EXPLORING THE AMELIORATIVE EFFECTS OF HYPERICUM SCABRUM L. ON A SURGICALLY-INDUCED ENDOMETRIOSIS RAT MODEL AND ITS PHYTOCHEMICAL PROFILING BY LC-MS/MS

Endometriosis is described as the existence and expansion of functional endometrial -like tissues in another part of the uterine cavity. It affects not only postmenopausal women but also teenagers after menarche, causing subfertility, dysmenorrhea and severe pelvic pain. Since no specific treatment is acknowledged for this disease, the present study aimed to investigate the efficacy of H. scabrum in a surgically -induced endometriosis rat model. In order to test the efficacy of H. scabrum on endometriosis, n-hexane, ethyl acetate (EtOAc), methanol (MeOH) and aqueous extracts were prepared from the aerial parts of H. scabrum. The cytokine levels, antioxidant enzyme activities, the contents of malondialdehyde (MDA) and glutathione (GSH) and histopathological ana lyses were evaluated. In order to reveal the potential active compounds, liquid chromatography-mass/mass spectroscopy (LC -MS/MS) analysis was performed on the MeOH extract who presented the highest activity. The MeOH extract significantly decreased the adhesion scores, endometriotic implant volumes, cytokine levels and the antioxidant parameters. Furthermore, histological procedures supported those findings. According to LC -MS/MS analysis of the MeOH extract, the major compounds were mostly phenolic acids and flavonoids. We concluded that the MeOH extract from H. scabrum aerial parts could be used for the treatment of endometriosis, and its activity could be attributed to the chlorogenic acid and quercetin, detected in the highest amounts. Rezumat Endometrioza se caracterizează prin prezența și proliferarea țesuturilor funcționale asemănătoare endometrului în locații din afara limitelor cavității uterine. Această patologie afectează femeile aflate în postmenopauză și adolescentele după menstruație. Deoarece nu există un tratament specific pentru endometrioză, am investigat în acest studiu eficacitatea speciei Hypericum scabrum într-un model de endometrioză indusă chirurgical la șobolan. Astfel au fost preparate diferite extracte, în n -hexan, acetat de etil (EtOAc), metanol (MeOH) și apă, din părțile aeriene de H. scabrum. După tratament, au fost evaluate scorurile de aderență, volumele de implant endometrial, nivelurile de citokine, activitățile enzimelor antioxidante, conținutul de malondialdehidă (MDA) și glutation (GSH) și au fost întreprinse analize histopatologice. Pentru a descoperi potențialii compuși activi, s-a realizat o analiză de cromatografie lichidă -spectroscopie de masă(LC-MS/MS) folosind extractul metanolic, care a prezentat cea mai mar e activitate. Extractul MeOH din părțile aeriene de H. scabrum poate fi utilizat în tratamentul endometriozei, iar activitatea sa ar putea fi atribuită acizilor fenolici, respectiv acidului clorogenic, și flavonoidelor, r espectiv quercetinei, compuși majoritari identificați.

antioxidant, cytokines, endometriosis, ethnopharmacology, Hypericum scabrum, LC-MS/MS

The genus Hypericum L., which belongs to the Hypericaceae family, includes approximately 500 accepted species. The flowers of the Hypericum genus exhibit bisexual characteristics, with five sepals and five petals. The petals are usually yellow, sometimes tinged red. In Türkiye, there are 69 species of Hypericum [1]. Our plant material, Hypericum scabrum L., is widely distributed in Türkiye [2]. The stems of the plant are glabrous, scabrid with unbranched red-gland- tipped emergences. The dimensions of the plant's leaves range from 7 to 20 mm and exhibit an oblong to lanceolate shape. Similarly, the sepals are oblong in form, while the petals measure between 5 and 7 mm in length. H. scabrum grows usually on dry rocky slopes, open woodland or steppe [1]. To date, various compounds such as tannins, phloroglucinols, phenyl- propanes, flavonoids and proanthocyanins have been extracted and identified from the Hypericum genus [3, 4]. FARMACIA, 2023, Vol. 71, 4 711 Pseudohypericin and hypericin were detected only in the flowers of H. scabrum . In addition to these compounds, kaempferol, quercetin and amentoflavone were observed in the flowers of the plant [5]. Quercetin, chlorogenic acid, apigenin -7-0-glucoside, rutin and hyperoside were detected in the leaves of the plant [5, 6]. H. scabrum is traditionally used for peptic ulcer, gastritis, haemorrhoids, rheumatism, constipation and jaundice [4, 7]. Furthermore, Yeşil and Akalın reported that the infusion prepared fro m the aerial part of H. scabrum is used for menstrual ailments in Malatya, Türkiye [8]. Endometriosis is described as the existence and expansion of functional endometrial -like tissues in another place than the uterine cavity [9]. Both its aetiology and pathogenesis have not been completely explained until now. It affects not only postmenopausal women but also teenagers after menarche [10]. It has been observed that this condition leads to subfertility, dysmenorrhea and intense pelvic pain [11]. It ranks third in terms of hospitalisations for gynaecological conditions in the United States. Furthermore, it is leading to an economic burden due to healthcare costs worldwide [9]. Recently, the acceptable tre atment

