{"paper_id":"e789599f-aeaa-4bb4-a9bc-72a16da05dfa","body_text":"* Corresponding author: Khafidhiyah Nur Insaniyah \n E-mail: khafidiyahn.unair20@yahoo.com  \n© 2023 by SPC (Sami Publishing Company)  \n \nJournal of Medicinal and Chemical Sciences 6 (2023) 2941-2951 \n \n \nJournal homepage: http://www.jmchemsci.com/ \n \n \n \n \n \nOriginal Article \nImpact of Ethanol Extract of Basil Leaves (Ocimum basilicum L.) \non Mast Cell Reduction and Prostaglandin E2 Levels in Female \nMice as a Model for Endometriosis Development \nKhafidhiyah Nur Insaniyah*1 \n , Ashon Sa’adi2 \n , Widjiati Widjiati3 \n  \n1Graduate Student of of Reproductive Health  Sciences, Faculty of Medicine, Universitas Airlangga, Surabaya, \nIndonesia \n2Department of Obstetrics and Gynecology, Faculty of Medicine, Universitas Airlangga, RSUD Dr. Soetomo, Surabaya, \nIndonesia \n3Department of Veterinary Embryology, Faculty of Veterinary Medicine, Universitas Airlangga,  Surabaya, Indonesia \nA R T I C L E     I N F O  A B S T R A C T \nArticle history \nReceive: 2023-05-21 \nReceived in revised: 2023-07-17 \nAccepted: 2023-07-23 \nManuscript ID: JMCS-2306-2121 \nChecked for Plagiarism: Yes \nLanguage Editor:  \nDr. Fatima Ramezani \nEditor who approved publication:  \nDr. Yasser Fakri Mustafa  \nDOI:10.26655/JMCHEMSCI.2023.12.9 \n Endometriosis, a painful inflammatory condition, arises from the growth of \nendometrial tissue beyond the uterus. The anti -inflammatory properties of \nbasil (Ocimum basilicum L.) su ggest its poten tial as a treatment for \nendometriosis. This study aimed to examine how basil ethanol extract \naffects mast cells and prostaglandin E2 (PGE2) levels in endometriosis. To \nconduct the study, 28 female mice were divided into four groups. One \ngroup served as the  untreated control, while the remaining three groups \nwere treated with varying doses of ethanol extract from basil leaves for 14 \ndays. On the 15th day, both endometriosis lesions and serum were collected \nand subjected to analysis. The group treated with ba sil ethanol extract \nexhibited a significant reduction in mast cell count when compared to the \ncontrol group. However, there was no notable difference in PGE2 levels \nbetween the intervention and control groups. Therefore, the \nadministration of ethanol extract of basil leaves has the potential to reduce \nmast cell numbers, but it does not significantly affect PGE2 levels. This \nstudy suggests that basil may have potential as an alternative treatment for \nendometriosis, but further research is need ed to investiga te its full \npotential. \nK E Y W O R D S \nBasil \nOcimum basilicum L \nSel mast \nProstaglandin E2 \nEndometriosis  \n \n \nG R A P H I C A L A B S T R A C T \n \n \n\nInsaniyah K.N., et al. / J. Med. Chem. Sci. 2023, 6(12) 2941-2951 \n2942 | P a g e  \n \nIntroduction \nEndometriosis is a gynecological conditio n \ncharacterized by the abnormal growth and \ndysfunction of endometrial glands, stroma, or \ntissue outside the uterus. This benign and \nestrogen-dependent disease also involves \ninflammatory processes, sometimes accompanied \nby reactive fibrosis and extrauterine  muscle \nmetaplasia [1]. \nEndometriosis affects 6 -10% of women of \nchildbearing age with a prevalence rate of 176 -\n190 million women worldwide [2, 3]. \nWomen with endometriosis often experience \npainful symptoms such as dysuria, dyschezia, \ndyspareunia, and dysmenorrhea to infertility, but \nendometriosis can be asymptomatic [4]. \n78.7% of women with endometriosis experience \nsecondary dysmenorrhea, namely pain or cramps \nbefore or during menstruation. This symptom is \none of the causes of late diagnosis [5]. \nDysmenorrhea can have an impact on aspects of \nlife including physical, psycho logical, sexual  \nrelations, social, economic, and reproductive \nfunctions [6]. \nWomen with endo metriosis will experience an \nincrease in local estrogen production [7]. \nEstrogen production is  due to the activity of \nprostaglandin E2 (PGE2) where the \ncyclooxygenase (COX) -2 enzyme induces its \nproduction [8]. \nIncreased p roduction of PG E2 will induce local \nestrogen synthesis and cause inflammation \nresulting in pain. Expression of COX -2 and PGE2 \nproduction can be increased in the uterus and \nendometriosis tissue because they are stimulated \nby PGE2 (autocrine action), IL -1β, VEGF, and \nestradiol (via the estrogen receptor), so that this \ncomplex mechanism maintains high levels of \nPGE2 production in endometriosis tissue [9]. \nMast cells and degranulated mast cells will \nexperience an increase in endometriosis lesions \n[10]. \nMedicinal plants and botanical products have \nobtained popularity in recent years as a meth od \nof complementary and alternative medicine for \nsome gynecological di sorders, such as \nendometriosis. \nTreatment using herbal products has the \nadvantage of being natural, comfortable, \naffordable, and minimal side effects, especially \nnot interfering with ovulation [11, 12]. \nBasil ( Ocimum basilicum  L.) is a widely utilized \nculinary herb and traditional medicinal plant in \nItaly and Southeast Asia [13]. Ocimum basilicum  \nL. has been shown to be b eneficial as anti -\nproliferative, anti -inflammatory, anti -angiogenic, \nanti-microbial