KR20250161293A - A pharmaceutical composition for preventing or treating endometriosis comprising transcrocetin - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating endometriosis, including transcrocetin. When using the pharmaceutical composition for preventing or treating endometriosis or complications of endometriosis, including transcrocetin or a saffron extract containing transcrocetin, as an active ingredient, compared to surgery and hormone therapy, the composition has fewer side effects and effectively manages endometriosis, and thus can be usefully used as a treatment for endometriosis or complications of endometriosis.

Description

{A pharmaceutical composition for preventing or treating endometriosis comprising transcrocetin}

The present invention relates to a pharmaceutical composition for preventing or treating endometriosis comprising transcrocetin.

Endometriosis is a chronic gynecological disease that affects 5-10% of women of reproductive age. It is characterized by the presence of abnormal endometrial-like tissue outside the female reproductive organs. Endometriosis is believed to be caused by implantation of shed endometrial tissue back into the abdominal cavity due to retrograde menstrual blood flow. The main symptoms of endometriosis include pelvic pain, dysmenorrhea, and dyspepsia. These symptoms not only impair the patient's quality of life, but half of those with endometriosis are also infertile. While the prevalence of endometriosis has recently increased in women of reproductive age, the exact cause of endometriosis remains unclear, making accurate diagnosis difficult.

Treatment approaches for endometriosis include surgery, hormone therapy, and anti-inflammatory treatments, primarily focusing on symptom relief. However, currently available endometriosis treatments are limited by high recurrence rates and side effects such as bone loss, amenorrhea, weight gain, and depression.

Therefore, to improve the quality of life of patients with endometriosis, treatments with minimal side effects and effective management of the condition are needed. Accordingly, extensive research is currently being conducted based on phytochemicals, which exhibit various physiological effects.

Transcrocetin, an apocarotenoid extracted from saffron ( Crocus sativus L. ), is all-trans retinoic acid (ATRA) with neuroprotective and antioxidant properties. Transcrocetin is known to possess neuroprotective and anti-inflammatory effects, and it is known to exhibit anticancer effects by suppressing the invasiveness of breast cancer cells by inhibiting the expression of matrix metalloproteases. However, there is currently no research on the therapeutic efficacy of transcrocetin in endometriosis, a gynecological disease.

The present inventors studied plant-derived components that significantly contribute to the prevention, improvement, or treatment of endometriosis. They discovered that transcrocetin, isolated from saffron, inhibits endometrial cell proliferation, arrests the cell cycle, and induces endoplasmic reticulum stress and intracellular calcium accumulation. Furthermore, they confirmed that it increases mitochondrial membrane potential depolarization and reactive oxygen species production, demonstrating its potential as a therapeutic agent or supplement for the prevention or treatment of endometriosis. This discovery led to the completion of the present invention.

U.S. Patent No. US10842805 (registered on November 24, 2020)

The present inventors have diligently researched and discovered plant-based compounds that not only alleviate the symptoms of endometriosis but also inhibit its progression and provide significant therapeutic and preventive effects. They have also developed pharmaceutical compositions containing these compounds. As a result, they have confirmed that transcrocetin, isolated from saffron, inhibits endometrial cell proliferation, arrests the cell cycle, and induces endoplasmic reticulum stress and intracellular calcium accumulation. Furthermore, they have confirmed that transcrocetin increases mitochondrial membrane potential depolarization and reactive oxygen species production in endometrial cells. This has led to the completion of the present invention, demonstrating its potential use as a therapeutic agent or supplement for the prevention or treatment of endometriosis.

Accordingly, the purpose of the present invention is to provide a pharmaceutical composition for preventing or treating endometriosis or complications of endometriosis, which comprises transcrocetin or a saffron extract containing transcrocetin as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or improving endometriosis or complications of endometriosis, which comprises transcrocetin or a saffron extract containing transcrocetin as an active ingredient.

Other objects and advantages of the present invention will become more apparent from the detailed description, claims and drawings below.

According to one aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating endometriosis or complications of endometriosis, comprising transcrocetin or a saffron extract containing transcrocetin as an active ingredient.

The present inventors have diligently researched and discovered plant-based compounds that not only alleviate the symptoms of endometriosis but also inhibit its progression and provide significant therapeutic and preventive effects. They have also developed pharmaceutical compositions containing these compounds. As a result, they have confirmed that transcrocetin, isolated from saffron, or a saffron extract inhibits cell proliferation in endometrial cells, arrests the cell cycle, and induces endoplasmic reticulum stress and intracellular calcium accumulation. Furthermore, they have been shown to increase mitochondrial membrane potential depolarization and reactive oxygen species production, demonstrating their potential use as therapeutic agents or supplements for the prevention or treatment of endometriosis. This has led to the completion of the present invention.

In the present invention, the "transcrocetin" is an apocarotenoid that can be extracted from saffron and is represented by the following chemical formula 1:

[Chemical Formula 1]

.

In the present invention, the "saffron" is a spice made by drying the style of the flower of the saffron crocus ( Crocus sativus ), a plant belonging to the Iridaceae family, and is native to southern Europe and Southwest Asia, and is one of the most expensive spices in the world.

Saffron is rich in vitamins and minerals and contains antioxidant properties, known to help alleviate asthma, insomnia, arteriosclerosis, high blood pressure, and memory loss. It is also reported to be effective in protecting brain cells, reducing inflammation, detoxifying, and activating cell function. In the Middle East and India, saffron is known to be effective in treating diabetes, headaches, menstrual cramps, and anxiety.

The transcrocetin according to the present invention can be used in the form of a pharmaceutically acceptable salt derived from an inorganic acid or an organic acid, and the preferred salt may be at least one selected from the group consisting of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, acetic acid, glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid, mandelic acid, tartaric acid, citric acid, ascorbic acid, palmitic acid, maleic acid, hydroxymaleic acid, benzoic acid, hydroxybenzoic acid, phenylacetic acid, cinnamic acid, salicylic acid, methanesulfonic acid, benzenesulfonic acid, and toluenesulfonic acid, but is not limited thereto.