for endometriosis have been medication, surgery or a combination of medication and surgery. However, the recurrence rate of the endometriosis is around 7 - 30% following the surgery [9, 12]. Due to the severe undesirable effects, the economic burden of endometriosis, and the recent interest in this pathology, the use of natural sources to treat itis considered an alternative approach. Because of its usage in folk medicine, H. scabrum was selected for this purpose. Therefore, the aim of this s tudy is to evaluate the efficacy of H. scabrum aerial parts on the endometriosis rat model.

Plant material H. scabrum was collected from the Van-Başkale road- side, Güzeldere district, Türkiye, on July 6 th, 2020 (38°11'04.7"N 43°54'34.5"E). It was authenticated by Hüseyin Eroğlu, PhD from the Department of Botany, Faculty of Science, Van Yüzüncü Yıl University, Türkiye. The herbarium specimens were kept in the herbarium of Van Yüzüncü Yıl University, Türkiye (VANF165216). Extraction method The aerial part of H. scabrum, weighing 600 g, was subjected to a drying process in a shaded environment. Subsequently, the plant material was pulverised using a grinder. A sample weighing 600 g of powdered and dried H. scab rum was subjected to extraction using n-hexane (5 L), ethyl acetate (EtOAc) (5 L) and methanol (MeOH) (5 L), respectively. The plant was extracted three times with each solvent. The extraction was evaporated at 40°C under reduced pressure by a rotary evaporator until dryness. A sample of 100 g of H. scabrum aerial parts was extracted with hot water (1 L) for the preparation of the infusion. The extraction was frozen at -20°C and lyophilized. After all of these procedures, n-hexane (HSH), EtOAc (HSE), MeOH ( HSM) and aqueous (HSA) extracts were obtained with yields of 4.3%, 2.2%, 13.7% and 16.2%, respectively. Animals and ethic approval For the purpose of this experiment, female rats of the Wistar albino strain weighing between 200 - 250 g were selected. Durin g the experiment, the animals were housed in appropriate cages. The temperature, humidity, and luminosity were controlled in this process. This study was approved by the Experimental Animal Ethics Committee of Van Yüzüncü Yıl University, Türkiye (Approval No. 2020/01). All experimental and surgical procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Experimental procedure of rat endometriosis model This procedure was conducted according to Vernon and Wilson [13]. All the rats were anaesthetized with intramuscular administration of the combination of ketamine and xylazine. Under anaesthesia, the abdomen was opened by creating a 3 cm incision using a scalpel blade, and the subcutaneous and muscle layers were separated from each other. The right uterine cornu were ligated with polyglactin absorbable suture (USP 4/0) from its two margins. The right uterine cornu was cut and a piece of 1.5 cm tissue was taken. The tissue fragment was cleaned in 0.9% saline solution and opened lengthwise. A fragment of endometrial tissue was stitched with polyglactin absorbable suture (USP 4/0) to the abdominal wall of the same rat around the blood vessel. The abdomen was sewn with a silk suture (USP 3/0). Twenty -eight days after the surgical procedure, a second surgery was done. In this surgical procedure, adhesion scores and endometriotic implant volumes were evaluated. In addition, peritoneal fluids were collected, and t he abdomen was closed with a silk suture (USP 3/0). For the treatment procedure, 42 rats were divided randomly into 6 groups (n = 7), namely control, n-hexane, EtOAc, MeOH, aqueous and reference. Treatment procedure Two days after the second experiment, 0.5% carboxy- methylcellulose was administered to the control group. Letrozole (0.2 mg/kg) was applied to the reference group. The n-hexane, EtOAc, MeOH and aqueous extracts were administered to the treatment groups at a dosage of 100 mg/kg. After the treatment procedure, the 3rd operation was done. In this surgery procedure, adhesion scores and endometrial foci areas were re - calculated, and peritoneal fluids were collected for the evaluation of cytokine levels. FARMACIA, 2023, Vol. 71, 4 712 The evaluation of adhesion scores and the calculation of endometriotic foci areas The adhesion scores were evaluated according to the scoring system of Blauer and Collins [14] as follows: 0, no adhesions; 1, thin adhesions; 2, thick adhesion in one area; 3, widespread thick adhesions; 4, adhesions of internal organs to the abdominal wall. The endometrial implant volumes were calculated according to the following formula: π/6 x length x width x height [15]. The detection of cytokine levels Tumour necrosis factor alpha (TNF -α), vascular endothelial growth factor (VEGF) and interleukin -6 (IL-6) levels in the peritoneal fluids were assessed quantitatively by enzyme-linked immunosorbent assay (ELISA) kits. The catalogue numbers of the ELISA kits were E-EL-R0019 (Elabscience®), E-EL-R2603 (Elabscience®) and E -EL-R0015 (Elabscience ®) for TNF-α, VEGF and IL-6, respectively. All procedures were performed according to the ELISA kit user manuals. The detection of malondialdehyde (MDA), glutathione (GSH) levels and antioxidant enzyme activities The levels of MDA, which is a marker for oxidative stress, were determined according to a previous published

