CN106562998A - Applications of lactobacillus crispatus in treatment or prevention of uterus myoma related diseases - Google Patents

Abstract

The invention provides the use of Lactobacillus crispatus in treating or preventing diseases related to uterine fibroids, and also provides pharmaceutical compositions, hygiene products and food compositions containing Lactobacillus crispatus. By providing Lactobacillus crispatus to experimental subjects, it can effectively prevent the development of fibroids, reduce the volume of fibroids, inhibit the recurrence of fibroids, and can be effectively used to treat or prevent diseases related to uterine fibroids.

Description

Application of Lactobacillus crispatus in the treatment or prevention of diseases related to uterine fibroids

technical field

The invention relates to the field of microbiology, in particular to the use of Lactobacillus crispatus in treating or preventing diseases related to uterine fibroids, and also to pharmaceutical compositions, hygiene products and food compositions containing Lactobacillus crispatus.

Background technique

Uterine fibroids are non-cancerous smooth muscle tumors that occur on the uterine wall. It is a common gynecological disease, and the incidence rate of women is as high as 20-50%. A significant number of patients with uterine fibroids are accompanied by painful pelvic recession, heavy and prolonged periods (which may lead to anemia or iron deficiency), bowel and bladder dysfunction, and infertility. Uterine fibroids can also cause symptoms such as low back pain, frequent urination, urgency, and painful sexual intercourse. Therefore, uterine fibroids have a great impact on the health of women of childbearing age.

Since the pathogenic mechanism of uterine fibroids is still unclear, it is difficult to prevent them. At present, there are many treatment methods, but the only curative treatment method is hysterectomy. Obviously, hysterectomy will make the patient permanently lose fertility, and the cost of hysterectomy is high, the postoperative recovery time is long, there is the possibility of serious postoperative complications, and the patient also suffers from physical pain. Therefore, hysterectomy is hardly an ideal treatment for uterine fibroids.

There are also many non-surgical drug-based treatments, but most of these drugs only target the symptoms caused by fibroids, not the fibroids themselves. In the early stages of the disease, doctors usually do not take treatment measures. However, fibroids can cause infertility even without a hysterectomy. Drugs that shrink fibroids or prevent them from growing are often unsatisfactory and often have serious side effects. In fact, many drugs have been found to be able to reduce the size of fibroids, but they cannot be used clinically due to serious side effects.

US Patent Publication No. US 5468741 A discloses that oral low-dose Mifepristone can treat the uterine femoral tract; European Patent Publication No. EP 2197415 B1 discloses vaginal administration of Mifepristone for the treatment of uterine fibroids. Although mifepristone can improve the size of fibroids, it will cause damage to the endocrine system. Side effects such as nausea, vomiting, dizziness, and uterine cramps will occur during the course of taking the medicine, and the fibroids will relapse and grow rapidly after stopping taking the medicine.

At present, in terms of various treatment methods for this disease, surgical treatment is often the last choice, hormone therapy has large side effects, and conservative treatment is often ineffective. Existing no matter Chinese and Western medicine, also do not have a kind of medicine that can fully and effectively suppress the growth of uterine fibroids, dwindle or eliminate tumor body, improve clinical symptoms, prevent the recurrence and malignant transformation of fibroids.

Contents of the invention

The present invention aims to solve one of the technical problems in the related art at least to a certain extent. Therefore, an object of the present invention is to propose a use of Lactobacillus crispatus in treating or preventing diseases related to uterine fibroids.

In the first aspect of the present invention, the present invention provides a use of Lactobacillus crispatus for preparing a composition for treating or preventing uterine fibroids. The inventors found that by providing the composition to animals, the development of fibroids can be effectively prevented, the volume of fibroids can be reduced, and the recurrence of fibroids can be inhibited, that is, uterine fibroids or related diseases can be effectively treated or prevented.

According to an embodiment of the present invention, the Lactobacillus crispatus ST1, Lactobacillus crispatus CIP 103604, Lactobacillus crispatus CIP 104459 and Lactobacillus crispatus LMG 12005 are at least one selected from. Therefore, the effect of treating or preventing diseases related to uterine fibroids is better.

