{"paper_id":"0a59f614-c776-42e6-a7b4-c4801c4c691f","body_text":"FARMACIA, 2023, Vol. 71, 4 \n 710 \nhttps://doi.org/10.31925/farmacia.2023.4.7 ORIGINAL ARTICLE \nEXPLORING THE AMELIORATIVE EFFECTS OF HYPERICUM \nSCABRUM L. ON A SURGICALLY-INDUCED ENDOMETRIOSIS RAT \nMODEL AND ITS PHYTOCHEMICAL PROFILING BY LC-MS/MS \n  \nMERT ILHAN 1,2*, ABDULHAMIT BATTAL 3, BURAK KAPTANER 4, ABDULAHAD DOGAN 5, \nFATIH DONMEZ 5, MUSTAFA ABDULLAH YILMAZ 6, HUSEYIN EROGLU 4 \n \n1Department of Pharmacognosy, Faculty of Pharmacy, Düzce University, 81620, Düzce, Türkiye \n2Department of Pharmacognosy, Faculty of Pharmacy, Van Yüzüncü Yıl University, 65080, Tusba, Van, Türkiye \n3Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Van Yüzüncü Yıl University, 65080, Tusba, Van, Türkiye \n4Department of Biology, Faculty of Science, Van Yüzüncü Yıl University, 65080, Tusba, Van, Türkiye \n5Department of Biochemistry, Faculty of Pharmacy, Van Yüzüncü Yıl University, 65080, Tusba, Van, Türkiye \n6Department of Analytical Chemistry, Faculty of Pharmacy, Dicle University, 21280, Diyarbakır, Türkiye \n \n*corresponding author: mertilhan@duzce.edu.tr \nManuscript received: March 2023 \n \nAbstract  \nEndometriosis is described as the existence and expansion of functional endometrial -like tissues in another part of the uterine \ncavity. It affects not only postmenopausal women but also teenagers after menarche, causing subfertility, dysmenorrhea and \nsevere pelvic pain. Since no specific treatment is acknowledged for this disease, the present study aimed to investigate the \nefficacy of H. scabrum  in a surgically -induced endometriosis rat model. In order to test the efficacy of H. scabrum on \nendometriosis, n-hexane, ethyl acetate (EtOAc), methanol (MeOH) and aqueous extracts were prepared from the aerial parts \nof H. scabrum. The cytokine levels, antioxidant enzyme activities, the contents of malondialdehyde (MDA) and glutathione \n(GSH) and histopathological ana lyses were evaluated. In order to reveal the potential active compounds, liquid \nchromatography-mass/mass spectroscopy (LC -MS/MS) analysis was performed on the MeOH extract who presented  the \nhighest activity. The MeOH extract significantly decreased the adhesion scores, endometriotic implant volumes, cytokine levels \nand the antioxidant parameters. Furthermore, histological procedures supported those findings. According to LC -MS/MS \nanalysis of the MeOH extract, the major compounds were mostly phenolic acids and flavonoids. We concluded that the MeOH \nextract from H. scabrum aerial parts could be used for the treatment of endometriosis, and its activity could be attributed to the \nchlorogenic acid and quercetin, detected in the highest amounts. \n \nRezumat \nEndometrioza se caracterizează prin prezența și proliferarea țesuturilor funcționale asemănătoare endometrului în locații din  \nafara limitelor cavității uterine. Această patologie afectează femeile aflate în postmenopauză și adolescentele după menstruație. \nDeoarece nu există un tratament specific pentru endometrioză, am investigat în acest studiu eficacitatea speciei Hypericum \nscabrum într-un model de endometrioză indusă chirurgical la șobolan. Astfel au fost preparate diferite extracte, în n -hexan, \nacetat de etil (EtOAc), metanol (MeOH) și apă, din părțile aeriene de H. scabrum. După tratament, au fost evaluate scorurile \nde aderență, volumele de implant endometrial, nivelurile de citokine, activitățile enzimelor antioxidante, conținutul de \nmalondialdehidă (MDA) și glutation (GSH) și au fost întreprinse analize histopatologice. Pentru a descoperi potențialii compuși \nactivi, s-a realizat o analiză de cromatografie lichidă -spectroscopie de masă(LC-MS/MS) folosind extractul metanolic, care a \nprezentat cea mai mar e activitate. Extractul MeOH din părțile aeriene de H. scabrum  poate fi utilizat în tratamentul \nendometriozei, iar activitatea sa ar putea fi atribuită acizilor fenolici, respectiv acidului clorogenic, și flavonoidelor, r espectiv \nquercetinei, compuși majoritari identificați. \n \nKeywords: antioxidant, cytokines, endometriosis, ethnopharmacology, Hypericum scabrum, LC-MS/MS \n \nIntroduction \nThe genus Hypericum L., which belongs to the \nHypericaceae family, includes approximately 500 \naccepted species. The flowers of the Hypericum genus \nexhibit bisexual characteristics, with five sepals and \nfive petals. The petals are usually yellow, sometimes \ntinged red. In Türkiye, there are 69 species of Hypericum \n[1]. Our plant material, Hypericum scabrum L., is \nwidely distributed in Türkiye [2]. The stems of the \nplant are