Patent Application
20180353553
The present invention in general relates to the therapeutic/biological effects of probiotic supplements. More specifically, the present invention relates to the hypolipidemic effect of Bacillus coagulans. Still more specifically, the present invention pertains to the cholesterol assimilation potential of Bacillus coagulans MTCC 5856 and therapeutic/biological indications thereof.
The hypolipidemic effects of probiotic supplementation have been previously documented in the art as follows.
United States patent documents U.S. Pat. No. 7,232,571, U.S. Pat. No. 8,349,337, U.S. Pat. No. 6,811,786 also disclose the cholesterol reducing property of Bacillus coagulans (Hammer strain) in combination with other cholesterol reducing agents and oligosaccharides. However, scientific literature also documents the inefficacy or poor efficacy of probiotic supplementation to exert hypocholesterolemic effects. Important ones include,
While in general, the major mechanisms for hypolipidemic effects of probiotics include reduction of cholesterol absorption in the gut, enzymatic deconjugation of bile salts, incorporation of cholesterol into probiotic cell membranes, and conversion of cholesterol to coprostanol [Lye H S, Kuan C Y, Ewe J A, Fung W Y, Liong M T, “The improvement of hypertension by probiotics: effects on cholesterol, diabetes, renin and phytoestrogens. Int J Dairy Sci. 2009:10(9):3755-75; and Ooi L G, Liong M T, “Cholesterol-lowering effects of probiotics and prebiotics: a review of in vivo and in vitro findings”, Int J Mol Sci. 2010; 11(6):2499-522], the inconsistent findings of hypocholesterolemic effects of probiotic microorganisms is a technical problem in the art. The technical solution to this problem lies in identifying a standalone strain-specific probiotic with lipid profile benefits much in line with the teachings that therapeutic benefits of probiotics is strain-specific and cannot be generalised. See, Indian Council of Medical Research-Department of Biotechnology (ICMR-DBT), “Guidelines for the evaluation of probiotics in food (2011), see section 2.3; and Joint FAO/WHO Working Group Report on Drafting Guidelines for the Evaluation of Probiotics in Food London, Ontario, Canada, April 30 and May 1, 2002, emphasising under Section 3.1 that “The current state of evidence suggests that probiotic effects are strain specific. Strain identity is important to link a strain to a specific health effect as well as to enable accurate surveillance and epidemiological studies”.
It is thus the principle objective of the present invention to disclose the novel and non-obvious hypolipidemic effects of Bacillus coagulans.
It is yet another principle objective of the present invention to disclose the novel and non-obvious cholesterol assimilation effect of Bacillus coagulans MTCC 5856.
The present invention fulfils the aforesaid objectives and provides further related advantages.
The present invention discloses the hypocholesterolemic potential of Bacillus coagulans MTCC 5856 and therapeutic/biological indications thereof.
The advantages of the present invention include the identification of probiotic strain specific lipid profile benefits, in specific the novel and non-obvious hypocholesterolemic [cholesterol assimilation] effect of Bacillus coagulans MTCC 5856.
Other features and advantages of the present invention will become apparent from the following more detailed description, taken in conjunction with the accompanying images, which illustrate, by way of example, the principle of the invention.
In the most preferred embodiment, the present invention relates to a method of cholesterol assimilation by probiotic bacteria Bacillus coagulans, said method comprising step of administering effective dose regimens of compositions containing said probiotic bacteria to mammals to bring about the effect of cholesterol reduction in the mammals. In a related embodiment, the Bacillus coagulans strain is Bacillus coagulans MTCC 5856. In another preferred embodiment, the Bacillus coagulans administered is a vegetative cell or spore. In yet another related embodiment, the spores include viable or heat killed or dead spores of Bacillus coagulans. In yet another related embodiment, the vegetative cells include viable or heat killed or dead cells of Bacillus coagulans. In another related embodiment, the Bacillus coagulans is administered orally in the form of tablets, capsules, syrups, gummies, powders, suspensions, emulsions, chewables, candies and eatables. In another related embodiment, the effective dose of Bacillus coagulans is about 1×106 to 1×1014 colony forming units (CFU). In another related embodiment, the effective dose of Bacillus coagulans is preferably 2×109 cfu. In another related embodiment, the mammal is preferably human.