anti -oxidant, and \nimmunomodulatory activity [14]. \nBasil leaves comprise flavonoids like quercetin, \nisoquercetrin, kaempferol, and rutin, as well as \nglycosides such as esculin and syringin which are \nresponsible for the anti -inflammatory activity. \nThe anti -inflammatory activity of basil l eaf \nextract can be throug h various mechanisms, \namong which the first is through the inhibition of \narachidonic acid through the lipoxygenase (LOX) \nand COX pathways [15], the second mechanism is \ninfluencing TLR -NF-KB signaling [16], and the \nthird mechanism inhibits C OX-2 activity and \nPGE2 production [17]. \nGiven the beneficial anti-inflammatory properties \nof basil, it holds promise in complementary and \nalternative medicine for endometriosis. As such, \nthis study seeks to assess the impact of an \nethanol extract of basil leaves on PGE2 levels and \nmast cell counts in female mice as an \nexperimental model. \nMaterials and Methods \nEthics approval \nThis research is an experimental study on female \nmice and has obtained ethical approval from the \nAnimal Ethics Commission, Faculty  of Veterinary \nMedicine, Airlangga University (No. \n2.KEH.021.03.2022). \nAnimal \nThe re search sample was twenty -eight female \nBalb/c mice aged two months, weighing 25 -30 \ngrams, who were not pregnant. The mice \nreceived treatment for two weeks. Adequate \nanimal care and usage protocols were observed \nfor the experimental mice [18]. \nAnimals during the study were kept in cages \ncontrolled by light in the room 12 h ours of \n\nInsaniyah K.N., et al. / J. Med. Chem. Sci. 2023, 6(12) 2941-2951 \n2943 | P a g e  \n \ndarkness and 12 hours of light . The environment \nwhere the mice were placed was regulated by \ntemperature and humidity according to the \ntemperature between 22-25 ᵒC and humidity \nbetween 45-65%. Feeding and access to drinking \nwater during the study were given ad libitum [15, \n19]. \nMaking animal models of endometriosis \nThe use of endometriosis model mice refers to \nprevious studies using heterologous techniques \nwith a success rate of 95.7%. Endometrial tissue \nwas collected from women who had undergone \nhysterectomy for non -cancerous uterine \nconditions and had not received hormonal \ntherapy for a duration of three months. The \ncollected endometrial tissue was rinsed twice \nwith phosphate buffered saline (PBS) through \ncentrifugation at 2,500 rpm. Subsequently, the \ntissue was combin ed as large fragments in a \nsolution of PBS, penicillin (200 IU/ml), and \nstreptomycin (200 µg/ml). On the first day, mice \nwere injected intraperitoneally with 0.1 ml of \nendometrial wet tissue, followed by \nintramuscular injection of 0.2 ml of cyclosporine \nA, and intramuscular injection of 5.4 µg ethynil \nestradiol. Ethynil estradiol injection was repeated \non the fifth day [20]. \nPreparation of ethanol ext ract of basil (Ocimum \nbasilicum L.) leaves \nFresh basil ( Ocimum basilicum  L.) leaves were \ntaken from the same area and garden, namely the \nBinjai garden, North Sumatra, with the same \nplant age and hours. Basil leaves are then dried \nand macerated at the Pharm acology Laboratory \nof Airlangga University. Fresh basil leaves were \nthen dried for 2 days at 30-35 ᵒC and crushed \nusing a blender to obtain powder. As much as 100 \ng of basil leaf powder was added to 100 ml of \n96% ethanol solvent, put into a jar  and added \nanother 1 liter of 96% ethanol, and then closed \nand left for 48 hours protected from sunlight. The \nmixture was filtered to obtain maserate. The \nresidue was subjected to maceration using 96% \nethanol following the identical procedure. \nMaceration w as performed with a digital shaker \nset at 50 rpm until a clear macerate was obtained. \nThe liquid extract obtain ed was subjected to \nevaporation using a rotary evaporator for 2 hours \nat 50 ᵒC until a concentrated extract with a thick \nconsistency was achieved. \nExtract administration \nThe study involved twenty -eight mice that were \nrandomly divided into four groups, each group \nconsisting of seven mice. Three of the groups \nreceived treatment with  ethanol extract of basil \nleaves, while the fourth group served as the \nendometriosis control group and was given a \nplacebo. The second group was administered the \nextract at a dose of 0.21 mg/g -BW, the third \ngroup accpted a dose of 0.42 mg/g -BW, and the \nfourth group with 0.84 mg/g-BW. Because there \nis no standard  dose of bacilli against \nendometriosis, the dosage we used refers to the \nprevious studies, then carried out the conversion \nand implications for endometriosis inflammatory \ndisease [15]. \nSample collection \nOn the 1st to 7 th day, the mice were acclimatized, \nfollowed by making the endomet riosis model \nuntil the 14 th day, then for 14 days the mice wer e \ntreated. After 14 days of treatment, the mice were \nsacrificed using ketamine 100 mg/kgBW while \nintracardiac blood samples were taken for PGE2 \nserum examination. The rats were then \nterminated and continued with the opening of the \nperitoneal cavity to take  endometriosis lesions \nfor mast cell examination. \nToluidine blue staining \nAfter the 14 -day treatment, the endometriosis \ntissue underwent preparation for analysis. It