LACTOBACILLUS REUTERI GMNL-263 FOR IMPROVING HYPERTENSION AND ITS COMPOSITIONS THEREOF

Abstract

Present invention discloses a Lactobacillus composition comprising Lactobacillus reuteri GMNL-263 which is a strain of heat-killed dead bacteria and has the effect of lowering blood pressure by inhibition of proinflammatory cytokine IL-1 and enhancement the growth of Bifidobacterium, and said Lactobacillus composition is a pharmaceutical composition, nutritional supplement, health food or a combination thereof.

Claims

1. A Lactobacillus composition for the improvement of hypertension, comprising Lactobacillus reuteri GMNL-263 (referred to as L. reuteri GMNL-263), wherein said L. reuteri GMNL-263 is dead bacteria.

2. The Lactobacillus composition as recited in claim 1, wherein the accession number of said L. reuteri GMNL-263 is CCTCC M 209263.

3. The Lactobacillus composition as recited in claim 1, wherein the L. reuteri GMNL-263 is applied for population with diabetes or those who need to control sugar intake.

4. The Lactobacillus composition as recited in claim 3, wherein said population with diabetes refers to type 2 diabetes mellitus (T2DM).

5. The Lactobacillus composition as recited in claim 1, wherein the Lactobacillus composition is a pharmaceutical composition, nutritional supplement, health food or a combination thereof.

6. The Lactobacillus composition as recited in claim 1, wherein the Lactobacillus composition is given by oral administration.

7. The Lactobacillus composition as recited in claim 5, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

8. The Lactobacillus composition as recited in claim 1, wherein the Lactobacillus composition further comprises edible materials including water, fluid dairy product, milk, concentrated milk, yogurt, sour milk, frozen yogurt, lactobacillus fermented beverage, milk powder, ice cream, cheese, sold or semi-solid cheese, soy milk, fermented soy milk, fruit and vegetable juices, fruit juices, sports drinks, desserts, jellies, confectionery, baby foods, health foods, animal feeds, Chinese herbal medicines or dietary supplements.

9. A method for improvement of hypertension, which comprises administering to a subject an effective amount of a composition, said composition including L. reuteri GMNL-263.

10. The method as recited in claim 9, wherein the improvement of hypertension is achieved by inhibition of a proinflammatory cytokine and enhancement of the growth of probiotics.

11. The method as recited in claim 10, wherein the proinflammatory cytokine is IL-1.

12. The method as recited in claim 10, wherein the probiotics are Bifidobacterium.

13. The method as recited in claim 9, wherein improvement of hypertension is specifically contributed to a population with diabetes.

14. The method as recited in claim 13, wherein the diabetes refers to type 2 diabetes mellitus (T2DM).

Description

BRIEF DESCRIPTION OF THE DRAWING

[0017] FIG. 1A shows the groups in the clinical trial.

[0018] FIG. 1B shows the steps of the clinical trial.

[0019] FIG. 2 shows the relevance analysis of the mean arterial pressure (MAP) and probiotic strains of Bifidobacterium.

DETAILED DESCRIPTION OF THE INVENTION

[0020] The invention is exemplified by the following embodiments but is not limited by thereof. The materials used in the invention, unless specified otherwise, are all commercially available materials in the market and Lactobacillus reuteri GMNL-263 [here in after referred to as GMNL-263] is deposited in Taiwan Food Industry Research and Development Institute with accession number of BCRC 910452 and in China Center for Type Culture Collection (CCTCC) with accession number of CCTCC M 209263.

Embodiment 1: Heat-Killed Dead GMNL-263 has the Effect of Lowering Blood Pressure

[0021] This embodiment is a human clinical trial. Due to the fact that patients with type 2 diabetes mellitus (T2DM) have high risk for hypertension, so patients are diagnosed with type 2 diabetes for more than 6 months and the other inclusion criteria include: 7%<HbA1c (glycated hemoglobin) 10%; age: 25-70 years; BMI>18.5; the exclusion criteria are: pregnancy/pregnant women; with a serious disease during past 3 years, such as cancer (except for well-controlled benign tumor), kidney failure/dialysis, heart disease, stroke, autoimmune disease and ingestion of health foods except for hypoglycemic drugs for improving blood sugar level 4 weeks before and during the trial period, fixed edible probiotic product and fixed use of antibiotics, as well as liver/kidney abnormalities, poor gastrointestinal function and those who cannot take oral medications.

