Oral Administration Evaluation of the Hydro-Ethanolic Extract of Ginger (Rhizome of Zingiber officinale) against Postoperative-Induced Peritoneal Adhesion: Investigating the Role of Anti-Inflammatory and Antioxidative Effects

Peritoneal adhesions (PAs) occur and develop after abdominal surgery. Abdominal adhesions are common and often develop after abdominal surgery. Currently, there are no effective targeted pharmacotherapies for treating adhesive disease. In this regard, ginger is wildly used in traditional medicine because of its anti-inflammatory and antioxidant effects and has been investigated for peritoneal adhesion treatment. This study analyzed ginger ethanolic extraction via HPLC to have a 6-gingerol concentration. Four groups induced peritoneal adhesion to evaluate ginger's effects on peritoneal adhesion. Then, ginger extract (50, 150, and 450 mg/kg) was administered by gavage in various groups of male Wistar rats (220 ± 20 g, 6–8 weeks). After scarifying the animals for biological assessment, macroscopic and microscopic parameters were determined via scoring systems and immunoassays in the peritoneal lavage fluid. Next, the adhesion scores and interleukin IL-6, IL-10, tumor necrosis factor-(TNF-) α, transforming growth factor-(TGF-) β1, vascular endothelial growth factor (VEGF), and malondialdehyde (MDA) were elevated in the control group. The results showed that ginger extract (450 mg/kg) notably decreased inflammatory (IL-6 and TNF-α), fibrosis (TGF-β1), anti-inflammatory cytokine (IL-10), angiogenesis (VEGF), and oxidative (MDA) factors, while increased antioxidant factor glutathione (GSH), compared to the control group. These findings suggest that a hydro-alcoholic extract of ginger is a potentially novel therapeutic strategy for inhibiting adhesion formation. Also, it might be considered a beneficial anti-inflammatory or antifibrosis herbal medicine in clinical trials. However, further clinical studies are required to approve the effectiveness of ginger.


Introduction
Peritoneal adhesions (PAs), with an estimated incidence rate of around 66% in surgeries worldwide [1], are caused by trauma, abdominal surgery, intraperitoneal (IP) infections [2], and various factors related to the types of operation, organs undergoing surgery, and suture types [3]. As a substantial reason, peritonitis and open procedure (laparotomy) can trigger PA progression. Also, various surgical and operation approaches are directly related to PA formation [4,5]. Statistically, in the UK, a study on 72,270 patients who underwent diferent types of surgery showed that 17.6% of them were estimated for disorders related to adhesion, 13.1% for surgical operations possibly caused by adhesions, and 3.5% for problems directly accompanied by adhesions [6]. In addition, PA can be a reason for female infertility [7], intestinal obstruction, and further chronic abdominal complications [3,8].
Ginger, the rhizome of Zingiber ofcinale Roscoe, has been consumed in food as a supplement, spice, and favoring agent. Te medicinal use history of ginger dates back to 2500 years ago in China, Iran, and India for some disorders, such as nausea, diarrhea, vomiting, dyspepsia, rheumatism, colds, and fu [26,27]. Also, it is commonly used for its benefcial characteristics such as aroma, nutrients, pungency, and pharmacological activities. Moreover, its rhizome has benefcial efects on diabetes [28] and metabolic syndrome [27], cholesterol levels [29], lipid metabolism [30], and infammation (as a replacive target to nonsteroidal antiinfammatory drugs for osteoarthritis disease conditions) [31,32]. Ginger has many proven biological (e.g., apoptosis, cell cycle/DNA damage, chromatin/epigenetic regulation, cytoskeletal regulation and adhesion, immunology and infammation, and neuroscience) and pharmacological properties. Te biological and pharmacological properties of ginger, as functional food, dietary supplement, and natural immunomodulatory, are owing to the presence of monoterpenes (cineole, citral, limonene, and α/β-pinenes), sesquiterpenes (β-elemene, farnesene, and zerumbone), and phenolics (gingerols, [6]-Shogaol, [6]-paradol, and zingerone). Tese properties are induced through specifc signaling pathways such as autophagy, mitogen-activated protein kinase, and cellular metabolism [27,33]. In addition, research has shown that ginger can induce estrogen activity (e.g., the chemoprevention of cancers) and the improvement of menopausal syndromes, osteoporosis, endometriosis, prostatic hyperplasia, and polycystic ovary syndrome [33,34]. In vitro studies have shown that the antiinfammatory efects of ginger are induced by the inhibition of arachidonic acid metabolism in both lipoxygenase (LOX) and cyclooxygenase (COX) pathways [35], which may show fewer side efects than NSAID [36]. Another study has shown that ginger inhibits the production of the genes participating in the infammatory reactions (e.g., chemokines, genes encoding cytokines, and the inducible enzyme nitric oxide synthase (iNOS) and COX-II) [27,37].
Te preclinical and clinical surveys have used several natural and synthetic bio-components for PA treatment. However, there still is not enough knowledge to express a "gold standard" for curative efects on postoperative PA. Terefore, surgeons and researchers still investigate impressive new methods [38,39], although none is extensively applied clinically due to a missing long-term efcacy. Terefore, in the present study, we investigate the protective efects of ginger against surgical-induced peritoneal adhesion in a rat model.