In another preferred embodiment, the present invention relates to a method of BSH production and bile salts deconjugation by using probiotic bacteria Bacillus coagulans, said method comprising step of administering effective dose regimens of compositions containing said probiotic bacteria to said mammals to bring about the desired effect of BSH production and bile salt deconjugation. In a related embodiment, production of BSH and deconjugating bile salts by the probiotic bacteria Bacillus coagulans results in effective cholesterol reduction in mammals. In a related embodiment, the Bacillus coagulans strain is Bacillus coagulans MTCC 5856. In another preferred embodiment, the Bacillus coagulans administered is a vegetative cell or spore. In yet another related embodiment, the spores include viable or heat killed or dead spores of Bacillus coagulans. In yet another related embodiment, the vegetative cells include viable or heat killed or dead cells of Bacillus coagulans. In another related embodiment, the Bacillus coagulans is administered orally in the form of tablets, capsules, syrups, gummies, powders, suspensions, emulsions, chewables, candies and eatables. In another related embodiment, the effective dose of Bacillus coagulans is about 1×106 to 1×1014 cfu. In another related embodiment, the effective dose of Bacillus coagulans is preferably 2×109 cfu. In another related embodiment, the mammal is preferably human.
In yet another most preferred embodiment, the present invention relates to a method of preventing or treating hypercholesterolemia in mammals using probiotic bacteria Bacillus coagulans, said method comprising step of administering to said mammals effective dose regimens of compositions containing said probiotic bacteria to bring about the effect of lowering levels of circulating cholesterol in the blood of said mammals. In a related embodiment, the Bacillus coagulans strain is Bacillus coagulans MTCC 5856. In another preferred embodiment, the Bacillus coagulans administered is a vegetative cell or spore. In yet another related embodiment, the spores include viable or heat killed or dead spores of Bacillus coagulans. In yet another related embodiment, the vegetative cells include viable or heat killed or dead cells of Bacillus coagulans. In another related embodiment, the Bacillus coagulans is administered orally in the form of tablets, capsules, syrups, gummies, powders, suspensions, emulsions, chewables, candies and eatables. In another related embodiment, the effective dose of Bacillus coagulans is about 1×106 to 1×1014 cfu. In another related embodiment, the effective dose of Bacillus coagulans is preferably 2×109 cfu. In another related embodiment, the mammal is preferably human.
In yet another most preferred embodiment, the present invention relates to a method of therapeutic management of dyslipidemia in mammals using probiotic bacteria Bacillus coagulans, said method comprising step of administering to said mammals effective dose regimens of compositions containing said probiotic bacteria to bring about the effect of lowering levels of circulating cholesterol in the blood, decreasing concentration of Low density lipoprotein (LDL), Low density lipoprotein (LDL)-cholesterol and lowering levels of circulating triglycerides in blood. In a related embodiment, the Bacillus coagulans strain is Bacillus coagulans MTCC 5856. In another preferred embodiment, the Bacillus coagulans administered is a vegetative cell or spore. In yet another related embodiment, the spores include viable or heat killed or dead spores of Bacillus coagulans. In yet another related embodiment, the vegetative cells include viable or heat killed or dead cells of Bacillus coagulans. In another related embodiment, the Bacillus coagulans is administered orally in the form of tablets, capsules, syrups, gummies, powders, suspensions, emulsions, chewables, candies and eatables. In another related embodiment, the effective dose of Bacillus coagulans is about 1×106 to 1×1014 cfu. In another related embodiment, the effective dose of Bacillus coagulans is preferably 2×109 cfu. In another related embodiment, the mammal is preferably human.
In yet another most preferred embodiment, therapeutic management of dyslipidemia is effected before the onset and for preventing disease conditions selected from the group consisting of high cholesterol induced atherosclerosis, coronary artery disease, hepatic steatosis and associated non alcoholic fatty liver disease, diabetes mellitus and obesity.
In another preferred embodiment, the invention discloses a method of reducing the cholesterol content in food stuff by probiotic bacteria Bacillus coagulans, said method comprising step of bringing into contact the prebiotic bacteria, together with bile salts and bile acids, with foods rich in cholesterol, to bring about the reduction and assimilation of cholesterol from the food stuff by the probiotic bacteria. In a related embodiment, the Bacillus coagulans strain is Bacillus coagulans MTCC 5856. In another related embodiment, foods rich in cholesterol is selected from the group consisting of, but not limited to, chicken liver, egg yolk, fish and fish oils, fast foods, animal fats and oil, seafood, processed meat, red meat, cheese, milk fat, butter and confectioneries.
