Polymolecular formulation for prevention of diabetes progression and a method for preparation thereof

Abstract

The present invention provides a polymolecular formulation and method for preparation thereof. The polymolecular formulation comprises naturally occurring ingredients specific to diabetes, which targets multiple pathways of the disease. The polymolecular formulation comprises synergistically effective proportions of combination of both whole herb and extracts, including Guava (Psidium guajava), Jamun (Syzygium cumini), Bitter gourd (Momordica charantia), Amla (Phyllanthus emblica), Tulsi (Ocimum sanctum) and Daruharidra (Berberis aristata), to provide optimal efficacy in a dose standardized form. Each of the herb in the formulation, target and act individually on multiple diabetic pathways to obtain the anti-diabetic effect. Hence, the polymolecular formulation helps in the reduction of the dose of diabetes medications in the patients and, helps to prevent insulin resistance, thereby preventing diabetes progression.

Description

POLYMOLECULAR FORMULATION FOR PREVENTION OF DIABETES PROGRESSION AND A METHOD FOR PREPARATION THEREOFA) TECHNICAL FIELD

[0001] The present invention is generally related to the field of pharmaceutical science. The present invention is particularly related to polymolecular formulation and method of preparation thereof. The present invention is more particularly related to a naturally derived polymolecular formulation based on composite pharmacology comprising whole herbs and extracts of the herbs targeting multiple molecular pathways, and achieving efficacy because of an aggregate molecular impact at lower toxicity for the treatment of diabetes.B) BACKGROUND OF THE INVENTION

[0002] Diabetes mellitus usually known as diabetes is a chronic disease associated with hyperglycemia, a condition of abnormally high levels of glucose in the blood caused by the absence or insufficient or impaired secretion of insulin (Type 1), a hormone produced by the pancreas, or inability of the body to use or respond to insulin appropriately (Type 2) such as managing hepatic glucose production and peripheral glucose utilization, which over time leads to serious damage to many of the body's system, especially the neural, vascular and organs such as heart, eyes and kidneys. Apart from the above systemic condition, recent research shows that some more conditions like adipocyte insulin resistance, reduced incretin secretion, increased glucagon secretion, enhanced glucose re-absorption, and central nervous system insulin resistance due to neurotransmitter dysfunction also contribute to diabetes.

[0003] Currently, the first line of treatment for prediabetes and type 2 diabetes is Metformin, a biguanide that suppresses glucose levels by the reduction of hepatic glucose output. Metformin’s primary site of action is the liver. The liver plays a pivotal role in glucose management in the body by regulating glucose storage (as glycogen) or hepatic glucose production (by glycogenolysis, gluconeogenesis, glycogen synthesis, glycolysis), thereby minimizing blood glucose fluctuations. In prediabetes, there is instability in glucose levels but the ability to lower blood glucose is not gone. Therefore, repurposing metformin as a prediabetic drug is not logical as metformin will solely reduce blood glucose levels through inhibition of hepatic glucose production without a concomitant increase in insulin concentrations or insulin-stimulated glucose uptake. Artificial glucose suppression at this stage will exhaust the molecular mechanisms trying to achieveglucose homeostasis resulting in a coordinated dysregulation of the metabolic pathways and lifelong dependence on anti-diabetics. We want to conserve and preserve the existing ability of the liver and body to manage blood glucose and minimize blood glucose fluctuations. Even though Metformin is a relatively safe and cheap drug, it has poor patient compliance due to its side effects which include gastrointestinal issues including nausea, bloating, abdominal pain, and loss of appetite and is not recommended for patients susceptible to kidney dysfunction. 30% of long-term metformin users develop vitamin Bl 2 deficiency. Besides, metformin does not improve glycemic control in some patients leading to hypoglycemia occasionally and lactic acidosis. Also, long-term usage of metformin leads to an increased risk of neurodegenerative diseases. One of the major drawbacks of metformin is it is more likely to induce life-long dependence with potential acceleration to higher anti-diabetes drugs. Metformin is purely a diabetes maintenance drug instead of a curative one. There is little to no evidence of metformin improving insulin sensitivity and no effect whatsoever when metformin treatment is not accompanied by a weight loss regime in type 2 diabetes. As the disease progresses, it becomes necessary to include other drugs for diabetes management.

