CN114350547A

CN114350547A - Bifidobacterium lactis strain B-622 and application thereof in preparation of medicines for treating diabetes

Info

Publication number: CN114350547A
Application number: CN202111550440.0A
Authority: CN
Inventors: 王轶; 王海莲
Original assignee: Sichuan Academy Of Medical Sciences Sichuan Provincial People's Hospital
Current assignee: Sichuan Min'an Zhichuang Technology Co ltd
Priority date: 2021-12-17
Filing date: 2021-12-17
Publication date: 2022-04-15
Anticipated expiration: 2041-12-17
Also published as: CN114350547B

Abstract

The invention discloses a bifidobacterium lactis strain B-622 and application thereof in preparing a medicament for treating diabetes. The preservation number of the strain B-622 is CCTCC NO.M: 20211355, deposited at the China center for type culture Collection on 11/2/2021. The strain obtained by screening plays a role in reducing blood sugar by promoting the secretion of glucagon-like peptide-1 (GLP-1), repairing the insulin secretion function, regulating blood fat metabolism, reducing chronic low-grade inflammation and inhibiting the activity of dipeptidyl peptidase-4, and can also inhibit a type 2 diabetes model mouse and stimulate the proliferation of islet beta cells of the type 2 diabetes model mouse.

Description

Bifidobacterium lactis strain B-622 and application thereof in preparation of medicines for treating diabetes

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a bifidobacterium lactis strain B-622 and application thereof in preparation of a medicament for treating diabetes.

Background

Diabetes (diabetes) is an endocrine metabolic disease associated with abnormal production and action of insulin, characterized primarily by hyperglycemia, and primarily includes type I and type II diabetes. The diabetes mellitus people in China mainly take II type diabetes mellitus, and account for more than 90 percent of the total number of diabetes mellitus patients. Type II diabetes, which is dominated by relative insulin deficiency and insulin resistance, eventually leads to hyperglycemia, and chronic hyperglycemia symptoms can cause a series of chronic complications, such as foot disease (foot ulcers, infection and gangrene), nephropathy (renal failure, uremia), eye disease (retinopathy, blurring, blindness), encephalopathy (cerebrovascular disease), heart disease, skin disease, venereal disease, and the like. Type II diabetes is a slowly progressive disease whose central link in onset is insulin resistance and a defect in islet beta cell function. At present, the number of people with type II diabetes mellitus in China is the first in the world, so that the development of prevention and treatment measures for type II diabetes mellitus is not slow. Many studies found that probiotics have a good biological effect on improving type II diabetes, but the mechanism of action is not clearly elucidated, and many strains of functional probiotics, leavening agents, and core technologies are monopolized abroad.

Incretins are a hormone secreted by small intestinal secretory cells that helps the body to produce a postprandial insulin response after eating carbohydrates, and their insulinotropic effect accounts for about 60% of the total amount of postprandial insulin secretion. To date, 2 incretins, glucose-dependent insulin release peptide (GIP) and Glucagon-like peptide-1 (GLP-1), have been isolated. Clinical researches show that GLP-1 and analogues thereof have important application values in the treatment process of type 2 diabetes, and GLP-1 has physiological functions of promoting insulin secretion, proliferating and inhibiting islet beta cells, inhibiting apoptosis, slowing down gastric emptying, inhibiting secretion of postprandial glucagon, reducing synthesis of glycogen, improving insulin sensitivity, controlling appetite and the like. Thus, GLP-1 is receiving increasing attention as a safe and effective insulinotropic secretion. However, GLP-1 is degraded by Dipeptidyl peptidase-4 (DPP-4) in vivo very rapidly, the half-sadness period is only 1-2min, and when the endogenous GLP-1 secretion amount is insufficient, the dose for activating the GLP-1 receptor cannot be accumulated, so that the application in clinic is difficult. The DPP-4 inhibitor inhibits the degradation of GLP-1 by DPP-4, so that endogenous GLP-1 is accumulated in vivo to achieve the effect of reducing blood sugar. The two medicines have the functions of protecting islet cells, reducing weight and the like, rarely cause hypoglycemia response, and are increasingly widely applied clinically. The most common adverse reactions of the two medicines are nausea, vomiting and other gastrointestinal reactions.

