KR20240097394A - Composition for preventing, ameliorating or treating dysmenorrhea containing extract of Aster spathulifolius as effective component - Google Patents
Composition for preventing, ameliorating or treating dysmenorrhea containing extract of Aster spathulifolius as effective component Download PDFInfo
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- KR20240097394A KR20240097394A KR1020220179410A KR20220179410A KR20240097394A KR 20240097394 A KR20240097394 A KR 20240097394A KR 1020220179410 A KR1020220179410 A KR 1020220179410A KR 20220179410 A KR20220179410 A KR 20220179410A KR 20240097394 A KR20240097394 A KR 20240097394A
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- menstrual pain
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Classifications
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- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
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- A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
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- A61K36/18—Magnoliophyta (angiosperms)
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- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2250/00—Food ingredients
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
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Abstract
본 발명은 해국 추출물을 유효성분으로 포함하는 생리통의 예방, 개선 또는 치료용 조성물에 관한 것으로, 에스트라디올을 투여하여 자궁내막을 두껍게한 후, 옥시토신을 투여하여 생리통을 유발시킨 동물모델에 해국 추출물 투여시, 생리통이 완화되는 효과가 우수하였고, 체중 및 자궁의 무게는 변화 없이 과도한 자궁의 수축을 효과적으로 억제하였으며, 자궁근육세포에서 PGF2α 및 IL1β 처리에 의해 증가된 NF-κB의 활성 및 p65(NF-κB의 subunit 중 하나)의 핵내 이동을 효과적으로 억제하였고, 비만세포의 과도한 히스타민 방출을 억제하였으므로, 해국 추출물을 유효성분으로 포함하는 본 발명의 조성물은 생리통의 개선 또는 치료용 건강기능식품 또는 의약외품, 생리통의치료제로 유용하게 사용될 수 있다.The present invention relates to a composition for preventing, improving, or treating menstrual pain containing a sea chrysanthemum extract as an active ingredient, which involves administering the sea chrysanthemum extract to an animal model in which menstrual pain is induced by administering oxytocin after thickening the endometrium by administering estradiol. It was excellent in alleviating menstrual pain, effectively suppressed excessive uterine contraction without changing body weight and uterine weight, and increased NF-κB activity and p65 (NF-) in uterine muscle cells by treatment with PGF2α and IL1β. Since it effectively inhibited the intranuclear movement of (one of the subunits of κB) and suppressed excessive histamine release from mast cells, the composition of the present invention containing the extract of sea chrysanthemum as an active ingredient is a health functional food or quasi-drug for improving or treating menstrual pain, and menstrual pain. It can be useful as a medical treatment.
Description
본 발명은 해국 추출물을 유효성분으로 포함하는 생리통의 예방, 개선 또는 치료용 조성물에 관한 것이다. The present invention relates to a composition for preventing, improving, or treating menstrual pain containing a sea chrysanthemum extract as an active ingredient.
생리통은 사춘기가 지난 여성의 약 50%가 경험하는 가장 흔한 부인과 질병으로서, 그 중 약 10 내지 15%의 여성은 매달 1 내지 3일 동안 일상생활을 하기 힘들 정도의 통증을 느끼게 된다. 이로 인해, 생리통은 젊은 여성들이 직장 또는 학교를 쉬게 되는 주요 원인이 되고 있으며 작업시간의 손실이나 학습장애를 일으킨다.Menstrual pain is the most common gynecological disease experienced by about 50% of women after puberty, of which about 10 to 15% of women feel pain that is difficult to carry out their daily lives for 1 to 3 days every month. As a result, menstrual pain is a major cause of young women taking time off from work or school, causing loss of work time and learning disabilities.
생리통은 그 원인에 따라 크게 원발성(1차성) 생리통과 속발성(2차성) 생리통으로 분류된다. 원발성 생리통은 골반이나 자궁내에 특별한 병변없이 발생하는 체질적인 생리통으로, 대부분의 생리통이 이에 해당한다. 자궁수축이 항진되어 나타나는 현상으로서, 한의학적인 측면은 통하지 않기 때문에 압력이 높아져서 통증이 발생한다는 '불통즉통(不通則痛)'으로 인식하고 있다. 보통의 경우 통증은 생리를 시작하기 수 시간 전이나 시작한 직후부터 시작되어 2~3일간 증세가 지속된다. 방광위쪽에서 진통과 비슷한 양상으로 느껴지며, 허리 및 천골 통증과 허벅지까지 통증이 전파될 수 있고, 오심 및 구토와 함께 설사를 동반할 수도 있다. Depending on the cause, menstrual pain is broadly classified into primary (primary) menstrual pain and secondary (secondary) menstrual pain. Primary dysmenorrhea is constitutional dysmenorrhea that occurs without any specific lesion in the pelvis or uterus, and most types of dysmenorrhea fall into this category. It is a phenomenon that occurs due to increased uterine contractions, and in oriental medicine, it is recognized as 'non-communicating pain', which means that pain occurs due to increased pressure because there is no flow. In most cases, pain begins a few hours before or immediately after menstruation begins and lasts for 2 to 3 days. It is felt similar to labor pain above the bladder, and pain may spread to the lower back, sacrum, and thighs, and may be accompanied by nausea and vomiting, as well as diarrhea.
한편, 속발성 생리통은 자궁이나 주위 장기에 특별한 병변이 생겨서 발생하는 통증으로서, 그 원인이 되는 병변으로는 자궁근종, 자궁선근종, 자궁내막증, 난소낭종, 골반염, 골반 유착, 자궁내 피임장치, 종양 등이 있다. 그 통증은 초경이 지난 몇년 후에 이러한 병변이 발생함에 따라 나타나는데, 통증이 생리 시작 1~2주전부터 나타나기 시작하여 생리가 끝난 이후에도 며칠간 지속된다는 점이 원발성 생리통과 다른 특징이다.Meanwhile, secondary dysmenorrhea is pain caused by a special lesion in the uterus or surrounding organs. The lesions that cause it include uterine fibroids, adenomyosis, endometriosis, ovarian cysts, pelvic inflammatory disease, pelvic adhesions, intrauterine contraceptive devices, tumors, etc. There is. The pain appears as these lesions develop several years after menarche, and is different from primary dysmenorrhea in that the pain begins 1 to 2 weeks before menstruation and continues for several days even after menstruation ends.