glabrous, scabrid with unbranched red-gland-\ntipped emergences. The dimensions of the plant's \nleaves range from 7 to 20 mm and exhibit an oblong \nto lanceolate shape. Similarly, the sepals are oblong \nin form, while the petals measure between 5 and 7 mm \nin length. H. scabrum  grows usually on dry rocky \nslopes, open woodland or steppe [1]. To date, various \ncompounds such as tannins, phloroglucinols, phenyl-\npropanes, flavonoids and proanthocyanins have been \nextracted and identified from the Hypericum genus [3, 4]. \n\nFARMACIA, 2023, Vol. 71, 4 \n 711 \nPseudohypericin and hypericin were detected only in \nthe flowers of H. scabrum . In addition to these \ncompounds, kaempferol, quercetin and amentoflavone \nwere observed in the flowers of the plant [5]. Quercetin, \nchlorogenic acid, apigenin -7-0-glucoside, rutin  and \nhyperoside were detected in the leaves of the plant \n[5, 6]. H. scabrum is traditionally used for peptic ulcer, \ngastritis, haemorrhoids, rheumatism, constipation and \njaundice [4, 7]. Furthermore, Yeşil and Akalın reported \nthat the infusion prepared fro m the aerial part of H. \nscabrum is used for menstrual ailments in Malatya, \nTürkiye [8]. \nEndometriosis is described as the existence and \nexpansion of functional endometrial -like tissues in \nanother place than the uterine cavity [9]. Both its \naetiology and pathogenesis have not been completely \nexplained until now. It affects not only postmenopausal \nwomen but also teenagers after menarche [10]. It has \nbeen observed that this condition leads to subfertility, \ndysmenorrhea and intense pelvic pain [11]. It ranks \nthird in terms of hospitalisations for gynaecological \nconditions in the United States. Furthermore, it is \nleading to an economic burden due to healthcare costs \nworldwide [9]. Recently, the acceptable tre atment \nmethods for endometriosis have been medication, \nsurgery or a combination of medication and surgery. \nHowever, the recurrence rate of the endometriosis is \naround 7 - 30% following the surgery [9, 12]. Due to \nthe severe undesirable effects, the economic burden of \nendometriosis, and the recent interest in this pathology, \nthe use of natural sources to treat itis considered an \nalternative approach. Because of its usage in folk \nmedicine, H. scabrum was selected for this purpose. \nTherefore, the aim of this s tudy is to evaluate the \nefficacy of H. scabrum aerial parts on the endometriosis \nrat model. \n \nMaterials and Methods \nPlant material \nH. scabrum was collected from the Van-Başkale road-\nside, Güzeldere district, Türkiye, on July 6 th, 2020 \n(38°11'04.7\"N 43°54'34.5\"E). It was authenticated by \nHüseyin Eroğlu, PhD from the Department of Botany, \nFaculty of Science, Van Yüzüncü Yıl University, \nTürkiye. The herbarium specimens were kept in the \nherbarium of Van Yüzüncü Yıl University, Türkiye \n(VANF165216). \nExtraction method \nThe aerial part of H. scabrum, weighing 600 g, was \nsubjected to a drying process in a shaded environment. \nSubsequently, the plant material was pulverised \nusing a grinder. A sample weighing 600 g of \npowdered and dried H. scab rum was subjected to \nextraction using n-hexane (5 L), ethyl acetate (EtOAc) \n(5 L) and methanol (MeOH) (5 L), respectively. The \nplant was extracted three times with each solvent. The \nextraction was evaporated at 40°C under reduced \npressure by a rotary evaporator until dryness. A sample \nof 100 g of H. scabrum aerial parts was extracted with \nhot water (1 L) for the preparation of the infusion. \nThe extraction was frozen at -20°C and lyophilized. \nAfter all of these procedures, n-hexane (HSH), EtOAc \n(HSE), MeOH ( HSM) and aqueous (HSA) extracts \nwere obtained with yields of 4.3%, 2.2%, 13.7% and \n16.2%, respectively. \nAnimals and ethic approval \nFor the purpose of this experiment, female rats of the \nWistar albino strain weighing between 200 - 250 g \nwere selected. Durin g the experiment, the animals \nwere housed in appropriate cages. The temperature, \nhumidity, and luminosity were controlled in this process. \nThis study was approved by the Experimental Animal \nEthics Committee of Van Yüzüncü Yıl University, \nTürkiye (Approval No. 2020/01). All experimental \nand surgical procedures were conducted in accordance \nwith the National Institutes of Health Guide for the \nCare and Use of Laboratory Animals. \nExperimental procedure of rat endometriosis model \nThis procedure was conducted according to Vernon \nand Wilson [13]. All the rats were anaesthetized with \nintramuscular administration of the combination of \nketamine and xylazine. Under