Probiotics (probiotic) is a micro-ecological preparation which has a promoting effect on the health of organisms, mainly comprises lactobacillus and bifidobacterium, and is a microorganism which is generally recognized as safe to eat. The probiotic has the effects of improving intestinal tract, enhancing immunity, resisting allergy, resisting oxidation, reducing cholesterol, lowering blood pressure, reducing weight, etc. In recent years, some lactic acid bacteria have been found to have hypoglycemic effects. The mechanism for reducing blood sugar mainly comprises: improving the barrier function of the intestinal mucosa; enhancing immunoregulatory activity; repairing the oxidative damage of the organism and improving the oxidation resistance; promoting insulin secretion and improving insulin sensitivity; inhibiting or delaying the absorption of glucose by intestinal tract, and promoting the utilization of glucose by peripheral tissues and target organs.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a bifidobacterium lactis strain B-622 and application thereof in preparing a medicament for treating diabetes, wherein the strain plays a role in reducing blood sugar by promoting secretion of glucagon-like peptide-1 (GLP-1), repairing insulin secretion function, regulating blood lipid metabolism, reducing chronic low-grade inflammation and inhibiting activity of dipeptidyl peptidase-4, and can also inhibit a type 2 diabetes model mouse and stimulate proliferation of islet beta cells of the type 2 diabetes model mouse.

In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:

a Bifidobacterium lactis (Bifidobacterium animalis subsp.Lactis) strain B-622, wherein the preservation number of the strain B-622 is CCTCC NO.M: 20211355, deposited at the China center for type culture Collection on 11/2/2021, address: wuhan university in Wuhan, China.

Further, the strain B-622, disrupted product or culture of the strain B-622 according to claim 1 is contained as an active ingredient.

Further, the probiotic preparation for reducing blood sugar is powder, tablets or capsules.

The hypoglycemic probiotic preparation is applied to the preparation of medicaments for preventing or assisting in reducing blood sugar.

The application of the strain B-622 in preparing hypoglycemic drugs.

The application of the strain B-622 in preparing a medicament for treating diabetes.

The application of the probiotic preparation for reducing blood sugar in the preparation of medicaments for treating diabetes is provided.

Further, the strain B-622 or the hypoglycemic probiotic preparation can inhibit apoptosis of islet beta cells and promote proliferation of the islet beta cells.

Further, diabetes is type ii diabetes.

The invention has the beneficial effects that:

the invention takes lactobacillus which is separated and identified from a 2-year-old healthy excrement sample which is breast-fed in the west-Tibet region and is not contacted with antibiotics as a research object, and obtains a new lactobacillus strain named as bifidobacterium lactis B-622 through a large number of experimental screens. The invention adopts high-fat feed and streptozotocin to establish a diabetes model, and the bifidobacterium lactis B-622 is continuously administered to a mouse by intragastric administration for 28 days to discuss the treatment effect of the bifidobacterium lactis B-622 on the diabetes model mouse. Research results show that the bifidobacterium lactis B-622 plays a role in reducing blood sugar by promoting the secretion of glucagon-like peptide-1 (GLP-1), repairing the secretion function of insulin, regulating blood fat metabolism, reducing chronic low-grade inflammation and inhibiting the activity of dipeptidyl peptidase-4.

The bifidobacterium lactis B-622 strain has the effects of preventing and treating diabetes, so that the bifidobacterium lactis B-622 strain can be used for preparing foods, health-care products or medicines and the like with the function of reducing blood sugar, and meanwhile, the bifidobacterium lactis B-622 strain provided by the invention can also be used as functional probiotics to be applied to preparing foods, health-care products or medicines, so that the bifidobacterium lactis B-622 strain is safe, free of toxic and side effects and wide in application prospect. The screening of the lactobacillus with the blood sugar reducing effect is developed, so that the audience population is wide, and the market space is huge.

Drawings

FIG. 1 shows the effect of Bifidobacterium lactis B-622 on blood glucose in type 2 diabetic mice;

FIG. 2 shows that Bifidobacterium lactis B-622 inhibits apoptosis of islet beta cells in type 2 diabetes model mice;

FIG. 3 shows that Bifidobacterium lactis B-622 stimulates the proliferation of islet beta cells in type 2 diabetes model mice.