The present invention may include not only the above transcrocetin or a pharmaceutically acceptable salt thereof, but also hydrates, solvates and optical isomers that can be prepared therefrom.

The term "hydrate" as used herein refers to a compound of the present invention or a salt thereof containing a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces. The hydrate of the compound represented by the above chemical formula 1 of the present invention may contain a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces. The hydrate may contain at least 1 equivalent, preferably 1 to 5 equivalents of water. Such a hydrate may be prepared by crystallizing the compound represented by the above chemical formula 1 of the present invention, an isomer thereof, or a pharmaceutically acceptable salt thereof from water or a solvent containing water.

The term "solvate" as used herein refers to a compound of the present invention or a salt thereof that contains a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. Preferred solvents include those that are volatile, non-toxic, and/or suitable for human administration.

The term "isomer" as used herein refers to a compound of the present invention or a salt thereof that has the same chemical formula or molecular formula but is structurally or sterically different. Such isomers include structural isomers such as tautomers, stereoisomers such as R or S isomers having an asymmetric carbon center, geometric isomers (trans, sis), and optical isomers (enantiomers). All of these isomers and mixtures thereof are also included in the scope of the present invention.

The term "extract" used herein has the meaning commonly used in the art as a crude extract, but in a broader sense, it also includes fractions obtained by further fractionation of the extract. That is, the saffron extract includes not only those obtained using an extraction solvent, but also those obtained by additionally applying a purification process thereto. For example, fractions obtained by passing the extract through an ultrafiltration membrane having a certain molecular weight cut-off value, separation by various chromatographies (designed for separation according to size, charge, hydrophobicity, or affinity), and other fractions obtained through various additional purification methods are also included in the extract of the present invention.

When the extract used in the composition of the present invention is obtained by treating saffron with an extraction solvent, various organic solvents may be used. Preferably, a polar solvent or a non-polar solvent may be used.

Suitable polar solvents include, but are not limited to, (i) water, (ii) lower alcohols having C1 to C6 (specifically, methanol, ethanol, propanol, butanol, n-propanol, iso-propanol, n-butanol, 1-pentanol, 2-butoxyethanol or ethylene glycol), (iii) acetic acid, (iv) dimethyl-formamide (DMFO) and (v) dimethylsulfoxide (DMSO).

Suitable non-polar solvents include, but are not limited to, acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkanes, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, 1-pentene, 1-chlorobutane, 1-chloropentane, o-xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, chlorobenzene, benzene, diethyl ether, diethyl sulfide, chloroform, dichloromethane, 1,2-dichloroethane, aniline, diethylamine, ether, carbon tetrachloride, and THF (Tetrahydrofuran).

In one embodiment of the present invention, the saffron extract is extracted with any one extraction solvent selected from the group consisting of water, an organic solvent, and a mixed solvent thereof, wherein the organic solvent may be selected from the group consisting of lower alcohols having 1 to 4 carbon atoms, petroleum ether, hexane, benzene, chloroform, methylene chloride, ether, ethyl acetate, and acetone, but is not limited thereto.

In one specific example of the present invention, the alcohol is selected from the group consisting of methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, normal-butanol, and mixed solvents thereof.

In the present invention, saffron can be extracted using, but is not limited to, a hot water extraction method, a pressurized hot water extraction method, a cold extraction method, a reflux extraction method, a room temperature extraction method, or an ultrasonic extraction method.

To prepare an extract according to one embodiment of the present invention, the amount of solvent used for extraction may be appropriately selected depending on the amount of saffron used. Specifically, for example, the volume may be 1 to 20 times the weight of the powder used for preparing the extract, more specifically, 1 to 15 times the volume, 1 to 10 times the volume, or 1 to 5 times the volume, but is not limited thereto.

The extraction temperature of the ethanol extract according to one embodiment of the present invention is not particularly limited, and may be, for example, 4°C to 30°C, and specifically, 10°C to 30°C, 15°C to 30°C, 20°C to 30°C, 20°C to 25°C, or 15°C to 25°C, but is not limited thereto.

The extraction temperature of the hot water extract according to one embodiment of the present invention is not particularly limited, and may be, for example, 50°C to 130°C, and specifically, 70°C to 130°C, 80°C to 130°C, 90°C to 130°C, 100°C to 130°C, or 110°C to 130°C, but is not limited thereto.

The extraction time of the ethanol extract according to one embodiment of the present invention is not particularly limited, and may be, for example, 3 hours to 2 days, 3 hours to 24 hours, and specifically, 3 hours to 18 hours, 3 hours to 12 hours, or 3 hours to 6 hours, but is not limited thereto.

The extraction time of the hot water extract according to one embodiment of the present invention is not particularly limited, and for example, the hot water extraction may be performed by heating for 30 minutes or more. Specifically, for example, the extraction time may be 30 minutes to 24 hours, 30 minutes to 18 hours, specifically 30 minutes to 12 hours, 30 minutes to 10 hours, 30 minutes to 8 hours, 30 minutes to 6 hours, and more specifically 30 minutes to 3 hours, but is not limited thereto.

The term "comprising as an active ingredient" used in the present invention means including an amount sufficient to achieve the pharmacological efficacy or activity of the transcrocetin or the saffron extract containing transcrocetin, and includes that various ingredients may be additionally added for drug delivery, stabilization, and formulation.

The term "endometriosis" as used herein refers to a disease in which endometrial tissue proliferates in locations other than the surface of the uterine cavity. Endometriosis occurs in approximately 10-15% of women of reproductive age and is a significant cause of pelvic inflammatory disease, pelvic adhesions, ovarian cysts, uterine fibroids, ectopic pregnancy, and infertility. The pathophysiology of endometriosis is quite similar to that of tumor cells in that endometrial tissue infiltrates other tissues or metastasizes beyond its original site.

