AU2021105135A4 - Probiotic compound preparation and use thereof in regulation of vaginal flora and hpv resistance - Google Patents

Abstract

OF THE DISCLOSURE The present disclosure discloses a probiotic compound preparation consisting of Lactobacillus acidophilus, Bifidobacterium and Lactobacillus crispatus. The aforementioned lactic acid bacteria have strong acid resistance, good adhesion to vaginal epithelial cells, and can be colonized in vagina. Moreover, the probiotic compound preparation has an inhibitory effect on conditional pathogens and HPVs in the vagina, can adjust the structure of vaginal flora, and plays a role in treating and preventing vaginitis and cervical cancer. ABSTRACT DRAWING - FIG. 6 11 17942232_1 (GHMatters) P116938.AU - 3/4 6 Group administrated with Lactobaci/us ac/dohi/us ormal group 4. Natural recovery group x Model group . :3 0 Co 2 C/) 0 3000 6000 9000 12000 Sequencing amount FIG 5 Others u Firmicutes Fusobacteriu 0 > B Bacteroides xProteobacteri a Normal Model Natural Group group group recovery administrated group with Lactobacillus acidophilus FIG6 17942207_1 (GHMatters) P116938.AU

Description

- 3/4

6

Group administrated with Lactobaci/us ac/dohi/us ormal group 4. Natural recovery group x Model group

. :3 0

Co 2 C/)

0 3000 6000 9000 12000

Sequencing amount FIG 5

Others u Firmicutes Fusobacteriu 0 > B Bacteroides xProteobacteri a

Normal Model Natural Group group group recovery administrated group with Lactobacillus acidophilus

FIG6

17942207_1 (GHMatters) P116938.AU

PROBIOTIC COMPOUND PREPARATION AND USE THEREOF IN REGULATION OF VAGINAL FLORA AND HPV RESISTANCE TECHNICAL FIELD

[01] The present disclosure belongs to the field of microorganisms, and in particular to a probiotic compound preparation and use thereof in regulation of vaginal flora and HPV resistance.

BACKGROUNDART

[02] A female vaginal microenvironment is a unique and complex dynamic system composed of various flora. In the vagina of a healthy woman, probiotics such as Lactobacillus, Bifidobacterium and Eubacterium are main strains, and meanwhile conditional pathogens causing gynecological diseases, such as Candida albicans, Gardnerella, Escherichia coli, Enterococcus, Peptostreptococcusand Staphylococcus epidermidis, also exist. Under normal circumstances, the pathogens and the probiotics are in a state of dynamic balance, and the probiotics can effectively inhibit the proliferation of the pathogens and have a regulation effect of realizing maintenance of the dynamic balance of the vaginal flora. However, some environmental factors can lead to imbalance of the vaginal flora, cause local abnormal inflammatory responses and immune responses, result in a variety of gynecological diseases, and ultimately endanger the reproductive health of women. Currently, antibiotics are still the main means to treat gynecological diseases of vagina, but long-term use of the antibiotics can lead to drug resistance of a host against the antibiotics, and the antibiotics can change the normal microecological environment of vagina and cause flora disequilibrium, resulting in a poor treatment effect, a high recurrence rate, and the like problems.

[03] Human papilloma virus (HPV) is a spherical DNA virus. After its DNA is integrated with the DNA of a squamous epithelial cell of skin mucosa, it can cause a hyperplasia disorder of the skin mucosa and induce a cancer. Therefore, HPV infection is involved in the occurrence and development process of cervical lesions. HPV has high epitheliotropic characteristics, and mainly infects epithelial cells of a gastrointestinal tract, a reproductive tract, a urinary tract, etc. Currently, about 200 kinds of HPV have been found, of which more than 10 are closely related to the occurrence and development of cervical lesions, cervical cancer and external genital cancer, and are called high-risk human papilloma viruses (HR-HPVs). Although HPV has been studied for more than 30 years, currently there is still no definite therapy for HPV. For the treatment of HR-HPV infection, the current treatment strategy is still "cure the disease rather than cure the virus". That is, treatment is conducted by destroying or excising transformed cells containing HR-HPV and associated pathological tissues, instead of treating the HR-HPV infection itself, which cannot completely eliminate the HR-HPV infection.