was \ncut and stained with tol uidine blue to assess the \nnumber of mast cells that had underg one \ndegranulation. The tissue was initially fixed in \n10% buffered formalin, dehydrated, and then \nplaced in paraffin. Sections of the paraffin -\nembedded tissue blocks, measuring 4 μm in \nthickness, were obtained using a Leica RM2135 \nMicrotome from Leica, Germany, and mounted on \nglass slides. The tissue was then thoroughly \ndeparaffinized with xylene, followed by \n\nInsaniyah K.N., et al. / J. Med. Chem. Sci. 2023, 6(12) 2941-2951 \n2944 | P a g e  \n \nrehydration in graded alc ohol for 5 minutes per \nstep. \nSubsequently, the tissue was soaked in water for \n5 minutes and exposed to toluidine blue stain in a \njar for 30 minutes before being carefully \nremoved. Finally, the tissue was immersed in \nabsolute alcohol for 1 minute, cleaned with \nxylene, and mounted on a slide using Entellan. \nMast cell granules appeared purple, while the \nrest of the tissue appeared b lue. Mast cell counts \nwere performed in all visual fields using a Nikon \nE100 light microscope with magnification \nranging from 100x to 400x [21]. \nThe Enzyme-Linked Immunosorbent Assay (ELISA) \nwas utilized in this study. Blood samples were \nobtained from the mice's hearts using a plain \nvacutainer and left to clot naturally without the \nuse of anticoagulants. The serum was the n \nseparated from the blood by centrifugation at \n1500x g for 20 minutes at 4 °C, and subsequently \nstored at -20 °C. PGE2 levels were determined \nusing the Mice PGE2 ELISA Kit (Cat. No \nEA0028Mo, Bioassay Technology Laboratory) \nfollowing the manufacturer's ins tructions. The \noptical density was measured using an ELISA \nreader (TC 96 microplate reader, Teco Company, \nCanada). \nStatistic analysis \nThe results were presented as the mean ± \nstandard error of the mean. Statistical analysis \nwas carried out using a One-way ANOVA test, and \nthen by a Tukey HSD post hoc test to determine \nsignificant variations between the groups. A \nsignificance level of p<0.05 was regarded \nstatistically significant. Data analysis was \nperformed utilizing the SPSS application. \nResults and Discussion \nOn the twent y-eighth day, all mice from all \ntreatment groups were still alive, had no \nabnormal appearance, and then were entered for \nexamination and data analysis. \nMast cell count \nFigure 1 in the study presents the count of mast  \ncells in the co ntrol group of endometriosis tissue \nstained with toluidine blue. The average mast cell \ncounts in the control group and the groups \ntreated with ethanol extract of basil leaves at \ndoses of 0.21 mg/g -BW, 0.42 mg/g -BW, and 0.84 \nmg/g-BW were (3.86 ± 1.21), (4.0 0 ± 2.31), (1.71 \n± 1.11), and (0.86 ± 0.90), respectively, as \ndepicted in Figure 2. The data followed a  normal \ndistribution, and a One -way ANOVA test revealed \na significant difference among the groups \n(p=0.001). A Tukey's post hoc test was conducted \nto compare the groups, indicating that the group \nreceived dosage of 0.84 mg/g -BW of ethanol \nextract of basil leaves exhibited a significant \ndistinction (p=0.005) compared to the control \ngroup. Furthermore, the group treate d with 0.21 \nmg/gBW showed a significant di stinction \n(p=0.039) compared to the groups administered \ndoses of 0.42 mg/g -BW and 0.84 mg/g -BW \n(p=0.003). \n \n \nFigure 1: Histology of endometriosis tissue stained with toluidine blue staining in each group. Mast cell s shown \nin bluish color are indicated by red arrows (Nikon E100 light microscope with 400x magnification)  \n\nInsaniyah K.N., et al. / J. Med. Chem. Sci. 2023, 6(12) 2941-2951 \n2945 | P a g e  \n \n \nFigure 2: Mast cell count in endometriosis mice model \nThe One -way ANOVA analysis demonstrated a \nnoteworthy distinction with a p -value of 0.001. \nThe subsequent exam ination using Tukey's HSD \ntest for the average mast cell count produced the \nfollowing results: \n(1) There was no notable distinction between the \ncontrol group and the intervention group with a \ndose of 0.21 mg/g -BW (p=0.998), (2) A minor \ndistinction was obse rved between the control \ngroup and the group treated with a dose of 0.42 \nmg/g-BW (p=0.057), and (3) A significant \ndistinction was found between the control group \nand the intervention group of 0.84 mg/g -BW \n(p=0.005). \nFurthermore, the followi ng nota ble disti nctions \nwere observed: \n(1) There existed a significant distinction \nbetween the intervention group treated with a \ndose of 0.21 mg/g -BW and the intervention \ngroup treated with 0.42 mg/g -BW (p=0.039), (2) \nA significant distinction was found be tween the \nintervention group treated with of 0.21 mg/g-BW \nand the intervention group treated with 0.84 \nmg/g-BW (p=0.003); however, (3) No significant \ndistinction was detected between the \nintervention group treated with 0.42 mg/g -BW \nand the intervention gro up treated with 0.84 \nmg/g-BW (p=0.706). \nPGE2 levels \nThe mean PGE2 levels in the control group, th at \ngiven ethanol extract of basil leaves at doses of \n0.21 mg/gBW, 0.42 mg/gBW, and 0.84 mg/gBW \nwere (44.52 ± 11.99), (41.95 ± 5.36), (50.73 ± \n4.44), and (44.39 ±6.53), respecti vely ( Figure 3). \nThe One -way ANOVA test was used because the \ndata were normally distributed and the results \nshowed no significant distinction between groups \n(p=0.199). This non -significant difference was \ndue to variations in the expressed PGE2 values. In \nthe control group, 2/7 rats had lower PGE2 levels \nthan the group given ethanol extract of basil \nleaves with 0.21 mg/g -BW, 0.42 mg/g -BW, and \n0.84 mg/g -BW. In the data proportion on PGE2 \nlevels, the 0.42 mg/g -BW dose gr oup had the \nhighest average compared to the other groups . \nThis study examined the mast cell count in the \ncontrol group and observed an ele vation in both \ndegranulated and overall mast cell numbers \nwithin endometriosis lesions, indicating their \ninvolvement i n inflammatory disease \ndevelopment [10]. \nEndometriosis patients displayed signific antly \nhigher mast cell counts and increased \ndegranulation in endometriotic lesions compared \nto the other tissues. This  phenomenon was \nparticularly prominent in deep infiltrating lesions \nand regions proximate to nerve fibers [22], \naligning with Kempuraj et al . (2004)  findings of \nelevated mast cell density per square mill imeter \nand 89% activation among endometriosis \npatients [23]. In addition, Borelli et al. (2020)  \nfound that mast cell degrees in the peritoneal \nfluid were significantly higher in women with \nendometriosis (2.8 ± 3.2) compar ed to healthy \nwomen (0.5 ± 0.5) (p < 0.05) [24].  \n\nInsaniyah K.N., et al. / J. Med. Chem. Sci. 2023, 6(12) 2941-2951 \n2946 | P a g e  \n \n \nFigure 3: PGE2 levels in the endometriosis mouse model. The One-way ANOVA test exposed no significant \ndistinction p=0.199 \nEndometriosis lesions contain degranulated mast \ncells that release proteases, leading to the \nproduction of histamine -releasing peptides and \ncytokines [10]. \nMast cell activation is implicated in the formation \nof fibrous adhesions that contribute to \nendometriosis lesions, scarring, and fibrosis [25]. \nActivated mast cells also produce mediators like \nhistamine and TNF-α that indirectly contribute to \nneuropathic pain by recruiting leukocytes [26]. In \npatients with endometriosis, the number of \ndegranulated and activated mast cells \nsignificantly increases in deep infiltrating lesions \nand those located near ne rves, which are the \nmost painful lesions. Anaf et al . (2006)  showed \nthat deep infiltrating lesions are associated with \nhigher pain scores, suggesting a link between \nmast cells and pain in endometriosis [27]. \nIn the present study, treating the subjects with \nethanol extract of basil leaves at a dosage of 0.84 \nmg/g-BW led to a notable decrease in mast cell \ncount in comparison to the control group, with a \nsignificant difference observed (p=0.005). Basil \nleaves contain the flavonoid quercetin and \nrosmaniric acid, which act as mast cell stabilizers. \nThe stabilization mechanism of mast cells in basil \nleaves suppresses the inhibition of IgE \nproduction in preventing cross-linking of the IgE-\nFcεRI complex to prevent the development of \nmast cell degranulation [28]. \nMast cells can be activated by interacting directly \nwith antigens (IgE -dependent pathway), where \nIgE production requires the release of T -helper2 \n(Th2) cytokines, namely IL -4, IL -5, and IL -13. \nAlthough there is increased expression of IL -4 in \nthe peritoneal fluid of endomet riosis patients, \nfurther studies regarding mast cell activation in \nIgE-dependent endometriotic lesions have not \nbeen confirmed [22]. \nThe reduction in mast cell count can be \ncharacterized to the inhibitory effects of \nquercetin present in basil leaves. Quercetin \ninhibits mast cell activation by blocking calcium \nion influx, sup pressing the release of histamine, \nleukotrienes, and prostaglandins, and inhibiting \nprotein kinase activation [29]. \nRelease of Ca 2+ from the extracellular matrix \nactivates mast cells and causes NFkB to \ntranslocate to the cell nu cleus, which results in \ncytokine transcription [30, 31]. \nResearch conducted by Ding et al. (2019), proved \nthat quercetin was significantly dose -dependent \non being able to stabilize peritoneal mast cells by \nreducing Ca2+ expenditure [32]. \nBasil leaves co ntain a variety of antioxidants, \nsuch as vitamin C, vitamin E, carotenoids, and \nflavonoids, which help safeguard against \noxidative stress and the harmful effects of free \nradicals induced by hepatotoxic substances. \nThese substances have the potential to har m \ncellular structures and trigger the manufacturing \nof Reactive Oxygen Species (ROS) [33]. \nFlavonoids can reduce mast cell secretion by \nadding a hydroxyl group at position 2', which  can \ninteract with oxygen at position 1 to form a cyclic \nstructure that could interfere with various \nbiological events [34]. \n\nInsaniyah K.N., et al. / J. Med. Chem. Sci. 2023, 6(12) 2941-2951 \n2947 | P a g e  \n \nFurthermore, flavonoids can decrease the \nnumber of mast cells by inhibiting the activity of \nphosphatidylinositol-3-phosphate kinase (PI3K) \nenzyme and activating AMP -activated protein \nkinase (AM PK). This mechanism provides anti -\ninflammatory and anti -cancer effects. Mast -cell \ndegranulation and activation can be through the \nPI3K enzyme pathway. Flavonoids contained in \nbasil leaves such as quercetin can also inhibit \nmast cell degranulation in acute inflammation by \ninhibiting PGE2 production via arachidonic acid \n[35].  \nIn a study conducted by Park et al. (2008), it was \nshown that flavonoids have inhibitory properties \non mast cell degranulation and activation. These \nspecific compounds were observed to hinder the \nrelease of histamine and inflammatory \nsubstances generated by mast cells. This \ninhibition resulted in the suppression of NF -Kb \nbinding, the release of pro -inflammatory \ncytokines (IL -6, IL -8, TNF-α, and IL-1β), and \nhistamine release (p<0.05) [36]. \nIn patients diagnosed with endome triosis, there \nis an increase in macrophage count. These \nmacrophages are responsible for enhancing the \nsecretion of cytokines, which play a significant \nrole in the development of lesions, angiogenesis, \nand proliferation. Endometriosis patients also \nexhibit elevated levels of proin flammatory \ncytokines such as interleukins -1, -8, -33, nuclear \nfactor kappa B (NF -κB), and tumor necrosis \nfactor alpha (TNF-α) [37]. \nIn estrogen -induced endometriosis (E2), the \nsignaling pathway involving cyclooxygenase -2 \n(COX-2), omega -3 PUFA, and IL -1β leads to an \nincrease in NF -κB activity. Consequently, COX -2 \npromotes the manufacturing of prostaglandin E2 \n(PGE2) [38]. \nPGE2, in turn, regulates steroidogenesis by \ninducing the expression of  steroidogenic acute \nregulatory protein (StAR) and aromatase. This \nenables the production of de novo estrogen from \ncholesterol without the need for intermediate \nmetabolite transport from other organs. \nMoreover, estrogen plays an important role in \nangiogenesis by stimulating VEGF expression and \ninducing endothelial cell proliferation [7].  \nProstaglandin E2 also inhibits apoptosis and \nincreases cell proliferation by inducing the \nexpression of fibroblast growth factor 9 (FGF -9) \nand matrix metalloproteinase -2 (MMP -2) [39]. \nIncreased levels of PGE2 can cause inflammatory \nmediators to escape from blood vessels to local \nareas, causing dysmenorrhea [40]. \nAdministration of ethanol extract of basil leaves \nas a complementary treatment showed no \nsignificant reduction in PGE2 levels (p=0.199), \nbut in doses of 0.21 mg/g -BW (41.95±5.36) and \n0.84 mg/g -BW (44.39±6.53) it reduced the \naverage levels of PGE2 compared to the control \ngroup (44.52±11.99). Administration of  ethanol \nextract of basil leaves at a dose of 0.21 mg/g -BW \nand a dose of 0.84 mg/g -BW reduced the average \nPGE2 level although not statistically significant. \nThe mechanism of ethanol extract of basil leaves \nin reducing the average PGE2 level is by reducing \nthe transfer of Nf -kb into the nucleus by \ninhibiting the Nfkb1 gene, which in turn \nsuppresses inflammatory cytokine genes through \ninhibition of Nf -kB function which will affect \nTLR4-Nf-kB signaling [16]. \nThe mechanism of basil leaves in anti -\ninflammation is through the inhibition of \narachidonic acid through the lipoxygenase and \ncyclooxygenase pathways thereby suppressing  \nthe production of PGE2 levels [15]. Numerous \nstudies have confirmed the efficacy of different \ntypes of flavonoids in suppressing endometriosis, \na condition that impacts over 5.5 million women \nin the United States , and 176 million women in \nworldwide [41]. \nA meta -analysis conducted by Jalali et al.  found \nthat the flavonoid quercetin has the potential to \ndecrease symptoms and levels of PGE2 (42). \nAnother study by Umar et al.  (2014) supported \nthis finding, demonstrating that the ethanol \nextract of basil leaves (Ocimum basilicum L.) \ncould decrease PGE2 production and inhibit COX \nenzymes (COX -1 and COX -2) by reducing \nmacrophage activation [17]. \nThe group that received the ethanol extract of \nbasil leaves with 0.42 mg/g -BW showed an \nabnormal increase in PGE2 levels compared to \nthe other treatment groups, which experienced a \ndecrease. The atypical rise can be ascribed to \ngenetic and protein variations and mutations, \nleading to alterations in observable \ncharacteristics. Mutations can arise from either \n\nInsaniyah K.N., et al. / J. Med. Chem. Sci. 2023, 6(12) 2941-2951 \n2948 | P a g e  \n \ninherited genetic changes passed down from \nparents (germline mutations) or acquired \nmutations that occur during an individual's \nlifetime (somatic mutations), with the latter \nbeing a major contributor to disease [43]. \nIn Wang & Song's study (2015) where the genetic \nvariation of COX -1195 is at risk for \nendometriosis. The COX -2 1195 AA genotype \nfrequency and the A allele frequency were \nsignificantly higher than the control group. Allele \nA is consistent in the function of the -1195 G>A \npolymorphism, which can form a c -Myb binding \nsite and significantly increase the activity of the \nCOX-2 gene promoter in regulating the balance \nbetween cell division, survival, and \ndifferentiation resulting in endometriosis [44]. \nAdministration of basil leaf extract ( Ocimum \nbasilicum L.) to endometriosis mice experienced \nan increase in PGE2 levels with increasing doses. \nThis could be due to the content of phytochemical \ncompounds such as euginal, euginol, sitosterol, \nursolic acid , and stigmasterol in basil