[16]. GSH levels were calculated according to the spectrophotometric method of Beutler et al. [17]. The activity determination of glutathione-S-transferase (GST) was based on the changes in absorbance read at 340 nm because of the creation of the GSH-CDNB complex [18]. Glutathione peroxidase (GPx) enzyme activity was measured spectrophotometrically at 450 nm by a commercially available ELISA kit following the manufacturer’s instructions (SunRed Biotechnology Company, catalogue no. DZE201-11-5104). Superoxide dismutase (SOD) enzyme activity was determined by using SOD (Ransod) assay kit (Randox catalogue no. SD125). Total phenolic (TPC) and flavonoid content (TFC) measurement of the extracts For the total phenolic content determination, the extracts were prepared at a concentration of 2 mg/mL in ethanol (75%). A 20 μL sample was transferred in the test tube and distilled water (1580 μL) was a dded. Then 100 μL Folin-Ciocâlteu phenol reagent and 20% sodium carbonate (300 μL) were added, respectively. All samples were incubated at 40°C for 30 min. The absorbances of the samples were read at 765 nm in the spectro - photometry. For the calibration curve, gallic acid (GA) was used, and the total phenolic contents of the extracts were calculated as mg GA per g of extract [19]. For the total flavonoid content determination, the extracts were prepared at a concentration of 2 mg/mL in 75% ethanol. A 500 μL sample was put in the test tube along with 75% ethanol (1500 μL), 10% aluminium chloride (100 μL), 1 M sodium acetate (100 μL) and distilled water (2800 μL), respectively. All samples were incubated at room temperature for 30 min. The absorbances of the samples were read at 415 nm in the spectrophotometry. For the calibration curve, quercetin was used, and the total flavonoid contents of the extracts were calculated as mg quercetin per g of extract [20]. LC-MS/MS studies LC-MS/MS studies were carried out according to Yilmaz’s method, which is a validated study [21]. Histological procedures The endometrium samples were excised and immediately fixed in 10% neutral buffered formalin for 48 h at 4°C. After rinsing the samples with phosphate buffered saline solution at a pH of 7.4, a series of ethanol, with increasing concentrations, was employed to remove water from the tissues, which were subsequently immersed in paraffin wax for embedding. As a next step, 5 µm thick tissue sections were taken using a MICROM HM 325 manual microtome (Waldorf, Germany) and then the sections were put onto adhesive- coated slides (Marienfeld GmbH, Lauda-Königshofen, Germany). Following deparaffinization and rehydration, the sections were stained with haematoxylin and eosin (H&E) or Mallory’s triple (MT) [22]. Histopathological examination of the sections and photography were conducted using Leica DMI 6000B and DM500 model microscopes (Leica Microsystems, Wetzlar, Germany). Photographs were captured at 100×, 200× or 400× magnifications. The histopathological alterations in endometriotic tissues were determined according to the criteria described by Demirel et al. and Elgamal et al. [23, 24]. Statistical analysis The Kolmogoro v-Smirnov test was carried out to determine whether the distribution is normal or not. The Kruskal-Wallis test was performed for the evaluation of the adhesion scores. For the other parameters, a one- way ANOVA-Dunnett’s post hoc test was performed. Statistical significance was determined by comparing all groups to the control group, with a significance level of p < 0.05. When the groups were compared with each other especially pre -treatment values, the ANOVA-Tukey post hoc test was used. An ANOVA- Tukey post hoc test was used for the total phenolic content tests whereas Student's t-test was used for the total flavonoid content tests.