일반적으로, 자궁은 생리주기에 따라 자궁수축의 강도가 다르다. 생리통이 없는 정상인의 경우, 생리 중에는 분만시와 같은 과도한 자궁수축이 일어나므로 자궁내 압력이 100㎜Hg 이상으로 상승하기도 하고, 증식기에는 자궁수축의 강도와 빈도가 떨어져 자궁내 압력이 약 10~30㎜Hg를 나타내며, 황체기에는 자궁내 압력이 60~90㎜Hg 정도로 약간 증가하여 생리 수일 전에는 분만의 시작 전과 비슷한 자궁수축이 일어난다. 비배란주기, 즉, 프로게스테론(progesterone)이 부족한 경우에는 증식기와 비슷한 자궁수축만을 나타낸다. 그러나, 원발성 생리통 환자는 정상인에 비하여 자궁수축이 지속되는 시간이 길며, 자궁수축이 없는 동안의 자궁내 압력 또한 더 높고, 수축시에는 자궁내 압력이 약 200~300㎜Hg까지 상승하기도 한다. 이러한 과도한 자궁수축으로 자궁으로의 혈류 공급이 줄어들어 자궁내에 빈혈이 초래되어 원발성 생리통이 나타나게 되는 것으로 여겨진다.In general, the intensity of uterine contractions varies depending on the menstrual cycle. In normal people without menstrual cramps, excessive uterine contractions, similar to those during childbirth, occur during menstruation, so the intrauterine pressure may rise to over 100 mmHg, and during the proliferative phase, the intensity and frequency of uterine contractions decrease, resulting in intrauterine pressures of approximately 10 to 30 mmHg. It represents mmHg, and during the luteal phase, the intrauterine pressure slightly increases to about 60-90 mmHg, causing uterine contractions similar to those before the start of labor a few days before menstruation. Non-ovulatory cycles, that is, when progesterone is insufficient, only show uterine contractions similar to the proliferative phase. However, in patients with primary dysmenorrhea, uterine contractions last longer than normal people, and the intrauterine pressure during periods without uterine contractions is also higher, and during contractions, the intrauterine pressure may rise to about 200-300 mmHg. It is believed that these excessive uterine contractions reduce blood flow to the uterus, causing anemia within the uterus and causing primary menstrual pain.
생리통의 대부분을 차지하는 원발성 생리통의 완화 및 치료 요법으로는 하복부를 따뜻하게 하고, 차가운 음식은 피하며, 가벼운 운동을 하고, 몸의 혈액순환을 돕는 따뜻한 샤워를 하는 대증요법(對症療法); 배란을 억제하여 무배란 상태를 유지하는 에스트로겐(estrogen) 요법, 프로게스테론(progesterone) 요법, 테스트로스테론(testrosterone) 요법 및 자궁수축 억제제를 투여하는 방법이 널리 알려져 있지만, 진통제에 너무 의존하거나 장기복용하면 부작용이 따를 수 있어 바람직하지 못하다. 따라서 생리통 완화 효과가 있고, 인체적용 안전성 측면에서 선호될 수 있는 천연물 소재를 발굴하는 것이 필요하다. Symptomatic treatment for relieving and treating primary menstrual pain, which accounts for most of menstrual pain, includes warming the lower abdomen, avoiding cold foods, doing light exercise, and taking a warm shower to help blood circulation in the body; Methods of administering estrogen therapy, progesterone therapy, testosterone therapy, and uterine contraction inhibitors to maintain anovulatory state by suppressing ovulation are widely known, but side effects can occur if you rely too much on painkillers or take them for a long period of time. This is undesirable as it can lead to this. Therefore, it is necessary to discover natural materials that have the effect of relieving menstrual pain and are preferred in terms of safety for human application.
한편, 해국(Aster spathulifolius)은 국화과(Asteraceae)에 속하는 식물이다. 해국은 항비만, 항 인플루엔자 바이러스 및 항암 활성을 갖는 것으로 보고되어 있다.Meanwhile, Aster spathulifolius is a plant belonging to the Asteraceae family. Sea chrysanthemum is reported to have anti-obesity, anti-influenza virus and anti-cancer activities.
천연물의 생리통 완화 효과에 관한 선행연구로는 한국등록특허 제0912912호에 귀전우, 계지, 의이인, 창출, 향부자, 진피, 우슬, 모과, 빈랑, 강활, 회향, 목향, 감초 및 부자를 포함하는 한방재를 함유하는 원발성 생리통 완화 및 치료용 한약 조성물이 개시되어 있고, 한국공개특허 제2022-0130496호에 육계, 복령, 천궁, 지각, 작약, 당귀 및 진피를 포함하는 생리통 완화 및 치료를 위한 조성물이 개시되어 있지만, 본 발명의 해국 추출물을 유효성분으로 포함하는 생리통의 예방, 개선 또는 치료용 조성물에 관해 전혀 개시된 바 없다. Previous research on the effect of natural products in relieving menstrual pain includes Korean Patent No. 0912912, which includes oriental medicine including Gwijeonwoo, Gyeji, Uiin, Changjang, Hyangbuja, Jinpi, Useul, Quince, Betel Nut, Ganghwal, Fennel, Wood Incense, Licorice and Buja. A herbal medicine composition containing ashes for relieving and treating primary menstrual pain is disclosed, and in Korea Patent Publication No. 2022-0130496, a composition for relieving and treating menstrual pain containing broiler, bokryeong, cheongung, crust, peony, angelica root and dermis is disclosed. Although disclosed, there is no disclosure at all regarding a composition for preventing, improving, or treating menstrual pain containing the extract of the present invention as an active ingredient.
본 발명은 상기와 같은 요구에 의해 도출된 것으로, 해국 추출물을 유효성분으로 포함하는 생리통의 예방, 개선 또는 치료용 조성물을 제공하고, 옥시토신을 투여하여 생리통을 유발시킨 동물모델에 상기 해국 추출물 투여시, 생리통을 완화하는 효과가 우수하고, 체중 및 자궁의 무게는 변화없이 과도한 자궁의 수축을 억제하며, 자궁근육세포에서 PGF2α 및 IL1β 처리에 의해 증가된 NF-κB의 활성 및 p65(NF-κB의 subunit 중 하나)의 핵내 이동을 효과적으로 억제하고, 비만세포의 과도한 히스타민 방출을 억제하는 것을 확인함으로써, 본 발명을 완성하였다.The present invention was developed in response to the above-mentioned needs, and provides a composition for preventing, improving, or treating menstrual pain containing a sea chrysanthemum extract as an active ingredient, when the sea chrysanthemum extract is administered to an animal model in which menstrual pain is induced by administering oxytocin. , has an excellent effect in alleviating menstrual pain, suppresses excessive uterine contraction without changing body weight and uterine weight, and increases the activity of NF-κB and p65 (NF-κB) in uterine muscle cells by treatment with PGF2α and IL1β. The present invention was completed by confirming that it effectively inhibits the intranuclear movement of (one of the subunits) and inhibits excessive histamine release from mast cells.