The following illustrative examples are included to understand the technical features and advantages of the present invention.
Methodology
Bile-Salt Hydrolase (BSH) Activity
Bacterial Growth and Cholesterol Assimilation
Growth of Bacillus coagulans MTCC 5856 in the presence of cholesterol was assessed at different time intervals till 24 h. Bacillus coagulans MTCC 5856 was inoculated in the sterilized MRS broth media supplemented with 0.30% ox-bile and 100 μg/ml water soluble cholesterol (polyoxyethanylcholesteryl sebacate). This was then incubated at 37° C. with gentle shaking (100 rpm) for 24 h. Samples were taken at different time intervals (0, 6, 12, 18 and 24 h) and the bacterial growth was measured at 600 nm using UV-spectrophotometer (Shimadzu UV-1601PC). Cholesterol assimilation determined according to the method described by the Rudel and Morris (1973) with some modifications proposed by Liong and Shah (2005). After the incubation, cells were centrifuged (8,000 rpm, at 4° C. for 10 min) and the remaining cholesterol concentration in the supernatant was determined using spectrophotometer (Shimadzu UV-1601PC) method. One ml of the aliquot was added with 1 ml of KOH (33% w/v) and 2 ml of absolute ethanol, vortexed for 1 min, followed by heating at 37° C. for 15 min. After cooling, 2 ml of distilled water and 3 ml of hexane were added and vortexed for 1 min. One ml of the hexane layer was transferred into a glass tube and evaporated. The residue was immediately dissolved in 2 ml of o-phthalaldehyde reagent. After complete mixing, 0.5 ml of concentrated sulfuric acid was added and the mixture was vortexed for 1 min. Absorbance was read at 550 nm after 10 min as described by Rudel L L, Morris M D. Determination of cholesterol using o-phthalaldehyde. J Lipid Re. 1973; 14 (3):364-6. All experiments were performed in triplicate and repeated twice. The cholesterol concentration was read off a standard curve prepared using the cholesterol stock solution (
Cholesterol assimilation/removal (%)=100−[(residual cholesterol after fermentation/Initial cholesterol added)×100].
Cholesterol Removal/Assimilation by Growing Cells, Resting Cells and Heat Killed (Dead) Cells of Bacillus coagulans MTCC 5856
The ability to assimilate/remove the cholesterol by the non-growing cells such as resting cells and heat killed (dead) cells of Bacillus coagulans MTCC 5856 was determined and compared with growing cells. Resting cells were prepared by growing Bacillus coagulans MTCC 5856 in MRS media overnight followed by centrifugation (8,000 rpm at 4° C. for 10 minutes) to obtained cell mass. This cell mass was further suspended into sterile phosphate buffer (0.05 M, pH 6.5) containing 0.3% (w/v) oxbile and 100 μg/ml of water-soluble cholesterol and then incubated for 24 h at 37° C. Heat killed (dead) cells were prepared by growing Bacillus coagulans MTCC 5856 in MRS media overnight followed autoclaving at 121° C. for 15 min. After autoclave, cells were collected by centrifugation (8,000 rpm at 4° C. for 10 minutes) and suspended aseptically in MRS broth supplemented with 0.3% (w/v) oxbile acid and 100 μg/ml of water-soluble cholesterol and then incubated for 24 h at 37° C. Growing cells were prepared by growing Bacillus coagulans MTCC 5856 in MRS media overnight and then 1 ml of this was added to the MRS broth supplemented with 0.3% (w/v) oxbile acid and 100 gig/ml of water-soluble cholesterol followed by incubation for 24 h at 37° C. After incubation, all the samples were centrifuged (8,000 rpm at 4° C. for 20 minutes) and cholesterol concentrations in the supernatants were measured following the Colorimetric method using Spectrophotometer as described above. All experiments were performed in triplicate and assayed twice.