anaesthesia, the abdomen \nwas opened by creating a 3 cm incision using a scalpel \nblade, and the subcutaneous and muscle layers were \nseparated from each other. The right uterine cornu  \nwere ligated with polyglactin absorbable suture (USP \n4/0) from its two margins. The right uterine cornu  \nwas cut and a piece of 1.5 cm tissue was taken. The \ntissue fragment was cleaned in 0.9% saline solution \nand opened lengthwise. A fragment of endometrial \ntissue was stitched with polyglactin absorbable suture \n(USP 4/0) to the abdominal wall of the same rat \naround the blood vessel. The abdomen was sewn with \na silk suture (USP 3/0). Twenty -eight days after the \nsurgical procedure, a second surgery was done. In this \nsurgical procedure, adhesion scores and endometriotic \nimplant volumes were evaluated. In addition, peritoneal \nfluids were collected, and t he abdomen was closed \nwith a silk suture (USP 3/0). For the treatment \nprocedure, 42 rats were divided randomly into 6 groups \n(n = 7), namely control, n-hexane, EtOAc, MeOH, \naqueous and reference. \nTreatment procedure \nTwo days after the second experiment, 0.5% carboxy-\nmethylcellulose was administered to the control group. \nLetrozole (0.2 mg/kg) was applied to the reference \ngroup. The n-hexane, EtOAc, MeOH and aqueous \nextracts were administered to the treatment groups at a \ndosage of 100 mg/kg. After the treatment procedure, \nthe 3rd operation was done. In this surgery procedure, \nadhesion scores and endometrial foci areas were re -\ncalculated, and peritoneal fluids were collected for \nthe evaluation of cytokine levels. \n \n\nFARMACIA, 2023, Vol. 71, 4 \n 712 \nThe evaluation of adhesion scores and the calculation \nof endometriotic foci areas \nThe adhesion scores were evaluated according to the \nscoring system of Blauer and Collins [14] as follows: \n0, no adhesions; 1, thin adhesions; 2, thick adhesion in \none area; 3, widespread thick adhesions; 4, adhesions \nof internal organs to the abdominal wall. \nThe endometrial implant volumes were calculated \naccording to the following formula: π/6 x length x \nwidth x height [15]. \nThe detection of cytokine levels \nTumour necrosis factor alpha (TNF -α), vascular \nendothelial growth factor (VEGF) and interleukin -6 \n(IL-6) levels in the peritoneal fluids were assessed \nquantitatively by enzyme-linked immunosorbent assay \n(ELISA) kits. The catalogue numbers of the ELISA \nkits were E-EL-R0019 (Elabscience®), E-EL-R2603 \n(Elabscience®) and E -EL-R0015 (Elabscience ®) for \nTNF-α, VEGF and IL-6, respectively. All procedures \nwere performed according to the ELISA kit user \nmanuals. \nThe detection of malondialdehyde (MDA), glutathione \n(GSH) levels and antioxidant enzyme activities \nThe levels of MDA, which is a marker for oxidative \nstress, were determined according to a previous published \nmethod [16]. GSH levels were calculated according to \nthe spectrophotometric method of Beutler et al. [17]. \nThe activity determination of glutathione-S-transferase \n(GST) was based on the changes in absorbance read \nat 340 nm because of the creation of the GSH-CDNB \ncomplex [18]. Glutathione peroxidase (GPx) enzyme \nactivity was measured spectrophotometrically at 450 \nnm by a commercially available ELISA kit following \nthe manufacturer’s instructions (SunRed Biotechnology \nCompany, catalogue no. DZE201-11-5104). Superoxide \ndismutase (SOD) enzyme activity was determined by \nusing SOD (Ransod) assay kit (Randox catalogue no. \nSD125). \nTotal phenolic (TPC) and flavonoid content (TFC) \nmeasurement of the extracts \nFor the total phenolic content determination, the extracts \nwere prepared at a concentration of 2 mg/mL in ethanol \n(75%). A 20 μL sample was transferred in the test \ntube and distilled water (1580 μL) was a dded. Then \n100 μL Folin-Ciocâlteu phenol reagent and 20% sodium \ncarbonate (300 μL) were added, respectively. All samples \nwere incubated at 40°C for 30 min. The absorbances \nof the samples were read at 765 nm in the spectro -\nphotometry. For the calibration curve, gallic acid \n(GA) was used, and the total phenolic contents of the \nextracts were calculated as mg GA per g of extract [19]. \nFor the  total flavonoid content determination, the \nextracts were prepared at a concentration of 2 mg/mL \nin 75% ethanol. A 500 μL sample was put in the test \ntube along with 75% ethanol (1500 μL), 10% aluminium \nchloride (100 μL), 1 M sodium acetate (100 μL) and \ndistilled water (2800 μL), respectively. All samples \nwere incubated at room temperature for 30 min. The \nabsorbances of the samples were read