Meanwhile, endometriosis is a difficult disease to diagnose because it presents with a variety of symptoms depending on the location of the lesion, the affected organ, and the severity of the lesion. Currently, clinical tests used to diagnose endometriosis include transvaginal ultrasound, magnetic resonance imaging (MRI), blood tests, and laparoscopic surgery. Recently, various diagnostic methods are being developed, such as using genetic markers that can diagnose endometriosis by utilizing the genetic characteristics of each patient or predict recurrence after treatment.

Endometriosis, as mentioned above, primarily occurs in the abdominal organs or peritoneum, but can also develop in other organs far from the uterus, such as the intestines and lungs. Endometriosis that occurs in the ovaries is called endometrioma. Those with endometriosis in the ovaries are at a higher risk of developing ovarian cancer than the general population.

The term "prevention" as used herein refers to any protective action that inhibits or delays the onset of endometriosis, complications of endometriosis, or at least one symptom thereof by administering a pharmaceutical composition according to the present invention. It also includes treatment of a subject suffering from the disease to prevent or mitigate recurrence.

The term "treatment" used in the present invention means any act of improving or beneficially changing the symptoms of endometriosis, complications of endometriosis, or at least one or more symptoms thereof, such as alleviating, reducing, or eliminating the symptoms by administering a pharmaceutical composition according to the present invention.

The term "pharmaceutical composition" as used in the present invention refers to a composition administered for a specific purpose, and for the purposes of the present invention, means administered to prevent or treat endometriosis, complications of endometriosis, or at least one symptom thereof. The pharmaceutical composition comprises a pharmaceutically effective amount of transcrocetin or a saffron extract containing transcrocetin.

The pharmaceutical composition of the present invention may be formulated as a powder, granule, tablet, coated tablet, pill, sugar tablet, capsule, liquid, suspension, gel, syrup, slurry, suppository, enema, emulsion, paste, ointment, cream, lotion, powder, spray or suspension.

The pharmaceutical composition of the present invention may further comprise a suitable carrier, excipient or diluent commonly used in the manufacture of pharmaceutical compositions. Examples thereof include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, mannitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate or mineral oil.

In addition to the above ingredients, the pharmaceutical composition of the present invention may further include excipients, stabilizers, diluents, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers, vehicles, excipients, stabilizers, or diluents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount.

In the present invention, "pharmaceutically effective amount" means an amount sufficient to achieve preventive, alleviative, or therapeutic efficacy for endometriosis, complications of endometriosis, or at least one symptom thereof. The effective dosage level may be determined based on factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route, and excretion rate, treatment duration, concurrently used drugs, and other factors well known in the medical field. The amount of the composition used may vary depending on the patient's age and body weight, but may be administered once or several times daily in an amount sufficient to achieve a blood concentration of the compound useful for the treatment of endometriosis.

The pharmaceutical composition of the present invention can be manufactured in a unit dose form or can be manufactured by inserting it into a multi-dose container by formulating it using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains, and the method can be performed. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granules, tablet or capsule, and may additionally include a dispersing agent or stabilizer.

The dosage of the above composition may be increased or decreased depending on the route of administration, severity of the disease, body weight, age, etc. Therefore, the above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention can be administered to a subject via various routes. All modes of administration are conceivable, including intracerebral administration, oral ingestion, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, intrathecal injection, sublingual administration, buccal mucosa administration, rectal insertion, vaginal insertion, otic administration, nasal administration, inhalation, oral or nasal spraying, dermal administration, and transdermal administration.

The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents. It can be administered sequentially or simultaneously with conventional therapeutic agents, or in single or multiple doses. Taking all of the above factors into account, it is important to administer an amount that achieves maximum efficacy with minimal side effects. This can be readily determined by those skilled in the art.

In one embodiment of the present invention, the transcrocetin or the saffron extract containing transcrocetin inhibits proliferation of endometrial cells.

In the present invention, the endometrial cells may be cells derived from the vaginal mucosa, uterus, cervix, and endocervix.

In the present invention, the endometrial cells may specifically be VK2/E6E7 cell lines derived from vaginal mucosa, and End1/E6E7 cell lines derived from uterus, cervix, and endocervix.

According to one embodiment of the present invention, it was confirmed that the transcrocetin of the present invention inhibits proliferation of endometrial cells by inducing cell cycle arrest of endometrial cell lines.

According to one embodiment of the present invention, the transcrocetin of the present invention was confirmed to induce G0/G1 cell cycle arrest of an endometrial cell line.

In one embodiment of the present invention, the transcrocetin or the saffron extract containing transcrocetin increases endoplasmic reticulum stress in endometrial cells.

In the present invention, the endoplasmic reticulum (ER) plays a key role in stress sensing, signaling, and regulating organelle homeostasis. ER stress is associated with the export of calcium ions into the cytoplasm. Excessive accumulation of calcium ions in the cytoplasm disrupts the normal autophagy pathway and activates the apoptotic pathway. Recently, ER stress has been considered a novel therapeutic target for endometriosis.

According to one embodiment of the present invention, it was confirmed that the transcrocetin increases intracellular stress by increasing the expression of proteins such as GRP78, IRE1α, p-EIF2α, and ATF6α.

According to one embodiment of the present invention, the transcrocetin was found to be involved in the gradual increase in calcium ion levels in cells.

Thus, we confirmed that transclotidin-induced endoplasmic reticulum stress and calcium release into the cytoplasm of End1/E6E7 and VK2/E6E7 cells resulted in impaired autophagy flux and activation of the apoptotic pathway.

In one embodiment of the present invention, the transcrocetin or the saffron extract containing transcrocetin causes dysfunction of mitochondria in endometrial cells.

The term "mitochondrial dysfunction" as used in the present invention means loss of normal mitochondrial biological function, including, but not limited to, loss of electron transport chain efficiency, reduced synthesis of high-energy molecules such as ATP, increased production of reactive oxygen species, interference with cellular respiration, reduced mitochondrial membrane potential, mitochondrial fragmentation, mutations in mitochondrial DNA, etc.