상기 과제를 해결하기 위하여, 본 발명은 해국 추출물을 유효성분으로 포함하는 생리통의 예방 또는 개선용 건강기능식품 조성물을 제공한다. In order to solve the above problems, the present invention provides a health functional food composition for preventing or improving menstrual pain containing a sea chrysanthemum extract as an active ingredient.
또한, 본 발명은 해국 추출물을 유효성분으로 포함하는 생리통의 예방 또는 치료용 약학 조성물을 제공한다. In addition, the present invention provides a pharmaceutical composition for preventing or treating menstrual pain containing a sea chrysanthemum extract as an active ingredient.
또한, 본 발명은 해국 추출물을 유효성분으로 포함하는 생리통의 예방 또는 개선용 의약외품을 제공한다. In addition, the present invention provides a quasi-drug for preventing or improving menstrual pain containing a sea chrysanthemum extract as an active ingredient.
본 발명은 해국 추출물을 유효성분으로 포함하는 생리통의 예방, 개선 또는 치료용 조성물에 관한 것으로, 에스트라디올을 투여하여 자궁내막을 두껍게 한 후, 옥시토신을 투여하여 생리통을 유발시킨 동물모델에 해국 추출물 투여시, 생리통을 완화하는 효과가 우수하였고, 체중 및 자궁의 무게는 변화 없이 과도한 자궁의 수축을 효과적으로 억제하였으며, 자궁근육세포에서 PGF2α 및 IL1β 처리에 의해 증가된 NF-κB의 활성 및 p65(NF-κB의 subunit 중 하나)의 핵내 이동을 효과적으로 억제하였고, 비만세포의 과도한 히스타민 방출을 억제하는 효과가 우수하였다. The present invention relates to a composition for preventing, improving, or treating menstrual pain containing a sea chrysanthemum extract as an active ingredient, which involves administering the sea chrysanthemum extract to an animal model in which menstrual pain is induced by administering oxytocin after thickening the endometrium by administering estradiol. It was excellent in alleviating menstrual pain, effectively suppressed excessive uterine contraction without changing body weight and uterine weight, and increased NF-κB activity and p65 (NF-) in uterine muscle cells by treatment with PGF2α and IL1β. It effectively inhibited the movement of κB (one of the subunits of κB) into the nucleus and was excellent at suppressing excessive histamine release from mast cells.
도 1은 생리통 유발 동물모델의 확립에 관한 것으로, (A)는 본 발명의 실험 디자인을 도식화한 것이고, (B)는 본 발명의 생리통 유발 동물모델의 몸부림 횟수(Writhing numbers)를 측정한 결과이다. CON은 에스트라디올만 투여한 정상 대조군이고, OXY50, OXY100은 옥시토신을 50 또는 100Unit/kg으로 복강 투여하여 생리통을 유발한 군이다.
도 2는 생리통 유발 동물모델에서 해국 추출물 투여에 의한 생리통 완화 효과를 확인한 것으로, (A)는 체중 변화를 확인한 결과이고, (B)는 몸부림 횟수(Writhing numbers)를 확인한 행동실험 결과이며, (C)는 부동시간을 확인한 행동실험 결과이다. CON은 에스트라디올만 투여한 정상 대조군이고, PD는 에스트라디올 투여 후 옥시토신을 투여한 생리통 유발군이고, PD+해국은 생리통 유발 및 해국 추출물 투여군이며, PD+ibuprofen은 생리통 유발 및 양성대조군인 이부프로펜을 투여한 군이다. ###은 정상 대조군(CON) 대비 생리통 유발군(PD)의 몸부림 횟수 또는 부동시간이 통계적으로 유의미하게 증가하였다는 것을 의미하며, p<0.001이다. *, **, ***은 생리통 유발군(PD) 대비 생리통 유발 및 해국 추출물 투여군(PD+해국) 또는 생리통 유발 및 이부프로펜 투여군(PD+ibuprofen)의 몸부림 횟수 또는 부동시간이 통계적으로 유의미하게 감소하였다는 것을 의미하며, *은 p<0.05, **은 p<0.01, ***은 p<0.001이다.
도 3은 생리통 유발 동물모델에서 해국 추출물 투여에 의한 자궁 조직의 변화를 확인한 것으로, (A)는 적출된 자궁 조직의 사진, (B)는 적출된 자궁 조직의 길이를 정량한 결과이고, (C)는 적출된 자궁 조직의 무게를 정량한 결과이다. CON은 에스트라디올만 투여한 정상 대조군이고, PD는 에스트라디올 투여 후 옥시토신을 투여한 생리통 유발군이고, PD+해국은 생리통 유발 및 해국 추출물 투여군이며, PD+ibuprofen은 생리통 유발 및 양성대조군인 이부프로펜을 투여한 군이다. ###은 정상 대조군(CON) 대비 생리통 유발군(PD)의 자궁 길이가 통계적으로 유의미하게 감소하였다는 것을 의미하며, p<0.001이다. ***은 생리통 유발군(PD) 대비 생리통 유발 및 해국 추출물 투여군(PD+해국) 또는 생리통 유발 및 이부프로펜 투여군(PD+ibuprofen)의 자궁 길이가 통계적으로 유의미하게 증가하였다는 것을 의미하며, p<0.001이다.
도 4는 생리통 유발 동물모델에서 해국 추출물 투여에 의한 자궁 내막의 두께 변화를 H&E(Hematoxylin and Eosin) 염색으로 확인한 결과(A) 및 이를 정량한 결과(B)이다. CON은 에스트라디올만 투여한 정상 대조군이고, PD는 에스트라디올 투여 후 옥시토신을 투여한 생리통 유발군이고, PD+해국은 생리통 유발 및 해국 추출물 투여군이며, PD+ibuprofen은 생리통 유발 및 양성대조군인 이부프로펜을 투여한 군이다. ###은 정상 대조군(CON) 대비 생리통 유발군(PD)의 자궁 내막 두께가 통계적으로 유의미하게 증가하였다는 것을 의미하며, p<0.001이다. **은 생리통 유발군(PD) 대비 생리통 유발 및 해국 추출물 투여군(PD+해국) 또는 생리통 유발 및 이부프로펜 투여군(PD+ibuprofen)의 자궁 내막 두께가 통계적으로 유의미하게 감소하였다는 것을 의미하며, p<0.01이다.