at 415 nm in the \nspectrophotometry. For the calibration curve, quercetin \nwas used, and the total flavonoid contents of the extracts \nwere calculated as mg quercetin per g of extract [20]. \nLC-MS/MS studies \nLC-MS/MS studies were carried out according to \nYilmaz’s method, which is a validated study [21]. \nHistological procedures \nThe endometrium samples were excised and immediately \nfixed in 10% neutral buffered formalin for 48 h at 4°C. \nAfter rinsing the samples with phosphate buffered \nsaline solution at a pH of 7.4, a series of ethanol, with \nincreasing concentrations, was employed to remove \nwater from the tissues, which were subsequently \nimmersed in paraffin wax for embedding. As a next  \nstep, 5 µm thick tissue sections were taken using a \nMICROM HM 325 manual microtome (Waldorf, \nGermany) and then the sections were put onto adhesive-\ncoated slides (Marienfeld GmbH, Lauda-Königshofen, \nGermany). Following deparaffinization and rehydration, \nthe sections were stained with haematoxylin and eosin \n(H&E) or Mallory’s triple (MT) [22]. Histopathological \nexamination of the sections and photography were \nconducted using Leica DMI 6000B and DM500 model \nmicroscopes (Leica Microsystems, Wetzlar, Germany). \nPhotographs were captured at 100×, 200× or 400× \nmagnifications. The histopathological alterations in \nendometriotic tissues were determined according to \nthe criteria described by Demirel et al. and Elgamal \net al. [23, 24]. \nStatistical analysis \nThe Kolmogoro v-Smirnov test was carried out to \ndetermine whether the distribution is normal or not. \nThe Kruskal-Wallis test was performed for the evaluation \nof the adhesion scores. For the other parameters, a one-\nway ANOVA-Dunnett’s post hoc test was performed. \nStatistical significance was determined by comparing \nall groups to the control group, with a significance \nlevel of p < 0.05. When the groups were compared \nwith each other especially pre -treatment values, the \nANOVA-Tukey post hoc test was used. An ANOVA-\nTukey post hoc test was used for the total phenolic \ncontent tests whereas Student's t-test was used for \nthe total flavonoid content tests. \n \nResults and Discussion \nThe evaluation of adhesion scores \nThe adhesion scores were evaluated according to the \nmethod of Blauer and Collins [14]. The adhesion scores \nwere significantly decreased in the MeOH and letrozole \nadministered groups. The MeOH extract decreased \nadhesion score from 3.900 ± 0.100 to 2.214 ± 0.395 \n(p < 0.05) while the letrozole decreased adhesion score \nfrom 3.900 ± 0.100 to 1.643 ± 0.325 (p < 0.001) when \ncompared to the control group. There was no significant \ndifference between the control and the remaining \ngroups in the adhesion scores (Table I). \n\nFARMACIA, 2023, Vol. 71, 4 \n 713 \nTable I \nAdhesion scores of tested groups in endometriosis \nmodel \nGroups Mean adhesion scores ± S.E.M. \nPre-Treatment Post-Treatment \nControl 3.857 ± 0.142 3.900 ± 0.100 \nHSH 3.571 ± 0.250 3.083 ± 0.358 \nHSE 3.500 ± 0.174 3.167 ± 0.366 \nHSM 3.538 ± 0.183 2.214 ± 0.395* \nHSA 3.857 ± 0.097 3.462 ± 0.243 \nReference 3.714 ± 0.163 1.643 ± 0.325*** \nThe values were given as Mean ± S.E.M. *: p < 0.05, **: p < \n0.01, ***: p < 0.001,  \n \nThe evaluation of endometriotic foci areas \nThe endometriotic implant volumes were significantly \nreduced in the HSM and reference groups. The MeOH \nextract decreased endometriotic implant volume from \n188.1 ± 30.9 to 53.4 ± 16.7 mm3 (p < 0.001) whereas \nletrozole decreased endometriotic implant volume from \n188.1 ± 30.9 to 11.2 ± 1.5 mm 3 (p < 0.0001). The \ndecreases were observe d in endometriotic implant \nvolumes in the HSH, HSE and HSA groups, but those \ndecreases were not statistically significant. Furthermore, \nthere was no statistical difference among the pre -\ntreatment endometriotic implant volumes of the groups \n(Figure 1). \n \n \nFigure 1. \nEndometriotic implant volumes of extracts administered groups (A: Pre-treatment, B: Post-treatment) \nThe values were given as Mean ± S.E.M. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001 \n \nThe evaluation of cytokine levels in peritoneal fluids \nFirstly, the cytokine levels of the pre-treatment were \ncompared with each other. Any significant difference \nwas not observed among the cytokine levels of pre -\ntreatment (Figure 2). Then, post -treatment cytokine \nlevels were compared. \nThe TNF-α levels of HSM and reference groups were \nsignificantly reduced. The MeOH extract decreased \nTNF-α levels from 1613.2 ± 190.4 to 1145.2 ± 56.9 \npg/mL (p < 0.05) while letrozole decreased TNF -α \nlevels from 1613.2 ± 190.4 to 1121.2 ± 99.0 pg/mL \n(p < 0.05). The decrease in TNF -α levels in other \ngroups was detected, but it was not statistically \nsignificant.  \nThe IL-6 levels of HSM and reference groups were \nfound to be decreased when compared to the control \ngroup. The IL -6 level of HSM was reduced from \n329.5 ± 2.2 to 280.7 ± 7.8 pg/mL (p < 0.01) whereas \nthe IL-6 level of letrozole was reduced from 329.5 ± \n2.2 to 254.5 ± 9.3 pg/mL (p < 0.0001). \nThe VEGF levels in HSM, HSA and reference groups \nwere found to be significantly decreased. The VEGF \nlevels of HSM were reduced from 684.8 ± 5.1 to \n453.5 ± 21.4 pg/mL (p < 0.0001). Similarly, the VEGF \nlevels of the reference group were reduced from 684.8 \n± 5.1 to 428.5 ± 8.1 pg/mL (p < 0.0001). Furthermore, \nthe VEGF levels of HSA were reduced from 684.8 ± \n5.1 to 584.8 ± 13.5 pg/mL (p < 0.01) (Figure 2). \nThe results of antioxidant parameters \nA significant decrease was detected in the MDA levels \nof HSM and the reference groups. HSM shrank the \nMDA levels from 8.754 ± 1.709 to 5.707 ± 0.476 \nnmol/g protein (p < 0.05). In addition, letrozole \ndecreased the MDA levels from 8.7 54 ± 1.709 to \n5.681 ± 0.338 (p < 0.05). Significant increases were \ndetected in the GSH levels of HSH, HSE, HSM, \nHSA and reference groups. The highest increase was \nmonitored in HSM among the administered extracts. \nThe GST activities of HSE, HSA and reference groups \nalso significantly increased when compared to the \ncontrol group. There was no significant change in the \nGPx activities of groups. The SOD activity of the \nreference group increased significantly (Table II). \n \n    (A)      (B) \nCONTROL\nHSH\nHSE\nHSM\nHSA\nREFERENCE\n0\n50\n100\n150\n200\n250\nEndometriotic volume (mm3)\n\nS.E.M.\nCONTROL\nHSH\nHSE\nHSM\nHSA\nREFERENCE\n0\n50\n100\n150\n200\n250\nEndometriotic volume (mm3)\n\nS.E.M.\n***\n****\n\nFARMACIA, 2023, Vol. 71, 4 \n 714 \n \n  \n \n(A) \n  \n \n(B) \nFigure 2. \nThe cytokine levels of extracts administered groups (A: Pre-treatment, B: Post-treatment) \nThe values were given as Mean ± S.E.M. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001 \n \nCONTROL\nHSH\nHSE\nHSM\nHSA\nREFERENCE\n0\n500\n1000\n1500\n2000\nTNF-α Levels (pg/mL)\nCONTROL\nHSH\nHSE\nHSM\nHSA\nREFERENCE\n0\n100\n200\n300\n400\nIL-6 Levels (pg/mL)\nCONTROL\nHSH\nHSE\nHSM\nHSA\nREFERENCE\n0\n200\n400\n600\nVEGF Levels (pg/mL)\nCONTROL\nHSH\nHSE\nHSM\nHSA\nREFERENCE\n0\n500\n1000\n1500\n2000\nTNF-α Levels (pg/mL)\n* *\nCONTROL\nHSH\nHSE\nHSM\nHSA\nREFERENCE\n0\n100\n200\n300\n400\nIL-6 Levels (pg/mL)\n** ****\nCONTROL\nHSH\nHSE\nHSM\nHSA\nREFERENCE\n0\n200\n400\n600\n800\nVEGF Levels (pg/mL)\n**** ****\n**\n\nFARMACIA, 2023, Vol. 71, 4 \n 715 \nTable II \nMDA, GSH contents and antioxidant enzyme activities in endometriotic tissue \nGroups MDA \n(nmol/g protein) \nGSH \n(μmol/mg protein) \nGST \n(nmol/g protein) \nGPx \n(ng/mg protein) \nSOD \n(U/g protein) \nControl 8.754 ± 1.709 8.493 ± 1.007 2.688 ± 0.470 3.740 ± 0.426 196.100 ± 24.400 \nHSH 7.914 ± 0.715 22.480 ± 3.308* 5.001 ± 0.854 6.464 ± 2.139 226.500 ± 8.523 \nHSE 7.514 ± 0.703 18.860 ± 2.639* 5.227 ± 0.550* 4.615 ± 1.252 308.600 ± 40.450 \nHSM 5.707 ± 0.476* 23.590 ± 1.907** 4.097 ± 0.506 3.434 ± 0.286 282.200 ± 27.670 \nHSA 6.372 ± 0.470 22.170 ± 2.345** 5.269 ± 0.934* 5.217 ± 1.256 294.400 ± 25.800 \nReference 5.681 ± 0.338* 31.270 ± 3.323**** 5.416 ± 0.461* 4.492 ± 1.141 321.300 ± 38.400* \nThe 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 \nvalues of the same column \n \nHistological results \nAccording to the histopathological examinations, in \nthe control group, endometrial cysts were observed \nto have an enlarged lumen and stroma. Numerous \nglands of various sizes were detected in the enlarged \ncyst stroma. The lumens of cysts and endometrial \nglands were lined by well-defined columnar epithelium \n(Figure 3 and Figure 4). Following standard drug \nadministration in the reference control group, moderate \nto severe histopathological changes such as reduced \ncyst cavity, reduction of epithelial cells lining the cyst \ncavity displaying disordered arrangement or desquamation, \nsevere fibrosis in the cyst stroma, degenerated endometrial \nglands, desquamation in the gland epithelium, hemosiderosis \nand mononuclear cell infiltration were observed (Figure 3 \nand Figure 4). Similar regressive changes in endometrial \ntissues were determined in HSM -, HSA-, HSE- and \nHSH-treated groups. The severity of histopathological \nchanges increased in the reference group, HSM -, \nHSA-, HSE- and HSH groups, respectively. \nTPC