According to an embodiment of the present invention, the treatment or prevention of uterine fibroids includes: (i) reducing the volume of uterine fibroids; (ii) reducing the uterine coefficient; (iii) reducing the concentration of estradiol; (iv) reducing the concentration of progesterone ; (v) inhibit the growth of uterine fibroids.

In a second aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition includes: Lactobacillus crispatus; and pharmaceutically acceptable excipients. The inventors were surprised to find that by providing the pharmaceutical composition to animals, the development of fibroids can be significantly prevented, the volume of fibroids can be reduced, and the recurrence of fibroids can be inhibited, indicating that the pharmaceutical composition can effectively treat or prevent diseases related to uterine fibroids.

According to an embodiment of the present invention, the Lactobacillus crispatus is at least one selected from L.crispatus ST1, L.crispatus CIP 103604, L.crispatus CIP 104459 and L.crispatus LMG 12005. Therefore, the effect of treating or preventing diseases related to uterine fibroids is better.

According to an embodiment of the present invention, when the pharmaceutical composition is in a solid state, the pharmaceutical composition comprises 1×10-1×10 ²⁰ cfu/g of the Lactobacillus crispatus, and when the pharmaceutical composition is in a liquid state , the pharmaceutical composition comprises 1×10-1×10 ²⁰ cfu/mL of the Lactobacillus crispatus. Therefore, the effect of inhibiting the occurrence of fibroids and reducing the volume of fibroids is obvious. If the content of Lactobacillus crispatus is too low, the effect of treating or preventing uterine fibroids is unsatisfactory. If the content of Lactobacillus crispatus is too high, it can treat or prevent uterine fibroids The effect of this method is not significantly improved, resulting in waste.

According to a preferred embodiment of the present invention, when the pharmaceutical composition is solid, the pharmaceutical composition contains 1×10 ⁴ -1×10 ¹⁵ cfu/g of the Lactobacillus crispatus, when the pharmaceutical composition In a liquid state, the pharmaceutical composition comprises 1×10 ⁴ -1×10 ¹⁵ cfu/mL of the Lactobacillus crispatus.

According to another preferred embodiment of the present invention, when the pharmaceutical composition is in a solid state, the pharmaceutical composition contains 1×10 ⁶ -1×10 ¹¹ cfu/g of the Lactobacillus crispatus, when the drug When the composition is in a liquid state, the pharmaceutical composition contains 1×10 ⁶ -1×10 ¹¹ cfu/mL of the Lactobacillus crispatus.

According to an embodiment of the present invention, the pharmaceutically acceptable excipient is at least one selected from carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, and preservatives.

According to an embodiment of the present invention, the pharmaceutically acceptable auxiliary material is selected from lactose, glucose, sucrose, sorbitol, mannose, starch, acacia, calcium phosphate, alginate, gelatin, calcium silicate, fine At least one of crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil.

According to an embodiment of the present invention, the pharmaceutical composition is administered orally, vaginally or rectally, and preferably the carrier material is at least one pharmaceutically acceptable carrier.

According to an embodiment of the present invention, the pharmaceutical composition is administered orally and vaginally, orally and rectally, or a combination of oral, vaginal and rectal.

According to an embodiment of the present invention, the pharmaceutical composition is prepared in the form of tablets, sucking tablets, sweet food, chewing gum, capsules, enteric-coated tablets and capsules, suppositories, vaginal tablets, vaginal gelatin capsules, Vaginal administration is in the form of lozenges, creams, gels, ointments, lotions, tampons, sanitary napkins, diapers, pads, dissolving strips, condoms, pessaries, sprays, and clinical nutritionals.

According to the embodiments of the present invention, by providing the pharmaceutical composition to animals, the development of fibroids can be significantly prevented, the volume of fibroids can be reduced, and the recurrence of fibroids can be inhibited, indicating that the drug can effectively treat or prevent diseases related to uterine fibroids.

According to an embodiment of the present invention, the medicine is in the form of at least one preparation selected from granules, capsules, tablets, powders, oral liquids, suspensions and emulsions. Thus, administration is facilitated.

According to an embodiment of the present invention, a sanitary product containing the pharmaceutical composition according to claim 4 is selected from the group consisting of tablets, capsules, lozenges, creams, gels, ointments, lotions, cotton Tampons, pads, diapers, pads, dissolving strips, condoms, pessaries, sprays.