17942232_1 (GHMatters) P116938.AU

SUMMARY

[04] The present disclosure aims to provide a probiotic compound preparation consisting of Lactobacillus acidophilus, Bifidobacterium and Lactobacillus crispatus, which has remarkable curative effects on treating vaginitis and resisting HPV, and lays a foundation for the development and application of novel microecological preparations in the future.

[05] According to one aspect of the present disclosure, a probiotic compound preparation is provided, which includes 2 parts of Lactobacillus acidophilus, 1 part of Bifidobacterium and 2 parts of Lactobacilluscrispatus based on the total number of colonies.

[06] The present disclosure also provides use of the probiotic compound preparation in a product for regulating the structure of vaginal flora.

[07] The present disclosure also provides use of the probiotic compound preparation in a product for treating HPV infection.

[08] The present disclosure also provides a product for treating HPV infection, which includes the aforementioned probiotic compound preparation.

[09] Also provided is a product for treating HPV infection, which also includes an interferon.

[10] The present disclosure also provides a product for preventing and treating vaginitis, which includes the aforementioned probiotic compound preparation.

[11] The present disclosure also provides use of the probiotic compound preparation in a product for preventing and treating vaginitis.

[12] The present disclosure also provides a product for preventing and treating cervical cancer, which includes the aforementioned probiotic compound preparation.

[13] The present disclosure also provides use of the probiotic compound preparation in a product for preventing and treating cervical cancer.

[14] Aiming at the defects existed in the prior art, the present disclosure provides a probiotic compound preparation consisting of Lactobacillus acidophilus, Bifidobacterium and Lactobacillus crispatus, wherein aforementioned lactic acid bacteria have strong acid resistance, have good adhesion to vaginal epithelial cells and can be colonized in the vagina; and the probiotic compound preparation has an inhibitory effect on conditional pathogens and HPVs in the vagina, can adjust the structure of vaginal flora, and plays a role in treating and preventing vaginitis and cervical cancer.

BRIEFT DESCRIPTION OF THE DRAWINGS

[15] FIG. 1 shows the adhesion of Lactobacillusacidophilus to vaginal epithelial cells;

[16] FIG. 2 shows the adhesion of Bifidobacterium to vaginal epithelial cells;

[17] FIG. 3 shows the adhesion of Lactobacilluscrispatus to vaginal epithelial cells; 2 17942232_1 (GHMatters) P116938.AU

[18] FIG. 4 is a rarefaction curve of rat vaginal flora;

[19] FIG. 5 is Shannon curve of rat vaginal flora;

[20] FIG. 6 shows the structure of rat vaginal flora at a phylum level; and

[21] FIG. 7 shows the structure of rat vaginal flora at a genus level.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[22] The present disclosure will be explained in detail with reference to the attached drawings.

[23] Example 1 Strain Culture

[24] Lactobacillus acidophilus, Bifidobacterium and Lactobacillus crispatus were lactic acid bacteria isolated from Koumiss and human vagina. It was found by research that the aforementioned strains had strong adhesion to vaginal epithelial cells, could inhibit the growth of conditional pathogens and HPVs, and provided a basis for developing living lactic acid bacteria preparations capable of treating vaginitis and cervical cancer.

[25] Lactobacillus acidophilus had been deposited in China General Microbiological Culture Collection Center (CGMCC) at the address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing on December 14, 2010 with the accession number of CGMCC No. 4472, and the preservation result was that it was alive.

[26] Bifidobacterium animalis had been deposited in China General Microbiological Culture Collection Center (CGMCC) at the address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing on December 14, 2010 with the accession number of CGMCC No. 4473, and the preservation result was that it was alive.

[27] Lactobacillus crispatus had been deposited in China General Microbiological Culture Collection Center (CGMCC) at the address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing on September 1, 2020 with the accession number of CGMCC No. 20585, and the preservation result was that it was alive.