Extract Standardisation Using High-Performance Liquid
Chromatography. Te high-performance liquid chromatography (HPLC) analysis was performed using a C18 column (5 μm particle size, 250 × 4.6 mm) from Capital (Broxburn, UK) with a 20 μl injection volume. Te HPLC analysis system was equipped with a Knauer HPLC system (Berlin, Germany), UV detector K-2600, HPLC pump (K-1001), and a Knauer K-500 degasser. Te chromatograms were registered at 280 nm wavelength. Te mobile phase included distilled acidifed water with trifuoroacetic acid (TFA, 0.05%) and methanol at a 1 ml/min fow rate at the controlled room temperature (22-25°C). We prepared a standard stock solution of 6-gingerol (500 μg/ml) in methanol. It was then diluted with the same solvent to make 100 and 50 μg/ml concentrations for quantitative analysis, and the standard curves were plotted. A linear gradient was used as follows: at 0 min (80% solvent A and 20% solvent B); at 7 min (100% solvent); 7-11 min (100% solvent B); at 13 min (80% solvent A and 20% solvent B); and 13-15 min (returning to initial conditions). In this study, 40 healthy mature male Wistar albino rats with weights of 250 ± 30 g and ages of 6-8 weeks were purchased from the animal laboratory of the Faculty of Medicine, Mashhad University of Medical Sciences, and Mashhad, Iran. For adaptation, they were acclimatized to the laboratory conditions and had ad libitum access to tap water and diet for one week before starting the experiments. In addition, more appropriate hygiene was provided with continuous cleaning and removal of feces and spilled feeds from cages daily. Te experimental rats were all alive until being sacrifced with the asphyxia method using a CO 2 chamber. All animals received human care in compliance with institutional guidelines.

Experimental Design and Interventions.
Te rats were randomly and equally divided into fve groups, each with 8 rats, as follows: (i) Group 1: sham group underwent surgical procedures but was not induced by PA. (ii) Group 2: control group underwent surgical procedures, and PA was induced. (iii) Groups 3 to 5: ginger groups underwent surgical procedures, and PA was induced. Next, the groups received the oral administration of the ginger extract (50, 150, and 450 mg/kg), respectively, for seven constitutive days.
According to our previously published articles [10,11,14,15], the experimental procedure was performed as follows: after anesthetization with 100 mg/kg of ketamine (intraperitoneally; IP) and 10 mg/kg of xylazine (IP) [11,[13][14][15], the rats' abdomen was shaved. In this process, alcohol and iodine solution was used for skin disinfection. Next, a 3 cm gap was carefully cleaved in the abdominal midline to achieve the abdominal cavity for inducing the adhesion aim. Ten, for peritoneal abrasion, one side of the middle abdominal hole was softly abraded via soft sterilized sandpaper until the cecum displayed a fne petechia and turbid appearance. Afterward, 4-0 polyglactin sutures were performed to close the abdomen wall after inducing the adhesion. After ending the surgery to inhibit the possible wound infection, a single dose of antibiotic cefazolin (300 mg/kg intramuscularly; IM, [11,13,14]) was immediately given to all rats. Finally, the rats were reserved in their cages for seven days to recover.

Assessment of Adhesion Grade.
Te laparotomy was performed on day 7 after the surgery. Two scoring systems, i.e., Nair et al. [11,14,40] and adhesion scoring scheme [11,14,41], were then used to determine PA scores (Tables 1  and 2, respectively), with two independent researchers blinded on the procedures and the grouping.

Preparation of Peritoneal Lavage Fluid.
After the rats' laparotomy, the peritoneal lavage fuid was prepared using 2.5 mL sterilized phosphate-bufered saline (PBS). In detail, the whole area of the peritoneum was washed twice. Ten, Evidence-Based Complementary and Alternative Medicine the collected fuid was centrifuged at 3000 RPM for 5 min at 4°C. Finally, the supernatant was separated for further investigation.