Normal vaginal flora ascend from the rectal mucosa (Reid G, Bruce AW. Urogenital infections in women: can probiotics help? Postgraduate Medical Journal 2003; 79:428-432.), which means that after surviving through the gastrointestinal Orally administered microorganisms will emerge in the vagina after a certain period of time, indicating that vaginal probiotics can be orally administered to the host. therefore,

In a third aspect of the invention, the invention provides a food product. According to an embodiment of the present invention, the food comprises: Lactobacillus crispatus; and food acceptable auxiliary materials. The inventors found that by providing the food to animals, the development of fibroids can be significantly prevented, the volume of fibroids can be reduced, and the recurrence of fibroids can be inhibited, indicating that the drug can effectively treat or prevent diseases related to uterine fibroids.

It should be noted that the type of the term "food" used in the present invention is not particularly limited, and may be any known food, including but not limited to dairy products, biscuits, cakes, beverages, health products, etc.

According to an embodiment of the present invention, the Lactobacillus crispatus is at least one selected from L.crispatus ST1, L.crispatus CIP 103604, L.crispatus CIP 104459 and L.crispatus LMG 12005. Therefore, the effect of treating or preventing diseases related to uterine fibroids is better.

According to an embodiment of the present invention, when the food is solid, the food contains 1×10-1×10 ²⁰ cfu/g of the Lactobacillus crispatus, and when the food is liquid, the food contains 1 ×10-1×10 ²⁰ cfu/mL of the Lactobacillus crispatus. Therefore, the effect of inhibiting the occurrence of fibroids and reducing the volume of fibroids is obvious. If the content of Lactobacillus crispatus is too low, the effect of treating or preventing uterine fibroids is unsatisfactory. If the content of Lactobacillus crispatus is too high, it can treat or prevent uterine fibroids The effect is not significantly improved, resulting in waste.

According to a preferred embodiment of the present invention, when the food is solid, the food contains 1×10 ⁴ -1×10 ¹⁵ cfu/g of the Lactobacillus crispatus; when the food is liquid, the The food contains 1×10 ⁴ -1×10 ¹⁵ cfu/mL of said Lactobacillus crispatus.

According to another preferred embodiment of the present invention, when the food is solid, the food contains 1×10 ⁶ -1×10 ¹¹ cfu/g of the Lactobacillus crispatus; when the food is liquid, The food contains 1×10 ⁶ -1×10 ¹¹ cfu/mL of the Lactobacillus crispatus.

According to an embodiment of the present invention, the food acceptable auxiliary material is selected from carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners and at least one of the spices.

According to an embodiment of the present invention, the food acceptable auxiliary material is selected from lactose, glucose, sucrose, sorbitol, mannose, starch, acacia, calcium phosphate, alginate, gelatin, calcium silicate, At least one of fine crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate and mineral oil.

According to an embodiment of the present invention, the food is in the form of at least one formulation selected from solids, dairy products, solution products, powder products and suspension products.

According to an embodiment of the present invention, the use of the food composition or the pharmaceutical composition is characterized in that the composition is used to reduce the volume of uterine fibroids in experimental subjects, or to inhibit the uterine fibroids recurrence.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

Figure 1 shows the therapeutic effect of Lactobacillus crispatus on the guinea pig model of uterine fibroids. A, volume change of fibroids in each group before and after treatment; B, uterine coefficient in each group after treatment. The ones on the error bars represent significant differences compared with CK1 group (P<0.01); ▲ represent significant differences compared with CK2 group (P<0.01).

Figure 2 shows the therapeutic effect of Lactobacillus crispatus on the Eker rat model of uterine fibroids. A, volume change of fibroids in each group before and after treatment; B, uterine coefficient in each group after treatment; C, concentration of estradiol (E2) in each group after treatment; D; concentration of progesterone (P) in each group after treatment. The ones on the error bars represent significant differences compared with CK1 group (P<0.01); ▲ represent significant differences compared with CK2 group (P<0.01).

Figure 3 shows the preventive effect of Lactobacillus crispatus on uterine fibroids in Eker rats. A, volume of fibroids in each group after treatment; B, uterine coefficient in each group after treatment. The ones on the error bars represent significant differences compared with CK1 group (P<0.01); ▲ represent significant differences compared with CK2 group (P<0.01).