Assimilation of Cholesterol from the Different Food Source by the Bacillus coagulans MTCC 5856 Under Gastric Stress
The effect of cholesterol assimilation by the Bacillus coagulans MTCC 5856 on the cholesterol rich food source was investigated by the method described earlier by de Palencia P F, Lopez P, Corbi A L, Peliez C, Requena T. Probiotic strains: survival under simulated gastrointestinal conditions, in vitro adhesion to Caco-2 cells and effect on cytokine secretion. Eur Food Res Technol 2008; 227: 1475-1484 and by Ritter P, Kohler C, von Ah U. Evaluation of the passage of Lactobacillus gasseri K7 and Bifidobacteria from the stomach to intestine using a single reactor model. BMC Microbiol 2009; 9: 87-95 with some modifications. A known weight of Egg yolk, butter (cow milk fat) and cleaned and processed chicken liver was taken in sterile beaker. Chicken liver was washed with the water and soaked into the 70% ethanol for 30 min and rinsed with sterile distilled water and then homogenized by adding into 0.2 M phosphate buffer pH 7.0. To mimic the in-vivo conditions, all samples were added to the 10 ml of a sterile electrolyte solution (6.2 g/L NaCl, 2.2 g/L KCl, 0.22 g/L CaCl2 and 1.2 g/L NaHCO3) and lysozyme (0.01%, Sigma-Aldrich) and pepsin (0.3%, Sigma-Aldrich). The pH of the samples was decreased to 1.5 by adding 1.0 mol/L HCl. Bacillus coagulans MTCC 5856 was added to each group and incubated at 37° C. with gentle shaking (100 rpm) for 3 h. After the incubation, pH of each sample was adjusted aseptically to 7.0 using sterile sodium bicarbonate saturated solution (8%, w/v). Oxygen reducing enzyme Oxyrase (Oxyrase® for Broth, Oxyrase Inc, Ohio, USA) was added to each flask to induce anaerobic environment. Sterile bile salt (0.3%, w/v, Sigma Aldrich) and pancreatin from porcine (0.1%, w/v, Sigma Aldrich) were also added to the each group. Sterile electrolyte solution containing 0.45% bile salts and 0.1% pancreatin (final concentration, both from Sigma Aldrich) were added to all the samples. All the samples were incubated at 37° C. with gentle shaking (100 rpm) for 24 h. After incubation, samples were centrifuged (8,000 rpm at 4° C. for 10 minutes) and cholesterol content was estimated in the supernatant by the method described above.
Results:
Bile Salt Hydrolase Activity of Bacillus coagulans MTCC 5856
Bacillus coagulans MTCC 5856 growth was observed on the agar plate containing ox bile and calcium carbonate which indicated its tolerance against ox bile and presence of bile salt hydrolase activity. There was 18 mm clear zone in the soft agar plate (
Assimilation/Removal of Cholesterol by the Bacillus coagulans MTCC 5856
There was a time dependant assimilation/removal of cholesterol by the Bacillus coagulans MTCC 5856 (
Assimilation/Removal of Cholesterol by the Non-Growing Cells of Bacillus coagulans MTCC 5856
The ability of Bacillus coagulans MTCC 5856, either growing or non-growing (resting or heat killed dead cells) to remove cholesterol was investigated. The removal of cholesterol varied remarkably in growing cells (48.42%) compared to resting and dead cells 36.46% and 8.5% in 24 h respectively (
Assimilation/Removal of Cholesterol by Bacillus coagulans MTCC 5856 in Cholesterol Rich Foods
The effect of Bacillus coagulans MTCC 5856 to assimilate/remove the cholesterol in cholesterol rich food source such as egg yolk, chicken liver and butter under gastric stress (acid and bile acids) was investigated with the aim to understand the in-vivo efficacy of Bacillus coagulans MTCC 5856. The significant reduction/assimilation of cholesterol was observed in all cholesterol rich food sources after 24 h of Bacillus coagulans MTCC 5856 fermentation (
Ethics
Institutional Ethics Committee
All pertinent study documents were reviewed by the Institutional Ethics Committee (IEC), Life Care Hospital, Bangalore prior to study initiation and gave a favourable opinion. This IEC functions independently of Sami Labs Limited or ClinWorld Private Limited (CRO) and the operations are in compliance with Part 56 of Title 21 of the Code of Federal Regulations (CFR) and International Conference on Harmonization (ICH) guidelines. The aforementioned Ethics Committee was registered under CDSCO as per the Gazette Notification Number F.28-10/45-H(1), dated 21 Dec. 1945 and last amended vide notification number G.S.R. 76(E) dated 8 Feb. 2012.