Statistical
Analysis. Graph Pad Prism (version 8.01) software was used for data analysis according to the nature of parametric or nonparametric analysis and expressed as means ± SD or median ± IQR. Brown-Forsythe one-way analysis of variance (ANOVA) was performed via Dunnett's T3 multiple comparisons post hoc test for parametric data (Table 3). Also, for the nonparametric data (adhesion scores), a Kruskal-Wallis test was utilised via Dunn's multiple comparison post hoc test. Data of wound size were then analyzed using repeated-measurement two-way ANOVA with Dunnett multiple comparisons post hoc tests. P values P ≤ 0.01 were considered statistically significant [20,21].

HPLC Analysis of the Ginger Extract.
HPLC is the most standard application method for the quality control and analysis of ginger and ginger-related marketed products [42,40]. Tis method is performed for quantitative and qualitative analysis of pungent ginger principles. First, the peak areas (y) against the standard solution (x) concentration are plotted, and the calibration curve equation is calculated as follows: y � 7576x + 144875, with R 2 � 0.998 (Figure 1(a)). Ten, the extract solution was prepared in methanol with a concentration of 5 mg/ml and injected into the HPLC-diode array detection-evaporative light scattering system for analysis (Figures 1(b) and 1(c)). All standard samples and extract samples were analyzed in triplicate. Te concentrations of the 6-gingerols were calculated using the plotted calibration curve (Figure 1(a)). Also, the content of 6-gingerol was calculated at 39.05 mg in 1 g of the dried extract (Figures 1(b) and 1(c)).

Evaluating Ginger Extract Efects on the Body and Spleen
Weights and Wound Healing. Tis experiment was conducted to evaluate the efects of ginger extraction on the rat's body and spleen weights (Table 4), spleen size (Table 4), and wound healing ( Figure 2). Ginger extract with 50, 150, and 450 mg/kg doses had no signifcant efect on body and spleen weights compared with the control and sham groups at the end of the study period. However, the wound size of groups treated with 150, 450 mg/kg was signifcantly smaller than at the beginning of the study (Figure 2; P < 0.001 for all cases). However, the ginger extract had a remarkable efect on wound healing compared with the control group at the end of during (Figure 2; P < 0.01 and P < 0.001, respectively).

Evaluating Ginger Extract Efects on Peritoneal Adhesion
(PA) Scoring. At the end of the experiment, the incisions of all rats were primarily ameliorated without infection or other complications. Peritoneal adhesions are represented in Figure 3. Te results demonstrated that the PA scores were signifcantly elevated in the control group compared to the sham group (Figure 4; P < 0.001 for both scoring systems: Naier et al. and adhesion scheme scoring). Meanwhile, Table 1: According to Nair et al. [11,14,40], the adhesion score is categorised.

Grade
Description of adhesive bands 0 Te complete absence of adhesions 1 A single band of adhesion between viscera or from viscera to the abdominal wall 2 Two bands, either between the viscera or from the viscera to the abdominal wall 3 More than two bands, between viscera or viscera to the abdominal wall or whole intestines form a mass without being adherent to the abdominal wall 4 Viscera directly adherent to the abdominal wall, irrespective of the number and extent of adhesive bands  Figure 4; P < 0.05 for both scoring systems).

Evaluating Ginger Extract on Fibrosis and Angiogenesis
Parameters. Following the PA, the angiogenesis and fbrosis levels were notably increased by determining VEGF (Figure 6(a)) and TGF-β1 ( Figure 6(b)), respectively, in the control group compared to the sham group (P < 0.001 for     Evidence-Based Complementary and Alternative Medicine both cases). Nevertheless, the ginger extract at the dose of 450 mg/kg signifcantly diminished the levels of VEGF (Figure 6(a)), and TGF-β1 (Figure 6(b)) compared to the control group (P < 0.001 for both cases).

Evaluating Ginger Extract on Oxidant and Antioxidant
Parameters. As an oxidative marker, the MDA level was signifcantly increased in the control group compared to the normal group (Figure 7(a); P < 0.001). In contrast, the results indicated that the GSH level, as an antioxidative marker, was strongly down-regulated in the control group compared to the normal group (Figure 7(b); P < 0.001). Nevertheless, administration of the ginger extract (450 mg/ kg/day) remarkably decreased the MDA level (Figure 7(a)) and increased the GSH level (Figure 7(b)) compared to the control group (P < 0.001 for both cases). In addition, the ginger extract (150 mg/kg/day) could signifcantly enhance the GSH level compared to the control group (Figure 7(b); P < 0.05).