Fig. 4 shows the changes in the volume of the fibroids during and after the treatment of the mouse model of uterine fibroids with Lactobacillus crispatus. The solid marks in L group represent significant difference (P<0.01) compared with CK2 group.

Figure 5 shows the therapeutic effect and recurrence inhibition effect of Lactobacillus crispatus on the transplanted mouse model of uterine fibroids. On the error bars, there is a significant difference between the 6th week and the CK1 group (P<0.01); ▲ represents the significant difference between the 6th week and the CK2 group (P<0.01); ☆represents the significant difference between the 10th week and the CK1 group Significantly (P<0.01); △ represents a significant difference (P<0.01) compared with the CK2 group at the 10th week.

detailed description

The present invention will be further explained below in conjunction with specific examples, but specific examples do not limit the present invention in any way, and unless otherwise specified, the reagents and methods involved in the examples are commonly used reagents and methods in the art.

Embodiment 1 Determination of Beneficial Bacteria

Vaginal scraping samples were collected from female volunteers, including 30 volunteers without uterine fibroids and 65 volunteers with uterine fibroids. The collected samples were put into sterilized collection tubes, frozen in liquid nitrogen, and stored in -80°C refrigerator. Transported to Shenzhen Huada Institute of Genomics with dry ice.

Take 0.2g of each sample for extraction of genomic DNA, using the QIAamp DNA Mini Kit (QIAGEN), referring to the instructions provided by the manufacturer.

Specific primers for the V4-V5 hypervariable region of the 16S rRNA gene were used for amplification. The primer sequences are as follows:

The obtained PCR product was purified using AxyPrep ^TM PCR Cleaning Kit (Axygen Scientific, Inc. US), and the specific steps were referred to the instructions provided by the manufacturer. use SizeSelect ^TM 2% agarose gel was used to purify the PCR products containing adapters and Ion Xpress barcode sequences, and concentrated using AMPure Beads 1.2× (Beckman Coulter); using Ion OneTouch 2 ^TM and Ion Template PGM ^TM OT2 400 kit ( Life Technologies) for emulsification PCR; using the Ion 318 ^TM Chip kit for sequencing on the Torrent PGM system.

Sequencing results were removed by PGM software to remove low-quality sequences and polyclonal sequences, and Mothur software was used for quality control (Schloss, P.D., Westcott, S.L., Ryabin, T., et al. (2009). Introducing mothur: open-source, platform- independent, community-supported software for describing and comparing microbial com). The obtained high-quality sequences were clustered to obtain OUT, and the OTUs were annotated with species. The correlation analysis between the abundance of OTUs and the diseases of the volunteers showed that the abundance of OTU#1 in the vaginal samples of non-uterine fibroids volunteers was significantly higher than that in the vaginal samples of uterine fibroids volunteers (p<0.05), After annotation, it was found that the similarity between the OTU and the V4-V5 region of the 16S rRNA gene of Lactobacillus kitasatonis and Lactobacillus crispatus was higher than 97%. In order to accurately annotate this OTU, the inventors cloned the full-length sequence of the 16S rRNA gene from a sample containing 99% of OTU#1 (sample number C056CU), and cloned a 289bp fragment of a conserved single-copy gene, which encodes phenylpropanoid Amino acid-tRNA ligase beta subunit (PF03147). Sanger sequencing was performed on the cloned fragments using Biosystems 3730 DNA sequencer, and the sequencing results were compared with the database. It was found that these two genes had the highest similarity with the corresponding genes of L.crispatus ST1 (NCBI gene ID: 9107847 and 9107287), so the invention Humans annotated OTU#1 as L. crispatus.