Detailed Description

The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.

Example 1 isolation screening and characterization of Bifidobacterium lactis B-622

1. Collection of samples

A 2 year old healthy stool sample from the tibetan region that was breast-fed and not exposed to antibiotics was collected.

2. Strain isolation

Placing 1g of sample into 9mL of physiological saline, shaking and uniformly mixing, carrying out gradient dilution on the bacterial suspension, sucking 100 mu L of appropriate gradient bacterial liquid on a bifidobacterium isolation medium agar plate added with mupirocin lithium salt, uniformly coating, replacing air with anaerobic air, and then standing at 37 ℃ for constant-temperature culture for 48 hours. And (3) selecting a single bacterial colony which is opaque, milky white, round, glossy, neat in edge, convex in surface and wet when the bacterial colony grows out, performing repeated streak purification culture on an MRS solid culture medium to obtain 1 bacterial strain of the infant fecal bacteria, and storing the bacterial strain at the temperature of 4 ℃ for later use. Bifidobacterium isolation medium (g/L): 10 parts of beef extract, 10 parts of peptone, 5 parts of yeast extract, 5 parts of glucose, 5 parts of sodium acetate, 2 parts of diammonium hydrogen citrate, 801 parts of Tween and K parts of₂HPO₄ 2、MgSO₄·7H₂ O 0.58、 MnSO₄·H₂O 0.25、CaCO₃Sterilizing at 20 and 121 deg.C for 20min, adding 1.5% agar powder into MRS solid culture medium, the agar has stable chemical structure, and only acts as coagulant in the culture medium, and melts at 100 deg.CThe mixture is formed into liquid and cooled to below 45 ℃ to be solidified again. The bacterial colony of the strain is opaque and milky white, round and glossy, neat in edge, convex and moist in surface. The strain is sensitive to oxygen and is strictly anaerobic, and the air is replaced by anaerobic air during culture. The optimal growth temperature of the strain is 37 ℃, and the optimal initial pH value is 6.8.

3. Molecular biological identification of bifidobacterium lactis B-622

And (3) purifying and culturing the primary screened thalli, collecting the primary screened thalli, extracting the strain genome DNA by adopting a genome DNA extraction kit (TIANGEN company), and performing the steps of: AGTTTGATCMTGGCTCAG (SEQ ID NO.1) and a downstream primer 1492R: GGTTACCTTGTTACGACTT (SEQ ID NO.2), and the 16S rDNA sequence is amplified to obtain a PCR product. And sequencing the PCR product. Wherein the PCR reaction system comprises: 2 mu L of each of the upstream and downstream primers, 2 mu L of the DNA template, 2 mu L of ddH2O 21 mu L, of Super PfX MasterMix 25 mu L (CW2965M, kang century, China). And (3) PCR reaction conditions: 10min at 95 ℃; 30s at 94 ℃, 30s at 55 ℃, 2min at 72 ℃ and 35 cycles; 10min at 72 ℃. The PCR product was detected by gel electrophoresis and sent to Beijing optimak company for sequencing. The determined gene sequences were submitted to NCBI database (www.ncbi.nlm.nih.gov) for BLAST analysis and alignment, using the 16S rDNA gene sequence homology of 99% or more as the identification standard.

Example 2 evaluation of tolerance of Bifidobacterium lactis B-622 to simulated artificial gastric and intestinal juices

Inoculating 100 μ L of test bacterial liquid into nitrogen-filled anaerobic glass tube filled with 7mL of MRS liquid culture medium, standing at 37 deg.C for culture and activation for 2 times. And centrifuging 200 mu L of activated bifidobacterium lactis B-622 bacterial liquid, collecting thalli, respectively adding 1mL of simulated artificial gastric juice with the pH value of 2.5 and simulated artificial intestinal juice with the pH value of 8.0, fully mixing, digesting for 3.0h at 37 ℃, sampling, detecting the number of viable bacteria, and calculating the survival rate, wherein the results are shown in table 1. Wherein the artificial gastric juice and the artificial intestinal juice are prepared immediately and stored at 4 ℃ for standby in a short time. The survival rate is the viable count of the activated bacteria liquid/the viable count after digestion is multiplied by 100 percent.

