CROSSREFERENCE TO RELATED APPLICATION

[ParaOOOlj This is a PCT application claiming priority from Indian Provisional application no. 201741030866 filed on 31 August 2017, the details of which are being incorporated herein by reference.

BACKGROUND OF THE INVENTION

Field of the invention

[ParaOOOlj The present invention relates to therapeutic intervention for regulating carbohydrate breakdown and absorption in mammals. More specifically, the present invention relates to compositions comprising β-glucogallin and therapeutic applications thereof in controlled kinetics of carbohydrate breakdown and monosaccharide absorption.

Description of prior art

[Para0003] Hyperglycemia is characterized by presence of elevated levels of glucose in blood. If left untreated, the condition may lead to many diseases like diabetes, obesity, hyperlipoproteinemia, hyperlipidemia, cardiovascular complications, cancer, atherosclerosis, allergy, inflammation, and osteoporosis. Currently there are many drugs which are administered to control the blood glucose levels (e.g metformin). B ut increased intake of these drugs increases stress on the kidneys leading to damage and nephrotoxicity. Thus, current research is targeted at developing a natural plant based drug which is safe, cheap and effective in managing the blood glucose levels. While there are many treatment methods available to reduce the aberrant glucose levels, developing inhibitors against key enzymes that regulate the carbohydrate breakdown and absorption can be a potential target, as these enzymes catalyze the most important biochemical pathways. Controlled kinetics of carbohydrate digestion and monosaccharide absorption could be of great value in the avoidance of aforementioned disease conditions. In this aspect, amylase, glueosidase and dipeptidyi peptidase inhibitors are of particular importance (Kim et al. (2004) Isolation and characterization of a-glucosidase inhibitor from the fungus- Ganoderma lucidtim. Journal of Microbiology, 42, 223-227; Ahren et al. (2004) Inhibition of Dipeptidyi peptidase-4 Reduces Glycemia, Sustains Insulin Levels, and Reduces Glucagon Levels in Type 2 Diabetes, The Journal of Clinical Endocrinology: & Metabolism, 89 (5), 2078-2084).

| Para0004| a-Amylase (E.C.3.2.1.1) is a hydrolase enzyme catafysing the hydrolysis of cc-1 , 4- giycosidic linkages in carbohydrates (starch) to yield monosaccharide units like glucose and maltose, a-glucosidase, is essential for the degradation of glycogen to glucose. Both these enzymes act on complex carbohydrate molecules to yield monosaccharide units which are readily absorbed in the blood stream. Dipeptidyl peptidase-4 is another key enzyme which increases the blood glucose evel by inhibiting the action of Incretins (metabolic hormones that stimulate a decrease in blood glucose levels by promoting insulin secretion) Inhibiting the activities of the above key enzymes would help in regulating the breakdown and uptake of carbohydrates, thereby decreasing the hyperglycemic condition,

[ParaOOOS] There are many plant based inhibitory molec ules of these enzymes which are discussed in the following prior arts;

1. Alagesan et al, "Amylase inhibitors: Potential source of anti-diabetic drug discovery from medicinal plants", Int. J. of Pharm. & Life Sci. (IJPLS), Vol. 3, Issue 2: Feb.: 2012, HOT- MI .

2. Da Silva et al., "Inhibition of salivary and pancreatic a-amylases by a pinhao coat (Araucaria ang stifoiia) extract rich in condensed tannin" Food Research International. Volume 56, February 2014, Pages 1-8

3. Borde et al., "Dipeptidyl peptidase-iv inhibitory activities of medicinal plants: TerminaUa aijima, Commiphora mukul, Gymnema syhesire, Morinda ciirifolia, Emblica officinalis" Asian J Pharm Clin Res, Vol 9, Issue 3, 2016, 180-182

4. Thilagam et al., "a-Glucosidase and -Amyiase Inhibitory Activity of Senna surattetisis" Journal of Acupuncture and Meridian Studies, Volume 6, Issue 1, February 2013, Pages 24- 30

5. Poongunran et al., "a-Glucosidase and a- Am lase Inhibitory Activities of Nine Sri Lankan Antidiabetic Plants", British Journal of Pharmaceutical Research, 7(5): 365-374, 2015.