In one embodiment of the present invention, the mitochondrial dysfunction comprises any one of the following: (a) depolarization of the membrane potential of the mitochondria; (b) disturbance of calcium ion homeostasis; (c) reduction of ATP-linked cellular respiration; or (d) a combination thereof.

According to one embodiment of the present invention, the transcrocetin of the present invention was confirmed to induce depolarization of the mitochondrial membrane potential by reducing the membrane potential of mitochondria in endometrial cells, thereby causing abnormal calcium ions to accumulate in the cytoplasm and mitochondrial matrix, causing calcium ion homeostasis disorder, and inhibiting cellular respiration including basal respiration, maximal respiration, and ATP production of endometriosis cells, thereby causing mitochondrial dysfunction.

The term “mitochondrial membrane potential (ΔΨm)” used in the present invention refers to the potential generated by the proton pump, which is essential for the energy storage process in oxidative phosphorylation. The mitochondrial membrane potential can be measured using a conventional method widely known in the field to which the present invention pertains, for example, JC-1, Rhodamine B hexyl ester, MitoTracker Red CMXRos, Rhodamine 6G, DiS-C3(3), HRB & HR101 or TMRE (tetramethylrhodamine, methyl ester) staining method, but is not limited thereto.

In the present invention, the intracellular electrochemical potential of calcium ions is maintained by various ion channels, pumps, and exchangers, and plays a role in regulating various cellular signaling pathways. In particular, calcium ion signaling is closely related to the production of reactive oxygen species (ROS), which regulate cell proliferation and apoptosis. Disrupted calcium ion homeostasis not only causes severe oxidative stress, but also exerts calcium ion stress on mitochondria, which act as calcium ion stores, and has detrimental effects on mitochondria and their membranes. Therefore, disruption of calcium ion homeostasis induces destabilization of the mitochondrial outer membrane, resulting in serious mitochondrial dysfunction.

In one embodiment of the present invention, the transcrocetin or the saffron extract containing transcrocetin induces autophagy of endometrial cells.

The above transcrocetin or saffron extract containing transcrocetin specifically induces autophagy of endometrial cells and impairs the flux of the late autophagy stage.

In one embodiment of the present invention, the transcrocetin or the saffron extract containing transcrocetin induces oxidative stress in endometrial cells.

In one embodiment of the present invention, the transcrocetin or the saffron extract containing transcrocetin regulates the expression of inflammation-related proteins in endometrial cells.

According to one embodiment of the present invention, it was confirmed that the transcrocetin reduces the expression of MyD99, IRAK4, TAK1, and NFkB in endometrial cells.

According to one embodiment of the present invention, it was confirmed that the transcrocetin inhibits the development of endometriosis lesions by suppressing the expression of genes related to adhesion of endometrial cell lines, increasing the expression of genes related to apoptosis, increasing the expression of genes related to endoplasmic reticulum stress, and decreasing the expression of proteins related to mitochondrial function.

According to one embodiment of the present invention, it was confirmed that the transcrocetin inhibits the development of endometriosis by causing cell death due to mitochondrial dysfunction in endometrial cell lines.

In one embodiment of the present invention, the endometriosis complication may be at least one selected from the group consisting of pelvic inflammatory disease, pelvic adhesions, ovarian cysts, uterine fibroids, ectopic pregnancy, and infertility, but is not limited thereto.

According to one aspect of the present invention, the present invention provides a food composition for preventing or improving endometriosis or complications of endometriosis, comprising transcrocetin or a saffron extract containing transcrocetin as an active ingredient.

The term "improvement" used in the present invention means any action that at least reduces the degree of symptoms of endometriosis or endometriosis complications by administering a composition containing transcrocetin according to the present invention as an active ingredient.

When the composition of the present invention is manufactured as a food composition, it includes ingredients commonly added during food manufacturing, such as proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. Examples of the carbohydrates mentioned above include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, oligosaccharides, etc.; and polysaccharides such as dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavoring agents and synthetic flavoring agents can be used as flavoring agents. For example, when the food composition of the present invention is manufactured into a drink, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, etc. may be additionally included in addition to the effective ingredient of the present invention.

In one embodiment of the present invention, the food composition comprises a health functional food. The health functional food refers to a food manufactured and processed using raw materials or ingredients with functional properties useful to the human body as defined in Act No. 6722 on Health Functional Foods, and refers to a food consumed for the purpose of obtaining beneficial effects for health purposes, such as regulating nutrients for the structure and function of the human body or physiological functions.

The health functional food of the present invention may contain conventional food additives, and its suitability as a food additive is determined by the specifications and standards for the relevant item in accordance with the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety, unless otherwise specified. Items listed in the above 'Food Additives Codex' include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, sucrose pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, noodle additive alkaline agents, preservative preparations, and tar color preparations. For example, a health functional food in the form of a tablet may be prepared by mixing the active ingredient of the present invention with an excipient, a binder, a disintegrant, and other additives, granulating the mixture using a conventional method, and then adding a lubricant, etc., and compression molding, or directly compression molding the mixture. In addition, the health functional food in the form of tablets may contain a fermenting agent, etc., if necessary. Among the health functional foods in the form of capsules, hard capsules can be manufactured by filling a mixture of the active ingredient of the present invention with additives such as excipients into a conventional hard capsule, and soft capsules can be manufactured by filling a mixture of the active ingredient of the present invention with additives such as excipients into a capsule base such as gelatin. The soft capsules may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative, etc., if necessary. The health functional food in the form of a ring can be prepared by molding a mixture of the active ingredient of the present invention with an excipient, a binder, a disintegrant, etc., by a conventionally known method, and, if necessary, can be coated with white sugar or another coating agent, or the surface can be coated with a material such as starch or talc. A health functional food in granular form can be manufactured into a granular form by mixing a mixture of the active ingredient of the present invention, excipients, binders, disintegrants, etc., using a conventionally known method, and may contain a flavoring agent, a flavoring agent, etc., as needed.