The formula of the artificial gastric juice comprises: preparing 0.85% physiological saline, adjusting pH to 2.5 with dilute hydrochloric acid, adding 0.3% pepsin, dissolving completely, and filtering with 0.22 μm microporous membrane for sterilization.

The formula of the artificial intestinal juice comprises: preparing 0.85% physiological saline, adjusting pH to 8.0 with sodium hydroxide, adding 0.1% trypsin, dissolving completely, and filtering with 0.22 μm microporous membrane for sterilization.

TABLE 1 Experimental data table for Bifidobacterium lactis B-622 simulation digestive tract environment

The results in Table 1 show that the survival rate of the Bifidobacterium lactis B-622 in both simulated artificial gastric juice and artificial intestinal juice is high, which indicates that the Bifidobacterium lactis B-622 can effectively resist the influence of the gastrointestinal tract and still has high activity after passing through the gastrointestinal tract.

Example 3 evaluation of bacteriostatic Activity of Bifidobacterium lactis B-622

And inoculating 100 mu L of test bacterial liquid into a nitrogen-filled anaerobic glass tube filled with 7mL of MRS liquid culture medium, and standing and culturing at the constant temperature of 37 ℃ for activation. Respectively inoculating 100 mu L of pathogenic indicator strains of escherichia coli, staphylococcus aureus and salmonella typhi into 10mL of liquid LB culture medium, and culturing and activating by a constant temperature shaker at 37 ℃ at 100 rpm. Diluting the activated pathogenic bacteria liquid to bacterial suspension with the concentration of 108CFU/mL, respectively adding 300 mu L of each pathogenic bacteria suspension into 30mL of MRS solid culture medium with the appropriate temperature (about 50 ℃), uniformly mixing (indicating the viable count of the bacteria is 106CFU/mL), pouring into a flat plate with 4 sterile oxford cups placed in the flat plate in advance, taking out the oxford cups by using tweezers after the culture medium is solidified, adding 160 mu L of test bacteria liquid (the viable count is 108CFU/mL) into holes left after the oxford cups are pulled out, and performing 3 parallels and 1 control, wherein the control is replaced by the MRS liquid culture medium. The plate was placed upright after being covered with a light lid, and after replacing the air with oxygen-free air, the plate was cultured in a constant temperature incubator at 37 ℃ for an appropriate time to observe, and the diameter of the zone of inhibition was measured with a vernier caliper, and the results are shown in Table 2. LB culture medium: 10g of tryptone, 5g of yeast extract powder, 10g of sodium chloride and 1000ml of distilled water, wherein the pH value is 7.0-7.2, and the sterilization is carried out at 121 ℃ for 15 min.

TABLE 2 antagonistic activity of Bifidobacterium lactis B-622 against pathogenic bacteria data Table

The results in Table 2 show that Bifidobacterium lactis B-622 has strong inhibitory effect on common enteric pathogenic bacteria Escherichia coli, Staphylococcus aureus and Salmonella typhi.

Example 4 evaluation of antibiotic susceptibility of Bifidobacterium lactis B-622

After test bacteria are streaked and activated on an MRS solid plate, bacterial mud is picked and placed in physiological saline to prepare bacterial suspension, the concentration of the bacterial suspension is adjusted to be 108CFU/mL, 100 mu L of the bacterial suspension is added to the MRS solid plate poured in advance, the bacterial suspension is uniformly coated on the plate by using an aseptic cotton swab, an antibiotic drug sensitive sheet is attached, air is replaced by anaerobic air, then the plate is cultured at the positive position of 37 ℃, the diameter of the sensitivity of the bacterial strain to the antibiotic is measured by using a vernier caliper after 24h, and the result is shown in Table 3.

TABLE 3 data table of Bifidobacterium lactis B-622 sensitivity to antibiotics

The results in table 3 show that bifidobacterium lactis B-622 is not resistant to 17 common antibiotics, demonstrating that bifidobacterium lactis is safe in use.

Example 5 establishment and experiment of animal models

One, packet processing

100 male C57BL/6J mice, randomly divided into 4 groups of 20 mice each, were treated as follows:

1. blank control group: feeding the test by adopting common feed from the beginning to the end of the test; at the 9 th week, the mice are injected with 30mg/kg of physiological saline at one time in the abdominal cavity after fasting for 12 hours; administration was performed from week 10 to week 13 at the following doses: 12mL of sterile PBS buffer was administered per kilogram of body weight.