[Para000(>| However, a plant based molecule that effectively inhibits key enzymes in carbohydrate metabolism, especialiy, amylase, glucosidase and dipeptidyl peptidase is lacking and there exists an unmet industrial need to find an active plant based molecule that is cheap, non-toxic and effective. The present invention solves the disclosing compositions comprising β- glucogallin for the management of aberrant carbohydrate metabolism in mammals.

[ParaOOO?] It is the principle objective of the invention to disclose compositions containing β- glucogallin the maintenance of carbohydrate homeostasis in mammals by the inhibition of enzymes amylase, glucosidase and dipeptidyl peptidase

[ParaOOOS] The present invention fulfils aforesaid objectives and provides further related advantages. SUMMARY OF THE INVENTION

|Para00G9] The present invention discloses compositions for the effective regulation of carbohydrate breakdown and absorption; More specifically, the invention discloses compositions containing at least 10% w/w or above of l-O-galloyl-p-D-glucose (β-glucogallin) and additionally comprising of about 10% w/w to greater than 60% w/w total mucic acid galiates including mucic acid 1,4-lactone 5- O-gallate, mucic acid 2-Ogallate, mucic acid 6-Methyl ester 2-O-gallate, mucic acid 1 -Methyl ester 2-O-gai!ate and ellagic acid for the effective regulation of carbohydrate breakdown and absorpti on by the inhibition of enzymes amylase, glueosidase and dipeptidyl peptidase.

[Para OlO] Other features and advantages of the present invention will become apparent from the following more detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, the principle of the invention.

BRIEF DESCRIPTION OF DRAWINGS

[ParaOOllj Fig. 1 shows the graphical representation of pancreatic amylase inhibition activity of the compositions comprising at least 10% w/w of i -O-galloyl-fi-D-glucose (β-glucogalHn) and 10% w/w to 60% w/w mucic acid galiates .

[ParaOOllj Fig. 2 shows the graphical representation of human salivary a amylase inhibition activity of the compositions comprising at least 10% w/w of I -O-galioyl- -D-glucose (β-glucogallin) and 10% w/w to 60% w/w mucic acid galiates .

[ParaOOUj Fig. 3 shows the graphical representation of a glueosidase inhibition activity of the compositions comprising at least 10% w/w of i-O-galloyl- -D-giueose (β-glucogallin) and 10% w/w to 60% w/w mucic acid galiates .

[Para0014j Fig. 4 shows the graphical representation of Dipeptidyl peptidase-4 Inhibition activity of the compositions comprising at least 10% w/w of l-O-galloyf-^VD-glucose (β-glucogallin) and 10% w/w to 60% w/w mucic acid galiates.

DESCRIPTION OF THE MOST PREFERRED EMBODIMENTS

[ParaOOlS] In a most preferred embodiment, the invention discloses a method of inhibiting key- enzymes involved in carbohydrate metabolism, said method comprising steps of:

i) Bringing into contact one of the key enzymes in carbohydrate metabolism with a suitable substrate; ii) Incubating with an effective concentration of a composition containing at least 10% w/w of 1-

O-galioyf-p-D-glucose (β-glucogallin) and 10%) w/w to 60% w/w mucic acid gaiiates under optimal conditions;

iii) Reading the change in absorbance using spectrophotometric and fluorinietrie methods iv) Comparing the absorbance with a control blank and determining the percentage enzyme inhibition (IC _5e ) by the said composition containing at least 10% w/w of 1 -O-galloyi-p-D- giucose (β-giueogallin) and 10% _. w/w to 60% w/w mucic acid gaiiates using the formula: % h ibition = [(absorbance of control - absorbance of inhibitor ) absorbance of control] ^y - 100.

[ParaOOlS] In a related embodiment, the mucic acid gaiiates are selected from the group consisting of mucic acid 1,4-lactone 5-O-gallate, mucic acid 2-O-gallate, mucic acid 6-Meihyl ester 2-O-gallate, mucic acid ί -Methyl ester 2-O-gallate and ellagic acid. In another related embodiment, the enzymes are selected from the list consisting of pancreatic a amylase, salivary a amylase, a glucosidase and dipeptidyl peptidase-4.