[0004] Numerous herbal medications are available in the market and are used by many patients and have claimed are known to provide benefits in the control of diabetes. However, availability of specific molecular combinations in herbals are highly variable, vary with seasons and soils and the production is often not scalable without a decline of vital components. Therefore, even when chemical classes such as polyphenols, glycosides, etc. are standardized, highly specific molecular groups such as ellagic acid, gallic acid, berberine, etc. which are essential to guarantee efficacy can be missing or grossly deficient leading to inconsistent clinical outcomes. In addition, some molecular clusters undergo degradation and hence will not remain stable over time. Therefore, assuring availability of actives within the predetermined ranges to provide dose consistency along with scalability is essential. The formulation needs to be constituted in tablets, capsules, etc. forms guaranteeing stability. This requires novel methods of standardization of formulation, sourcing, and manufacturing. To establish plant-derived molecules as prescribable “standard of care” medication for prediabetes / diabetes, it is necessary to align the formulation to mechanism of action of its components to specific molecular pathways sufficient to explain its efficacy and predict potential toxicity. It is also important to establish qualitative and quantitative consistency of its active molecular composition as the basis for dose standardization like its pharmaceutical counterparts linked to measurable clinical outcomes.

[0005] Hence, there is a need for development of a diabetes-specific polymolecular formulation derived from plants that can target multiple pathways of the disease.

[0006] The above-mentioned shortcomings, disadvantages and problems are addressed herein, and which will be understood by reading and studying the following specification.C) OBJECTS OF THE INVENTION:

[0007] The primary object of the present invention is to provide a polymolecular formulation and method for preparation thereof.

[0008] Another object of the present invention is to provide a polymolecular formulation comprising both whole herb and extracts of Psidium guajava, Syzygium cumini, Momordica charantia, Phyllanthus emblica, Ocimum sanctum and Berberis aristata in a qualitatively and quantitatively reproducible range of molecular composition targeting multiple pathways of diabetes.

[0009] Yet another object of the present invention is to provide a method for preparation of polymolecular formulation comprising both whole herb and extracts of Psidium guajava, Syzygium cumini, Momordica charantia, Phyllanthus emblica, Ocimum sanctum and Berberis aristata with a reproducible consistency of specific composition obtained through a combination of selective extraction and combination with whole herb optimized for targeting multiple pathways of diabetes with the best clinical outcomes.

[0010] Yet another object of the present invention is to provide a polymolecular formulation comprising a combination of whole herbs and extracts, and pharmaceutical excipients to enhance the actives present in the herb and to reduce the dosage to provide optimal efficacy.

[0011] Yet another object of the present invention is to provide a polymolecular formulation comprising a combination of whole herbs and extracts, in a qualitatively and quantitatively reproducible range of molecular composition to prevention of insulin resistance thereby preventing diabetes progression during the nascent or prediabetes stage.

[0012] Yet another object of the present invention is to provide a polymolecular formulation prepared as a tablet or capsule for translating molecules sourced from plants to match pharmaceutical expectations in clinical outcomes and dose standardization.

[0013] Yet another object of the present invention is to provide a polymolecular formulation the polymolecular formulation and extraction method depicted the presence of active ingredients,such as gallic acid, pedunculagin, catechin, ellagic acid, quercetin di-glycoside, isoquercetin hyperoside, guaijaverin, rosmarinic acid and berberine.

[0014] Yet another object of the present invention is to provide a polymolecular formulation comprising active ingredients effective against multiple diabetic pathways.

[0015] These and other objects and advantages of the embodiments herein will become readily apparent from the following detailed description taken in conjunction with the accompanying drawings.D) SUMMARY OF THE INVENTION

[0016] The following details present a simplified summary of the embodiments herein to provide a basic understanding of the several aspects of the embodiments herein. This summary is not an extensive overview of the embodiments herein. It is not intended to identify key / critical elements of the embodiments herein or to delineate the scope of the embodiments herein. Its sole purpose is to present the concepts of the embodiments herein in a simplified form as a prelude to the more detailed description that is presented later.

[0017] The other objects and advantages of the embodiments herein will become readily apparent from the following description taken in conjunction with the accompanying drawings. It should be understood, however, that the following descriptions, while indicating preferred embodiments and numerous specific details thereof, are given by way of illustration and not of limitation. Many changes and modifications may be made within the scope of the embodiments herein without departing from the spirit thereof, and the embodiments herein include all such modifications.