2. Model group: feeding with high fat feed in 1-8 weeks, and feeding with common feed in 9-9 weeks; at 9 weeks, 30mg/kg of streptozotocin is injected into the abdominal cavity once after the mice fasting for 12 hours; administration is performed from week 10 to week 13; the administration dose is as follows: 12mL of sterile PBS buffer was administered per kilogram of body weight.

3. Positive control group: feeding with high fat feed in 1-8 weeks, and feeding with common feed in 9-9 weeks; at 9 weeks, 30mg/kg of streptozotocin is injected into the abdominal cavity once after the mice fasting for 12 hours; administration is performed from week 10 to week 13; the administration dose is as follows: a0.1 mg/ml solution of metformin in PBS was administered at 12ml/kg body weight once a day.

Group B-622: feeding with high fat feed in 1-8 weeks, and feeding with common feed in 9-9 weeks; at 9 weeks, 30mg/kg of streptozotocin is injected into the abdominal cavity once after the mice fasting for 12 hours; administration is performed from week 10 to week 13; the administration dose is as follows: 12mL of the bacterial suspension was administered per kg of body weight. The preparation method of the bacterial liquid comprises the following steps: suspending Bifidobacterium lactis B-622 with PBS buffer to obtain Bifidobacterium lactis B-622 with concentration of 1.0 × 10⁹CFU/mL of bacterial liquid.

Week 1 to week 8, i.e., days 1 to 56 of the test, week 10 to week 13, i.e., days 64 to 91 of the test, and so on. Intraperitoneal injection of streptozotocin or saline was performed on day 57 of the experiment.

Second, judging and establishing diabetes model standard

At week 9 (day 63 of the experiment), the mice were bled for Fasting Blood Glucose (FBG). The mice meeting the following criteria are type 2 diabetes model mice: FBG is measured for two times continuously and is more than or equal to 11.1 mmol/L. The results show that on the 57 th day of the experiment, the mice of the model group, the positive control group and the B-622 group are type 2 diabetes model mice.

Third, blood sugar detection

The fasting blood glucose level of the mice was measured by tail vein blood sampling at week 10, week 11, week 12, week 13 and week 14, respectively (the average value in the week was calculated by daily measurement). Test day 99 a glucose tolerance test was performed.

The fasting blood glucose results are shown in FIG. 1. Compared with the model group, the fasting blood glucose value of the mice in the B-622 group is in a descending trend. The fasting blood glucose values of the mice in the B-622 group are obviously reduced at 12 weeks, 13 weeks and 14 weeks, and the blood glucose value at the 14 week is reduced to 12.35mmol/L, which are very obviously different from those of the mice in the model group (P is less than 0.01). The result shows that the bifidobacterium lactis B-622 has a treatment effect on hyperglycemia of type 2 diabetes and can obviously reduce the fasting blood glucose value.

Detection of blood lipid, insulin and inflammatory cytokine

At the end of the test (day 100), each group of mice was collected, the eyeballs were removed, blood was collected, the mixture was coagulated at room temperature for 60min and centrifuged at 3000r/min for 15min, and the supernatant (serum) was collected. Detecting the content of total cholesterol, triglyceride, high density lipoprotein cholesterol, low density lipoprotein cholesterol, insulin, endotoxin, anti-tumor necrosis factor-alpha and interleukin-6 in serum. The results of the measurement of serum lipid-related factors are shown in Table 1. Compared with the model group, the serum TC and TG contents of the mice in the B-622 group are reduced. The result shows that the bifidobacterium lactis B-622 can obviously improve the hyperlipemia symptom of type 2 diabetes.

TABLE 1 Effect of Bifidobacterium lactis B-622 on blood lipids in type 2 diabetic mice

Note: significant differences (P < 0.05) and very significant differences (P < 0.01) were compared to the model group.