Discussion
Te main fnding of our study was that ginger extract could lower the PA following surgeries. To the best of our knowledge, this is the frst report describing the signifcance of the ginger efect on PA from both clinical medicine and basic science viewpoints. Our results suggested that ginger extract may be considered a herbal medicine that can improve the PA in patients by suppressing the infammatory cytokines such as IL-6 and TNF-α, and TGF-β1 and VEGF as fbrotic and angiogenesis factors. In this study, the ginger extract reduced the upregulated-IL-10 in the control group. Also, it has been shown that ginger extract had a curative efect on macroscopic parameters (wound healing). However, it was not efective on the body and spleen weight change, despite increasing spleen weight in the broiler chickens [43]. In addition, it could increase the weight change in Japanese quail [44]. Regarding these discrepancies, further research is needed for the conclusion.   Evidence-Based Complementary and Alternative Medicine 7 Local injuries (e.g., peritoneal surgery) lead to a growth in the number and infltration of leucocytes in the bloodstream to the injured area. As a result, infammatory cytokines such as TNF-α, IL-1β, IL-6, and interferon (IFN)-c are overproduced and majorly appear in both bloodstream and the injured area [11,45,46]. Moreover, infections and tissue injuries cause temporary secretion of IL-6, thereby simulating acute phase responses, hematopoiesis, and immune reactions. However, IL-6 expression is tightly regulated by transcriptional and posttranscriptional mechanisms, and uncontrolled synthesis of IL-6 shows a pathological efect on chronic infammation and immunity [47]. In line with this result, ginger extract at 50 mg/kg remarkably attenuated the serum levels of TNF-α, IL-6, and IL-17 in the ginger group than in the type 2 collagen and Freund's adjuvant-induced arthritis group [48]. Also, Adegbola et al. demonstrated that the ginger extract at a dose of 500 mg/kg alone or in combination with Allium sativum 1000 mg/kg signifcantly declines the infammation following the high-fat diet-induced obesity in rats by decreasing the IL-6 and TNF-α levels and lipid profle [49]. Another study showed that oral administration of Zingiber ofcinalis extract at doses of 100 and 1000 mg/kg strongly diminishes LPS-Induced Infammation by downregulating the release of proinfammatory cytokines such as IFN-c and IL-6 [50].
Tese studies could support our fndings regarding the antiinfammatory efects of ginger extract.
Furthermore, IL-6 facilitates the diferentiation of naïve CD4 + T cells. Terefore, it plays an essential function in relating innate to acquired immune responses. In addition, IL-6, incorporated with TGF-β, is critical for T17 diferentiation from naïve CD4 + T cells [51]. Research has shown that the activation of IL-6 signaling entirely relates to the presence of sIL-6R in malignant mesothelioma cells [52]. As a result, the signal transduction cascade is initiated by IL-6. TGF-β1, secreted from most immune cells, is essential in regulating the immune system [53]. Also, muscle injury causes the expression of TGF-β1, which triggers chronic infammation, fbrosis, and extracellular-matrix accumulation [54]. TNF-α, as a proinfammatory cytokine, activates intracellular signaling pathways (e.g., MAPKs, transcription factors, or apoptosis) that contribute to vasodilatation and leukocyte adhesion to epithelium through the efect on adhesion molecules expression. IL-10, as an antiinfammatory biomarker, is secreted by various cells (T cells, B cells, and monocytes/macrophages) triggered by innate immune cells. Besides, it lowers the concentration levels of IL-6, IL-8, and TNF-α [55].
Terefore, neutralizing TGF-β1 and TNF-α expression in injured tissue could inhibit the formation of infammation and fbrosis [54,56]. Consistent with our fndings, Abdi et al. showed that the ginger extract at doses of 100, 200, and 400 mg/kg provides sufcient cardioprotection in the diabetic group by reducing infammatory mediators and decreasing the fbrotic markers gene expression, including angiotensin II type 1 receptor and TGF-β1 and TGF-β3 [57]. Furthermore, the extract induces its efect by regulating the SMAD/TGF-β signaling pathway [57].