[0022] The Clinical Trial Registration Number of this embodiment is NCT02274272 on Clinicaltrial.gov and was conducted in Changhua Christian Hospital; the Institutional Review Board (IRB) No. is 140703. FIG. 1A and FIG. 1B illustrate the groups and the process of the trail, which is a parallel research having a control group and using a double-blind and randomized allocation method that includes 74 subjects enrolled and divided into three groups randomly. After deducting the number of subjects leaving the trial, the total number of final analysis data was 68 subjects which includes the placebo group (N=22) and two probiotic trial groups: live L. reuteri GMNL-89 group (here in after referred to as GMNL-89) (N=22) and heat-killed dead L. reuteri GMNL-263 group (here in after referred to as GMNL-263) (N=24); in addition, after elimination of one subject who lost the fecal sample, the fecal samples of 67 subjects were analyzed based on their bacterial flora and the metabolic indicators such as baseline characteristics, blood sugar values, intestinal flora and proinflammatory cytokines before ingestion, 6 months after ingestion and 3 months after stopping using the probiotic product, that under the condition of concomitant use of current prescriptions.

[0023] The powder of live GMNL-89 bacteria and heat-killed dead GMNL-263 were prepared by GenMont biotech Incorporation via fermentation, the dose of live GMNL-89 was 410.sup.9 cfu (colony forming unit)/day and the dose of dead GMNL-263 was 210.sup.10 cells/day, the powder was given every day for a total of 6 months.

[0024] In the various statistical analyses, the comparison of the basic information was analyzed with Chi-square test or two-sample t-test, while the medication records and hypertension comparison were analyzed with Fisher exact test and the others use two-sample t-test.

[0025] First, the basic data of subjects were analyzed. Table 1 showed that there was no significant difference among the three groups of subjects at the beginning of the trial. Table 2 showed no significant difference in the proportion of hypertension among the three groups of subjects, indicating that the background conditions of the three groups of subjects were similar. Further, in terms of blood pressure, although these subjects all used drugs to control their blood pressure during the trial period, there were no statistical difference in the type of drugs used in the three groups. Therefore, there was no possibility of drug induced experimental error, but the systolic blood pressure (SBP) of all groups is still relatively high, indicating poor blood pressure control.

TABLE-US-00001 TABLE 1 Basic information of the three groups of subjects Placebo GMNL-263 GMNL-89 Baseline N = 22 N = 24 P-value N = 22 P-value Male 13 (59.1%) 13 (54.2%) 0.7365 12 (54.5%) 0.7609 Female 9 (40.9%) 11 (45.8%) 10 (45.5%) Age 55.77 8.55 53.88 7.78 0.4346 52.32 10.2 0.2302 Height (cm) 161.8 7.28 162 7.83 0.9176 163 7.92 0.6173 Weight (kg) 72.4 11.64 73.77 12.54 0.7043 74.97 15.73 0.5408 BMI (kg/m.sup.2) 27.53 3.15 28.03 3.88 0.6374 28.04 4.29 0.6587 SBP (mmHg) 126.8 9.93 132.9 16.93 0.1378 126.2 13.08 0.8668 DBP (mmHg) 75.32 9.09 76.08 6.93 0.7484 76.68 8.87 0.6172 Waist circumference (cm) 95.45 10.59 96.19 9 0.7998 95.94 12 0.8873 Hip circumference (cm) 100 7.66 101.1 7.95 0.6476 101.9 10.86 0.5073 GOT (U/L) 31.73 14.65 32.38 17.84 0.8941 30.91 10.39 0.8319 GPT (U/L) 39.59 23.11 43.71 27.67 0.5885 38.77 15.88 0.8918 HbA1c (%) 7.91 0.62 8.07 0.67 0.4252 7.91 0.68 >0.999