leaves \nwhich act as phytoandrogens or phytoestrogens \n[45]. \nBasil leaves (Ocimum basilicum L.) contain \norientin, vicenin, and isoflavones which are \nestrogenic which can increase exogenous \nestrogen, bind to estrogen receptors in the body, \nand proliferate the uterus [19, 46, 47]. \nIsoflavones can cause an estrogen -raising effect \nin many tissues. The content of systesterol and \nstigmasterol in basil leaves is androgenic. \nSitosterol and stigmasterol are androgen \nprecursors that can be converted into \ntestosterone, resulting in higher testosterone \nlevels and suppressing the development of \novarian follicles [45]. \nGiving ocimum basilicum L. leaf extract which has \nestrogenic content has caution in the use of \nendometriosis treatment, where estrogen is a key \nhormone for the growth and persistence of \nendometriosis tissue as well as inflammation and \npain [48]. \nThe rise in PGE2 can also be triggered by mast \ncells that have been activated. When women with \nendometriosis lesions have activated mast cells, \nthey produce soluble products like TNF -α, IL-4, \nIL-5, and IL-6. These products then stimulate \nfibroblasts to produce collagenase and PGE2, and \ninduce macrophages t o produce pro -\ninflammatory factors in the endometrium [25]. \n \nDisclosure Statement \nNo potential conflict of interest was reported by \nthe authors. \nFunding \nThis research did n ot receive any specific grant \nfrom funding agencies in the public, commercial, \nor not-for-profit sectors. \nAuthors' Contributions \nAll authors contributed to data analysis, drafting, \nand revising of the paper and agreed to be \nresponsible for all the aspects of this work. \nOrcid  \nKhafidhiyah Nur Insaniyah \nhttps://orcid.org/0009-0007-4770-8216 \nAshon Sa’adi \nhttps://orcid.org/0000-0003-2682-3139 \nWidjiati Widjiati \nhttps://orcid.org/0000-0002-8376-1176 \nReferences \n[1]. Laganà A .S., Garzon S ., Götte M ., Viganò P ., \nFranchi M ., Ghezzi F ., Martin D.C., The \npathogenesis of endometri osis: Molecular and \ncell biolo gy insights , International journal of \nmolecular sciences., 2019, 20:1 [Crossref], [Google \nScholar], [Publisher] \n[2]. Moen M .H., Endometriosis, an everlasting \nchallenge, Acta Obstetricia et Gynecologica \nScandinavica., 2017, 96:783 [Crossref], [Google \nScholar], [Publisher] \n[3]. Zondervan K .T., Becke r C .M., Missmer.  SA. \nEndometriosis, Review Article , N Engl J Med ., \n2020, 382:56 [Crossref], [Publisher] \n[4]. Parasar P., Ozcan P ., Terry K. , Endometriosis: \nEpidemiology, Diagnosis and Clinical \nManagement, Current obstetrics and gyne cology \nreports., 2016, 26:196 [Crossref], [Google \nScholar], [Publisher] \n[5]. Guo Y., Liu F.Y., Shen Y., Xu J.Y., Xie L.Z., Li S.Y., \nDing D. N., Zhang D.Q., Han  F.J., Complementary \nand Alternative Medicine for Dysmenorrhea \n\nInsaniyah K.N., et al. / J. Med. Chem. Sci. 2023, 6(12) 2941-2951 \n2949 | P a g e  \n \nCaused by Endometriosis: A Review of Utilization \nand Mechanism , Evidence-Based Complementary \nand Alternative Medicine ., 2021, 2021 [Crossref], \n[Google Scholar], [Publisher] \n[6]. Moradi M ., Parker M ., Sne ddon A ., Lopez V ., \nEllwood D., Impact of endometriosis on women’s \nlives: A qualitative study , BMC women's health ., \n2014, 14:1 [Crossref], [Publisher] \n[7]. Hsiao K. Y., Wu M .H., Tsai S .J., Roles of \nProstaglandin E 2 in Endometriosis , \nEndometriosis: Pathogenesis a nd Treatment ., \n2014, 1 25 [Crossref], [Google Scholar ], \n[Publisher] \n[8]. Tsuge K., Inazumi T., Shimamoto A., Sugimoto \nY., Molecular mechanisms underlying \nprostaglandin E2 -exacerbated inflammation and \nimmune diseases , International immunology ., \n2019, 31:597 [Crossref], [Google Scholar ], \n[Publisher] \n[9]. Taylor H .S., Pal L ., Seli E. , Speroff’s Clinical \nGynecologic Endocrinology and Infertility , \nConnecticut: Wolters Kluwer ., 2020, 580 \n[Publisher] \n[10]. McCallion A., Nasirzadeh Y ., Lingegowda H ., \nMiller J .E., Khalaj K ., Ahn S ., Monsanto S .P., \nBidarimath M.,  Sisnett D.J., Craig A.W., Young S.L., \nLessey B.A., Koti M., Tayade C., Estrogen mediates \ninflammatory role of mast cells in endometriosis \npathophysiology, Frontiers in Immunology ., 2022, \n13:961599 [Crossref], [Google Scholar ], \n[Publisher] \n[11]. a) Ilhan M ., Gürağaç Dereli F .T., Akkol E .K., \nNovel Drug Targets with Traditional Herbal \nMedicines for Overcoming End ometriosis, \nCurrent Drug Delivery ., 2018, 16:386 [Crossref], \n[Google S cholar], [ Publisher]; b) Hamood Al -\nBehadili W.K., Jawad Y.M., Hasan H.J. Study the \nEffect of pH on Absorption and Fluorescence \nSpectra of Eosin Y Dye in Ethanol. Asian Journal of \nGreen Chemistry , 2023, 7:163 [Crossref], \n[Publisher]; c) Ahmadyousefi Y., A brief overview  \nof plant -derived chemotherapeutic agents for \ncancer therapy. Asian Journal of Green Chemistry , \n2023, 7:175 [Crossref], [Publisher] \n[12]. a) Nimesh S., Ashwlayan V.D., R ani R., \nPrakash O., Advantages of Herbal Over Allopathic \nMedicine in the Management of Kidney and \nUrinary Stones Diseas , Borneo Journal of \nPharmacy., 2020,  3:179 [Crossref], [Google \nScholar], [ Publisher]; b) Monjezi A., Karimian P., \nYousofvand V. Therapeutic applications of \nSalvadora persica