Embodiment 2 strain source and preparation

2.1 Source of strain

Lactobacillus crispatus is a Gram-positive bacterium with a slender rod shape, slightly bent into chains, no flagella, and no spores; the colony is white, with irregular edges, flaky, and convex in the center. It is chemoheterotrophic and concurrently Anaerobic, non-liquefying gelatin; available mannose, galactose, glucose, maltose, melibiose, raffinose, ribose, sucrose, trehalose, fructose; catalase negative, oxidase negative; acidogenic, thermophilic . The embodiment of the present invention adopts four strains of Lactobacillus crispatus isolated from human vagina, and its information is as follows:

strain source separate source L. crispatus ST1 University of Helsinki chick crop L.crispatus CIP 103604 CIP human cervix vagina L. crispatus CIP 104459 CIP human vagina L. crispatus LMG 12005 BCCM/LMG pregnant vagina

L.crispatus ST1 comes from the University of Helsinki, Finland (Division of General Microbiology, Department of Biosciences, FIN-00014University of Helsinki) (reported in Edelman S, B, S et al. In vitro adhesion specificity of indigenous lactobacilli within the avian intestinal tract. Applied and Environmental Microbiology 2002; 68:5155-5159.)

CIP: The Collection of Institut Pasteur

BCCM/LMG: Belgian Coordinated Collections of Microorganisms, Laboratorium voor Microbiologie, Universiteit Gent

Obtain the freeze-dried bacterial powder of above-mentioned bacterial strain, at first in MRS medium (peptone 10g, beef extract 10g, yeast extract 5g, diammonium hydrogen citrate 2.0g, glucose 20g, dipotassium hydrogen phosphate 2g, sodium acetate 5g, magnesium sulfate 0.20 g, manganese sulfate 0.05g, Tween 801mL, add distilled water to 1000mL, pH 6.2～6.5, add 0.05% cysteine hydrochloride) to activate.

After activation, each strain was inoculated, inserted into MRS liquid medium, and vibrated at 37°C and 200r/min to obtain strain fermentation broth. The fermentation broth was centrifuged at 4200r/min for 15min, and the supernatant was discarded to obtain bacterial cells, which were dried under low temperature and negative pressure conditions to obtain bacterial powder, and the viable bacterial unit (CFU/mg) of the bacterial powder was measured.

2.2 Preparation of bacteria preparation for vagina

The obtained bacterial powder is mixed with a pharmaceutically acceptable excipient, so that the bacterial content in the mixture is 1×10 ⁸ CFU/mg. Capsules were prepared by techniques known to those skilled in the art, and each capsule contained 15 mg of the mixture.

2.3 Preparation of bacterial preparations for oral administration

Fresh milk was pasteurized, and a certain amount of bacterial powder was dissolved into the sterilized milk so that the bacterial content in the milk was 1×10 ⁹ CFU/mL.

2.4 Control drugs

Mifepristone capsules (trade name "Nuoluting", Beijing Zizhu Pharmaceutical Co., Ltd.), tablets are used for feeding experimental animals, the contents of the capsules are taken out and mixed with aseptic milk.

The CCI-779 preparation used in the examples is "Sirolimus Oral Solution" (trade name "Rapaming", Wyeth Pharmaceutical Co., Ltd.).

The treatment of embodiment 3 Lactobacillus crispatus to guinea pig uterine leiomyoma

Adult female guinea pigs (Dunkin Hartley) were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. Guinea pigs can spontaneously develop uterine fibroids as they age. Feeding guinea pigs estradiol, tretinoin, or troglitazone promotes this spontaneous process. Guinea pigs were injected with estradiol 3-5 times a week for 2-4 months until uterine fibroids formed.

After ultrasonic diagnosis, the guinea pigs with uterine fibroids were divided into 6 groups, 15 in each group. Among them, the first group administered excipients through the vagina, which was the negative control group (hereinafter referred to as "CK1 group"). The second group was administered mifepristone capsules vaginally, which was the positive control group (hereinafter referred to as "CK2 group"). The other four groups received one capsule containing L.crispatus ST1, L.crispatus CIP 103604, L.crispatus CIP 104459, or L.crispatus LMG 12005 through vaginal administration, respectively, and were L1, L2, L3, and L4 groups, collectively referred to as L group.

According to the method described by Borahay et al. (Borahay MA, Vincent K, Motamedi M et al. Novel effects of simvastatin on uterine fibroid tumors: in vitro and patient-derived xenograft mouse model study. American Journal of Obstetrics and Gynecology 2015; 213:196. e191-196.e198.), before treatment and after 6 weeks of treatment, the size of myoma was measured by high-resolution ultrasound system (Vevo 2100; Visualsonics, Canada), and the volume change of myoma was calculated according to the formula (A) ( %):

Among them, X represents the volume of fibroids after 6 weeks of treatment, and Y represents the volume of fibroids before treatment.