and Discussion The evaluation of adhesion scores The adhesion scores were evaluated according to the

of Blauer and Collins [14]. The adhesion scores were significantly decreased in the MeOH and letrozole administered groups. The MeOH extract decreased adhesion score from 3.900 ± 0.100 to 2.214 ± 0.395 (p < 0.05) while the letrozole decreased adhesion score from 3.900 ± 0.100 to 1.643 ± 0.325 (p < 0.001) when compared to the control group. There was no significant difference between the control and the remaining groups in the adhesion scores (Table I). FARMACIA, 2023, Vol. 71, 4 713 Table I Adhesion scores of tested groups in endometriosis model Groups Mean adhesion scores ± S.E.M. Pre-Treatment Post-Treatment Control 3.857 ± 0.142 3.900 ± 0.100 HSH 3.571 ± 0.250 3.083 ± 0.358 HSE 3.500 ± 0.174 3.167 ± 0.366 HSM 3.538 ± 0.183 2.214 ± 0.395* HSA 3.857 ± 0.097 3.462 ± 0.243

3.714 ± 0.163 1.643 ± 0.325*** The values were given as Mean ± S.E.M. *: p < 0.05, **: p < 0.01, ***: p < 0.001, The evaluation of endometriotic foci areas The endometriotic implant volumes were significantly reduced in the HSM and reference groups. The MeOH extract decreased endometriotic implant volume from 188.1 ± 30.9 to 53.4 ± 16.7 mm3 (p < 0.001) whereas letrozole decreased endometriotic implant volume from 188.1 ± 30.9 to 11.2 ± 1.5 mm 3 (p < 0.0001). The decreases were observe d in endometriotic implant volumes in the HSH, HSE and HSA groups, but those decreases were not statistically significant. Furthermore, there was no statistical difference among the pre - treatment endometriotic implant volumes of the groups (Figure 1). Figure 1. Endometriotic implant volumes of extracts administered groups (A: Pre-treatment, B: Post-treatment) The values were given as Mean ± S.E.M. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001 The evaluation of cytokine levels in peritoneal fluids Firstly, the cytokine levels of the pre-treatment were compared with each other. Any significant difference was not observed among the cytokine levels of pre - treatment (Figure 2). Then, post -treatment cytokine levels were compared. The TNF-α levels of HSM and reference groups were significantly reduced. The MeOH extract decreased TNF-α levels from 1613.2 ± 190.4 to 1145.2 ± 56.9 pg/mL (p < 0.05) while letrozole decreased TNF -α levels from 1613.2 ± 190.4 to 1121.2 ± 99.0 pg/mL (p < 0.05). The decrease in TNF -α levels in other groups was detected, but it was not statistically significant. The IL-6 levels of HSM and reference groups were found to be decreased when compared to the control group. The IL -6 level of HSM was reduced from 329.5 ± 2.2 to 280.7 ± 7.8 pg/mL (p < 0.01) whereas the IL-6 level of letrozole was reduced from 329.5 ± 2.2 to 254.5 ± 9.3 pg/mL (p < 0.0001). The VEGF levels in HSM, HSA and reference groups were found to be significantly decreased. The VEGF levels of HSM were reduced from 684.8 ± 5.1 to 453.5 ± 21.4 pg/mL (p < 0.0001). Similarly, the VEGF levels of the reference group were reduced from 684.8 ± 5.1 to 428.5 ± 8.1 pg/mL (p < 0.0001). Furthermore, the VEGF levels of HSA were reduced from 684.8 ± 5.1 to 584.8 ± 13.5 pg/mL (p < 0.01) (Figure 2). The results of antioxidant parameters A significant decrease was detected in the MDA levels of HSM and the reference groups. HSM shrank the MDA levels from 8.754 ± 1.709 to 5.707 ± 0.476 nmol/g protein (p < 0.05). In addition, letrozole decreased the MDA levels from 8.7 54 ± 1.709 to 5.681 ± 0.338 (p < 0.05). Significant increases were detected in the GSH levels of HSH, HSE, HSM, HSA and reference groups. The highest increase was monitored in HSM among the administered extracts. The GST activities of HSE, HSA and reference groups also significantly increased when compared to the control group. There was no significant change in the GPx activities of groups. The SOD activity of the