[0018] The various embodiments of the present invention provides a polymolecular formulation for preventing diabetes progression and method for preparation thereof.

[0019] In one aspect, a polymolecular formulation for preventing diabetes progression is disclosed. The polymolecular formulation includes synergistically effective proportions of a whole herb, and extracts comprising guava leaf powder at a concentration of 175 mg, guava leaf extract at a concentration of 28 mg, jamun fruit powder at a concentration of lOOmg, jamun seed extract at a concentration of 20mg, bitter gourd fruit powder at a concentration of 150 mg, amla fruit powderat a concentration of 100 mg, tulsi leaf extract at a concentration of 20 mg, and Daruharidra root extract at a concentration of 4 mg.

[0020] According to an embodiment, the guava leaf extract is obtained by 75% ethanol / water extraction method.

[0021] According to an embodiment, the jamun seed extract is obtained by 70% ethanol / water extraction method.

[0022] According to an embodiment, the Tulsi leaf extract is obtained by water extraction method.

[0023] According to an embodiment, the Daruharidra root extract is obtained by 70% ethanol / water extraction method.

[0024] According to an embodiment, the whole herb and the extracts are in the ration of 1 : 1 at the raw material stage.

[0025] According to an embodiment, the formulation further includes one or more pharmaceutical excipients comprising at least one of: povidone K 30, microcrystalline cellulose powder, dicalcium phosphate, cross carmellose sodium, magnesium stearate, silicon dioxide and coating materials comprising opadry white and opadry maroon.

[0026] According to an embodiment, the polymolecular formulation is prepared in the form of a tablet or a capsule.

[0027] According to an embodiment, the active ingredients of the polymolecular formulation are effective against multiple diabetic pathways.

[0028] According to an embodiment, the polymolecular formulation helps in amelioration of the disease phenotype and additionally a prevention of insulin resistance / diabetes progression.

[0029] According to an embodiment, the polymolecular formulation helps in glucose maintenance rather than glucose suppression.

[0030] In another aspect, a method of preparation of a polymolecular formulation for preventing diabetes progression is provided. The method includes combining a combining synergistically effective proportions of whole herb and extracts comprising guava leaf powder at a concentration of 175 mg, guava leaf extract at a concentration of 28 mg, jamun fruit powder at aconcentration of lOOmg, jamun seed extract at a concentration of 20mg, bitter gourd fruit powder at a concentration of 150 mg, amla fruit powder at a concentration of 100 mg, tulsi leaf extract at a concentration of 20 mg, and Daruharidra root extract at a concentration of 4 mg.

[0031] According to an embodiment, the method includes extraction of guava leaf powder for preparation of the polymolecular formulation by mixing Guava leaf powder at a concentration of 5 gm with 100 ml of 75% Ethanol / Water in a 250ml round bottom flask. The method also includes heating the mixture at a temperature of 80 degree Celsius for 6 hours with reflux condenser and stirring the mixture at 400 rpm. The method also includes transferring the mixture into falcon tubes and centrifuging at 5000 rpm for 10 minutes. The method also includes filtering the supernatant obtained after centrifuge using filter paper and discarding the residue. The method also includes removing the ethanol from the filtrate on rotavapor under vacuum at 36 degree Celsius and removing the remaining water on a freeze dryer. The method also includes storing the extract obtained in an airtight container at 4 degree Celsius till further use.

[0032] According to an embodiment, the method includes extraction of Jamun seed for preparation of the polymolecular formulation by mixing Jamun seed powder at a concentration of 5 gm with 100 ml of 70% ethanol / water in a 250ml round bottom flask and heating the mixture at 60 degree Celsius for 3 hours with reflux condenser under stirring at 400 rpm. The method also includes transferring the contents of the flask into falcon tubes and centrifuged at 5000 rpm for 10 minutes. The method also includes obtaining supernatant after centrifuge is filtered using filter paper and discarding the residue. The method also includes removing Ethanol from the filtrate on rotavapor undervacuum at 36 degree Celsius removing remaining water on a freeze dryer. The method also includes storing the extract obtained in an airtight container at 4 degree Celsius for subsequent use.