도 5는 생리통 유발 및 촉진 조건에서 자궁근육세포의 NF-κB 활성을 분석한 결과이다. PGF2α(Prostaglandin F2, 자궁근육수축유발호르몬, 생리통을 유발하는 호르몬의 일종)와 염증성 사이토카인 IL1β를 처리하여 생리통을 유발하였다. ####은 아무것도 처리하지 않은 군 대비 PGF2α+IL1β 처리군의 NF-κB 활성이 통계적으로 유의미하게 증가하였다는 것을 의미하며, p<0.0001이다. *, ***, ****은 PGF2α+IL1β 처리군 대비 PGF2α+IL1β 및 해국 추출물 처리군의 NF-κB 활성이 통계적으로 유의미하게 감소하였다는 것을 의미하며, *은 p<0.05, ***은 p<0.001, ****은 p<0.0001이다.
도 6은 생리통 유발 및 촉진 조건에서 p65 단백질의 자궁근육세포 핵내로의 이동을 확인한 결과이다. PGF2α(Prostaglandin F2, 자궁근육수축유발호르몬, 생리통을 유발하는 호르몬의 일종)와 염증성사이토카인 IL1β를 처리하여 생리통을 유발하였다. Control은 아무것도 처리하지 않은 군이고, PGF2α+IL1β+DMSO는 PGF2α 및 IL1β를 처리하여 생리통 유발 및 촉진 환경을 조성한 군이고, PGF2α+IL1β+해국은 PGF2α 및 IL1β를 처리하여 생리통과 유사한 환경을 조성하고, 해국 추출물을 처리한 군이다. p65 단백질은 붉은색으로 관찰된다.
도 7은 비만세포에서 해국 추출물의 히스타민 방출 억제 효과를 확인한 결과이다. PMA(Phorbol 12-myristate 13-acetate) 및 A23187(Ionophore)을 처리하여 히스타민 방출을 유도하였다. ####은 아무것도 처리하지 않은 군 대비 PMA+A23187 처리군의 히스타민 방출량이 통계적으로 유의미하게 증가하였다는 것을 의미하며, p<0.0001이다. ****은 PMA+A23187 처리군 대비 PMA+A23187 및 해국 추출물 처리군의 히스타민 방출량이 통계적으로 유의미하게 감소하였다는 것을 의미하며, p<0.0001이다.Figure 1 relates to the establishment of an animal model inducing menstrual pain, (A) is a schematic diagram of the experimental design of the present invention, and (B) is the result of measuring the number of wrestling numbers of the animal model inducing menstrual pain of the present invention. . CON is a normal control group administered only estradiol, and OXY50 and OXY100 are groups in which menstrual pain was induced by intraperitoneal administration of oxytocin at 50 or 100 Unit/kg.
Figure 2 confirms the effect of relieving menstrual pain by administration of Haeguk extract in an animal model inducing menstrual pain. (A) is the result of confirming the change in body weight, (B) is the result of a behavioral experiment confirming the number of struggling, and (C) ) is the result of a behavioral experiment that confirmed dead time. CON is a normal control group administered only estradiol, PD is a group that induces menstrual pain by administering oxytocin after administration of estradiol, PD+Haechuk is a group that induces menstrual cramps and is administered a Haechuk extract extract, and PD+ibuprofen is a group that induces menstrual cramps and administers ibuprofen as a positive control. It's a group. ### indicates a statistically significant increase in the number of struggles or immobility time in the dysmenorrhea-induced group (PD) compared to the normal control group (CON), p<0.001. *, **, *** showed a statistically significant decrease in the number of struggles or immobility time in the group that induced menstrual pain and received Haechi extract (PD+Haechik) or the group that induced menstrual pain and administered ibuprofen (PD+ibuprofen) compared to the group that induced menstrual pain (PD). means, * means p<0.05, ** means p<0.01, and *** means p<0.001.
Figure 3 shows the changes in uterine tissue caused by administration of sea chrysanthemum extract in an animal model inducing menstrual pain. (A) is a photograph of the extracted uterine tissue, (B) is the result of quantifying the length of the extracted uterine tissue, and (C) ) is the result of quantifying the weight of the extracted uterine tissue. CON is a normal control group administered only estradiol, PD is a group that induces menstrual pain by administering oxytocin after administration of estradiol, PD+Haechuk is a group that induces menstrual cramps and is administered a Haechuk extract extract, and PD+ibuprofen induces menstrual pain and administers ibuprofen as a positive control group. It's one group. ### means that the uterine length of the dysmenorrhea-induced group (PD) was statistically significantly reduced compared to the normal control group (CON), p<0.001. *** means that there was a statistically significant increase in uterine length in the menstrual pain-inducing and Haechuk extract-administered group (PD+Haekuk) or menstrual pain-induced and ibuprofen-administered group (PD+ibuprofen) compared to the menstrual pain-induced group (PD), p<0.001 am.
Figure 4 shows the results of confirming the change in thickness of the endometrium due to the administration of the sea chrysanthemum extract in an animal model inducing menstrual pain by H&E (Hematoxylin and Eosin) staining (A) and the results of quantifying this (B). CON is a normal control group administered only estradiol, PD is a group that induces menstrual pain by administering oxytocin after administration of estradiol, PD+Haechuk is a group that induces menstrual cramps and is administered a Haechuk extract extract, and PD+ibuprofen induces menstrual pain and administers ibuprofen as a positive control group. It's one group. ### means that the endometrial thickness of the dysmenorrhea-induced group (PD) increased statistically significantly compared to the normal control group (CON), p<0.001. ** means that compared to the menstrual pain inducing group (PD), there was a statistically significant decrease in the endometrial thickness in the menstrual pain inducing and Haechi extract administration group (PD+Hae chrysanthemum extract) or the menstrual pain inducing and ibuprofen administration group (PD+ibuprofen), p<0.01 am.