group increased significantly (Table II). (A) (B) CONTROL HSH HSE HSM HSA

0 50 100 150 200 250 Endometriotic volume (mm3)  S.E.M. CONTROL HSH HSE HSM HSA

0 50 100 150 200 250 Endometriotic volume (mm3)  S.E.M. *** **** FARMACIA, 2023, Vol. 71, 4 714 (A) (B) Figure 2. The cytokine levels of extracts administered groups (A: Pre-treatment, B: Post-treatment) The values were given as Mean ± S.E.M. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001 CONTROL HSH HSE HSM HSA

0 500 1000 1500 2000 TNF-α Levels (pg/mL) CONTROL HSH HSE HSM HSA

0 100 200 300 400 IL-6 Levels (pg/mL) CONTROL HSH HSE HSM HSA

0 200 400 600 VEGF Levels (pg/mL) CONTROL HSH HSE HSM HSA

0 500 1000 1500 2000 TNF-α Levels (pg/mL) * * CONTROL HSH HSE HSM HSA

0 100 200 300 400 IL-6 Levels (pg/mL) ** **** CONTROL HSH HSE HSM HSA

0 200 400 600 800 VEGF Levels (pg/mL) **** **** ** FARMACIA, 2023, Vol. 71, 4 715 Table II MDA, GSH contents and antioxidant enzyme activities in endometriotic tissue Groups MDA (nmol/g protein) GSH (μmol/mg protein) GST (nmol/g protein) GPx (ng/mg protein) SOD (U/g protein) Control 8.754 ± 1.709 8.493 ± 1.007 2.688 ± 0.470 3.740 ± 0.426 196.100 ± 24.400 HSH 7.914 ± 0.715 22.480 ± 3.308* 5.001 ± 0.854 6.464 ± 2.139 226.500 ± 8.523 HSE 7.514 ± 0.703 18.860 ± 2.639* 5.227 ± 0.550* 4.615 ± 1.252 308.600 ± 40.450 HSM 5.707 ± 0.476* 23.590 ± 1.907** 4.097 ± 0.506 3.434 ± 0.286 282.200 ± 27.670 HSA 6.372 ± 0.470 22.170 ± 2.345** 5.269 ± 0.934* 5.217 ± 1.256 294.400 ± 25.800