[28] Lactobacillus acidophilus and Lactobacillus crispatus were inoculated into a MRS liquid medium and cultured at 37°C for 24 h, and Bifidobacterium was inoculated into a TPY liquid medium and cultured anaerobically at 37°C for 24 h. The aforementioned strains were collectively referred to as lactic acid bacteria.

[29] Example 2 Acid Resistance of Lactic Acid Bacteria

[30] The fermentation supernatant of lactic acid bacteria strains that had been cultured for 24 h was taken and centrifuged at 4,000 r/min for 10 min, and the supernatant was discarded. The precipitate was resuspended with PBS buffers with pH values of 3, 4 and 5 respectively. Viable count was conducted after treatment for 0 h, 1 h, 2 h and 3 h, and the viable count results of Lactobacillus acidophilus, Bifidobacterium and Lactobacillus crispatus in the acid resistance test 3 17942232_1 (GHMatters) P116938.AU were shown in Tables 1 to 3 respectively. The vaginal pH of a healthy woman was equal to 3.8-4.4, and the acid resistance of vaginal flora is an important index to test the probiotic characteristics of vaginal lactic acid bacteria. It could be seen from Tables 1 to 3 that the viable count was decreased by 1 order of magnitude when Lactobacillus acidophilus, Bifidobacterium and Lactobacillus crispatus were treated at a condition of pH = 3 for 3 hours, but the viable count was hardly changed when these bacteria were treated at conditions of pH = 4 and pH = 5 for 3 hours, indicating that the lactic acid bacteria had strong acid resistance and could survive in vagina.

[31] Table 1 Viable Count Result of Lactobacillus acidophilus in Acid Resistance Test Viable count (CFU/mL) pH Oh 1h 2h 3h 3 3.25 x 108 5.36 x 107 3.02 x 107 2.21 x 107 4 3.92 x 108 3.47 x 108 3.43 x 108 3.09 x 108 5 3.41 x 108 3.30 x 108 3.25 x 108 3.33 x 108

[32] Table 2 Viable Count Result of Bifidobacterium in Acid Resistance Test Viable count (CFU/mL) pH Oh 1h 2h 3h 3 7.31 x 10 9 4.16 x 108 4.22 x 108 3.51 x 108 4 6.94 x 10 9 4.73 x 10 9 2.51 x 10 9 4.32 x 109 5 2.34 x 10 9 4.31 x 10 9 5.23 x 10 9 3.49 x 109

[33] Table 3 Viable Count Result of Lactobacillus crispatus in Acid Resistance Test Viable count (CFU/mL) pH Oh 1h 2h 3h 3 5.21 x 108 4.62 x 10 7 4.07 x 10 7 3.15 x 10 7 4 4.81 x 108 5.28 x 108 3.44 x 108 5.21 x 108 5 4.56 x 108 4.15 x 108 3.01 x 108 2.29 x 108

[34] Example 3 Bacteriostasis of Lactic Acid Bacteria

[35] Culture of indicator bacteria: Candida albicans, Staphylococcus aureus, Escherichia coli., Beta Hemolytic Streptococcus, Pseudomonas aeruginosa and Shigella dysenteriae were activated in a LB medium and cultured at 37°C for 24 h. Lactobacillus jensenii, Lactobacillus gasseri and Lactobacillusgrignardwere activated in a MRS medium and cultured at 37°C for 24 h.

[36] Each 100 L of a solution of indicator bacteria was taken and evenly coated onto a

4 17942232_1 (GHMatters) P116938.AU corresponding solid medium. Wells were perforated with an Oxford cup (with a well diameter of 6 mm), added with the fermentation supernatant of the strain at a loading volume of 50 L/well, diffused at 4°C for 2 h, and put into a 37°C incubator for overnight culture, and then the inhibitory zone diameter was measured. The results were as shown in Table 4. The lactic acid bacteria could produce hydrogen peroxide, acidic substances (formic acid, acetic acid, propionic acid), bacteriocin, and the like, which had a good inhibitory effect on vaginal conditional pathogens, but had no inhibitory effect on probiotics such as Lactobacillus jensenii, Lactobacillus gasseri and Lactobacillusgrignardin the vagina.

