source of antioxidant polyphenols and flavonoids, can scavenge free radicals generated during ischemia and play a significant role in preventing these complications.

In this study, the low severity of inflammation observed in group A compared to group C also reinforces the above statement. Many researchers have proved the antioxidant impacts of R. damascena compounds.²⁶ Moreover, antioxidant compounds also have been shown to stimulate the expression of genes encoding the growth of connective tissue and inhibiting collagen type I gene expression.⁴³ Other constituents of R. damascena extract are vitamin C, carboxylic acids, tannins, and especially flavonoids and polyphenol compounds with high antioxidant activity such as kaempferol and quercetin.^18,19 These compounds may be useful in wound healing by inhibiting inflammation; and by lysis of fibrin, collagen production and accumulation⁴⁴ may prevent the formation of abdominal adhesions after abdominal and cesarean surgeries. Restorative effects of this extract or similar drugs with antioxidant effects also have improved skin lesions.⁴⁵ Although many studies have been done to determine the best practices to treat these complications, natural antioxidants or antioxidant components have not been fully studied. In one of these studies,¹⁴ the inhibitory effect of green tea (Camellia sinensis) extract on intra-abdominal adhesions was examined in rats, and its inhibitory effect was attributed to the antioxidants properties.¹⁴ In another study,⁴⁶ the positive effects of lipid peroxidation inhibitors were shown to prevent peritoneal adhesions in rabbits.⁴⁶ Thus, given the significant role of R. damascena extract on the prevention of free radical activity and lipid peroxidation,^22,47 the present study is consistent with the aforementioned research.

Based on the results of this study, and the composition of polyphenolic compounds with antioxidant and anti-inflammatory effects, 1% R. damascena extract could be effective in the healing process of wounds in the abdominal cavity in Wistar rats and could help to prevent the development of intra-abdominal adhesions. Consequently, further studies are needed to clarify its effectiveness in similar cases in humans and to define its toxic properties. The authors recommend further clinical studies evaluating the different doses and toxicity to find the most efficient and safe dosage that can be used in a human model. 

This article is derived from research project No. 1414 and MD thesis No. 1114, which were both approved by the Research Deputy of Shahrekord University of Medical Science. The authors would like to thank the Deputy and the Medical Plants Research Center, Shahrekord University of Medical Science, as well as all who helped throughout the study.

From the Department of Surgery, Shahrekord University of Medical Sciences, Shahrekord, Iran; Medical Plants Research Center, Faculty of Medicine, Shahrekord University of Medical Sciences, Shahrekord, Iran; Young Researchers and Elite Club, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran; Medical Plants Research Center, Shahrekord University of Medical Sciences, Shahrekord, Iran; and Department of Surgery, Isfahan University of Medical Sciences, Isfahan, Iran

Address correspondence to:
Sareh Ezzati, MD
Medical Plants Research Center, Faculty of Medicine
Shahrekord University of Medical Sciences
Shahrekord, Iran
sareh_ezzati@yahoo.com

Disclosure: The authors disclose no financial or other conflicts of interest.