Ethical Conduct of the Study
This research was conducted in accordance with the clinical research guidelines established by the Drugs and Cosmetics Act, 1940 of India. Drugs and Cosmetics Rules, 1945 of India, Ethical Guidelines for Biomedical Research on Human Participants, 2006 of Indian Council of Medical Research (ICMR) in India, the principles enunciated in the Declaration of Helsinki (Edinburgh, 2000) and the ICH—harmonized tripartite guideline regarding Good Clinical Practice (GCP).
Subject Information and Consent
Written and oral information about the study in a language easily understandable by the study volunteers was provided. Each volunteer was informed by the investigator, prior to the screening, of the purpose of this clinical trial, including possible risks and benefits and documented the informed consent process in the patient's file. Prior to entry into the study or initiation of any study related procedures, the patient read, signed and dated the IEC approved informed consent form. Sufficient time was provided for each subject to decide whether to participate in the study and all the questions and clarifications regarding the study were clarified by the investigator to the patient's satisfaction. The person executing the consent also signed and dated the final consent form page. The subject's willingness to participate in the study was documented in source notes by the Investigators. The original signed informed consent form has been summarized, in non-technical terms, the purpose of the study, the procedures to be carried out, and the potential hazards, if any.
Study Rationale
The current clinical study was planned to assess the efficacy and safety of Bacillus coagulans MTCC 5856 (2×109 cfu) in patients with dyslipidemia (n=30).
Study Objectives
Primary Objective
The main objective was to assess the efficacy of Bacillus coagulans MTCC 5856 in Patients with dyslipidemia
Efficacy Outcomes
Secondary Objective
The secondary objective was to assess the safety of Bacillus coagulans MTCC 5856 (2×109 cfu) in Patients with dyslipidemia.
Safety Outcomes
Selection of Study Population
Subjects were included in the study if indicated “Yes” to all of the inclusion criteria and “No” to all of the exclusion criteria
Inclusion Criteria
The Inclusion Criteria were as follows:
1. Patients with dyslipidemia
2. Age between 30 and 65 years.
3. Have not consumed any probiotic for the last 2 months prior to recruitment.
4. Ability to comply with the study protocol.
Exclusion Criteria
The Exclusion Criteria were as follows:
Treatment Line
Dose Administration and Treatment Regimen
Subjects were administered one capsule of Bacillus coagulans MTCC 5856 containing 2×109 cfu once a day orally for a period of 90 days.
Prior and Concomitant Therapy
History of any medications being used were elicited and documented. Any other medication that was used during the study duration was also documented. The subjects were followed up regularly and all concomitant dosing from the time of screening till the follow up visit was captured and recorded and co-morbid and concomitant medicine were captured in the study.
Treatment Compliance
Compliance with study medication was reviewed at each visit. The task was performed by examination of the returned medication and patient inquiry. All accountability records were incorporated into the investigator's study file.
Methodology for Assessment of Efficacy and Safety Variables
Changes in laboratory parameters from screening to day 90 were observed. Blood samples (12.0 ml) will be collected in centrifuge tubes at screening. Collection, sample shipment and processing will be performed as recommended or specified in the lab manual.
The following was assessed:
Hematology (Hemoglobin, Packed cell volume, White blood cell count-differential leukocyte count, Mean cell volume, Mean corpuscular hemoglobin. Mean corpuscular hemoglobin concentration, Mean platelet volume).
Complete lipid profile (Low density lipoprotein, Very low density lipoprotein, High density lipoprotein, Total Cholesterol, Triglycerides).
Urine Pregnancy test—All females who did not reach menopause will undergo a pregnancy test.
Statistical Methods
Laboratory data was summarized by presenting summary statistics of raw data and change from baseline values showing change from baseline to end of study. The statistical tools that was used and performed in this study will be detailed in the statistical analysis plan; the same will be followed for analysis. For continuous data such as age, the descriptive statistics Mean, Mode, Median, Q1, Q3, IQR, Whisker, 95% CI Mean and 95% CI was presented. For categorical data such as sex, the frequency and percentage was presented. For all the parameters like liver profile, anti-oxidant Enzymes (Biomarkers), Haematology, lipid Profile, and quality of life questionnaire were calculated from screening to visit 4. A p value was measured between screening and visit 4 over QOL domain. The p value was analyzed using Leven'sscale Test on MINITAB. These scores were obtained using two sample t-test and multiple comparisons using method of null hypothesis over Levene's scale of mean change. The analysis of efficacy of the product was estimated on lipid and diabetic profile which was performed through ANOVA and multiple comparisons t-Test and method of null hypothesis. A mean P-Value change was observed and calculated for these efficacy parameters from Visit 1 to Visit 4 which was advised as Day 0 to Day 90 in the protocol. An average mean and P value was calculated with 95% CI mean for the safety parameters like Hematology profile from day 0 to Day 90 through t-test and ANOVA. For demographic profile average and range was obtained by simple statistical methods.