[37] Table 4 Test Results of bacteriostasis Inhibitory zone diameter/mm Indicator bacteria Lactobacillus Bifidobacterium Lactobacillus acidophilus crispatus Candida albicans 18.35 0.21 17.31 0.12 18.63 0.24 Staphylococcus Aureus 21.61 0.35 18.32 0.25 20.17 0.23 Escherichiacoli 25.66 0.12 20.16 0.23 22.34 0.42 Beta Hemolytic 18.05 0.27 17.15 0.16 16. 95 0.31 Streptococcus Pseudomonas 22.53 0.32 20.13 0.23 21.41 0.11 aeruginosa Shigella dysenteriae 18.21 0.15 16.24 0.25 17.17 0.16 Lactobacillusjensenii Lactobacillusgasseri Lactobacillusgrignard

[38] Example 4 Adhesion of Lactic Acid Bacteria to Vaginal Epithelial Cells

[39] Preparation of lactic acid bacteria: the cultured bacteria solution was centrifuged, the supernatant was discarded, the bacteria solution was washed with PBS, the concentration of the bacteria solution was adjusted to 108 CFU/mL with a McIlvaine buffer (pH = 4.4), and the bacteria solution was placed at 4°C for later use.

[40] Preparation of vaginal epithelial cells: vaginal epithelial cells were suspended in 30 ml of the McIlvaine buffer (pH = 4.4), and washed by shaking with a vortex mixer to remove endogenous bacteria, and the cell concentration was adjusted to 105 cells/mL.

[41] Adhesion of strains to vaginal epithelial cells: each 1 mL of the bacterial suspension and the vaginal epithelial cell suspension were taken and mixed evenly, then cultured in a shaker at 37°C

5 17942232_1 (GHMatters) P116938.AU for 1 h, centrifuged at 1,000 r/min for 5 min, and washed with a PBS buffer, and the bacteria that did not adhere to the cells were discarded. The precipitate was smeared, dried and subjected to Gram staining, and the adhesion of the strains was observed under an oil immersion objective. The results were as shown in FIGs. 1 to 3. The number of bacteria adhering to the vaginal epithelial cells was more than 200, which indicated that Lactobacillus acidophilus, Bifidobacterium and Lactobacillus crispatus had good adhesion to the vaginal epithelial cells and could be colonized in vagina.

[42] Example 5 Establishment of Rat Vaginal Bacterial Infection Model

[43] 40 normal female rats were selected and randomly divided into a normal group, a model group, a natural recovery group and a group administrated with the probiotic compound preparation, with 10 rats in each group. Except the normal group, in other groups 50 L of an amoxicillin sodium solution at a concentration of 10 mg/mL was absorbed with medical absorbent gelatin sponge, and the sponge was stuffed into the vagina and uterine cavity of the rats, and these operations were conducted once a day for continuous 5 days. Then 50 L of a mixed bacterial solution (at a concentration of 3.0 x 109 CFU/mL formulated by mixing Beta Hemolytic Streptococcus, Staphylococcus aureus and Escherichia coli. in a ratio of 1:1:2) was absorbed with medical absorbent gelatin sponge, and the sponge was stuffed into the vagina and uterine cavity of the rats, and these operations were conducted once a day for continuous 6 days, so as to generate a rat vaginal bacterial infection model. In the normal group, the same volume of physiological saline was absorbed by medical absorbent gelatin sponge, and the sponge was stuffed into the vagina and uterine cavity of rats, and these operations were conducted once a day for continuous 11 days.

[44] Example 6 Treatment of Rat Vaginal Bacterial Infection Model

[45] The concentration of the probiotic compound bacteria solution was adjusted to 109 CFU/mL, wherein the quantity ratio of Lactobacillus acidophilus, Bifidobacterium and Lactobacillus crispatus was 2:1:2. 50 L of the bacterial solution was absorbed by medical absorbent gelatin sponge, and the sponge was stuffed into the vagina and uterine cavity of rats, and these operations were conducted once a day for continuous 6 days. The natural recovery group continued to receive the same volume of normal saline once a day for continuous 6 days. The model group and the normal group were not subjected to any intervention.