TABLE-US-00002 TABLE 2 History of disease and drug use records of the three groups of subjects placebo GMNL-263 GMNL-89 Variable N = 22 N = 24 P-value N = 22 P-value Medical History (N) Hypercholesterolemia 18 (81.8%) 22 (91.7%) 0.41 18 (81.8%) >0.99 Hypertension 16 (72.7%) 17 (70.8%) >0.99 18 (81.8%) 0.72 Medication for Hypertension (N) Diuretics 6 (27.3%) 8 (33.3%) 0.75 4 (18.2%) 0.72 Beta-blockers 6 (27.3%) 9 (37.5%) 0.54 7 (31.8%) >0.99 Alpha/Beta-blockers 0 (0.0%) 0 (0.0%) NA 1 (4.6%) >0.99 Angiotensin-Converting Enzyme Inhibitor (ACEI) 0 (0.0%) 1 (4.2%) >0.99 0 (0.0%) NA Angiotensin II Receptor Antagonist (All RA) 13 (59.1%) 9 (37.5%) 0.24 8 (36.4%) 0.23 Calcium channel blockers (CCB) 1 (4.6%) 1 (4.2%) >0.99 1 (4.6%) >0.99 Vasodilator 2 (9.1%) 3 (12.5%) >0.99 0 (0.0%) 0.49 Mixed-Drug 6 (27.3%) 9 (37.5%) 0.54 11 (50.0%) 0.22 Medication for diabetes (N) Insulin 5 (22.7%) 5 (20.8%) >0.99 3 (13.6%) 0.7 Metformin 20 (90.9%) 22 (91.7%) >0.99 20 (90.9%) >0.99 Sulphonylurea 16 (72.7%) 18 (75.0%) >0.99 14 (63.6%) 0.75 DDP-4 inhibitor Sitagliptin 4 (18.2%) 2 (8.3%) 0.41 2 (9.1%) 0.66 Vildagliptin 2 (9.1%) 2 (8.3%) >0.99 3 (13.6%) >0.99 Saxagliptin 12 (54.6%) 10 (41.7%) 0.56 9 (40.9%) 0.55 Linagliptin 7 (31.8%) 8 (33.3%) >0.99 5 (22.7%) 0.74 GLP-1 Receptor agonists Exenatide 0 (0.0%) 2 (8.3%) 0.49 1 (4.6%) >0.99 Liraglutide 0 (0.0%) 2 (8.3%) 0.49 3 (13.6%) 0.23 Acarbose 6 (27.3%) 2 (8.3%) 0.13 5 (22.7%) >0.99

[0026] The results showed that after 6 months, the net change of glycosylated hemoglobin A1c (HbA1c) in the diabetic subjects taking the live GMNL-89 (i.e., net change=the value of the sixth month deducts that of the first month=6M0M) was significantly reduced (0.390.80, P<0.05), while the heat-killed dead GMNL-263 group was not significantly different from the placebo group (0.240.93), indicating that taking the heat-killed dead GMNL-263 does not affect the glycemic value of the diabetic subjects.

[0027] In terms of blood pressure (see Table 3), the net change of the systolic blood pressure (SBP) and the mean blood pressure of the heat-killed dead GMNL-263 group were decreased and showed significant statistical difference (P<0.05), indicating that GMNL-263 had the effect of lowering blood pressure. However, there was no significant change in the blood pressure values of the GMNL-89, same as Lactobacillus reuteri strain, indicating that GMNL-89 did not have the effect of regulating blood pressure. The comparison results also indicate that the effect of lowering blood pressure on the specific bacterial colonies of the Lactobacillus reuteri strain is not common and easy to be known, and it must be confirmed by experiments.