[Para00l6j In another most preferred embodiment, the invention discloses a composition containing at least 10% w/w of 1 -O-galloyl-fl-D-glucose (β-glucogallin) and 10% w/w to 60% w/w mucic acid gaiiates for the management of hyperglycemic conditions in mammals by inhibiting carbohydrate absorption, and normalizing the metabolism of glucose. In a related embodiment, the mucic acid gaiiates are selected from the group consisting of mucic acid 1 ,4-lactone 5-O-gailate, mucic acid 2-O- gallate, mucic acid 6-Methyl ester 2-O-gallate, mucic acid 1 -Methyl ester 2-O-gallate and ellagic acid. In another related embodiment, the effective regulation of carbohydrate absorption and breakdown reduces hyperglycemic conditions present in disease states selected from the group consisting of diabetes, obesity, hyperlipoproteinemia, hyperlipidemia, cardiovascular complications, cancer, atherosclerosis, allergy, inflammation, and osteoporosis, in another related embodiment, the mammal is human. In another related embodiment, the composition is formulated with p armaceuticaliy nuiraceutieaily acceptable excipieats, adjuvants, diluents or carriers and administered orally in the form of tablets, capsules, syrups, gummies, powders, suspensions, emulsions, chewables, candies and eatables,

|ParaO©17] In another preferred embodiment, the invention discloses a method for the therapeutic management of hyperglycemia in mammals by inhibiting the activity of key enzymes, said method comprising steps of administering a composition containing at least 10% w/w of l-Q-galioyl-P-D- giucose (β-glueogaliin) and 10% w/w to 60% w/w mucic acid gaiiates, to bring about the inhibition of key enzymes involved in carbohydrate metabolism. In a related embodiment, the inhibition of enzymes results in decreasing blood glucose levels by regulating the release of glucose into the blood and cellular uptake of glucose from the blood stream. In a related embodiment, the mucic acid gallates are selected from the grou consisting of mucic acid i ,4-lactone 5-O-gallate, mucic acid 2-0- gallaie, mucic acid 6-Methyl ester 2-O-gallate, mucic acid I -Methyl ester 2-O-gallate and ellagic acid, in another related embodiment, the enzymes are selected from the list consisting of pancreatic a- amylase, salivary a-amylase, a-glucosidase and dipeptidyIpeptidase-4. In another related embodiment, the hyperglycemia condition is present in disease states selected from the group consisting of diabetes, obesity, hyperlipoproteiniemia, hyperlipidemia, cardiovascular complications, cancer, atherosclerosis, allergy, inflammation, and osteoporosis. In another related embodiment, the mammal is human. In another related embodiment, the composition is formulated with phffi'maeeutically/nutraceutieally acceptable excipients, adjuvants, diluents or carriers and administered orally in the form of tablets, capsules, syrups, gnmmies, powders, suspensions, emulsions, chewables, candies and eatables.

[ParaO S] In another preferred embodiment, the invention discloses a method for the therapeutic management of hyperglycemia by increasing secretion of insulin in mammals, using a composition containing at least 10% w/w of l-0-gailoyl-{3-D-glueose (β-giucogaliin) and 10% w/w to 60% w/w mucic acid gallates to said mammals, to bring about the inhibition of enzyme dipeptidyi peptidase-4 thereby promoting insulin secretion and increased cellular uptake of glucose from the blood. In a related embodiment the mucic acid gallates are selected from the group consisting of mucic acid 1 ,4- lactone 5-O-gallate, mucic acid 2-O-gallate, mucic acid 6-Methyl ester 2-O-gallate, mucic acid 1 - Methyl ester 2-O-gallate and ellagic acid. In another related embodiement the said composition is used for the management of hyperglycemia present in disease conditions selected from the group consisting of diabetes, obesity, hyperlipoproteinemia, hyperlipidemia, cardiovascular complications, cancer, atherosclerosis, allergy, inflammation, and osteoporosis. In a preferred embodiement, the mammal is human. In another preferred embodiment, the composition is formulated with pharmaceutically/nutraceutically acceptable excipients, adjuvants, diluents or carriers and administered orally in the form of tablets, capsules, syrups, gummies, powders, suspensions, emulsions, chewables, candies and eatables.