[46] Example 7 Determination of Vaginal Flora in Rat

[47] 7 days after recovery from the treatment, sterilized medical absorbent gelatin sponge was soaked in a sterile PBS solution, and the sponge was stuffed into the vagina of rats, and then after 2 to 5 min, the sponge was taken out and placed in a centrifuge tube filled with sterile PBS. The viable count of Beta Hemolytic Streptococcus, Staphylococcus aureus, Escherichia coli. and Lactobacillus and the high-throughput sequencing technique were respectively conducted to 6 17942232_1 (GHMatters) P116938.AU analyze the structure changes of the rat vaginal flora after recovery from treatment. The counting results were as shown in Table 5, and the structure changes of the rat vaginal flora were as shown in FIGs. 4 to 7.

[48] It could be seen from Table 5 that the number of Beta Hemolytic Streptococcus, Staphylococcus aureus and Escherichia coli. In the group administrated with the probiotic compound preparation was significantly less than that in other groups, while the number of Lactobacilluswas significantly higher than that in other groups.

[49] It could be seen from FIGs. 4 and 5 that the microbial information of the vagina of the rats could be reflected under the current sequencing amount. It could be seen from FIG. 4 that with the increase of the sequencing amount, the increase in the number of OUT in the rarefaction curve was gradually slowed down but still did not reach an equilibrium state, indicating that new bacterial species might be found in the vagina of the rats with the increase of the sequencing amount. However, it could be seen from the Shannon curve in FIG. 5 that the curve tended to be equilibrium with the increase of the sequencing quantity, and there was no obvious change with the continued increase of the sequencing amount, indicating that the current sequencing amount had reached a saturation state, which could reflect the structure of the rat vaginal bacterial flora.

[50] It could be seen from FIG. 6 that at the phylum level, the rat vaginal flora is mainly composed of Proteobacteria,Bacteroides, Fusobacteriumand Firmicutes. After treatment with the mixed bacterial solution, the content of each phylum in the vagina of the rats was changed, which indicated that the structure of the rat vaginal flora was changed, and the rat bacterial vaginal infection model was successfully established.

[51] It could be seen from FIG. 7 that, at a genus level, the main flora in each group was composed of Aggregatibacter, Bacteroides, Streptobacillus, Clostridium and Lactobacillus. Compared with the normal group, the model group and the natural recovery group, in the group administrated with the probiotic compound preparation the number of Lactobacillus, Clostridium and Streptobacillus had been increased significantly, while the number of Escherichia, Porphyromonas,Bacteroides and Aggregatibacterhad been decreased significantly, indicating that the group administrated with the probiotic compound preparation could regulate the structure of the rat vaginal flora.

[52] It could also be seen from FIGs. 6 and 7 that there were differences in the structure of rat vaginal flora in each experimental group, which indicated that the rat bacterial vaginal infection model had been successfully established, and the structure of the rat vaginal flora could be effectively regulated by the probiotic compound preparation.

[53] Table 5 Counting Results of Vaginal Flora in Rat Escherichi Staphylococcus Beta Hemolytic Lactobacill 7 17942232_1 (GHMatters) P116938.AU a coli Aureus (CFU/mL) Streptococcus us (CFU/mL) (CFU/mL) (CFU/mL) Normal group 3.42 x 104 2.98 x 104 2.31 x 104 5.27 x 106 Model group 4.51 x 108 2.05 x 108 1.63 x 108 3.24 x 104 Natural recovery group 2.58 x 106 1.93 x 106 4.21 x 106 6.31 x 105 Group administrated with the probiotic 4.13 x 103 4.36 x 103 2.17 x 103 2.92 x 108 compound preparation

[54] Example 8 Treatment of Patients Infected with HPV with Probiotic Compound Preparation

[55] 58 patients that were infected with HPV, all aged between 25-55, had regular menstrual cycles and normal menstrual periods, were non-pregnancy and non-lactation, and had normal and regular sexual lives, were incorporated into this study, including 31 cases in a group administrated with a combined therapy and 27 cases in a group administrated with interferon.