In one embodiment of the present invention, the endometriosis complication may be at least one selected from the group consisting of pelvic inflammatory disease, pelvic adhesions, ovarian cysts, uterine fibroids, ectopic pregnancy, and infertility, but is not limited thereto.

In the case of the food composition of the present invention, the transcrocetin and the active ingredient included in the pharmaceutical composition, which is an embodiment of the present invention described above, are the same, so the common contents are applicable as they are. Therefore, in order to avoid excessive complexity in the description of this specification, the overlapping contents are cited and the description thereof is omitted.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a pharmaceutical composition for preventing or treating endometriosis or complications of endometriosis, comprising transcrocetin or a saffron extract containing transcrocetin as an active ingredient.

(b) The present invention provides a health functional food composition for preventing or improving endometriosis or complications of endometriosis, which comprises transcrocetin or a saffron extract containing transcrocetin as an active ingredient.

(c) When a pharmaceutical composition for preventing or treating endometriosis or endometriosis complications containing transcrocetin or a saffron extract containing transcrocetin of the present invention as an active ingredient is used, endometriosis can be effectively managed with fewer side effects compared to surgery and hormone therapy, and thus can be usefully used as a treatment for endometriosis or endometriosis complications.

Figures 1a to 1e show the results of analyzing the effects of transcrocetin on cell proliferation, cell cycle, and p21 protein expression in endometrial cells (VK2/E6E7 and End1/E6E7).
Figure 1f shows the results of analyzing the effect of transcrocetin treatment on p21 protein expression in T-HESC cells.
Figures 2a to 2d show the results of analyzing the regulation of protein expression related to endoplasmic reticulum stress and calcium ion accumulation patterns according to transcrocetin treatment in endometrial cells.
Figures 3a to 3c show the results of analyzing the changes in mitochondrial membrane potential and the pattern of calcium accumulation in mitochondria following transcrocetin treatment in endometrial cells.
Figures 4a to 4e show the results of analyzing changes in mitochondrial cell respiration and OXPHOS gene expression by transcrocetin in endometrial cells.
Figures 5a to 5d show the results of analyzing the changes in the production of reactive oxygen species and the effects on cell proliferation according to transcrocetin alone or in combination with an inhibitor in endometrial cells.
Figures 6a and 6b show the results of measuring the regulation of inflammation and autophagy-related protein expression according to transcrocetin treatment in endometrial cells.
Figures 7a to 7d show the results of measuring the regulation of autophagy-related protein expression and acidic endoplasmic reticulum (AVO) accumulation according to single or combined treatment with transcrocetin and an autophagy inhibitor in endometrial cells.
Figure 8 shows the mechanism of cell proliferation inhibition by transcrocetin in endometrial cells.

Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

Example

Experimental Example 1: Cell Culture

Endometrial cell lines VK2/E6E7 and End1/E6E7 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Both cells were cultured in a 5% CO 2 , ₃₇ °C incubator using Keratinocyte Serum-Free Medium (Gibco, Waltham, MA, USA) supplemented with bovine pituitary extract (0.05 mg/mL) and human recombinant epidermal growth factor (EGF) (0.1 ng/mL) for cell monolayer culture. Subculture was also performed using DMEM/F12 (HyClone) 1:1 medium containing 10% inactivated fetal bovine serum (FBS).

T-HESC cells were purchased from ATCC and cultured in DMEM/F12 1:1 medium containing 3.1 g/L glucose and 1 mM sodium pyruvate without phenol red, supplemented with 1.5 g/L sodium bicarbonate, 1% Corning ^™ ITS+Premix Universal Culture Supplement (Cat No: CB-40352), 500 ng/ml puromycin (sigma, Cat No: D8833), and 10% charcoal/dextran-treated FBS.

Normal uterine stromal cells (HUtF, FC-0076) were purchased from Lifeline cell technology (Frederick, MD, USA) and used using ReproLife ^TM . Both endometrial cells and normal cell lines were cultured in a cell incubator at 5% CO ₂ and 37°C.

Experimental Example 2: Cytotoxicity and cell proliferation inhibition assay of transcrocetin using MTT and BrdU.

The present inventors conducted an MTT cell viability evaluation and a BrdU cell proliferation evaluation to determine the effect of transcrocetin treatment on the survival and proliferation ability of endometrial cells.

End1/E6E7 and VK2/E6E7 cells were seeded at a density of 5 × 10 ³ cells/well in 96-well plates, treated with transclorotin in a dose-dependent manner (0, 2, 5, 10, and 20 μM), and cultured for 48 h. Experiments were then performed using the MTT kit (Cat No: 11465007001, Roche) and Brd U kit (Cat No: 1167229001, Roche) according to the manufacturers' instructions.

For the MTT assay, MTT labeling reagent (1X) was dispensed into each well and incubated for 4 hours in a 37℃/5% _CO2 incubator. Then, solubilization buffer was treated to each well and incubated for more than 16 hours in a 37℃/5% _CO2 incubator. Cell viability was analyzed by measuring the absorbance at 560 nm and 650 nm using an ELISA reader.

For the BrdU assay, 10 μM BrdU (Bromodeoxyuridine) was added to each well and cultured for 2 hours in a 37°C, 5% CO ₂ incubator. Next, the cells were fixed, anti-BrdU-POD solution was added, and the cells were incubated at room temperature for 90 minutes. After the reaction was completed, the cells were washed three times. Finally, 100 μL of 3,3',5,5'-tetramethylbenzidine substrate was added and the reaction was performed, and the cell proliferation ability was analyzed by measuring the absorbance at wavelengths of 370 nm and 492 nm.

Experimental Example 3: Cell Cycle Analysis

To confirm the cell cycle changes in endometrial cells caused by transcrocetin, the inventors performed flow cytometry analysis.

Specifically, 5×10 ³ cultured VK2/E6E7, End1/E6E7, and T-HESC cells were treated with transclocetin in a dose-dependent manner (0, 2, 5, 10, and 20 μM) and cultured for 48 h at 37°C in a 5% CO ₂ incubator.