Figure 5 shows the results of analyzing NF-κB activity of uterine muscle cells under conditions that induce and promote menstrual pain. Menstrual pain was induced by treating PGF2α (Prostaglandin F2, uterine muscle contraction-inducing hormone, a type of hormone that causes menstrual pain) and inflammatory cytokine IL1β. #### indicates a statistically significant increase in NF-κB activity in the PGF2α+IL1β treated group compared to the untreated group, p<0.0001. *, ***, **** indicate a statistically significant decrease in NF-κB activity in the PGF2α+IL1β and sea chrysanthemum extract treatment group compared to the PGF2α+IL1β treatment group, * is p<0.05, ** * means p<0.001, **** means p<0.0001.
Figure 6 shows the results confirming the movement of p65 protein into the uterine muscle cell nucleus under conditions that induce and promote menstrual pain. Menstrual pain was induced by treating PGF2α (Prostaglandin F2, uterine muscle contraction-inducing hormone, a type of hormone that causes menstrual pain) and inflammatory cytokine IL1β. Control is a group that has not been treated with anything, PGF2α+IL1β+DMSO is a group that is treated with PGF2α and IL1β to create an environment that induces and promotes menstrual pain, and PGF2α+IL1β+Hae-Guk is a group that is treated with PGF2α and IL1β to create an environment similar to menstrual pain. , This is the group treated with sea chrysanthemum extract. p65 protein is observed in red.
Figure 7 shows the results of confirming the inhibitory effect of histamine release of the sea chrysanthemum extract in mast cells. Histamine release was induced by treatment with PMA (Phorbol 12-myristate 13-acetate) and A23187 (Ionophore). #### means that there was a statistically significant increase in histamine release in the PMA+A23187 treated group compared to the untreated group, p<0.0001. **** means that there was a statistically significant decrease in histamine release in the PMA+A23187 and sea chrysanthemum extract treatment group compared to the PMA+A23187 treatment group, p<0.0001.
본 발명의 목적을 달성하기 위하여, 본 발명은 해국 추출물을 유효성분으로 포함하는 생리통의 예방 또는 개선용 건강기능식품 조성물을 제공한다. In order to achieve the object of the present invention, the present invention provides a health functional food composition for preventing or improving menstrual pain containing a sea chrysanthemum extract as an active ingredient.
상기 해국 추출물의 추출 용매는 물, C1~C4의 저급 알코올 또는 이의 혼합물일 수 있고, 바람직하게는 에탄올인 것이지만, 이에 제한하는 것은 아니다. The extraction solvent for the Haechukgi extract may be water, a C 1 to C 4 lower alcohol, or a mixture thereof, preferably ethanol, but is not limited thereto.
상기 생리통은 원발성 생리통인 것이 바람직하고, 더 바람직하게는 자궁 근육의 과도한 수축에 의해 발생하는 NF-κB 활성에 의한 생리통인 것이지만, 이에 한정하지 않는다. The dysmenorrhea is preferably primary dysmenorrhea, and more preferably is dysmenorrhea caused by NF-κB activity caused by excessive contraction of uterine muscles, but is not limited to this.
상기 해국 추출물은 자궁 근육 세포의 NF-κB 활성을 억제하고, NF-κB 활성에 의한 p65 단백질의 세포 핵내로의 이동을 억제하며, 자궁 근육의 과도한 수축을 억제한다. The sea chrysanthemum extract inhibits NF-κB activity of uterine muscle cells, inhibits the movement of p65 protein into the cell nucleus by NF-κB activity, and inhibits excessive contraction of uterine muscles.
상기 조성물은 분말, 과립, 환, 정제, 캡슐, 캔디, 시럽 및 음료 중에서 선택된 어느 하나의 제형으로 제조될 수 있지만, 이에 제한되는 것은 아니다. The composition may be manufactured in any one formulation selected from powder, granule, pill, tablet, capsule, candy, syrup, and beverage, but is not limited thereto.
본 발명의 건강기능식품 조성물을 식품첨가물로 사용하는 경우, 상기 건강기능식품 조성물을 그대로 첨가하거나 다른 식품 또는 식품성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분은 그의 사용 목적에 따라 적절하게 사용될 수 있다. 일반적으로, 식품 또는 음료의 제조시 본 발명의 건강기능식품 조성물은 원료에 대하여 15 중량부 이하, 바람직하게는 10 중량부 이하의 양으로 첨가된다. 그러나 건강을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로 사용될 수 있다.When using the health functional food composition of the present invention as a food additive, the health functional food composition may be added as is or used together with other foods or food ingredients, and may be used appropriately according to conventional methods. The active ingredient may be used appropriately depending on its intended use. Generally, when manufacturing a food or beverage, the health functional food composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the raw materials. However, in the case of long-term intake for health purposes, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.
상기 건강기능식품의 종류에 특별한 제한은 없다. 상기 건강기능식품 조성물을 첨가할 수 있는 식품의 예로는 육류, 소시지, 빵, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차 드링크제, 알콜 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강식품을 모두 포함한다.There are no particular restrictions on the types of health functional foods. Examples of foods to which the health functional food composition can be added include meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, and tea. There are drinks, alcoholic beverages, and vitamin complexes, and it includes all health foods in the conventional sense.
또한, 본 발명의 건강기능식품 조성물은 식품, 특히 기능성 식품으로 제조될 수 있다. 본 발명의 기능성 식품은 통상적으로 첨가되는 성분을 포함할 수 있다. 예를 들어, 단백질, 탄수화물, 지방, 영양소 및 조미제를 포함한다. 예컨대, 드링크제로 제조되는 경우에는 유효성분 이외에 천연 탄수화물 또는 향미제를 추가 성분으로서 포함시킬 수 있다. 상기 천연 탄수화물은 모노사카라이드(예컨대, 글루코오스, 프럭토오스 등), 디사카라이드(예컨대, 말토스, 수크로오스 등), 올리고당, 폴리사카라이드(예컨대, 덱스트린, 시클로덱스트린 등) 또는 당알코올(예컨대, 자일리톨, 소르비톨, 에리쓰리톨 등)인 것이 바람직하다. 상기 향미제는 천연 향미제(예컨대, 타우마틴, 스테비아 추출물 등)와 합성 향미제(예컨대, 사카린, 아스파르탐 등)를 이용할 수 있다.Additionally, the health functional food composition of the present invention can be manufactured into food, especially functional food. The functional food of the present invention may contain commonly added ingredients. For example, it includes proteins, carbohydrates, fats, nutrients and seasonings. For example, when manufacturing a drink, natural carbohydrates or flavoring agents may be included as additional ingredients in addition to the active ingredient. The natural carbohydrates include monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g., dextrins, cyclodextrins, etc.), or sugar alcohols (e.g., , xylitol, sorbitol, erythritol, etc.) are preferred. The flavoring agent may be a natural flavoring agent (e.g., thaumatin, stevia extract, etc.) or a synthetic flavoring agent (e.g., saccharin, aspartame, etc.).