5.681 ± 0.338* 31.270 ± 3.323**** 5.416 ± 0.461* 4.492 ± 1.141 321.300 ± 38.400* The values were given as Mean ± S.E.M. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001. Statistical analysis was done in the values of the same column Histological results According to the histopathological examinations, in the control group, endometrial cysts were observed to have an enlarged lumen and stroma. Numerous glands of various sizes were detected in the enlarged cyst stroma. The lumens of cysts and endometrial glands were lined by well-defined columnar epithelium (Figure 3 and Figure 4). Following standard drug administration in the reference control group, moderate to severe histopathological changes such as reduced cyst cavity, reduction of epithelial cells lining the cyst cavity displaying disordered arrangement or desquamation, severe fibrosis in the cyst stroma, degenerated endometrial glands, desquamation in the gland epithelium, hemosiderosis and mononuclear cell infiltration were observed (Figure 3 and Figure 4). Similar regressive changes in endometrial tissues were determined in HSM -, HSA-, HSE- and HSH-treated groups. The severity of histopathological changes increased in the reference group, HSM -, HSA-, HSE- and HSH groups, respectively. TPC and TFC of the extracts According to the results, the highest TPC and TFC were detected in the MeOH extract obtained from H. scabrum aerial parts. The values of TPC and TFC of the MeOH extract were 213.54 ± 0.10 mg GAE/g extract, 88.30 ± 0.24 mg quercetin/g extract, respectively. When TPC of the extracts were sorted from high to low, the sorting was HSM, HSA, HSE and HSM, respectively. The TPCs of the extracts were statistically different from each other. The TFCs of HSH and HSE could not be detected. The TFC of HS M was significantly higher than that of HSA (Table III). Table III Total phenolic and flavonoid contents of the extracts Extracts Total phenolic content (mg GAE/g extract) Total flavonoid content (mg quercetin/g extract) HSH 42.79 ± 0.04d nd HSE 74.28 ± 0.05c nd HSM 213.54 ± 0.10a 88.30 ± 0.24a HSA 106.68 ± 0.04b 46.05 ± 0.07b The values were given as Mean ± S.E.M.; nd: not detected, GAE: Gallic acid; a, b, c, d represents statistically significance LC-MS/MS results According to all of our results, the MeOH extract was found to be the most active extract in the endometriosis rat model. Therefore, the LC -MS/MS studies were conducted on the MeOH extract to determine the compounds that could ameliorate the endometriosis. Quinic acid, gallic acid, protocatechuic acid, catechin, chlorogenic acid, protocatechuic aldehyde, tannic acid, epicatechin, caffeic acid, piceid, salicylic acid, rutin, iso- quercitrin, hesperidin, quercitrin, astragalin, nicotiflorin, quercetin, naringenin, hesperetin, luteolin, kaempferol, apigenin and amentoflavone were detected in the MeOH extract of H. scabrum. The major compounds of the extract were determined to be the quinic acid (103.287 mg/g extract), the chlorogenic acid (9.124 mg/g extract), the epicatechin (6.169 mg/g extract), the quercetin (4.023 mg/g extract), the quercitrin (3.770 mg/g extract), the isoquercitrin (3.122 mg/g extract) and the catechin (1.802 mg/g extract) (Table IV). Furthermore, fumaric acid, aconitic acid, epigallocatechin, gentisic acid, epigallocatechin gallate, cynarin, 4 -OH benzoic acid, vanilic acid, syringic acid, vanil lin, syringic aldehyde, daidzein, epicatechin gallate, p -coumaric acid, ferulic acid, sinapic acid, coumarin, cynaroside, miquelianin, o - coumaric acid, genistin, rosmarinic acid, ellagic acid, cosmosiin, fisetin, genistein, chrysin and acacetin were not detected in the MeOH extract of H. scabrum. FARMACIA, 2023, Vol. 71, 4 716 Figure 3. Views of histological sections of the endometrial tissues from the control (A, B) and reference (C, D, E, F, G and H) groups. A: An endometrial cyst from the control group with numerous glands (g) in various sizes in the stroma (H&E) (100×). B: Well-arranged simple columnar epithelium (ce) lining endometrial cyst lumen (CL) from the control group (H&E) (400×). C: Highly regressed endometrial cyst with narrowed lumen and degenerated gland (dg) from the reference group (H&E) (100×). D: Highly regressed endometrial tissue containing degenerated glands (dg) and fibrous stroma (f) (MT) (100×). E: Reduced epithelium containing exfoliated cells into the cyst lumen (CL) (MT) (400×). F: An endometrial cyst with a narrowed lumen and containing severe fibrosis (f) in the stroma from the reference group (MT) (100×). G, H: Hemosiderosis (hs), fibrosis (f) in subepithelial region and degenerated glands (dg) in cyst stroma (f) from the reference group (200×) (G, H&E; H, MT) FARMACIA, 2023, Vol. 71, 4 717 Figure 4. Views of