plant in medical sci ences: a \nreview article. Asian Journal of Green Chemistry , \n2023, 7:180 [ Crossref], [Publisher]; c) \nAkbarnejad F., Dermatology Benefits of Punica \ngranatum: A Review of the Potential Benefits of \nPunica granatum in Skin Disorders. Asian Journal \nof Green Chemistry , 2023, 7:208 [ Crossref], \n[Publisher] \n[13]. Filip S. , Internation f Valuable \nPhytonutrients, J Clin Nutr Diet ., 2017, 3 \n[Crossref], [Google Scholar], [Publisher] \n[14]. Shahrajabian M .H., Sun W ., Cheng Q. , \nChemical components and phar macological \nbenefits of Basil (Ocimum basilicum): a review , \nInternational Journal of Food Properties ., 2020, \n23:1961 [Crossref], [Google Scholar], [Publisher] \n[15]. Eftekhar N ., al Moghimi A ., Mohammadian \nRoshan N ., Sa adat S ., Boskabady M .H., \nImmunomodulatory and anti -inflammatory \neffects of hydro -ethanolic extract o f Ocimum \nbasilicum leaves and its effect on lung \npathological changes in an ovalbumin-induced rat \nmodel of asthma , BMC complementary and \nalternative medicin e., 2019, 19:1 [Crossref], \n[Publisher] \n[16]. Takeuchi H., Takahashi-Muto C., Nagase M ., \nKassai M ., Tanaka -Yachi R ., Kiyose C. , Anti-\ninflammatory effects of extracts of sweet basil \n(Ocimum b asilicum l.) on a co -culture of 3t3 -l1 \nadipocytes and raw264.7 macrophages, Journal of \noleo science ., 2020, 69:487 [Crossref], [Google \nScholar], [Publisher] \n[17]. Umar A., Zhou W ., Abdusalam E ., Tursun A ., \nReyim N ., Tohti I ., Moore N., Effect of Ocimum \nbasilicum L. on  cyclo-oxygenase isoforms and \nprostaglandins involved in thrombosis , Journal of \nethnopharmacology., 2014, 152:151 [Crossref], \n[Google Scholar], [Publisher] \n [18]. Scholten D., Trebicka  J., Liedtke  C., \nWeiskirchen R., The carbon tet rachloride model \nin mice ,  Laboratory Animals ., 2015, 49:4 \n[Crossref], [Google Scholar], [Publisher] \n [19]. Andriyanto A ., Widi L .N., Subangkit M ., \nTarigan E ., Irarang Y ., Nengsih R .F., Manalu W., \nPotential use of Ind onesian basil (Ocimum \nbasilicum) maceration to increase estradiol and \nprogesterone synthesis and secretion to improve \n\nInsaniyah K.N., et al. / J. Med. Chem. Sci. 2023, 6(12) 2941-2951 \n2950 | P a g e  \n \nprenatal growth of offspring using female albino \nrats as an ani mal model, Veterinary World., 2022, \n15:1197 [Crossref], [Google Scholar], [Publisher] \n[20]. Trisetiyono Y ., Widjiati W ., Hidayat S .T., \nPramono N., Antioxidant Herbs Supplementation \nInhibits Endometriosis Extension in Mice, Journal \nof Biomedicine and Translational Research ., 2019, \n5:53[Crossref], [Google Scholar], [Publisher] \n [21]. Iuvone T ., Affaitati G ., De Filippis D ., \nLopopolo M ., G rassia G ., Lapenna D, Negro L., \nCostantini R., Vaia M., Cipollone  F.,  Ialenti  A., \nUltramicronized palmitoylethanolamide reduces \nviscerovisceral hyperalgesia in a rat model of \nendometriosis plus ureteral calculosis: Role of \nmast cells, Pain., 2016, 157:80 [Google Scholar ], \n[Publisher] \n[22]. Binda M .M., Donnez J ., Dolmans M .M., \nTargeting mast cells: a new way to treat \nendometriosis, Expert opinion on therapeutic \ntargets., 2017, 21:67 [Crossref], [Google Scholar], \n[Publisher] \n[23]. Mariuzzi L., Domenis  R., Orsar ia M., \nMarzinotto S., Londero  A.P., Bulfoni M., Candotti \nV., Zanello A., Ballico M., Mimmi M.C., Calcagno  A.,   \nFunctional Expression of Aryl Hydroca rbon \nReceptor on Mast Cells Populating Human \nEndometriotic Tissues , Laboratory Investigation ., \n2016, 96:959 [Crossref], [Google Scholar ], \n[Publisher] \n[24]. Borelli V ., Martinelli M ., Luppi S ., Vita F ., \nRomano F ., Fanfani F ., Trevisan E., Celsi  F., \nZabucchi G., Zanconati F.,  Bottin  C., Mast Cells in \nPeritoneal Fluid From Women With \nEndometriosis and Their Possible Role i n \nModulating Sperm Function , Frontiers in \nPhysiology., 2020, 10 [Crossref], [Google Scholar], \n[Publisher] \n[25]. Vallvé-Juanico J., Houshdaran S., Giudice L.C., \nThe endometrial immune environment of women \nwith endometriosis, Human reproduction update., \n2019, 25:565 [Crossref], [Google Scholar ], \n[Publisher] \n[26]. Zhu T .H., Zou G ., Ding S .J., Li T .T., Zhu L .B., \nWang J.Z., Yao Y.X., Zhang X.M., Mast cell stabilizer \nketotifen reduces hyperalgesia in a  rodent model \nof surgically induced endometrio sis, Journal of \npain research ., 2019, 12:1359 [Google Scholar ], \n[Publisher] \n[27]. Maddern J ., Grundy L ., Castro J ., Brierley \nS.M., Pain in Endometriosis , Frontiers in cellular \nneuroscience., 2020, 14:590823 [Crossref], \n[Google Scholar], [Publisher] \n[28]. Sim L .Y., Rani N .Z.A., Husain K. , Lamiaceae: \nAn insight on their anti -allergic potential and its \nmechanisms of action, Frontiers in pharmacology., \n2019, 10:677 [Crossref], [Google Scholar ], \n[Publisher] \n[29]. Mlcek J., Jurikova T., Skrovankova S., Sochor \nJ., Quercetin and it s anti -allergic immune \nresponse, Molecules., 2016, 21:623 [Crossref], \n[Google Scholar], [Publisher] \n[30]. Komi D .E.A., Wöhrl S ., Bielory L. , Mast Cell \nBiology at Molecular Level: a Comprehensive \nReview, Clinical reviews in allergy & immunology ., \n2020, 58:342 [Crossref], [Google Scholar ], \n[Publisher] \n[31]. Krystel-Whittemore M., Dileepan