상기 건강기능식품 조성물 이외에 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산음료에 사용되는 탄산화제 등을 더 함유할 수 있다. 이러한 상기 첨가되는 성분의 비율은 크게 중요하진 않지만 본 발명의 건강기능식품 조성물 100 중량부에 대하여, 0.01 내지 0.1 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the above health functional food composition, various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonic acid. It may further contain carbonating agents used in beverages. The ratio of the ingredients added is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food composition of the present invention.
또한, 본 발명은 해국 추출물을 유효성분으로 포함하는 생리통의 예방 또는 치료용 약학 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating menstrual pain containing a sea chrysanthemum extract as an active ingredient.
본 발명의 약학 조성물은 상기 유효성분 이외에 약학적으로 허용 가능한 담체, 부형제 또는 희석제를 더 포함할 수 있지만, 이에 제한되는 것은 아니다. The pharmaceutical composition of the present invention may further include, but is not limited to, a pharmaceutically acceptable carrier, excipient, or diluent in addition to the above active ingredients.
본 발명의 약학 조성물은 유효성분 이외에 약학적으로 허용되는 담체를 포함할 수 있으며, 이러한 담체는 제제 시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 약학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier in addition to the active ingredient, and such carriers are commonly used in formulations, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, Calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and minerals. Includes, but is not limited to, oil. In addition to the above ingredients, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.
본 발명에 따른 약학조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있다. The appropriate dosage of the pharmaceutical composition according to the present invention is prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity. It can be.
본 발명의 조성물에 포함되는 유효성분의 농도는 치료 목적, 환자의 상태, 필요기간 등을 고려하여 결정할 수 있으며 특정 범위의 농도로 한정되지 않는다.The concentration of the active ingredient included in the composition of the present invention can be determined considering the purpose of treatment, patient condition, required period, etc., and is not limited to a specific concentration range.
또한, 본 발명은 해국 추출물을 유효성분으로 포함하는 생리통의 예방 또는 개선용 의약외품을 제공한다.In addition, the present invention provides a quasi-drug for preventing or improving menstrual pain containing a sea chrysanthemum extract as an active ingredient.
본 발명에서 사용되는 용어 "의약외품"은 사람이나 동물의 질병을 진단, 치료, 개선, 경감, 처치 또는 예방할 목적으로 사용되는 물품들 중 의약품보다 작용이 경미한 물품들을 의미하는 것으로, 예를 들어 약사법에 따르면 의약외품이란 의약품의 용도로 사용되는 물품을 제외한 것으로, 사람ㆍ동물의 질병 치료나 예방에 쓰이는 제품 또는 인체에 대한 작용이 경미하거나 직접 작용하지 않는 제품 등이 포함된다.The term "quasi-drug" used in the present invention refers to products with a milder effect than pharmaceuticals among products used for the purpose of diagnosing, treating, improving, alleviating, treating or preventing diseases in humans or animals. For example, according to the Pharmaceutical Affairs Act. According to it, quasi-drugs exclude products used for medicinal purposes, and include products used to treat or prevent diseases in humans and animals, or products that have a mild or no direct effect on the human body.
본 발명의 의약외품은 생리통의 개선을 목적으로 사용되는 것으로, 그 제형에 있어서 특별히 한정되는 바가 없고, 각 제형에 있어서 의약외품은 다른 성분들을 기타 의약외품의 제형 또는 사용목적 등에 따라 임의로 선정하여 배합할 수 있다. 유효성분의 혼합양은 사용 목적(억제 또는 완화)에 따라 적합하게 결정될 수 있다. 예를 들어, 점증제, 안정화제, 용해화제, 비타민, 안료 및 향료와 같은 통상적인 보조제, 및 담체 등을 포함할 수 있다.The quasi-drug of the present invention is used for the purpose of improving menstrual pain, and there is no particular limitation in its formulation. In each formulation, the quasi-drug can arbitrarily select and mix other ingredients depending on the formulation or purpose of use of the other quasi-drug. . The amount of active ingredients mixed can be appropriately determined depending on the purpose of use (suppression or alleviation). For example, it may include conventional auxiliaries such as thickeners, stabilizers, solubilizers, vitamins, pigments and flavors, and carriers.
이하, 제조예 및 실시예를 이용하여 본 발명을 더욱 상세하게 설명하고자 한다. 이들 제조예 및 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들에 의해 제한되지 않는다는 것은 당해 기술분야에서 통상의 지식을 가진 자에게 있어 자명한 것이다. Hereinafter, the present invention will be described in more detail using manufacturing examples and examples. These manufacturing examples and examples are only for illustrating the present invention in more detail, and it is obvious to those skilled in the art that the scope of the present invention is not limited thereto.
제조예 1. 해국 추출물의 제조Preparation Example 1. Preparation of sea chrysanthemum extract
해국 잎 1 중량부에 대하여 10 중량부의 70%(v/v) 에탄올을 첨가하여 3시간씩 2회에 걸쳐 환류 냉각 추출 및 여과하였다. 이후 상기 해국 추출액을 45℃에서 감압 농축하여 에탄올을 제거한 후, -80℃에서 동결건조하여 해국 추출물을 획득하였다.10 parts by weight of 70% (v/v) ethanol was added to 1 part by weight of Haeguk leaves, followed by reflux cooling extraction and filtration twice for 3 hours each. Afterwards, the Haeguk extract was concentrated under reduced pressure at 45°C to remove ethanol, and then freeze-dried at -80°C to obtain a Haeguk extract.