Afterwards, cells were detached from the culture dish using trypsin, washed with 0.1% BSA/PBS, and fixed with 70% ethanol for 24 hours. Afterwards, cells were washed with 0.1% BSA/PBS, and stained with PI (50 mg/mL) under RNase A (10 mg/mL, Sigma). The stained solution was transferred to a FACS tube, and the fluorescence intensity and cell cycle were analyzed using a flow cytometer.

Experimental Example 4: Immunofluorescence

The present inventors aimed to confirm the expression of p21 Waf1/Cip1 protein in endometrial cells following transcrocetin treatment, and performed immunofluorescence imaging for confocal imaging of p21 Waf1/Cip1 protein expression.

Specifically, endometrial cells were seeded at a density of 6 × 10 ⁴ cells/dish in a confocal dish (Cat No: 100350, SPL Life Science, Republic of Korea) with 300 μl of medium containing 10% FBS, cultured, and treated with 20 μM transclorotin for 48 h. After culture, the cells were fixed with methanol for 10 min, treated with p21 Waf1/Cip1 antibody diluted 1:400, and incubated at 4°C for 16 h. Afterwards, the cells were washed twice with PBS containing 0.1% BSA (bovine serum albumin), and goat anti-rabbit IgG Alexa 594 (Cat No: A-11001, Invitrogen, Carlsbad, CA, USA) was diluted 1:250 in antibody dilution buffer and incubated at room temperature for 1 h. After culture, endometrial cells were washed with 0.1% BSA-PBS and additionally stained with DAPI (4'6-diamidino-2-phenylindole) to simultaneously observe the target protein and nuclei within the endometrial cells. After the experiment, cells were observed and photographed using an LSM710 (Carl Zeiss, Oberkochen, Germany) confocal microscope.

Experimental Example 5: Protein Expression Analysis

The present inventors performed western blot analysis to analyze the expression of proteins associated with the development of endometriosis when treated with transcrocetin.

Specifically, endometrial cells were treated with transclocetin in a dose-dependent manner (0, 5, 10, and 20 μM) or in combination with a signal transduction pathway inhibitor (CQ), and then total proteins were extracted from the treated cells and quantified using the Bradford protein assay (Bio-red, Hercules, CA, USA).

Afterwards, the extracted proteins were denatured at 95°C for 5 minutes, electrophoresis was performed using a 10% SDS/PAGE gel, and then transferred to a nitrocellulose membrane. After sequential incubation with primary and secondary antibodies on the membrane, the expression of the target protein was analyzed using a chemiDoc EQ system and Quantity One software (Bio-rad) using a cheiluminescence detection reagent (Supersignal West Pico, Pierce, Rockford, IL, USA).

Experimental Example 6: Measurement of mitochondrial membrane potential using JC-1 staining.

To confirm the change in mitochondrial membrane potential (MMP) of endometrial cells by transclorotin, the inventors measured mitochondrial membrane potential using a mitochondria staining kit (Cat no: CS0390, sigma-aldrich).

Specifically, endometrial cell lines End1/E6E7 and VK1/E6E7 were cultured in 6 wells, and transclorotin was added to the wells in a dose-dependent manner (0, 2, 5, 10, and 20 μM) and cultured in a 37°C, 5% _CO2 incubator for 48 h. Afterwards, cells were detached using trypsin and centrifuged to obtain a cell pellet. The cell pellet was resuspended in JC-1 staining solution and cultured in a 37°C, 5% _CO2 incubator for 20 min. The stained cells were centrifuged again, washed with 1X JC-1 staining buffer, and then analyzed for changes in mitochondrial membrane potential using a flow cytometer.

Experimental Example 7: Analysis of Changes in Reactive Oxygen Species Levels

To determine the effect of transcrocetin on the production of reactive oxygen species in endometrial cells, the inventors attempted to determine the level of ROS using flow cytometry.

Specifically, cultured endometrial cells were detached using trypsin and centrifuged to obtain a cell pellet. After washing with PBS, the cells were stained with DCFH-DA (final concentration: 10 μM) for 30 minutes. The cells were then washed with PBS and treated with transclocetin dose-dependently or in combination with 2 mM N-acetyl stain (NAC) for 1 hour in a 37°C incubator. After the reaction, the cells were washed with PBS, and the fluorescent DCF, an indicator of ROS levels, was detected by flow cytometry.

Experimental Example 8: Analysis of Mitochondrial Respiration Regulation

To confirm the mitochondrial respiration regulation effect of transcrocetin on endometrial cells, the present inventors purchased an XF Cell mitostress kit (Agilent Technologies, Santa Clara, CA, USA) and conducted an experiment.

Specifically, endometrial cells were seeded in 24 wells at a density of 5.0 × 10 ^{4 -} cells/well, cultured in a 37°C, 5% CO2 incubator for 16 h, and then treated with 20 μM transclocetin for 20 h. Subsequently, both cell lines were sequentially treated with oligomycin, FCCP, rotenone, and antimycin A to calculate various mitochondrial respiration parameters.

Experimental Example 9: Analysis of Gene Expression Changes Using Real-Time Polymerase Chain Reaction

The present inventors performed quantitative RT-PCR analysis to analyze gene expression related to endometriosis development during transcrocetin treatment.

Specifically, total RNA of each cell line was extracted using TransZol Up reagent from cells that were not treated with transcrocetin and cells that were treated with transcrocetin in the endometrial cells, and the extracted RNA was synthesized into cDNA.

Primers were designed to be approximately 80 to 150 bp using the Primer 3 program, and a real-time PCR detection system using SYBR green (Sigma-Aldrich) was performed. The analysis of each gene and the GAPDH gene was repeated three times, and the standard curve and Ct values were normalized to the expression level of the GAPDH gene and analyzed to confirm the expression level. PCR conditions: 94°C, 3 min (denaturation step) and 40 cycles (94°C, 20 s - denaturation; 60°C, 40 s - annealing; 72°C, 1 min - extension). Melting curve program analysis was performed by obtaining continuous fluorescence measurements while increasing the temperature from 55°C to 95°C at a rate of 0.5°C per 10 s. Quantitative RT-PCR products were identified according to the melting curve obtained by the Ct value, and the relative gene expression pattern was analyzed according to the 2 ^-ΔΔCt method.