[Para0019j The aforesaid most preferred embodiments incorporating the technical features and technical effects of instant invention, are explained through illustrative examples herein under.

[Para0020] Example 1: Inhibition of a amylase

(Para0021] One unit of enzyme pancreatic a-amylase (Sigma- Aldrich, St. Louis, MO, USA) or salivary a- amylase (Sigma- Aldrich) was prepared using 0.IM sodium acetate buffer of pH 4.8. One ml of enzyme was mixed with one ml of 500, 250, 125, 62.5, 31.3, 15.6 and 7.8 ,ug ml of compositions comprising at least 10% w/w of I -Q~gaik>yl-B-D~glucose (β-glucogallin) and 10% w/w to 60% w/w mucic acid gallates including mucic acid 1 ,4-kctone 5-O-gallate, mucic acid 2-0- gallate, mucic acid 6-Methyl ester 2-0-gallate, mucic acid 1-Metbyl ester 2-O-gaiiate and eiiagic acid was used as test and Acarbose as internal experimental control, mixed and kept in water bath at 25°C for 10 miii. After 10 min one ml of reaction mixer was mixed with 1 mi of 0.5% starch and further mcubated at 25 °C for 30 min. To stop the reaction 1 ml of Drnitrosalicylic acid reagent was added mixed and placed in boiling water bath for 15 min, cooled to room temperature. After the boiling, 9 ml of DM water was added and cooled to room temperature. The absorbance was recorded at 540 nm.

[Para0022] The composition containing β glucogallin and 10% w/w to 60% w/w mucic acid gallates exhibited significant inhibition of pancreatic a amylase and salivary a amylase. Seven different concentration were tested, the said composition showed good inhibitory effect at all the tested concentrations (500, 250, 125, 62.5, 31.3 15.6 and 7.8 g/ml) but at a higher concentration of 500 the maximum inhibitory effect of said composition was showed significant pancreatic amylase and salivary a- amylase inhibitory activity (72.16%, 75.24%) with IC _¾ value of 252.6 μ&'ηιΐ, 180.62 ,iig/ml respectively (Fig land Fig 2). The experiment was repeated for three times and results expressed as mean value of inhibition activity percentage.

[Para0©23] Example 2: inhibition of a giucosidase

jPara0 24| For a giucosidase inhibition, a-glucosidase (Code G5003; Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 67 mM potassium phosphate buffer, pH 6.8, containing 8 containing 0.2% Bovine Serum Albumin (Sigma-Aldrich) & 0.02%) sodium azide (Sigma-Aldrich) which was used as enzyme source. Paranitrophenyi- a-d-giucopyranoside (Sigma-Aldrich) was used as substrate. Compositions comprising at least 10% w/w of i-O-galioyl-p-D-glucose (β-glucogallm) and 50% mucic acid gallates including mucic acid 1 ,4-lactone 5-O-galiate, mucic acid 2-O-gal ate, mucic acid 6-Methyl ester 2-O-gallate, mucic acid 1 -Methyl ester 2-O-galiate and eiiagic acid was weighed and serial dilutions of 62.5, 125, 250, 500, 1000 μ@¾η! were made up with equal volumes of distilled water. 50 μΐ of said composition was incubated for 5 mm with 50μ _. Ι enzyme source (0.15 U/ml). After incubation, 50 μΐ of suhstrate (1.25 mM) was added and further incubated for 20 min at room temperature. Presubstrate and post-substrate addition, absorbance was measured at 405 nm on a microplate reader (BMG FLUOslar OPTIMA Microplate Reader). The increase in absorbance on substrate addition was obtained. Each test was performed three times and the mean absorption was used to calculate percentage -glucosidase inhibition. Acarbose was used as positive control with various concentrations. The inhibitory activities of varying concentrations of said composition were expressed as 100 minus the absorbance difference (%) of the said composition relative to the absorbance change of the negative control (i.e., water used as the test solution). The measurements were performed in triplicate, and the lC ₅ o value (i.e., the concentration of said composition that result in 50% inhibition of maximal activity) was determined. [Para0025] The composition exhibited -concentration dependant inhibition of a giucosidase with maximum inhibition (93.92%) at a concentration of 1000 ug/ml (Fig 3).