Subsequently, the guinea pigs were sacrificed, the wet weight of the uterus was measured, and the uterine coefficient (%) was calculated using the formula (B):

Among them, W ₁ is the wet weight of the uterus, and W ₂ is the body weight of the guinea pig.

The therapeutic effect of table 1 Lactobacillus crispatus on guinea pig uterine fibroids

The data in the table are the mean ± standard deviation, using Tukey’s multiple test, it represents a significant difference with the CK1 group (P<0.01), ▲ represents a significant difference with the CK2 group (P<0.01), the same below.

It can be seen from Table 1 and Figure 1 that the fibroid volume in group CK1 was larger than that before treatment, while the volume of fibroids in guinea pigs in group L and CK2 was significantly reduced, and the reduction in the volume of fibroids in group L was significantly higher than that in group CK2. On the other hand, the uterine coefficients of L group and CK2 group were also significantly lower than those of CK1 group after treatment, and the uterine coefficients of L group were significantly lower than those of CK2 group. The above results show that Lactobacillus crispatus has a better therapeutic effect on uterine fibroids than mifepristone.

The treatment of embodiment 4 Lactobacillus crispatus to Eker rat hysteromyoma

Eker rats are well-studied uterine leiomyoma models (Walker CL, Hunter D, Everitt JI. Uterine leiomyoma in the Eker rat: A unique model for important diseases of women. Genes, Chromosomes and Cancer 2003; 38:349-356 .). Due to the mutation of Tsc-2 gene, about 65% of female Eker rats will spontaneously form uterine fibroids (about 12-16 weeks).

Eker rats were from the University of Texas MD Anderson Cancer Center. Eker rats with spontaneous uterine fibroids were selected and divided into 6 groups, 15 rats in each group. Among them, the CK1 group was fed with aseptic milk; the CK2 group was fed with mifepristone; the L1-L4 groups were fed with 100 mL of L.crispatus ST1, L.crispatus CIP103604, L.crispatus CIP 104459 or L.crispatus LMG 12005 respectively. milk.

After 6 weeks of treatment, the method described in Example 3 was used to measure and calculate the uterine volume change (%) and uterine coefficient (%) of Eker rats. 40 minutes after the last administration, the rats were intraperitoneally anesthetized with pentobarbital sodium 40 mg/kg, blood was collected from the abdominal aorta, and the serum was separated, and the serum estradiol (E2) and progesterone (P) contents were measured by radioimmunoassay.

Therapeutic effect of table 2 Lactobacillus crispatus on Eker rat uterine fibroids

It can be seen from Table 2 and Figure 2 that the volume of fibroids in group CK1 was larger than that before treatment, while the volume of fibroids in guinea pigs in group L and CK2 was significantly reduced, and the volume reduction of fibroids in group L was significantly higher than that in group CK2. On the other hand, the uterine coefficients of L group and CK2 group were also significantly lower than those of CK1 group after treatment, and the uterine coefficients of L group were significantly lower than those of CK2 group.

The results also showed that both Lactobacillus crispatus and mifepristone can reduce the concentration of estradiol and progesterone, and there is a definite dose-effect relationship, and the effect of Lactobacillus crispatus on reducing the concentration of estradiol and progesterone is higher than that of mifepristone. Since estradiol can promote the development of uterine fibroids, and uterine fibroids are known to grow in a progesterone-dependent manner, the reduction of estradiol and progesterone concentrations will inhibit the growth of uterine fibroids.

It can be seen that Lactobacillus crispatus has the effect of inhibiting the growth of uterine fibroids, and the therapeutic effect of Lactobacillus crispatus on uterine fibroids is better than that of mifepristone.

The prevention of embodiment 5 Lactobacillus crispatus to Eker rat hysteromyoma

Eker rats aged about 9 weeks were selected and randomly divided into 6 groups with 15 rats in each group. Among them, the CK1 group was fed with aseptic milk; the CK2 group was fed with CCI-779 oral solution at a dose of 25mg/kg; Or milk of L. crispatus LMG 12005.