Experimental Example 10: Statistical Analysis

The results of this experiment were analyzed by least-squares analysis of variance (ANOVA) using the general linear model procedure of the SAS (statistical analysis system) statistical program. All experiments were repeated at least three times, and the results were expressed as means and standard deviations to assess significant differences between the vehicle or naive group and the transclocetin group. Significance levels are indicated by asterisks (* P <0.05, ** P <0.01, and *** P <0.001).

Example 1: Analysis of the efficacy of transcrocetin in inhibiting endometrial cell proliferation and inducing cell cycle arrest.

To confirm the effect of transcrocetin on the proliferative ability of endometrial cells, the inventors conducted brdU cell proliferation evaluation and cell cycle analysis, and the results are shown in Figure 1.

As shown in Figures 1a and 1b, it was confirmed that the viability of endometrial cells was reduced by 61.1% in End1/E6E7 and 57.1% in VK2/E6E7 due to transcrocetin, and the proliferation ability of cells was reduced by 75.48% in End1/E6E7 and 56.58% in VK2/E6E7.

In addition, as a result of examining cytotoxicity by treating normal cell lines with transcrocetin, it was confirmed that it did not have a significant effect on cell survival, as shown in Figure 1c.

Additionally, cell cycle analysis was performed to determine whether transcrocetin could induce cell cycle arrest in endometriosis, and the results are shown in Fig. 1d.

According to Fig. 1d, the relative G0/G1 phase population of End1/E6E7 cells increased from 68.9% to 75.3% after transcrocetin treatment, and in VK2/E6E7 cells, it increased from 66.8% to 73.0%, confirming that transcrocetin treatment caused cell cycle arrest and induced cell death.

In addition, to confirm the effect of transcrocetin treatment on the expression of p21 Waf1, a cell cycle-related protein in endometrial cells, immunofluorescence was used, and the results are shown in Fig. 1e.

As shown in Fig. 1e, p21 Waf1/Cip1 expression was dramatically increased by 20 μM transcloxetin in both End1/E6E7 and VK2/E6E7 cells, which was consistent with the cell cycle arrest results.

Finally, we additionally confirmed that there was no change in the expression of p21 Waf1/Cip1, a cell cycle-related protein, in normal endometrial stromal cell lines (T-HESC) (Fig. 1f).

Therefore, these results suggest that transclocetin may inhibit the progression of endometriosis by affecting endometrial cell survival and proliferation.

Example 2: Analysis of transcrocetin's effects on endoplasmic reticulum stress and increased cytosolic calcium ion concentration.

To confirm the effect of transcrocetin on endoplasmic reticulum stress, the inventors analyzed changes in the expression of proteins related to endoplasmic reticulum stress, and the results are shown in Figures 2a and 2b.

As shown in Figures 2a to 2c, transcrocetin was found to increase GRP78 and p-eIF2α, among endoplasmic reticulum stress-related proteins, by up to 2.5-fold in End1/E6E7 and VK2/E6E7 cells. IRE1α was found to increase 1.6-fold in End1/E6E7 cells upon transcrocetin treatment, but was reduced by half in VK2/E6E7 cells. ATF6α was found to be reduced in both cell lines.

In addition, to confirm whether transcrocetin causes calcium ion accumulation in the cytoplasm, cytoplasmic calcium accumulation was analyzed using Fluo-4 dye, and the results are shown in Fig. 2d.

According to Fig. 2d, it was confirmed that when transcrocetin was treated, the calcium ion accumulation level increased by up to 169% in End1/E6E7 cells and up to 246% in VK2/E6E7 cells.

Therefore, these results suggest that transcrocetin may inhibit the progression of endometriosis by not only inducing endoplasmic reticulum stress in endometrial cells but also accumulating intracytoplasmic calcium ions.

Example 3: Analysis of changes in mitochondrial membrane potential and calcium ion levels in the mitochondrial matrix by transcrocetin.

The present inventors analyzed the change in mitochondrial membrane potential in endometrial cells by transcrocetin treatment using JC-1 staining, and the results are shown in Figure 3a.

As shown in Figures 3a and 3b, transcrocetin increased the rate of mitochondrial membrane potential loss by 279% in End1/E6E7 cells and by 412% in VK2/E6E7 cells. It was also confirmed that there was no change in mitochondrial membrane potential loss in normal endometrial stromal cells (T-HESC).

In addition, the inventors of the present invention attempted to confirm changes in the level of calcium ions in the mitochondrial matrix of endometrial cells following transcrocetin treatment using Rhod-2 staining, and the results are shown in Fig. 3c.

According to Figure 3c, as a result of transcrocetin treatment, calcium accumulation was observed to increase by 145% in End1/E6E7 cells and by 191% in VK2/E6E7 cells.

These results confirm that transcrocetin disrupts mitochondrial electrophysiological properties and induces a unique apoptotic pathway in endometrial cell lines.

Example 4: Analysis of the induction of mitochondrial-based bioenergetic changes in endometrial cells by transclocetin.

The present inventors analyzed the changes in mitochondrial bioenergy when transcrocetin was treated in endometrial cells using seahorse XFe, and the results are shown in Figures 4a to 4e.

As shown in Figures 4a to 4d, maximal respiration was reduced to the basal respiration level in both End1/E6E7 and VK2/E6E7 cells by transcrocetin treatment, and ultimately, the basal and maximal respiration rates were observed to be approximately 1/3 and 1/2, respectively, in End1/E6E7 and VK2/E6E7, respectively, compared to the control group. Accordingly, ATP production and cellular reserve capacity were also observed to be lower in transcrocetin-treated cells compared to the control group.