[Para0026j Example 3: Inhibition of Dipeptidyl Peptidase-4

j Para0 27| To determine tlie ability of the compositions comprising at least 10% w/w of 1-O-galioyl- β-D-glucose (β-glucogallin) and 10% w/w to 60% w/w mucic acid gallates includin mucic acid 1,4- lactone 5-O-gallate, mucic acid 2-O-gallate, mucic acid 6-Methyl ester 2-Q-gallate, mucic acid 1 - Methyl ester 2-O-gallate and e!lagic acid to inhibit dipeptidyl peptidase-4 (DPP-IV) enzyme, biochemical assay was performed. The assay was performed in 96- well plate. Recombinant Human DPPIV/CD26 (rhDPPIV) (Sigma- Aldrich) was used as the enzyme source. 2.5 nM Tris HCL, pH 8 was used as assay buffer. Gly-Pfo-7-a5aido-4-meihylcoumarin hydrobromide (H-Gly-Pro-AMC; Bachem, Catalog: 1-1225» was used as the substrate. Said composition was dissolved in water and concentrations ranging from 250 to 2000 μg/ml were taken for the assay. 50 μΐ. of 0.2 ng μΐ. of rhDPPIV was added to the plate and start the reaction by adding 50 f.iL of 20 μΜ .Substrate. As a Substrate Blank combine 50 μί, of Assay Buffer and 50 μΐ, of Substrate. The assay was run by mixing 70 μΐ of 0.1 M Tris buffer, 50 μ.Ι, of 0,2 ng/μί, of rhDPPIV and 30 μΐ of the test sample. After a 15 minute incubation period at 37°C, 50 uL of substrate solution (20 μΜ) was added to the appropriate wells to commence the reaction. Following an incubation period of 30 minutes at 37°C, fluorescence was determined at excitation and emission wavelengths of 380 nm and 460 am, respectively, in kinetic mode for 5 minutes-. The assay was performed in triplicate with appropriate blanks and standard. Diprotin A was used as internal experimental control for the study. The percentage of inhibition was calculated using the following formula: % Inhibition = [(absorbance of control - absorbance of inhibitor)/absorbance of control] * 100.

[Para0028] The said composition inhibited the activity of an enzyme Dipeptidyl peptidase-4 (DPP-4) in a concentration dependant manner with maximum inhibition (27.9%) at a concentration, of 2000 ug/nil (Fig, 4).

[Para0029] Example 4: Compositions / formulations containing β-g!ueogailm

[Para003 ] Given that β-glucogallin is useful in regulating blood glucose levels and its non toxic nature (LD _5P approx. 2000 mg/kg bodyweight),. it can be effectively blended in different compositions/formulations that has potential role in therapeutic indications wherein carbohydrate homeostasis play a vital role viz. maintenance and rejuvenation of general health, blood glucose support, for cardiac health, as an antioxidant, for regulating digestion, energy and sports endurance.

[Para0 1 | Tables 1-6 provide illustrative examples of food formulations containing β-giucogallin and mucic acid gallates for regulating carbohydrate homeostasis {Para0 32| Table 1: Rejuvenation blend (Premix) containing β-g!ucogallin

Active Ingredients

β-glucogallin and mucic acid gallates (50-500mg), Taurine

Excipients

Fructose , Citric Acid , Tartaric Acid , Sucralose , Lemon flavour and artificial Peppermint Flavour

Directions for use: Dissolve the required premix in 200-300 ml water and mix well before use

[Para0033] Table 2: Digestive support blend (Premix) containing β-giucogallin

Active Ingredients

β-glucogallm and mucic acid gallates (50-5O0mg), Bacillus coagulans MTCC 5856,

Fenumannans, Triphala Aquasol, 20 % Gingerols,

Excipients

Maltodextrin , Citric Acid , Malic Acid , Sucralose , Lime, Spearmint and Mangoginger flavours and artificial Mint Flavour, Cumin powder , Black Salt powder , Asafoetida

Directions for use: Dissolve the required premix in 200-300 ml water and mix well before use

[Para0034] Table 3: Blood glucose support blend (Premix) containing (5-glucogallin Active Ingredients