and TFC of the extracts \nAccording to the results, the highest TPC and TFC \nwere detected in the MeOH extract obtained from H. \nscabrum aerial parts. The values of TPC and TFC of \nthe MeOH extract were 213.54 ± 0.10 mg GAE/g \nextract, 88.30 ± 0.24 mg quercetin/g extract, respectively. \nWhen TPC of the extracts were sorted from high to \nlow, the sorting was HSM, HSA, HSE and HSM, \nrespectively. The TPCs of the extracts were statistically \ndifferent from each other. The TFCs of HSH and \nHSE could not be detected. The TFC of HS M was \nsignificantly higher than that of HSA (Table III). \nTable III \nTotal phenolic and flavonoid contents of the extracts \nExtracts Total phenolic content \n(mg GAE/g extract) \nTotal flavonoid content \n(mg quercetin/g extract) \nHSH 42.79 ± 0.04d nd \nHSE 74.28 ± 0.05c nd \nHSM 213.54 ± 0.10a 88.30 ± 0.24a \nHSA 106.68 ± 0.04b 46.05 ± 0.07b \nThe values were given as Mean ± S.E.M.; nd: not detected, GAE: Gallic acid; a, b, c, d represents statistically significance \n \nLC-MS/MS results \nAccording to all of our results, the MeOH extract was \nfound to be the most active extract in the endometriosis \nrat model. Therefore, the LC -MS/MS studies were \nconducted on the MeOH extract to determine the \ncompounds that could ameliorate the endometriosis. \nQuinic acid, gallic acid, protocatechuic acid, catechin, \nchlorogenic acid, protocatechuic aldehyde, tannic acid, \nepicatechin, caffeic acid, piceid, salicylic acid, rutin, iso-\nquercitrin, hesperidin, quercitrin, astragalin, nicotiflorin, \nquercetin, naringenin, hesperetin, luteolin, kaempferol, \napigenin and amentoflavone were detected in the \nMeOH extract of H. scabrum. \nThe major compounds of the extract were determined \nto be the quinic acid (103.287 mg/g extract), the \nchlorogenic acid (9.124 mg/g extract), the epicatechin \n(6.169 mg/g extract), the quercetin (4.023 mg/g extract), \nthe quercitrin (3.770 mg/g extract), the isoquercitrin \n(3.122 mg/g extract) and the catechin (1.802 mg/g \nextract) (Table IV). Furthermore, fumaric acid, aconitic \nacid, epigallocatechin, gentisic acid, epigallocatechin \ngallate, cynarin, 4 -OH benzoic acid, vanilic acid, \nsyringic acid, vanil lin, syringic aldehyde, daidzein, \nepicatechin gallate, p -coumaric acid, ferulic acid, \nsinapic acid, coumarin, cynaroside, miquelianin, o -\ncoumaric acid, genistin, rosmarinic acid, ellagic acid, \ncosmosiin, fisetin, genistein, chrysin and acacetin were \nnot detected in the MeOH extract of H. scabrum. \n \n\nFARMACIA, 2023, Vol. 71, 4 \n 716 \n \nFigure 3. \nViews of histological sections of the endometrial tissues from the control (A, B) and reference (C, D, E, F, G and \nH) groups. A: An endometrial cyst from the control group with numerous glands (g) in various sizes in the stroma \n(H&E) (100×). B: Well-arranged simple columnar epithelium (ce) lining endometrial cyst lumen (CL) from the \ncontrol group (H&E) (400×). C: Highly regressed endometrial cyst with narrowed lumen and degenerated gland \n(dg) from the reference group (H&E) (100×). D: Highly regressed endometrial tissue containing degenerated glands \n(dg) and fibrous stroma (f) (MT) (100×). E: Reduced epithelium containing exfoliated cells into the cyst lumen \n(CL) (MT) (400×). F: An endometrial cyst with a narrowed lumen and containing severe fibrosis (f) in the stroma \nfrom the reference group (MT) (100×). G, H: Hemosiderosis (hs), fibrosis (f) in subepithelial region and \ndegenerated glands (dg) in cyst stroma (f) from the reference group (200×) (G, H&E; H, MT)  \n \n\n\nFARMACIA, 2023, Vol. 71, 4 \n 717 \n \nFigure 4. \nViews of histopathological findings in the endometrial tissues from animal groups treated with HSM - (A, B), \nHSA- (C, D, E and F), HSE- (G) and HSH- (H) extracts. A: Reduced epithelium (arrows) lining the cyst lumen \nand degenerated glands (dg) in the cyst stroma from the group treated with HSM-extract (H&E) (200×). B: Severe \nfibrosis (f) in cyst stroma from the group treated with HSM-extract (MT) (200×). C: Reduced epithelium (arrow) \nlining the cyst lumen and degenerated glands (dg) in the cyst stroma from the group treated with HSA-extract \n(H&E) (200×). D: Hemosiderosis (hs) in the subepithelial stroma in the group treated with HSA-extract (H&E) \n(400×). E: Severe fibrosis (f) in stroma of a cyst with a narrowed lumen (NCL) from the group treated with \nHSA-extract (MT) (100×). F: Mononuclear cell infiltration (m) at surrounding areas of a narrowed cyst lumen \n(NCL) from the group treated with HSA-extract (MT) (200×). G: Reduced and exfoliated epithelium (arrows) \nlining