실시예 1. 생리통 유발 동물모델의 확립Example 1. Establishment of an animal model inducing menstrual pain
도 1A에 개시된 바와 같이, 8주령 ICR 마우스에 에스트라디올(Estradiol)을 1mg/kg의 농도로 4일에 걸쳐 복강 투여(Intraperitoneal injection, IP injection)하여 자궁내막의 두께를 최대한 두껍게한 후, 마지막 4일차에는 에스트라디올 투여 30분 뒤 자궁수축호르몬(옥시토신)을 100Unit/kg으로 복강 투여하여 원발성 생리통을 유발하였고, 도 1B에 개시된 바와 같이 몸부림 횟수(Writhing numbers) 측정을 통해 생리통이 잘 유발된 것을 확인하였다.As shown in Figure 1A, estradiol (Estradiol) was administered to 8-week-old ICR mice at a concentration of 1 mg/kg over 4 days to increase the thickness of the endometrium as much as possible, and then the last 4 days. On the first day, 100 Units/kg of uterine contraction hormone (oxytocin) was intraperitoneally administered 30 minutes after estradiol administration to induce primary menstrual pain, and it was confirmed that menstrual pain was well induced by measuring the number of writhing as shown in Figure 1B. did.
실시예 2. 생리통 유발 동물모델에서 해국 추출물 투여에 의한 생리통 완화 효과 확인Example 2. Confirmation of menstrual pain relief effect by administration of Haeguk extract in an animal model inducing menstrual pain
1) 해국 추출물의 투여1) Administration of sea chrysanthemum extract
상기 실시예 1의 방법으로 생리통 유발 동물모델을 구축하였고, 실험과정 동안 에스트라디올 투여 30분 전에 해국 추출물을 경구투여하여 총 4일간 해국 추출물을 투여하였다. 양성대조군으로 사용된 이부프로펜도 해국 추출물과 동일한 방법으로 동물모델에 투여되었다.An animal model inducing menstrual cramps was constructed using the method of Example 1, and during the experimental process, the extract of Haechukja was orally administered 30 minutes before administration of estradiol for a total of 4 days. Ibuprofen, used as a positive control, was also administered to the animal model in the same manner as the sea chrysanthemum extract.
실험과정 동안 1일 1회 체중을 측정하였으며, 그 결과 도 2A에 개시된 바와 같이 생리통 유발 및 시료 투여에 의한 체중의 변화는 없었다.Body weight was measured once a day during the experiment, and as a result, there was no change in body weight due to menstrual cramps or sample administration, as shown in Figure 2A.
2) 행동실험2) Behavioral experiment
생리통 유발 후, 30분 동안 몸부림 횟수(Writhing numbers) 및 부동시간을 측정하였다. After inducing menstrual pain, writing numbers and immobility time were measured for 30 minutes.
그 결과, 도 2B에 개시된 바와 같이 대조군에서는 몸부림이 나타나지 않았으나, 생리통 유발군에서는 30분 동안 15회 이상의 몸부림이 관찰되었고, 해국 추출물을 투여한 군에서는 5~8회로 몸부림 횟수가 감소하여 생리통 증상이 완화된 것을 확인할 수 있었다.As a result, as shown in Figure 2B, no struggle was observed in the control group, but more than 15 struggles were observed in 30 minutes in the group inducing menstrual pain, and in the group administered the sea chrysanthemum extract, the number of struggles was reduced to 5 to 8, showing menstrual pain symptoms. It was confirmed that it was alleviated.
또한 도 2C에 개시된 바와 같이 대조군에서는 부동시간이 없었으나, 생리통 유발군에서는 통증에 의한 부동시간이 30분 중 50% 이상 나타났다. 해국 추출물을 투여한 군에서는 30분 동안 20~30%의 부동시간을 나타내어 해국 추출물이 생리통을 줄여 부동시간 감소에 효과적인 것을 확인하였다.Additionally, as shown in Figure 2C, there was no immobility time in the control group, but in the menstrual pain-induced group, immobility time due to pain occurred for more than 50% of 30 minutes. In the group administered Haeguk extract, immobility time was 20-30% for 30 minutes, confirming that Haekuk extract was effective in reducing menstrual pain and reducing immobility time.
3) 자궁 조직의 변화3) Changes in uterine tissue
행동실험 종료 후, 마우스의 자궁 조직을 적출하여 조직의 무게 및 길이 변화를 분석하였다. After the behavioral experiment was completed, the uterine tissue of the mouse was removed and changes in weight and length of the tissue were analyzed.
그 결과, 도 3에 개시된 바와 같이 생리통 유발에 의해 자궁 조직의 길이가 짧아졌고, 해국 추출물 투여에 의해 길이가 회복되는 것을 확인하였다. 자궁의 무게는 생리통 유발 및 시료 투여에 따른 변화가 관찰되지 않아 자궁 조직의 길이변화는 세포사멸과 관련이 없음을 확인하였다.As a result, as shown in Figure 3, it was confirmed that the length of the uterine tissue was shortened due to the induction of menstrual pain, and the length was restored by administration of the sea chrysanthemum extract. The weight of the uterus was not observed to change due to menstrual pain induction or sample administration, confirming that the change in the length of the uterine tissue was not related to apoptosis.
적출된 자궁 조직을 H&E(Hematoxylin and Eosin) 염색하여 자궁 내막의 두께를 측정한 결과, 도 4에 개시된 바와 같이 생리통 유발에 의해 자궁의 수축이 이루어져 자궁내막이 두꺼워진 반면, 해국 추출물 투여군은 자궁 수축에 의해 두꺼워진 자궁 내막이 농도 의존적으로 정상 두께로 회복되는 것을 확인하였다.As a result of measuring the thickness of the endometrium by staining the extracted uterine tissue with H&E (Hematoxylin and Eosin), as shown in FIG. 4, the uterus contracted due to menstrual pain and the endometrium thickened, while the group administered the sea cucumber extract showed uterine contraction. It was confirmed that the thickened endometrium recovered to normal thickness in a concentration-dependent manner.
실시예 3. 자궁근육세포에서 NF-κB 활성 변화Example 3. Changes in NF-κB activity in uterine muscle cells
1) NF-κB 활성 분석(Luciferase assay)1) NF-κB activity analysis (Luciferase assay)
자궁근육세포(HutSMC)에서 PGF2α(Prostaglandin F2, 자궁근육수축유발 호르몬, 생리통을 유발하는 호르몬)와 염증성사이토카인 IL1β의 과도한 분비는 NF-κB의 활성을 증가시켜 생리통을 더욱 촉진한다. 따라서 자궁근육세포의 NF-κB의 활성 억제 소재 발굴은 생리통을 억제하는 새로운 완화 방법으로 이용될 수 있다. 그러므로 생리통 유발 환경에서 해국 추출물 처리에 의한 NF-κB 변화를 확인하였다.Excessive secretion of PGF2α (Prostaglandin F2, a hormone that induces uterine muscle contraction, a hormone that causes menstrual pain) and inflammatory cytokine IL1β in uterine muscle cells (HutSMC) increases the activity of NF-κB, further promoting menstrual pain. Therefore, discovery of materials that inhibit the activity of NF-κB in uterine muscle cells can be used as a new relief method to suppress menstrual pain. Therefore, changes in NF-κB due to treatment with Haeguk extract in an environment that induces menstrual pain were confirmed.