After 6 weeks of treatment, the volume of myoma was measured using the method described in Example 3, and the uterine coefficient (%) was measured and calculated.

Table 3 preventive effect of Lactobacillus crispatus on Eker rat uterine fibroids

As can be seen from Table 3 and Figure 3, all groups formed fibroids, but the fibroids in group CK1 were larger, while the fibroids in groups L and CK2 were smaller in guinea pigs, with significant differences (P<0.05). The volume of myoma in L group was significantly smaller than that in CK2 group. On the other hand, the uterine coefficients of L group and CK2 group were also significantly lower than those of CK1 group after treatment, and the uterine coefficients of L group were significantly lower than those of CK2 group.

The above results indicated that the preventive effect of Lactobacillus crispatus on uterine fibroids in Eker rats was better than that of CCI-779.

Example 6 Effect of Lactobacillus crispatus on Uterine Fibroid Transplantation Model Mice

6-week-old female immunodeficient NOG (NOD/Shi-scid/IL-2Rγ ^nul ) mice were purchased from Taconic Biosciences, Inc. (New York, USA). The deficient mice are more suitable for establishing human xenograft models.

The fibroid tissues were obtained from patients with uterine fibroids, cut into strips of 2×2×3 mm under sterile conditions, and immersed in basement membrane matrigel (BD). A small incision was made in the abdominal skin of the mouse with a sterile hand knife, and then a strip of fibroid tissue was implanted subcutaneously and wrapped with sterile surgical tape.

One week after transplantation, the surgical tape was removed and the graft was inspected. The transplanted mice were randomly divided into 6 groups, 15 in each group. Among them, the CK1 group administered excipients vaginally; the CK2 group administered mifepristone capsules vaginally; the L1-L4 groups administered one capsule containing L.crispatus ST1, L.crispatus CIP103604, L.crispatus CIP 104459 or L. .crispatus LMG 12005 capsules.

Stop application after 6 weeks, and then continue to observe for 4 weeks. From the beginning of the experiment, the volume of the uterus was measured every week using the method described in Example 3, taking the volume before treatment (that is, the 0th week) as 100%, and starting from the 1st week, the volume percentage relative to the 0th week was calculated.

In addition, the volume changes at weeks 6 and 10 relative to week 0 are summarized in the table.

Table 5 The therapeutic effect of Lactobacillus crispatus on transplanted fibroids and the effect of inhibiting recurrence

▲ represents a significant difference between the 6th week and the CK1 group (P<0.01); ▲ represents a significant difference between the 6th week and the CK2 group (P<0.01); ☆ represents a significant difference between the 10th week and the CK1 group (P<0.01) 0.01); △ represents a significant difference (P<0.01) compared with the CK2 group at the 10th week.

It can be seen from Table 4 and Figure 4 that during the treatment period (i.e. from the 1st week to the 6th week), the volume of the CK1 transplanted fibroids increased continuously, while the volume of the transplanted fibroids in the L group and CK2 group decreased continuously, and the volume of the transplanted fibroids in the L group decreased. The smaller range was larger; by the 6th week, the volume of transplanted fibroids in group L and CK2 was significantly lower than that in group CK1, and the volume of transplanted fibroids in group L was significantly lower than that in group CK2 (Table 5 and Figure 5).

It can also be seen from Table 4 and Figure 4 that after stopping the treatment, the volume of the transplanted fibroids in the CK1 group continued to increase; the volume of the transplanted fibroids in the CK2 group rebounded significantly, and at the 10th week, that is, the 4th week after stopping the drug, the volume of the transplanted fibroids recovered to CK1 level; while the L group also increased, but the increase was not obvious (Table 5 and Figure 5).

The above results show that although mifepristone treatment can treat the development of transplanted fibroids to a certain extent, it will relapse after stopping the drug, and Lactobacillus crispatus can not only effectively reduce the volume of transplanted fibroids, but also significantly inhibit the recurrence of fibroids after stopping the drug. .

Embodiment 7 contains the food composition of lactobacillus crispatus Lactobacillus crispatus

The ratio of raw materials is shown in Table 7.