K.N., Wood \nJ.G., Mast cell: A multi -functional master cell , \nFrontiers in immunology ., 2016, 6:1 [Crossref], \n[Google Scholar], [Publisher] \n[32]. Ding Y ., Che D ., Li C ., Ca o J ., Wang J ., Ma P ., \nZhao T., An H.,  Zhang  T., Quercetin inhibits \nMrgprx2-induced pseudo -allergic reaction via \nPLCγ-IP3R re lated Ca 2+ fluctuations , \nInternational immunopharmacology ., 2019, \n66:185 [Crossref], [Google Scholar], [Publisher] \n[33]. Renovaldi D., Adam A .K., Potential of Sweet \nBasil (Ocimum basilicum) as a Hepatoprotector \nAgent for Liver Injury Related to Drug s, \nMuhammadiyah Medical Journal ., 2020, 1:63 \n[Crossref], [Google Scholar], [Publisher] \n  \n[34]. Al-Khayri J.M., Sahana G.R. , Nagella  P., \nJoseph B.V., Alessa F.M.,  Al-Mssallem M.Q. , \nFlavonoids as Potential Anti -Inflammatory \nMolecules: A Review , Molecules., 2022, 27:2901 \n[Crossref], [Google Scholar], [Publisher] \n[35]. Shaik Y ., Caraffa A ., Ronconi G ., Lessiani G ., \nConti P., Impact of polyphenols on mast cells with \nspecial emphasis on the effect of quercetin and \nluteolin, Central European Journal of \nImmunology., 2018, 43:476 [Crossref], [Google \nScholar], [Publisher] \n[36]. Rakha A., Umar N., Rabail R., Butt  M.S., \nKieliszek M., Hassoun A., Aadil  R.M., Anti-\ninflammatory and anti -allergic potential of \ndietary flavonoids: A review , Biomedicine & \n\nInsaniyah K.N., et al. / J. Med. Chem. Sci. 2023, 6(12) 2941-2951 \n2951 | P a g e  \n \nPharmacotherapy., 2022, 156:113945 [Crossref], \n[Google Scholar], [Publisher] \n[37]. Donnez J., Cacciottola L. , Endometriosis: An \nInflammatory Disease That Requires New \nTherapeutic Options , International Journal of \nMolecular Sciences ., 2022, 23:1518 [Crossref], \n[Google Scholar], [Publisher] \n[38]. Lai Z.Z., Yang H.L., Ha S.Y., Chang K.K., Mei J., \nZhou W .J., Qiu X.M., Wang X.Q., Zhu R., Li D.J., Li  \nM.Q., Cyclooxygenase-2 in endometriosis , \nInternational journal of biological scie nces., 2019, \n15:2783 [Crossref], [Google Scholar], [Publisher] \n[39]. Nasry W.H.S., Rodr iguez-Lecompte J.C., \nMartin C.K., Role of COX -2/PGE2 Mediated \nInflammation in Oral Squamous Cell Carcinoma , \nCancers., 2018, 10:348 [Crossref], [Google \nScholar], [Publisher] \n[40]. Huang Q., Liu X ., Guo S. , Changing \nprostaglandin E2 (PGE 2 ) signaling during \nlesional progression and exacerbation of \nendometriosis by inhibitio n of PGE 2 receptor \nEP2 and EP4,  Reproductive Medicine and Biology ., \n2022, 21:12426 [Crossref], [Google Scholar ], \n[Publisher] \n[41]. Safe S., Jayaraman A ., Chapkin R .S., Howard \nM., Mohan kumar K ., Shrestha R. , Flavonoids: \nstructure–function and mechanisms of action and \nopportunities for drug development , \nToxicological Research ., 2021, 37:147 [Crossref], \n[Google Scholar], [Publisher] \n[42]. Jalali M., Mahmoodi M., Moosavian S.P., Jalali \nR., Ferns G ., Mosallanezhad A ., Imanieh M.H., \nMosallanezhad Z., The effects of ginger \nsupplementation on markers of inflammatory \nand oxidative stress: A systematic review and \nmeta-analysis of clinical trials , Phytotherapy \nResearch., 2020, 34:1723 [Crossref], [Google \nScholar], [Publisher] \n[43]. Karki R ., Pandya D ., Elst on R .C., Ferlini C. , \nDefining ‘mutation’ and ‘polymorphism’ in the era \nof personal genomics , BMC medical genomics ., \n2015, 8:1 [Crossref], [Google Scholar], [Publisher] \n[44]. Wang Y., Qu Y., Song W., Genetic variation in \nCOX-2 -1195 and the risk of endometriosis and \nadenomyosis, Clin Exp O bstet Gynecol ., 2015, \n42:168 [Google Scholar], [Publisher] \n[45]. Maryanti S ., Nasihun T ., Amalia H. , The \nEffect of Administering Lemon Basil ( Ocimum \nsanctum ) Leaves on FSH and  LH Estrogen Levels \nand Size of Ovarian Antral Follicle in  Female Balb \n/ C Mice,  Sains Medika: Jurnal Kedokteran dan \nKesehatan., 2019, 10:57 [Google Scholar ], \n[Publisher] \n[46]. Mousavi L ., Salleh R .M., Murugaiyah V. , \nPhytochemical and bioactive compounds \nidentification of Ocimum tenuiflorum leaves of \nmethanol extract and its fraction with an \nantidiabetic potential , International Journal of \nFood Properties ., 2018, 21:2390 [Crossref], \n[Google Scholar], [Publisher] \n[47]. Farhana A., Reddy A ., Bhavana K ., Mutha S ., \nBakshi V ., Assessment of Ocimum Sanctum To \nNormalize the Estrous Cycle in Letrazole Induced \nPolycystic Ovary, World J Pharm Res., 2018, 7:907 \n[Google Scholar], [Publisher] \n[48]. Antonescu A.I., Miere F ., Fritea L., Ganea M ., \nZdrinca M., Dobjanschi L., Antonescu A., Vicas S.I., \nBodog F., Sindhu R.K.,  Cavalu  S., Perspectives on \nthe combined effects of ocimum basilicum and \ntrifolium pratense extracts in terms of \nphytochemical prof ile and pharmacological \neffects, Plants., 2021, 10:1390 [Crossref], [Google \nScholar], [Publisher] \n \nHOW TO CITE THIS ARTICLE \nKhafidhiyah Nur Insaniy ah*, Ashon Sa’adi, Widjiati , Impact of Ethanol Extract of Basil Leaves (Ocimum basilicum L.) on \nMast Cell Reduction and Prostaglandin E2 Levels in Female Mice as a Model for Endometriosis Developmen t. J. Med. \nChem. Sci., 2023, 6(12) 2941-2951. \nDOI: https://doi.org/10.26655/JMCHEMSCI.2023.12.9 \nURL: https://www.jmchemsci.com/article_176669.html","source_license":"CC0","license_restricted":false}