HutSMC 세포를 웰당 5X106 개로 12웰 플레이트에 분주한 후, 24시간 뒤 해국 추출물을 30분 동안 전처리하였다. 그 후, PGF2α(20μM) 및 IL1β(10ng/ml)를 24시간 동안 처리한 다음 루시퍼라아제 분석을 수행하였다.HutSMC cells were distributed in a 12-well plate at 5×10 6 cells per well, and 24 hours later, they were pretreated with the Haeguk extract for 30 minutes. Afterwards, the cells were treated with PGF2α (20 μM) and IL1β (10 ng/ml) for 24 hours and then luciferase assay was performed.
그 결과, 도 5에 개시된 바와 같이 생리통 유발 및 촉진 조건에서 NF-κB의 루시퍼라아제 활성이 증가하였고, 해국 추출물 투여시 농도 의존적으로 NF-κB의 루시퍼라아제 활성을 억제하였다.As a result, as shown in Figure 5, the luciferase activity of NF-κB increased under conditions that induce and promote menstrual cramps, and the luciferase activity of NF-κB was inhibited in a concentration-dependent manner when the Hoechuk extract was administered.
2) p65 위치 변화 확인(형광현미경)2) Confirmation of change in p65 location (fluorescence microscope)
NF-κB 활성화에 의한 p65의 핵내 이동을 형광현미경으로 관찰하였다.The intranuclear movement of p65 due to NF-κB activation was observed using a fluorescence microscope.
구체적으로 1X106개의 HutSMC 세포를 콘포칼 접시(confocal dish)에 준비한 다음 24시간 후, 해국 추출물을 30분 동안 전처리하였다. 그 후, PGF2α(20μM) 및 IL1β(10 ng/ml)를 30분 동안 처리한 다음 세포를 4%(v/v) PFA(paraformaldehyde)로 5분간 고정하였고, 그 후 5분 동안 0.1%, Triton X100으로 세포 천공(cell permeabilization)한 후, PBS로 세척한 다음 세포 Blocking solution에 1시간 동안 방치하였다. 그 후, 1차 항체(anti-p65)를 처리한 후, 16시간 동안 반응시킨 다음 PBS로 세척한 후, 2차 항체(Alexa 555, red color)를 처리한 다음 1시간 동안 반응시킨 후, 형광현미경으로 관찰하였다. Specifically, 1X10 6 HutSMC cells were prepared in a confocal dish, and then 24 hours later, they were pretreated with the Haeguk extract for 30 minutes. Afterwards, cells were treated with PGF2α (20 μM) and IL1β (10 ng/ml) for 30 min, then the cells were fixed with 4% (v/v) PFA (paraformaldehyde) for 5 min, and then 0.1%, Triton for 5 min. After cell permeabilization with X100, the cells were washed with PBS and left in cell blocking solution for 1 hour. Afterwards, the primary antibody (anti-p65) was treated, reacted for 16 hours, washed with PBS, secondary antibody (Alexa 555, red color) was treated, and then reacted for 1 hour, followed by fluorescence Observed under a microscope.
그 결과, 도 6에 개시된 바와 같이 PGF2α+IL1β는 세포질에 존재하던 p65(NF-κB의 subunit 중 하나)를 핵내로 이동시키는데, 해국 추출물은 p65의 이동을 억제하는 것을 확인하였다.As a result, as shown in Figure 6, PGF2α+IL1β moves p65 (one of the subunits of NF-κB) existing in the cytoplasm into the nucleus, and it was confirmed that the Haeguk extract inhibits the movement of p65.
실시예 4. 비만세포에서 히스타민 방출 억제 효과Example 4. Inhibitory effect of histamine release from mast cells
비만세포(mast cell)의 히스타민 과다 방출은 생리통을 유발하는 하나의 원인으로 알려져 있다. 생리통증이 심한 사람은 자궁 주위 비만세포 분포가 일반인보다 높다. 따라서 PMA와 A23187로 히스타민 방출을 유도한 비만세포에 해국 추출물을 처리하여 히스타민 방출 억제 효과를 확인하였다.Excessive release of histamine from mast cells is known to be a cause of menstrual pain. People with severe menstrual pain have a higher distribution of mast cells around the uterus than the general population. Therefore, the effect of inhibiting histamine release was confirmed by treating mast cells that had induced histamine release with PMA and A23187 with a sea chrysanthemum extract.
마우스 비만세포인 MC9(mast cell 9, mouse)를 2mM L-글루타메이트, 0.05mM 2-머캅토에탄올, 10% RAT T-STIM 및 10% FBS(fetal bovine serum)가 포함된 DMEM으로 배양한 후, 30분 동안 해국 추출물 또는 양성 대조군인 CsA(Cyclosporine A)를 전처리한 다음, 100ng/mL의 PMA(Phorbol 12-myristate 13-acetate) 및 1μM의 A23187(Ionophore)을 6시간 동안 처리하여 히스타민 방출을 유도하였다. 그 후, ELISA로 방출된 히스타민의 양을 측정하였다.MC9 (mast cell 9, mouse), a mouse mast cell, was cultured in DMEM containing 2mM L-glutamate, 0.05mM 2-mercaptoethanol, 10% RAT T-STIM, and 10% FBS (fetal bovine serum). After pretreatment with Haeguk extract or CsA (Cyclosporine A), a positive control, for 30 minutes, histamine release was induced by treatment with 100 ng/mL PMA (Phorbol 12-myristate 13-acetate) and 1 μM A23187 (Ionophore) for 6 hours. did. Afterwards, the amount of histamine released was measured by ELISA.
그 결과, 도 7에 개시된 바와 같이 PMA 및 A23187 처리에 의해 증가된 히스타민 방출은 해국 추출물을 처리한 경우, 농도 의존적으로 감소하는 것을 확인하였다.As a result, as shown in FIG. 7, it was confirmed that the histamine release increased by PMA and A23187 treatment decreased in a concentration-dependent manner when treated with the sea chrysanthemum extract.
Claims (11)
Publications (1)
| Publication Number | Publication Date |
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| KR20240097394A true KR20240097394A (en) | 2024-06-27 |
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