Te STAT3 signaling pathway induces the VEGF by stimulating IL-6 in C22A cervical carcinoma cell lines [58]. Te increased VEGF levels caused by IL-6/sIL-6R are related to the STAT3 pathway. Hence, IL-6 can trigger VEGF and vascularisation in chronic infammatory diseases [59]. Moreover, a recent study demonstrated that ginger constituent 6-Shogaol inhibits the development of vascular endothelial growth factor (VEGF)-induced endothelial blossoms from human umbilical vein endothelial cells (HUVECs) spheroids and from murine aortic rings. Importantly, for the frst time, this research shows that 6-Shogaol disrupts aortic angiogenic sprouts in a murine model of vascular disruption [60]. Another recent study investigated the potential protective efects of ginger extract on rats with type 2 diabetic retinopathy. Te results showed that diabetic rats had altered ocular expression of e/iNOS, G6PDH, VEGF, NF-kB, and apoptosis-related genes. In contrast, ginger extract has been shown to reduce oxidative damage, infammation, iNOS, VEGF, and apoptosis and improve eNOS and G6PDH in diabetic retinopathy. Tese results also are consistent with our fndings [61].
Tis study showed a considerable diference in MDA and GSH in ginger-treated rats. Our results are similar to the survey reported by some researchers [13,62,63]. Te efect of ginger on lipid peroxidation is applied via Fe 2+ chelating properties, antioxidant activity, and OH scavenging ability. Te ginger administration has been displayed to strongly downregulate thiobarbituric acid-reactive substances (TBARS) levels, which expresses as an indicator of lipid peroxidation and oxidative stress damage. Accordingly, reducing TBARS levels upregulates the GPX function and impact, thereby inactivating the lipid peroxidation reactions [64]. ROS is generated in the postoperative site or infammatory diseases. Consequently, an antioxidant enzyme (e.g., catalase, superoxide dismutase (SOD), GSH, and glutathione peroxidase (GPx)) is needed to remove ROS [65,66]. Te GSH, as a nonprotein thiol, is a vital need for protecting cells from the cytotoxic efect of ROS. Also, the harmful efects of As were suppressed via GSH by elevating the converting the methylation form of As to low-toxic metabolites [67]. Many in vitro, in vivo, and clinical studies have proven the safety of ginger and shown that it has no adverse/toxic efects [68][69][70][71]. In a clinical study, patients associated with chemotherapy-induced nausea and vomiting (CINV) who used the ginger extract (40 mg/day) showed no further gastrointestinal disorders such as epigastric and abdominal pain, dyspepsia, and hiccups than the placebo group [68]. Moreover, it has been suggested that using the standardized ginger extract up to 2 g/day leads to no notable adverse efects on human health. Terefore, it is classifed as frst ranked on the toxicity scale of the National Cancer Institute [72]. In addition, clinical studies have demonstrated that ginger and its extract (up to 1.5 g/d) can be consumed to attenuate nausea and vomiting during early pregnancy [73].
In summary, this study demonstrated that oral ginger intake induced benefcial efects on postoperational adhesion without any adverse efects by improving the antioxidative factors and decreasing the fbrosis, infammatory cytokines, oxidative factors, and angiogenesis biomarkers. Hence, ginger extraction can be used to treat postoperative PA as a potential herbal medicine. However, further clinical studies are required to approve the efectiveness of ginger. Scoring systems; data were presented as median ± interquartile range, IQR (n � 8 for each group). Data were analysed using the kruskalwallis test with Dunn's multiple comparison post hoc test. * compared the treated group with the control group; * : P < 0.05. + compared the control group with the sham group; +++ : P < 0.001.

Sham
Control  Figure 6: Te efects of diferent doses of ginger extract on the peritoneal lavage levels of (a) VEGF and (b) TGF-β; data were obtainable as means ± SD (n � 6-8 for each group). Brown-Forsythe one-way analysis of variance (ANOVA) was utilised via Dunnett's-T3 multiple comparisons post hoc test for parametric data. * Compared the treated group with the control group; * * * : P < 0.001. + Compared the control group with the sham group; +++ : P < 0.001.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon reasonable request.

Ethical Approval
Te

Conflicts of Interest
Te authors declare that there are no conficts of interest.  Figure 7: Te efects of diferent doses of ginger extract on the peritoneal lavage levels of (a) MDA and (b) GSH; data were presented as means ± SD (n � 6-8 for each group). Brown-Forsythe one-way analysis of variance (ANOVA) was utilised via Dunnett's-T3 multiple comparisons post hoc test for parametric data. * Compared the treated group with the control group; * : P < 0.05 and * * * : P < 0.001. + Compared the control group with the sham group; +++ : P < 0.001.

12
Evidence-Based Complementary and Alternative Medicine