[0033] According to an embodiment, the method includes extraction of Tulsi leaf for preparation of the polymolecular formulation for preventing diabetes progression by mixing Tulsi leaf powder at a concentration of 5gm with 100 ml of distilled water in a 250ml round bottom flask and stirring the mixture at room temperature for 23 hours at 500 rpm. The method also includes transferring the contents of the flask into falcon tubes and centrifuging at 5000 rpm for 10 minutes. The method also includes filtering supernatant obtained after centrifuge using filter paper and discarding the residues. The method also includes removing the solvent from the filtrate on freeze dryer, obtaining the extract and storing in an airtight container at 4 degree Celsius for subsequent use.

[0034] According to an embodiment, the method includes extraction of Daruharidra root for preparation of the polymolecular formulation by mixing about 5 gm of the Daruharidra root powder at a concentration of 5gm with 100ml of 70% Ethanol / Water in a 250ml round bottom flask and heating the mixture at 60 degree Celsius for 3 hours with reflux condenser under stirring at 400 rpm. The method also includes transferring the heated mixture into falcon tubes and centrifuging at 5000 rpm for 10 minutes. The method also includes filtering the supernatant obtained after centrifuge using filter paper and discarding the residue. The method also includes removing Ethanol from the filtrate on rotavapor under vacuum at 36 degree Celsius and removing remaining water on freeze dryer till dry. The method also includes obtaining extract and storing in airtight container at 4 degree Celsius for subsequent use.

[0035] From the foregoing discussion, it is apparent that the present invention provides a polymolecular formulation, which serves as an effective alternative for metformin as it would function as a glucose maintenance drug rather than a suppressant. Maintenance of glucose levels in the blood would impose less burden on the pancreas to release insulin and combined with the ability of the polymolecular formulation to repair pancreatic damage due to its high antioxidant potential can provide a wholesome approach towards improving diabetic symptoms.

[0036] It is to be understood that the aspects and embodiments of the disclosure described above may be used in any combination with each other. Several of the aspects and embodiments may be combined to form a further embodiment of the disclosure.

[0037] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description.E) BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The other objects, features and advantages will occur to those skilled in the art from the following description of the preferred embodiment and the accompanying drawings in which:

[0039] FIG. 1 A illustrates a flowchart exhibiting the herbs, extract forms and their respective concentration, in the polymolecular formulation, according to an embodiment of the present invention.

[0040] FIG. IB depicts a table providing weightage of each active component in the polymolecular formulation and the amount per tablet, containing active components in the quantities along with the excipients, in accordance with an embodiment of the present invention.

[0041] FIG. 2 illustrates the elution peaks based on the retention time of the active ingredients of the formulation using Liquid Chromatography Mass Spectroscopy (LCMS), according to an embodiment of the present invention.

[0042] FIG. 3. illustrates an MTT assay measured cell viability when treated with the polymolecular formulation and doxycycline (positive control) in HepG2 cells, according to an embodiment of the present invention.

[0043] FIG. 4 illustrates an amylase assay measured percentage inhibition of amylase activity with the polymolecular formulation vs acarbose, according to an embodiment of the present invention.

[0044] FIG. 5A-5C illustrates glucose uptake measurement of a fluorescent glucose analog 2- NBDG, according to an exemplary scenario.

[0045] FIG. 6 illustrates an oral glucose tolerance test (OGTT) in rats using polymolecular formulation, according to an exemplary scenario.

[0046] FIG. 7 illustrates the graph of blood glucose levels in STZ treated rats, according to an exemplary scenario.

[0047] FIG. 8 illustrates a flow diagram of polymolecular process standardization for the development of a polymolecular formulation, according to an embodiment of the present invention.

[0048] FIG. 9 illustrates HPLC chromatograms of guava leaf extracts in five separate solvents, according to an embodiment of the present invention.

[0049] FIG. 10A illustrates an impact of guava leaf extracts on cellular viability by measurement using the MTT assay, according to an embodiment of the present invention.

[0050] FIG. 10B illustrates an impact of guava leaf extracts on glucose uptake by measurement using 2-NBDG, according to an exemplary scenario.

[0051] FIG. 11 illustrates an effect of guava leaf extract on glucose uptake in insulin-sensitive (IR-) and insulin-resistant (IR+) L6 myoblast cells, according to an embodiment of the present invention.

[0052] FIG. 12 illustrates HPLC chromatogram of jamun seed extraction process in five separate solvents, according to an embodiment of the present invention.

[0053] FIG. 13A shows an impact of jamun seed extracts on cellular viability by measurement using the MTT assay, according to an embodiment of the present invention.