Additionally, as a result of confirming the change in expression of mitochondrial complexes and their subunits (OXPHOS) by transcrocetin at the transcriptional level, as shown in Fig. 4e, it was confirmed that the expression of genes related to mitochondrial respiratory complexes I and III was all decreased by transcrocetin, and the expression of genes related to mitochondrial respiratory complex V was increased.

The above results imply that transcrocetin treatment of endometrial cells may affect mitochondrial dysfunction by reducing ATP-linked respiration.

Example 5: Analysis of the inhibition of cell proliferation by transcrocetin due to disruption of reactive oxygen species homeostasis in endometrial cells.

The present inventors conducted flow cytometry analysis to confirm the effect of transcrocetin on the production of reactive oxygen species in endometrial cells, and the results are shown in Figures 5a to 5c.

As shown in Figures 5a and 5b, when endometrial cells were treated with transclocetin in a dose-dependent manner (0, 2, 5, 10, 20 μM), a dramatic increase in intracellular ROS levels was observed in both cells, while no significant change was observed in the normal endometrial stromal cell line (T-HESC).

In addition, we sought to determine whether treatment of endometrial cell lines with NAC, a ROS scavenger, alone or in combination with transclocetin affected changes in ROS levels and cell proliferation, and the results are shown in Fig. 5c.

As shown in Fig. 5c, when NAC (5 mM) was treated alone, the elevated ROS level in END1/E6E7 cells was restored to normal levels, and in VK2/E6E7 cells, the ROS level was confirmed to be reduced from 493% to 349%. When cell proliferation ability was measured under the same conditions, it was also confirmed that cell proliferation ability was restored from 68% to 82% and 103% in both cell lines, respectively (Fig. 5d).

Through the above results, it was confirmed that oxidative stress induced by transcrocetin affects the proliferation of endometrial cells.

Example 6: Analysis of the efficacy of transcrocetin in regulating inflammation and autophagy in endometrial cells.

To confirm the effect of transclotidin on the expression of intracellular inflammation and autophagy-related proteins in endometrial cells, the inventors analyzed inflammation-related proteins MyD99, p-IRAK4, p-TAK1, p-NFkB, and autophagy-related proteins p-SQSTM1/p62, t-SQSTM1/p62, LC3B I/II using Western blot, and the results are shown in Figures 6a and 6b.

According to Figures 6a and 6b, the expression of inflammation-related proteins in endometrial cells End1/E6E7 and VK2/E6E7 was reduced in a dose-dependent manner with transcrocetin treatment, and it was confirmed that the expression of autophagy-related proteins was regulated by transcrocetin.

Additionally, to determine whether autophagy flux is impaired in endometrial cells by transclorotin, we attempted to determine whether autophagy inhibitor chloroquine was used alone or in combination, and the results are shown in Figures 7a and 7b.

As a result, in both End1/E6E7 and VK2/E6E7 cell lines, when transcrocetin was treated alone or in combination with chloroquine, the expression of p-SQSTM1/p62 and t-SQSTM1/p62 proteins increased, while the expression of LC3B protein did not change. In the case of LC3B protein expression, it was observed that the expression increased when chloroquine was treated alone. In addition, as a result of confirming whether autophagy induced by transcrocetin induces inhibition of cell proliferation ability and accumulation of acidic endoplasmic reticulum, it was confirmed that when transcrocetin was treated in combination with chloroquine, cell proliferation was reduced by 11% and 18% more, respectively, and more acidic endoplasmic reticulum was accumulated than when transcrocetin was treated alone (Figures 7c and 7d).

The above results indicate that transcrocetin induces autophagy in endometrial cells, and specifically, impairs the flux in late stage autophagy.

In summary, the present inventors sought to determine the effects of transcrocetin, isolated from saffron, on endometrial cells. As a result, the transcrocetin of the present invention was found to inhibit cell proliferation and arrest the cell cycle by inducing endoplasmic reticulum stress and intracellular calcium accumulation. Furthermore, the inventors demonstrated that transcrocetin can aid in the prevention or treatment of endometriosis by increasing mitochondrial membrane potential depolarization and reactive oxygen species production in endometrial cells and by impairing normal autophagy. Therefore, the proposed transcrocetin is expected to be useful in the development of potential therapeutics or supplements for the prevention or treatment of endometriosis.

Claims (10)

A pharmaceutical composition for preventing or treating endometriosis or complications of endometriosis, comprising transcrocetin represented by the following chemical formula 1 or a saffron extract containing transcrocetin as an active ingredient:
[Chemical Formula 1]
.
A pharmaceutical composition according to claim 1, characterized in that the transcrocetin or the saffron extract containing transcrocetin inhibits proliferation of endometrial cells.
A pharmaceutical composition according to claim 1, characterized in that the transcrocetin or the saffron extract containing transcrocetin increases endoplasmic reticulum stress in endometrial cells.
A pharmaceutical composition according to claim 1, characterized in that the transcrocetin or the saffron extract containing transcrocetin causes dysfunction of mitochondria in endometrial cells.
A pharmaceutical composition according to claim 1, characterized in that the transcrocetin or the saffron extract containing transcrocetin induces autophagy of endometrial cells.
A pharmaceutical composition according to claim 1, characterized in that the transcrocetin or the saffron extract containing transcrocetin induces oxidative stress in endometrial cells.
A pharmaceutical composition according to claim 1, characterized in that the transcrocetin or the saffron extract containing transcrocetin regulates the expression of inflammation-related proteins in endometrial cells.
A pharmaceutical composition, characterized in that in claim 1, the endometriosis complication is at least one selected from the group consisting of pelvic inflammatory disease, pelvic adhesions, ovarian cysts, uterine fibroids, ectopic pregnancy, and infertility.
A food composition for preventing or improving endometriosis or complications of endometriosis, comprising transcrocetin represented by the following chemical formula 1 or a saffron extract containing transcrocetin as an active ingredient:
[Chemical Formula 1]
.
A food composition according to claim 9, characterized in that the endometriosis complication is at least one selected from the group consisting of pelvic inflammatory disease, pelvic adhesions, ovarian cysts, uterine fibroids, ectopic pregnancy, and infertility.