the cyst lumen and degenerated glands (dg) in the cyst stroma from the group treated with HSE -extract \n(H&E) (200×). H: Reduced epithelium (arrow) and desquamation of gland epithelium (arrowhead) in the HSH \nextract treated group (MT) (400×) \n \n\n\nFARMACIA, 2023, Vol. 71, 4 \n 718 \nTable IV \nQuantitative analysis of H. scabrum MeOH extract \nby LC-MS/MS \n Compounds Quantity (mg/g extract) \n1 Quinic acid 103.287 \n2 Fumaric acid nd \n3 Aconitic acid nd \n4 Gallic acid 0.145 \n5 Epigallocatechin nd \n6 Protocatechuic acid 0.483 \n7 Catechin 1.802 \n8 Gentisic acid nd \n9 Chlorogenic acid 9.124 \n10 Protocatechuic aldehyde 0.032 \n11 Tannic acid 0.060 \n12 Epigallocatechin gallate nd \n13 Cynarin nd \n14 4-OH Benzoic acid nd \n15 Epicatechin 6.169 \n16 Vanilic acid nd \n17 Caffeic acid 0.051 \n18 Syringic acid nd \n19 Vanillin nd \n20 Syringic aldehyde nd \n21 Daidzin nd \n22 Epicatechin gallate nd \n23 Piceid 0.011 \n24 p-Coumaric acid nd \n25 Ferulic acid-D3 Internal Standard \n26 Ferulic acid nd \n27 Sinapic acid nd \n28 Coumarin nd \n29 Salicylic acid 0.033 \n30 Cynaroside nd \n31 Miquelianin nd \n32 Rutin 0.123 \n33 Rutin-D3 Internal Standard \n34 Isoquercitrin 3.122 \n35 Hesperidin 0.048 \n36 o-Coumaric acid nd \n37 Genistin nd \n38 Rosmarinic acid nd \n39 Ellagic acid nd \n40 Cosmosiin nd \n41 Quercitrin 3.770 \n42 Astragalin 0.128 \n43 Nicotiflorin 0.026 \n44 Fisetin nd \n45 Daidzein nd \n46 Quercetin-D3 Internal Standard \n47 Quercetin 4.023 \n48 Naringenin 0.010 \n49 Hesperetin 0.230 \n50 Luteolin 0.044 \n51 Genistein nd \n52 Kaempferol 0.093 \n53 Apigenin 0.022 \n54 Amentoflavone 0.313 \n55 Chrysin nd \n56 Acacetin nd \nnd: not detected \n \nH. scabrum is traditionally used for several diseases \nsuch as peptic ulcer, gastritis, haemorrhoids, rheumatism, \nconstipation and jaundice  [4, 7]. According to Yeşil \nand Akalın [8], the infusion prepared from the aerial \npart of H. scabrum is used against menstrual ailments \nin Malatya, Türkiye. \nEndometriosis is characterised by the presence of the \nendometrial layer with glands outside the uterine cavity. \nIt causes infertility, inflammatory reactions and pelvic \npain [25, 26]. The symptoms of endometriosis affect \nthe daily activities of women and cause a perception of \nworsening health status [27]. Furthermore, endometriosis \ngives rise to severe adhesions in the surrounding tissues \n[26]. In the present study, it was found that there were \nsevere adhesions in the control group. On the other \nhand, the adhesions in the HSM group were significantly \ndecreased. Current medical approaches for endometriosis \nconsist of combined oral contraceptives, gonadotropin \nreleasing hormone (GnRH) agonists and androgenic \nagents [12, 28], as well as some dietary and lifestyle \nchanges, which may relieve symptoms of endometriosis \nand decrease the severity of the disease [29]. Those \nmedical treatments have numerous undesirable side \neffects [12]. Therefore, alternative treatment options are \nneeded for the treatment of endometriosis. Letrozole, \nan aromatase enzyme inhibitor, is an effective compound \nin the treatment of endometriosis, as endometriosis is \nan oestrogen-dependent disease [26, 30]. However, \nit has several side effects including increased bone \nturnover and an unwanted blood lipid profile [31]. \nPrevious studies ind icated that TNF -α levels were \nelevated in the peritoneal fluid of women with \nendometriosis and as the severity of endometriosis \nincreases, TNF-α levels also increase [32-34]. In the \npresent study, the TNF-α levels in the peritoneal fluid \nwere significantly  decreased in the MeOH extract \nadministered group. Furthermore, Lu et al. reported \nthat anti TNF -α treatment could be effective for \nendometriosis [35]. \nAccording to Harada et al. [36], IL-6 levels in peritoneal \nfluid were found to be increased in cases of endometriosis. \nSimilarly, the present study exhibited that IL-6 levels \nin the control group were elevated. However, the \nMeOH extract significantly reduced the IL -6 levels \nin the peritoneal fluid. \nAccording to previous studies, VEGF induces angio-\ngenesis [37] and also increases the vascularization [38]. \nOther studies showed that women with endometriosis \nhave high VEGF levels in their peritoneal fluid [39]. \nFurthermore, numerous studies have shown that VEGF \nexpression increases in cases of endometriosis [40, 41]. \nSimilarly, VEGF levels in the peritoneal fluids of the \ncontrol group were increased in the present study. \nThe MeOH extract obtained from H. scabrum, which \nis the most active extract in our study