TABLE-US-00003 TABLE 3 Net blood pressure changes of the three groups of subjects after 6-month of trial placebo GMNL-263 GMNL-89 N = 22 N = 24 P-value N = 22 P-value SBP (mmHg) 6 M 0 M 1.95 13.93 7.54 13.77 0.0248* 2.62 11.03 0.2146 DBP (mmHg) 6 M 0 M 0.36 8.28 3.17 5.45 0.0921 0.91 6.71 0.5783 pulse pressure (mmHg) 6 M 0 M 1.59 11.66 4.26 12.54 0.1019 1.91 8.69 0.2664 mean pressure (mmHg) 6 M 0 M 0.89 8.95 4.63 6.94 0.0254* 1.55 7.34 0.3285

Embodiment 2: Heat-Killed Dead GMNL-263 Shows the Effect of Lowering Blood Pressure by Reducing the Proinflammatory Cytokine IL-1p

[0028] To examine which mechanism of GMNL-263 is mediated to the effect of lowering blood pressure, further changes in cytokine levels were analyzed. First, the blood samples of the subjects in three groups were collected at the beginning of trial (0M) and 6 months later (6M) and ELISA kits were used to analyze the content of various cytokines, including human IL-6 (Cat#900-K16, PeproTech, USA), human IL-10 (Cat#900-K21, PeproTech, USA), human TNF- (Cat#50-114-2609, eBioscience, USA) and human IL-1 (Cat#437005, Biolegend, USA); the data processing is the net change value obtained from the value of the sixth month (6M) deducting the initial value (0M), and further statistical analysis is carried out with two-sample t-test.

[0029] The results indicate that after taking GMNL-263 for 6 months, the IL-1 in subjects' blood showed a significant decrease in statistical significance when compared with the placebo group, whereas no significant differences of the other cytokines were observed between the two groups (see Table 4), indicating that GMNL-263 was regulated by the decline of IL-1 to adjust the chronic inflammation reaction of the whole body, which in turn achieves the goal of lowering blood pressure.

TABLE-US-00004 TABLE 4 Changes of cytokines in the blood of the three groups of subjects after 6 months of trial placebo GMNL-263 GMNL-89 N = 22 N = 24 P-value N = 22 P-value IL-1 (pg/ml) 6 M 0 M 0.21 1.52 1.43 2.7 0.0181* 0.72 1.94 0.1027 IL-6 (ng/ml) 6 M 0 M 0.9 1.8 1.55 2.41 0.3189 0.95 2.65 0.9461 IL-10 (ng/ml) 6 M 0 M 1.04 2.41 2.05 3.25 0.2469 1.48 3.09 0.6109 TNF- (pg/ml) 6 M 0 M 3.07 72.22 12.81 88 0.5191 .sup.32 81.24 0.2317

Embodiment 3: Heat-Killed Dead GMNL-263 has the Effect of Lowering Blood Pressure by Enhancement of the Expression of Bifidobacterium

[0030] To clarify whether the effect of GMNL-263 on lowering blood pressure is related to the regulation of intestinal probiotics, the fecal samples were collected from the subjects at the beginning (0M) and the sixth month (6M) of the trial to analyze the DNA of the stool flora by using quantitative polymerase chain reaction (Q-PCR) and calculate the changes of the flora after taking the probiotics for 6 months. The calculation method is: (the CT [Threshold cycle] value of the fecal DNA after taking the probiotics for 6 months obtained by the Q-PCRthe CT value of the total bacteria)=Ct, Ct(the CT value of the fecal DNA collected from those who have not taken the probiotics obtained by the Q-PCRthe CT value of the total bacteria)=Ct, which is converted to 2.sup.Ct that is the change of the flora after taking the probiotics for 6 months.

[0031] The analysis of stool flora by the Q-PCR can be divided into two parts: DNA extraction and the Q-PCR analysis. First, the QIAamp DNA Stool Mini Kit (QIAGEN, Lot. 51504) was used for extraction of DNA from fecal samples, the RNA later was removed from the patient's stool before Buffer ASL was added and the sample was then placed on a heat plate for heating at 70 C. for 5 minutes; next, added to the 0.1 mm sterilized microbeads (Model No.: BioSpec Products 0.1 MM ZIRCONIA/SILICA BEADS, Cat. No. 11079101z) and vortexed strongly for at least 15 seconds until the stool was mixed and crushed homogenously; the sample was then subjected to centrifugation at 13,000 rpm for 1 minute, the supernatant was collected for DNA extraction and the concentration of the extracted DNA was adjusted to 1 ng/l for future use.