[0054] FIG. 13B shows an impact of jamun seed extracts on glucose uptake by measurement using 2-NBDG, according to an embodiment of the present invention.

[0055] FIG. 14 illustrates an effect of Jamun fruit extract and jamun seed extract on glucose uptake in insulin-sensitive (IR-) and insulin-resistant (IR+) L6 myoblast cells, according to an embodiment of the present invention.

[0056] FIG. 15A illustrates an impact of tulsi extracts on cellular viability by measurement using the MTT assay, according to an embodiment of the present invention.

[0057] FIG. 15B illustrates an impact of tulsi extracts on glucose uptake by measurement using 2-NBDG, according to an embodiment of the present invention.

[0058] FIG. 16 illustrates an effect of tulsi extract on glucose uptake in insulin-sensitive (IR- ) and insulin-resistant (IR+) L6 myoblast cells, according to an embodiment of the present invention.

[0059] FIG. 17 illustrates HPLC chromatograms of daruharidra root extracts in five separate solvents, according to an embodiment of the present invention.

[0060] FIG. 18A illustrates an impact of daruharidra extracts on cellular viability by measurement using the MTT assay, according to an embodiment of the present invention.

[0061] FIG. 18B illustrates an impact of daruharidra extracts on glucose uptake by measurement using 2-NBDG, according to an embodiment of the present invention.

[0062] FIG. 19 illustrates an effect of daruharidra extract / berberine on glucose uptake in insulin-sensitive (IR-) and insulin-resistant (IR+) L6 myoblast cells, according to an embodiment of the present invention.

[0063] FIG. 20A illustrates a graph which represents glucose uptake measured in insulin- resistant L6 myoblasts in the presence of each of the five formulations, according to an embodiment of the present invention.

[0064] FIG. 20B illustrates a graph which represents glucose uptake measured in insulinsensitive L6 myoblasts in the presence of each of the five formulations, according to an embodiment of the present invention.

[0065] FIG. 21 illustrates a flow chart on soft targeting approach of polymolecular formulation on diabetes pathway, according to an embodiment of the present invention.

[0066] Although the specific features of the present invention are shown in some drawings and not in others. This is done for convenience only as each feature may be combined with any or all of the other features in accordance with the present invention.F) DETAILED DESCRIPTION OF THE DRAWINGS:

[0067] In the following detailed description, a reference is made to the accompanying drawings that form a part hereof, and in which the specific embodiments that may be practiced is shown by way of illustration. These embodiments are described in sufficient detail to enable those skilled in the art to practice the embodiments and it is to be understood that other changes may be made without departing from the scope of the embodiments. The following detailed description is therefore not to be taken in a limiting sense.

[0068] In the following description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the present disclosure. It will be apparent, however, to one skilled in the art that the present disclosure can be practiced without these specific details.

[0069] The detailed description of various exemplary embodiments of the disclosure is described herein with reference to the accompanying drawings. It should be noted that the embodiments are described herein in such detail as to clearly communicate the disclosure. However, the amount of details provided herein is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.

[0070] It is also to be understood that various arrangements may be devised that, although not explicitly described or shown herein, embody the principles of the present disclosure. Moreover, all statements herein reciting principles, aspects, and embodiments of the present disclosure, as well as specific examples, are intended to encompass equivalents thereof.

[0071] While the disclosure is susceptible to various modifications and alternative forms, specific embodiment thereof has been shown by way of example in the drawings and will be described in detail below. It should be understood, however, that it is not intended to limit thedisclosure to the forms disclosed, but on the contrary, the disclosure is to cover all modifications, equivalents, and alternatives falling within the scope of the disclosure.

[0072] In order to more clearly and concisely describe and point out the subject matter of the claimed invention, the following definitions are provided for specific terms, which are used in the following written description.

[0073] The term “formulation” means a material or mixture prepared using different ingredients in a specified ratio, as the context requires.

[0074] The term “Liquid Chromatography-Mass Spectrometry (LCMS)” refers to an analytical chemistry technique combining both physical separation capabilities of liquid chromatography and mass analysis capabilities of mass spectrometry.

[0075] As used herein, “%” refers to %w / w or the weight percent of a substance as it relates to the overall formulation unless otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. The term "a" and "an" refers to one or to more than one (i.e., to at least one) of the grammatical object of the article. The information provided in this document, and particularly the specific details of the described exemplary aspects, is provided primarily for clearness of understanding and no unnecessary limitations are to be understood from there.