Safety Evaluations
The assessments for safety were based mainly on reporting of adverse events by the subjects. Other safety data (e.g., vital signs, laboratory tests) was summarized by presenting the number and no adverse events have occurred. Laboratory data was summarized by presenting summary statistics of raw data and change from screening values as well as shift tables showing changes from screening to end of study in laboratory values relative to normal reference limits.
Analysis of Adverse Events:
No adverse events were reported by the patients who turned up to the clinical site on Day 90 and until 15 days of telephonic follow-up.
Deaths, Other Serious Adverse Events, and Other Significant Adverse Events:
There were no Serious Adverse Events or Significant Adverse Events noticed in this study.
Results
Total 30 Subjects were enrolled observing a strict line on inclusion and exclusion criteria. Principal Investigator and study team strictly followed the protocol. The study's overall safety and efficacy parameters were calculated based on the mean value change and p value. The efficacy parameter of the study was estimated over lipid profile and the safety parameters were blood investigation like hematology and demographic profile. The results were justified with mean change between screening to Day 90 for the efficacy parameters. The below table reports the demographic data in the screening visit of the patients
Safety Evaluation
Extent of Exposure: 90 days.
Brief summary of Adverse Event
For the safety evaluation the parameters considered in this study were physical examinations, vital signs namely blood Pressure, heart rate, Pulse rate and respiratory rate.
Analysis of Adverse Event
No statistically significant changes were observed with respect to vitals, laboratory parameters and clinical findings from screening to end of the study.
Listing of Individual Laboratory Measurements by the Patient and Each Abnormal Laboratory Value
No abnormal laboratory values were observed.
Deaths, Other Serious Adverse Events and Other Significant Adverse Events
None of the patients enrolled in this study had any serious or significant adverse events. There were no deaths reported in this study.
Safety Conclusion
On the screening visit and during the whole course of the study the vital signs were normal. No statistical significant changes were observed. No clinically significance concerning safety was observed with respect to laboratory parameters from screening to end of the study.
The tables 4 and 5 report the safety parameters for the screening and final visit.
Efficacy Evaluation
For efficacy parameters like total cholesterol, LDL, TG and quality of life the mean p value change for Bacillus coagulans MTCC 5856 was obtained as p<0.05 which means Bacillus coagulans MTCC 5856 had showed statistical significant effect on total cholesterol, LDL, TG and quality of life from day 0 to day 90.
Bacillus coagulans MTCC 5856
Tables 7, 8 and 9 provide illustrative examples of food formulations containing Bacillus coagulans for lowering cholesterol.
Bacillus coagulans MTCC 5856 (1 × 106 to 1 × 1014 cfu),
Bacillus coagulans MTCC 5856 (1 × 106 to 1 × 1014 cfu) Inulin,
Bacillus coagulans MTCC 5856 (1 × 106 to 1 × 1014 cfu),
Tables 10, 11 and 12 provide illustrative examples of nutraceutical formulations containing Bacillus coagulans for lowering cholesterol.
Bacillus coagulans Chewable Tablet (For Cholesterol Lowering)
Bacillus coagulans MTCC 5856
Bacillus coagulans has total viable count of 15 billion spores per gram.
Bacillus coagulans Tablet (For Cholesterol Lowering)
Bacillus coagulans MTCC 5856
Bacillus coagulans has total viable count of 15 billion spores per gram.
Bacillus coagulans Capsule (For Cholesterol Lowering)
Bacillus coagulans MTCC 5856
Bacillus coagulans has total viable count of 15 billion spores per gram.
The above formulations are just illustrative examples, any formulation containing the above active ingredient intended for the said purpose will be considered equivalent.
While the invention has been described with reference to a preferred embodiment, it is to be clearly understood by those skilled in the art that the invention is not limited thereto. Rather, the scope of the invention is to be interpreted only in conjunction with the appended claims.
This application is non-provisional filing of U.S. provisional application No. 62/517,320 filed on 9 Jun. 2017, the content of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 62517320 | Jun 2017 | US |