significantly, \ndecreased the VEGF levels in th e rat endometriosis \nmodel. In addition to MeOH extract, the aqueous extract \nof H. scabrum also reduced VEGF levels. However, \n\nFARMACIA, 2023, Vol. 71, 4 \n 719 \nthe decrease in the HSM group was higher than that \nof the HSA. \nOxidative stress (OS) is explained as an imbalance \nbetween antioxida nts and reactive oxygen species. \nRecently published articles exhibited that OS could be \nimplicated in the endometriosis pathophysiology and \nalso cause an inflammatory response in the peritoneal \ncavity [42, 43]. Previous studies investigated the correlation \nbetween MDA, a marker of lipid peroxidation and \nendometriosis, and found that MDA levels were \nincreased in serum and endometriotic lesions of women \nwith endometriosis when compared to healthy women \n[43, 44]. Similarly, the MDA levels were the highest in \nthe control group in our study and the MeOH extract \nsignificantly reduced the MDA levels in the endometriotic \nlesions. According to Prieto et al. [45], SOD activity \ndecreases in cases of endometriosis in women. In the \npresent study, the SOD activity of the control group \nwas lower than that of other groups. Mashayekhi et al. \n[46] and Mate et al. [47] indicated that the concentrations \nof GST significantly declined according to severity \nof endometriosis. The present study found that GST \nactivities in extracts and letrozole administered groups \nincreased. Singh et al. [48] stated that GPx activity \ndecreases in endometriosis tissue samples. All extracts \nand letrozole increased the GPx activity except the \nMeOH extract, but those increases were statistically \nsignificant in none of the groups. \nChlorogenic acid is one of the major compounds in \nthe MeOH extract prepared from H. scabrum. Ayan \net al. and Llorent-Martínez et al. reported the presence \nof chlorogenic acid in H. scabrum [5, 6]. Kim et al. [49] \nindicated that chlorogenic acid has anti -angiogenic \neffects on choroidal neovascularization and Saltan et \nal. [50] found that Viburnum opulus L. showed significant \nactivity in a rat endometriosis model, the main compound \nbeing the chlorogenic acid. Furthermore, quercetin, \nquercitrin and isoquercitrin were major compounds \nin the MeOH extract of H. scabrum. Park et al. [51] \ndemonstrated that quercetin could be used to reduce \nand treat human endometriosis. According to Oner \net al. [26], the medical therapy for endometriosis is \noften aimed at lowering estrogen levels. Le Bail et \nal. [52] and Collins-Burow et al. [53] indicated that \nflavonoids have anti-estrogenic activity. Furthermore, \nflavonoids have antiangiogenic activities [54]. Bina et \nal. found that Achillea cretica L. aerial part showed \npromising activity on endometriosis. According to the \nLC-MS analysis of A. cret ica aerial extract, some \nquinic acid derivatives were found in this extract [55]. \nXu et al. investigated the effect of epigallocatechin \ngallate (EGCG) on an experimental endometriosis \nmouse model. They found that EGCG significantly \ninhibits the development of experimental endometriosis \nthrough its anti-angiogenic effects [56]. In our study, \nepicatechin was detected as one of the major compounds \nin the MeOH extract. Therefore, epicatechin may have \nplayed a role in decreasing endometriotic implant \nvolumes. \n \nConclusions \nAs a conclusion, the MeOH extract obtained from H. \nscabrum, which is used against menstrual disorders \nin folk medicine, exhibited prominent activity in the \nsurgically induced endometriosis model. The MeOH \nextract significantly decreased adhesion scores, endometrial \nfoci areas, and cytokine levels and exhibited a good \nantioxidant activity. LC-MS/MS results showed the \nmain compounds of the MeOH extract are mainly \nphenolic acids and flavonoids. Therefore, this prominent \nactivity of the MeOH extract could be attributed to its \nphenolic acids and flavonoids. Chlorogenic acid was \ndetected as a major phenolic acid in the MeOH extract, \nwhereas quercetin was detected as a major flavonoid. \n \nAcknowledgement \nThis research was funded by Van Yüzüncü Yıl \nUniversity Scientific Research Project Commission \n(Grant number: TYD-2021-9253). \n \nConflict of interest \nThe authors declare no conflict of interest. \n \nReferences \n1. Robson NKB, Hypericum L. In: Flora of Turkey and \nthe East Aegean Islands Vol 2, Davis PH, Ed.; \nUniversity of Edinburgh: Edinburgh, 1967; 355-401. \n2. Tanker N, Studies on Hypericum scabrum L. J Fac \nPharm Ankara., 1971; 1: 10-15. \n3. 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