TABLE 2 Effect of Bifidobacterium lactis B-622 on ET, TNF- α, IL-6, INS, GLP-1, DPP-4 in serum of type 2 diabetes model mice

As shown in Table 2, the INS content in the serum of mice in each administration group was increased to different extents during the treatment period, and the INS content in the serum of mice in the positive control group and B-622 group was significantly increased (P < 0.01) and the ET level in the serum of mice in the B-622 group was significantly decreased (P < 0.01) as compared with the model group. B-622 can obviously reduce the serum of type 2 diabetic mice, reduce the contents of TNF-alpha and IL-6 and effectively relieve chronic inflammation of organisms. GLP-1 secretion of the B-622 group is obviously increased, and DPP-IV enzyme activity is obviously inhibited.

Fifth, the effect of Bifidobacterium lactis B-622 on islet beta cells of type 2 diabetes model mice

In the experiment, all mice are killed by removing cervical vertebrae after blood is taken on day 100, pancreas samples are taken and fixed by 4% formalin fixing solution, then wax blocks are made by dehydration and transparency, the thickness of the slices is 5um, and subsequent staining is carried out. Verification that bifidobacterium lactis B-622 inhibits apoptosis of islet beta cells of type 2 diabetes model mice and stimulates proliferation of islet beta cells of type 2 diabetes model mice

1. Bifidobacterium lactis B-622 for inhibiting apoptosis of islet beta cells of type 2 diabetes model mouse

Apoptosis test TUNEL apoptosis kit (cat 11684817910, Roche) was stained to show green fluorescence according to the kit's instructions, the primary antibody was polyclonal Guinea pig anti-insulin (cat A0564, DAKO), the secondary antibody was goat anti-guinea pig Texas Red stained to show Red fluorescence, the green fluorescence was apoptotic cells (white arrow mark), the Red fluorescence was islet beta cells (dotted line delineation region), group B-622 was clearly seen in FIG. 2, and the staining results of the positive control group showed that the expression of green fluorescence was significantly lower than that of the model group. Thus, B-622 can inhibit the apoptosis of islet beta cells.

2. Bifidobacterium lactis B-622 for stimulating proliferation of islet beta cells of type 2 diabetes model mouse

Experiment of stimulating proliferation of beta cells of pancreatic islets of mice model of type 2 diabetes by bifidobacterium lactis B-622 staining with double staining of insulin and PCNA, and labeling of beta cells of pancreatic islets: the primary antibody was polyclonal Guinea pig anti-insulin (cat # A0564, DAKO) and the secondary antibody was goat anti-guinea pig Texas Red stained with Red fluorescence (solid line delineation). Labeling of proliferating cells: the primary antibody is mouse monoclonal antibody [ PC10] to PCNA (cat: ab29, abcam), and the secondary antibody is goat anti-mouse FITC stained in green fluorescence (gray arrow mark). In FIG. 3, it can be seen that the green fluorescence expression of the staining results of the B-622 group and the positive control group is significantly higher than that of the model group, indicating that B-622 can stimulate the proliferation of islet beta cells.

Claims

1. A Bifidobacterium lactis (Bifidobacterium animalis subsp.Lactis) strain B-622 is characterized in that the preservation number of the strain B-622 is CCTCC NO.M: 20211355, deposited at the China center for type culture Collection on 11/2/2021.

2. A hypoglycemic probiotic preparation, comprising the strain B-622, disrupted product or culture of the strain B-622 according to claim 1 as an active ingredient.

3. The hypoglycemic probiotic formulation according to claim 2, wherein the hypoglycemic probiotic formulation is a powder, tablet or capsule.

4. Use of a probiotic formulation for lowering blood glucose according to claim 2 or 3, for the preparation of a medicament for the prophylactic or adjunctive lowering of blood glucose.

5. The use of the strain B-622 according to claim 1 for the preparation of a hypoglycemic medicament.

6. The use of the strain B-622 according to claim 1 for the preparation of a medicament for the treatment of diabetes.

7. Use of a probiotic formulation for lowering blood glucose according to any of claims 2 or 3 for the preparation of a medicament for the treatment of diabetes.

8. The use according to claim 6 or 7, wherein the strain B-622 or the hypoglycemic probiotic preparation inhibits apoptosis of islet beta cells and promotes proliferation of islet beta cells.

9. The use according to claim 6 or 7, wherein the diabetes is type II diabetes.