[56] In the group administrated with a combined therapy, both a recombinant human interferon-2a suppository (opin suppository) and the probiotic compound preparation produced by our company were put into the vagina every other day to treat for 3 treatment courses, with administration for 7 days as a treatment course. Administration was stopped during menstruation.

[57] The group administrated with interferon was treated by putting the recombinant human interferon 2a suppository into the vagina once every other day to treat for 3 treatment courses, with times of dosing as a treatment course.

[58] All patients were reexamined for HPV after menstruation in the sixth month after treatment. According to the negative conversion status of the HPV in the patients, the therapeutic effects on the patients were divided into three grades of negative conversion, effective and ineffective. Negative conversion: the examination results after return visit showed that all HPV subtypes turned negative; effective: some positive HPV subtypes turned negative, while > 1 HPV subtypes were still positive; ineffective: all HPVs were still positive. It could be seen from Table 6 that the effective rate and negative conversion rate of the group administrated with the combined therapy were 90.3% and 61.3% respectively, and the effective rate and negative conversion rate of the group administrated with interferon were 77.7% and 40.7% respectively. There were significant differences in the effective rate and negative conversion rate between the two groups, which indicated that the probiotic compound preparation had an obvious effect in treating HPV infection. The results showed that the group administrated with a combined therapy had a better therapeutic effect on patients infected with HPV than that when interferon was used alone, which indicated that the probiotic compound preparation had an obvious inhibitory effect on HPV.

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[59] Table 6 Effect Comparison of Two Therapies

Therapeutic effect on HPV Effective Negative Number - Groups Negative conversion of cases Ineffective Effective rate conversion ratio Group administrated with 31 3 9 19 90.3% 61.3% combined therapy Group administrated with 27 6 10 11 77.7% 40.7% interferon

[60] The effective rate x 2 = 0.290, and p = 0.591; and the negative conversion rate x2 = 4.453, and p = 0.034.

[61] The probiotic compound preparation provided by the present disclosure had a strong bacteriostatic effect, and had an inhibitory effect on most conditional pathogens in vagina, but had no inhibitory effect on the probiotic bacteria Lactobacillus in vagina.

[62] The probiotic compound preparation provided by the present disclosure had an inhibitory effect on HPV, and had an obvious effect in treating a patient infected with HPV when used in combination with interferon.

[63] The probiotic compound preparation provided by the present disclosure had strong tolerance to an acidic environment and high adhesion to vaginal epithelial cells, so that the bacteria could be colonized in vagina to regulate vaginal flora and played a role in treating and preventing vaginitis and cervical cancer.

[64] The aforementioned description is only some examples of the present disclosure. It should be noted that those of ordinary skills in the art can further make several variations and modifications without departing from the inventive concept of the present disclosure, and such variations and modifications all fall within the claimed scope of the present disclosure.

[65] It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country.

[66] In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

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Claims (4)

WHAT IS CLAIMED IS:

1. A probiotic compound preparation comprising 2 parts of Lactobacillus acidophilus, 1 part of Bifidobacterium and 2 parts of Lactobacillus crispatus based on the total number of colonies.

2. Use of the probiotic compound preparation according to claim 1 in a product for regulating the structure of vaginal flora, treating HPV infection, preventing and treating vaginitisor or preventing and treating cervical cancer..

3. A product for treating HPV infection, comprising the probiotic compound preparation according to claim 1; further comprising an interferon.

4. A product for preventing and treating vaginitis, comprising the probiotic compound preparation according to claim 1.

.5. A product for preventing and treating cervical cancer, comprising the probiotic compound preparation according to claim 1.

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FIG. 1

FIG. 2

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FIG. 3

Group administered with Lactobacillus Normal group acidophilus Natural recovery group

Model group

Sequencing amount FIG. 4

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Sequencing amount FIG. 5

administrated

FIG. 6

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group administrated

FIG. 7

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