Table 6

raw material Mass percentage (%) Lactobacillus crispatus 0.5 milk 90.0 white sugar 9.5

Mix the above formula ratio, stir until completely mixed, preheat, 20Mpa pressure homogenization, sterilize at about 90°C for 5-10 minutes, cool to 40-43°C, inoculate 1-100×10 ⁶ cfu/g Lactobacillus crispatus , Lactobacillus crispatus ST1, Lactobacillus crispatus CIP 103604, Lactobacillus.crispatus CIP 104459 and Lactobacillus crispatus LMG 12005 are at least one, that is, a food composition containing Lactobacillus crispatus is prepared.

Example 8

Pharmaceutical composition containing Lactobacillus crispatus

The ratio of raw materials is shown in Table 7.

Table 7

raw material Mass percentage (%) Lactobacillus crispatus 1.0% lactose 2.0% yeast 2.0% Peptone 1.0% pure water 94.0%

Mix lactose, yeast powder, and peptone evenly with purified water according to the proportion, preheat to 60-65°C, homogenize with 20Mpa pressure, sterilize at about 90°C for 20-30 minutes, cool to 36-38°C, and insert Lactobacillus crispatus Crispatus live bacteria (1-50×10 ⁶ cfu/mL), Lactobacillus crispatus is at least one selected from Lactobacillus crispatus ST1, Lactobacillus crispatus CIP 103604, Lactobacillus.crispatus CIP104459 and Lactobacillus crispatus LMG 12005, fermented at 36-38°C until the pH value is 6.0, centrifuge, and freeze-dry until the water content is less than 3%, that is, to prepare a freeze-dried product of Lactobacillus crispatus. Weighing 0.5 g of Lactobacillus crispatus freeze-dried product and mixing equal amounts of maltodextrin, then filling it into capsules to prepare the pharmaceutical composition containing Lactobacillus crispatus.

In the description of this specification, descriptions referring to the terms "one embodiment", "some embodiments", "example", "specific examples", or "some examples" mean that specific features described in connection with the embodiment or example , structure, material or feature is included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the described specific features, structures, materials or characteristics may be combined in any suitable manner in any one or more embodiments or examples. In addition, those skilled in the art can combine and combine different embodiments or examples and features of different embodiments or examples described in this specification without conflicting with each other.

Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention, those skilled in the art can make the above-mentioned The embodiments are subject to changes, modifications, substitutions and variations.

Claims (9)

1. the purposes of a kind of Lactobacillus crispatus, it is characterized in that, described Lactobacillus crispatus is used for preparing the composition of treating or preventing hysteromyoma. 2. The use according to claim 1, wherein the Lactobacillus crispatus is at least one selected from Lactobacillus crispatus ST1, Lactobacillus crispatus CIP 103604, Lactobacillus.crispatus CIP 104459 and Lactobacillus crispatus LMG 12005. 3. purposes as claimed in claim 1, is characterized in that, described treatment or prevention hysteromyoma comprise: (i) reduce hysteromyoma volume; (ii) reduce uterine coefficient; (iii) reduce estradiol concentration ; (iv) reduce the concentration of progesterone; (v) inhibit the growth of uterine fibroids. 4. A pharmaceutical composition, characterized in that the pharmaceutical composition contains an effective amount of Lactobacillus crispatus and/or its metabolites, and a pharmaceutically acceptable carrier. 5. The pharmaceutical composition according to claim 4, wherein the dosage form of the pharmaceutical composition is: granule, capsule, tablet, powder, oral liquid, suspension or emulsion. 6. A hygienic product containing the pharmaceutical composition of claim 4, characterized in that, the hygienic product is selected from the group consisting of tablet, capsule, lozenge, cream, gel, ointment, lotion, tampon, hygienic product Wipes, diapers, pads, dissolving strips, condoms, pessaries, sprays. 7. A food composition, characterized in that, the food composition contains an effective amount of Lactobacillus crispatus and/or its metabolites, and a balance of food-acceptable carriers. 8. The food composition according to claim 7, wherein the dosage form of the food composition is selected from solid, milk, solution products, powder products, or suspension products. 9. the purposes of a kind of pharmaceutical composition described in claim 4 or the described food composition of claim 7, it is characterized in that, described composition is used for reducing experiment object's hysteromyoma volume, or inhibits hysteromyoma Relapse after discontinuation of medication.