[0032] Next, the Q-PCR analysis of the stool was carried out. The above extracted DNA was used as a template for the Q-PCR and each reagent with 5 l was added to the 2 Rotor-Gene SYBR Green PCR Master Mix (QIAGEN, Cat. 204076), 2 l of the fecal DNA was added before addition of 3 l of the flora primers (0.66 M Forward (F)+Reverse primers (R)) to make up the total volume to 10 l and the Q-PCR is executed by the PCR machine (Model No. QIAGEN: Rotor-Gene Q 2Plex).

[0033] The data statistical analysis of the flora changes was carried out by using the two-sample t-test and the correlation analysis between the changes of the flora and blood pressure was carried out by using PASW Statistics 18 Software (SPSS Inc.) for conducting Spearman's rho correlation.

[0034] The results indicate that after the subjects taking the heat-killed dead GMNL-263 for 6 months, the expression of Bifidobacterium in the probiotics group of these subjects increased and showed a statistically significant difference as compared with the placebo group (see Table 5). In order to clarify the correlation between the changes of flora and blood pressure, by further utilization of correlation statistical analysis, it was found that the increasing amount of Bifidobacterium was negatively correlated with the net change of the mean blood pressure (R=0.268; P=0.028, see FIG. 2), indicating that blood pressure will be reduced when the amount of Bifidobacterium increases. Therefore, the result proves that heat-killed dead GMNL-263 can achieve the effect of lowering blood pressure by increasing the Bifidobacterium in the feces of the probiotics.

TABLE-US-00005 TABLE 5 Microbiological changes of the three groups of subjects after 6 months of trial Microbiota placebo GMNL-263 P-value GMNL-89 P-value Lactobacillu.sub.reuteri 6 M 0 M 1.53 1.77 6.23 11.25 0.0548 98.43 174.3 0.0165* Lactobacillus 6 M 0 M 2.51 3.66 14.53 52.73 0.2767 5.47 20.02 0.5025 Bifidobacterium 6 M 0 M 6.28 19.74 73.72 156.2 0.04868* 3441 15649 0.3149 Akkermansia muciniphila 6 M 0 M 25.84 59.38 .sup.170 618.3 0.3983 81.07 268.2 0.3559 Clostridium cluster I 6 M 0 M 20.43 81.26 5.57 10.21 0.4149 2.21 2.78 0.3167 Bacteroidetes 6 M 0 M 1.42 1.29 2.47 3.53 0.16 7.21 24.39 0.26 Firmicutes 6 M 0 M 1.57 2.71 3.02 3.35 0.12 1.62 1.73 0.94 Bacteroidetes/Firmicutes 6 M 0 M 99.37 338.03 87.98 213.26 0.9 22.28 168.75 0.15

[0035] The present invention has proved that after taking the heat-killed dead Lactobacillus reuteri GMNL-263 for 6 months can help the diabetic patients with hypertension symptoms reduce systolic blood pressure and mean blood pressure without affecting blood glucose values, which is beneficial to diabetic patients who need to control sugar or calorie intake. The L. reuteri GMNL-263 is subjected to heat sterilized treatment and can be prepared to products such as capsules or powder packs and has the advantages of high stability and convenience; in addition, because Lactobacillus is the probiotic bacteria and is considered as health food and thus safety is not a concern. Common people can use this product to prevent or control hypertension and there is no population limitation and thus the product can be used in many ways. The present invention further verified by correlation statistical analysis that heat-killed dead L. reuteri GMNL-263 can achieve the effect of lowering blood pressure simultaneously by reducing the proinflammatory cytokine IL-1 and regulating intestinal flora of the patients to increase Bifidobacterium of the probiotics. Furthermore, by using the same species of GMNL-89 as the control group, the results proved that not all L. reuteri can be used for lowering blood pressure, but only requires specific strains and should be confirmed by clinical trials. The L. reuteri GMNL-263 of the present invention is the first disclosure of using probiotics in the field of improving hypertension application, which breaks through the using forms and population limitation of the prior arts.