β-giucogallin and mucic acid gallates (iOO-lOOOmg), Fenumannans, Moringa leaf extract, Pterocarpus water soluble extract, Aloe Powder, Vitamin B6, Vitamin B12

Excipients

Maltodextrin , Taurine , Citric Acid , Malic Acid , Sucralose , Trisodiul Citrate , Contains Nature identical Apple flavour and artificial Peppermint Flavour and Bitterness masker flavor

Directions for use: Dissolve the required premix in 200-300 ml water and mix well before use

[Para0 35] Table 4: Energy blend (Premix) containing p-glHCOgallin

Active Ingredients

β-glucogalli and mucic acid gallates (100-iOOOmg), Taurine, Inositol, L-Arginine, Coeocin, GlucoronoLactotie, L-Selenomethiomne, Zinc Monomethionine, Magnesium Gluconate, Calcium Citrate Malate, Ascorbic Acid, B Vitamin Blend - Vitamin B3, Vitamin B6, Vitamin B9, Vitamin B 12

Excipients

Carageenan , Citric Acid , Tartaric Acid , Trisodium Citrate , Sucralose , Lime flavour and artificial Pepeprmint Flavour, Synthetic food colour.

Directions for use: Dissolve the required premix in 200-300 ml water and mix well before use

[Para0036] Table 5: Sports support blend (Premix) containing β-ghicogailin

Active Ingredients

β-glucogallin and mucic acid gallates (200-2000rng), Taurine, SaBeet ^® (beet root

extract), L-Arginine, Grapeseed Extract, Moringa leaf extract, B Vitamins - Vitamin

B6, Vitamin B 12

Excipients

Maltodextrin , Xylitol , Citric Acid , Malic Acid , Potassium Chloride , Sucraiose ,

Strawberry flavor, and also contains permitted Class II presrvative (Sodium Benzoate).

Directions for use: Dissolve the required premix in 200-300 ml water and mix well before use * ^" Registered TM of Sabinsa Corporation, USA

[Para0 37] Table 6: Anti-oxidant/Cardiac health blend (Premix) containing β-glyeogallin Active Ingredients

β-glucogalli and mucic acid gallates (50-500mg), Beetroot Extract, Citrin Crystals

(Anthocyanins), Moringa Leaf Extract, B Vitamins - Vitamin B6 and Vitamin B12,

Fruit Powder (Orange, Banana, Pineapple, Apple, Pomegranate, Jamun, Cranberry,

Grape)

Excipients

Maltodextrin , Xylitol , Citric Acid , Malic Acid , Potassium Chloride , Sucraiose ,

Calcium Silicate , Strawberry flavour.

Directions for use: Dissolve the required premix in 200-300 ml water and mix well before use

[Para0038] Tables 7 and 8 provides illustrative examples of nutraceutical formulations containing β- glucogallin and mucic acid gallates for regulating carbohydrate homeostasis jPara00391 Table 7: β-glacogaUift Tablet

Active Ingredients

β-glucogallin and mucic acid gallates (5O-50Omg)

Excipients

Microcrystalline cellulose, Hypromc!lose. Croscarrnellose Sodium, Colloidal silicon

dioxide. Magnesium stearate jPara0 4 | Table 8: β-glacogallin Capsule

Active Ingredients

β-glucogallin and mucic acid galiates (50-500mg)

Excipients

Microcrystalline cellulose, Croscarmellose Sodium, Magnesium stearate f Para0041| Table 9 provides ilhistrative example of a chewable gummy composition containing β- glucogallin and mucic acid galiates for regulating carbohydrate homeostasis

[Para0 42j Table 9: β-glucogallin Gummy composition

Active Ingredients

β-glucogalli and mucic acid galiates (50-5QGrng), Pectin, Glucose corn syrup

Excipients

Citiric acid, Lactic acid, Lemon peel oil (flavor), DL Tartaric acid, refinated sugar

[Para(M>43] Tire above formulations are just illustrative examples, any formulation containing the above active ingredient intended for the said purpose will be considered equivalent.

[Para0 44| Other modifications and variations to the invention will be apparent to those skilled in the art from the foregoing disclosure and teachings. Thus, while only certain embodiments of the invention have been specifically described herein, it will be apparent that numerous modifications may be made thereto without departing from the spirit and scope of the invention.