histopathological findings in the endometrial tissues from animal groups treated with HSM - (A, B), HSA- (C, D, E and F), HSE- (G) and HSH- (H) extracts. A: Reduced epithelium (arrows) lining the cyst lumen and degenerated glands (dg) in the cyst stroma from the group treated with HSM-extract (H&E) (200×). B: Severe fibrosis (f) in cyst stroma from the group treated with HSM-extract (MT) (200×). C: Reduced epithelium (arrow) lining the cyst lumen and degenerated glands (dg) in the cyst stroma from the group treated with HSA-extract (H&E) (200×). D: Hemosiderosis (hs) in the subepithelial stroma in the group treated with HSA-extract (H&E) (400×). E: Severe fibrosis (f) in stroma of a cyst with a narrowed lumen (NCL) from the group treated with HSA-extract (MT) (100×). F: Mononuclear cell infiltration (m) at surrounding areas of a narrowed cyst lumen (NCL) from the group treated with HSA-extract (MT) (200×). G: Reduced and exfoliated epithelium (arrows) lining the cyst lumen and degenerated glands (dg) in the cyst stroma from the group treated with HSE -extract (H&E) (200×). H: Reduced epithelium (arrow) and desquamation of gland epithelium (arrowhead) in the HSH extract treated group (MT) (400×) FARMACIA, 2023, Vol. 71, 4 718 Table IV Quantitative analysis of H. scabrum MeOH extract by LC-MS/MS Compounds Quantity (mg/g extract) 1 Quinic acid 103.287 2 Fumaric acid nd 3 Aconitic acid nd 4 Gallic acid 0.145 5 Epigallocatechin nd 6 Protocatechuic acid 0.483 7 Catechin 1.802 8 Gentisic acid nd 9 Chlorogenic acid 9.124 10 Protocatechuic aldehyde 0.032 11 Tannic acid 0.060 12 Epigallocatechin gallate nd 13 Cynarin nd 14 4-OH Benzoic acid nd 15 Epicatechin 6.169 16 Vanilic acid nd 17 Caffeic acid 0.051 18 Syringic acid nd 19 Vanillin nd 20 Syringic aldehyde nd 21 Daidzin nd 22 Epicatechin gallate nd 23 Piceid 0.011 24 p-Coumaric acid nd 25 Ferulic acid-D3 Internal Standard 26 Ferulic acid nd 27 Sinapic acid nd 28 Coumarin nd 29 Salicylic acid 0.033 30 Cynaroside nd 31 Miquelianin nd 32 Rutin 0.123 33 Rutin-D3 Internal Standard 34 Isoquercitrin 3.122 35 Hesperidin 0.048 36 o-Coumaric acid nd 37 Genistin nd 38 Rosmarinic acid nd 39 Ellagic acid nd 40 Cosmosiin nd 41 Quercitrin 3.770 42 Astragalin 0.128 43 Nicotiflorin 0.026 44 Fisetin nd 45 Daidzein nd 46 Quercetin-D3 Internal Standard 47 Quercetin 4.023 48 Naringenin 0.010 49 Hesperetin 0.230 50 Luteolin 0.044 51 Genistein nd 52 Kaempferol 0.093 53 Apigenin 0.022 54 Amentoflavone 0.313 55 Chrysin nd 56 Acacetin nd nd: not detected H. scabrum is traditionally used for several diseases such as peptic ulcer, gastritis, haemorrhoids, rheumatism, constipation and jaundice [4, 7]. According to Yeşil and Akalın [8], the infusion prepared from the aerial part of H. scabrum is used against menstrual ailments in Malatya, Türkiye. Endometriosis is characterised by the presence of the endometrial layer with glands outside the uterine cavity. It causes infertility, inflammatory reactions and pelvic pain [25, 26]. The symptoms of endometriosis affect the daily activities of women and cause a perception of worsening health status [27]. Furthermore, endometriosis gives rise to severe adhesions in the surrounding tissues [26]. In the present study, it was found that there were severe adhesions in the control group. On the other hand, the adhesions in the HSM group were significantly decreased. Current medical approaches for endometriosis consist of combined oral contraceptives, gonadotropin releasing hormone (GnRH) agonists and androgenic agents [12, 28], as well as some dietary and lifestyle changes, which may relieve symptoms of endometriosis and decrease the severity of the disease [29]. Those medical treatments have numerous undesirable side effects [12]. Therefore, alternative treatment options are needed for the treatment of endometriosis. Letrozole, an aromatase enzyme inhibitor, is an effective compound in the treatment of endometriosis, as endometriosis is an oestrogen-dependent disease [26, 30]. However, it has several side effects including increased bone turnover and an unwanted blood lipid profile [31]. Previous studies ind icated that TNF -α levels were elevated in the peritoneal fluid of women with endometriosis and as the severity of endometriosis increases, TNF-α levels also increase [32-34]. In the present study, the TNF-α levels in the peritoneal fluid were significantly decreased in the MeOH extract administered group. Furthermore, Lu et al. reported that anti TNF -α treatment could be effective for endometriosis [35]. According to Harada et al. [36], IL-6 levels in peritoneal fluid were found to be increased in cases of endometriosis. Similarly, the present study exhibited that IL-6 levels in the control group were elevated. However, the MeOH extract significantly reduced the IL -6 levels in the peritoneal fluid. According to previous studies, VEGF induces angio- genesis [37] and also increases the vascularization [38]. Other studies showed that women with endometriosis have high VEGF levels in their peritoneal fluid [39]. Furthermore, numerous studies have shown that VEGF expression increases in cases of endometriosis [40, 41]. Similarly, VEGF levels in the peritoneal fluids of the control group were increased in the present study. The MeOH extract obtained from H. scabrum, which is the most active extract in our study significantly, decreased the VEGF levels in th e rat endometriosis model. In addition to MeOH extract, the aqueous extract of H. scabrum also reduced VEGF levels. However, FARMACIA, 2023, Vol. 71, 4 719 the decrease in the HSM group was higher than that of the HSA. Oxidative stress (OS) is explained as an imbalance between antioxida nts and reactive oxygen species. Recently published articles exhibited that OS could be implicated in the endometriosis pathophysiology and also cause an inflammatory response in the peritoneal cavity [42, 43]. Previous studies investigated the correlation between MDA, a marker of lipid peroxidation and endometriosis, and found that MDA levels were increased in serum and endometriotic lesions of women with endometriosis when compared to healthy women [43, 44]. Similarly, the MDA levels were the highest in the control group in our study and the MeOH extract significantly reduced the MDA levels in the endometriotic lesions. According to Prieto et al. [45], SOD activity decreases in cases of endometriosis in women. In the present study, the SOD activity of the control group was lower than that of other groups. Mashayekhi et al. [46] and Mate et al. [47] indicated that the concentrations of GST significantly declined according to severity of endometriosis. The present study found that GST activities in extracts and letrozole administered groups increased. Singh et al. [48] stated that GPx activity decreases in endometriosis tissue samples. All extracts and letrozole increased the GPx activity except the MeOH extract, but those increases were statistically significant in none of the groups. Chlorogenic acid is one of the major compounds in the MeOH extract prepared from H. scabrum. Ayan et al. and Llorent-Martínez et al. reported the presence of chlorogenic acid in H. scabrum [5, 6]. Kim et al. [49] indicated that chlorogenic acid has anti -angiogenic effects on choroidal neovascularization and Saltan et al. [50] found that Viburnum opulus L. showed significant activity in a rat endometriosis model, the main compound being the chlorogenic acid. Furthermore, quercetin, quercitrin and isoquercitrin were major compounds in the MeOH extract of H. scabrum. Park et al. [51] demonstrated that quercetin could be used to reduce and treat human endometriosis. According to Oner et al. [26], the medical therapy for endometriosis is often aimed at lowering estrogen levels. Le Bail et al. [52] and Collins-Burow et al. [53] indicated that flavonoids have anti-estrogenic activity. Furthermore, flavonoids have antiangiogenic activities [54]. Bina et al. found that Achillea cretica L. aerial part showed promising activity on endometriosis. According to the LC-MS analysis of A. cret ica aerial extract, some quinic acid derivatives were found in this extract [55]. Xu et al. investigated the effect of epigallocatechin gallate (EGCG) on an experimental endometriosis mouse model. They found that EGCG significantly inhibits the development of experimental endometriosis through its anti-angiogenic effects [56]. In our study, epicatechin was detected as one of the major compounds in the MeOH extract. Therefore, epicatechin may have played a role in decreasing endometriotic implant volumes.

As a conclusion, the MeOH extract obtained from H. scabrum, which is used against menstrual disorders in folk medicine, exhibited prominent activity in the surgically induced endometriosis model. The MeOH extract significantly decreased adhesion scores, endometrial foci areas, and cytokine levels and exhibited a good antioxidant activity. LC-MS/MS results showed the main compounds of the MeOH extract are mainly phenolic acids and flavonoids. Therefore, this prominent activity of the MeOH extract could be attributed to its phenolic acids and flavonoids. Chlorogenic acid was detected as a major phenolic acid in the MeOH extract, whereas quercetin was detected as a major flavonoid.

This research was funded by Van Yüzüncü Yıl University Scientific Research Project Commission (Grant number: TYD-2021-9253). Conflict of interest The authors declare no conflict of interest.

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