[0076] The various embodiments of the present invention provides a polymolecular formulation and method for preparation thereof. The polymolecular formulation comprises naturally occurring ingredients specific for diabetes, which targets multiple pathways of the disease. The polymolecular formulation comprises of a combination of herbs in both whole herb and extract form, to provide optimal efficacy in a dose standardized form. Each of the herb in the formulation target and act individually on multiple diabetic pathways to obtain the anti-diabetic effect. Hence, the polymolecular formulation helps in reduction of dose of diabetes medications in the patients and also, helps to prevent insulin resistance, thereby preventing diabetes progression.

[0077] According to one embodiment of the present invention, a polymolecular formulation for preventing diabetes progression by targeting multiple diabetes pathway is disclosed. The polymolecular formulation comprises synergistically effective proportions of whole herb and extracts, including Guava (Psidium guajava), Jamun (Syzygium cumini), Bitter gourd (Momordicacharantia), Amla (Phyllanthus emblica), Tulsi (Ocimum sanctum) and Daruharidra (Berberis aristata). Specifically, the polymolecular formulation comprises guava leaf powder at a concentration of 175 mg, guava leaf extract obtained by 75% ethanol / water extraction method at a concentration of 28 mg, jamun fruit powder at a concentration of lOOmg, jamun seed extract obtained by 70% ethanol / water extraction method at a concentration of 20mg, bitter gourd fruit powder at a concentration of 150 mg, amla fruit powder at a concentration of 100 mg, tulsi leaf extract obtained by water extraction method at a concentration of 20 mg and daruharidra root extract obtained by 70% ethanol / water extraction method at a concentration of 4 mg.

[0078] FIG. 1A depicts the herbs 104, 114, 124, 130, 136 and 142, extract forms 106, 108, 126, 132, 138, 116, 118, and 144, and their respective concentrations 110, 112, 120, 122, 128, 134, 140 and 146, in the polymolecular formulation 102, according to an embodiment of the present invention.

[0079] FIG. IB depicts a table 148 providing weightage of each active component in the polymolecular formulation and the amount per tablet, containing active components in the quantities given in table 148 along with the excipients, in accordance with an embodiment. The weightage of each component in the formulation is provided, based on the mode of action relevant to each component, part of the plant used, and the combination of whole herb or extract for the formulation. Based on the ratio given for each herb, a 1 : 1 ratio of whole herb and extract is used wherever both are to be used, and the quantity of extract is determined based on the yield obtained from the extraction of that herb. Bitter gourd and Amla were used only in whole herb powder form, Guava and Jamun were used in both whole herb powder and extract form whereas Tulsi and Daruharidra were used only in their extract form as provided in the table 148.

[0080] According to one embodiment of the present invention, the polymolecular formulation 102 further includes pharmaceutical excipients, where the pharmaceutical excipients are pharmaceutical additives or ingredients which are utilized for enhancing stability, maintaining texture and appearance, preventing microbial growth of the final dosage formulation. The pharmaceutical excipients includes povidone K 30, microcrystalline cellulose powder, dicalcium phosphate, cross carmellose sodium, magnesium stearate, silicon dioxide and coating materials including opadry white and opadry maroon.

[0081] According to one embodiment, the polymolecular formulation 102 is prepared as tablet or capsule. Furthermore, the polymolecular formulation 102 and extraction method depicted thepresence of active ingredients, such as gallic acid, pedunculagin, catechin, ellagic acid, quercetin diglycoside, isoquercetin hyperoside and guaijaverin, rosmarinic acid and berberine. The active ingredients of the polymolecular formulation 102 are effective against multiple diabetic pathways.

[0082] As opposed to commercial drugs for diabetes targeting a specific pathway for instance, Metformin targets hepatic glucose metabolism, Piaglitazone is an insulin sensitizer, the polymolecular formulation 102 targets all the diabetes-related pathways. The targeting of multiple diabetic-pathway is a mild response rather than obliterating the entire pathway. Hence, the polymolecular formulation 102 helps in amelioration of the disease phenotype and additionally a prevention of insulin resistance / diabetes progression. Furthermore, the polymolecular formulation 102 helps in glucose maintenance rather than glucose suppression.

[0083] According to one embodiment of the present invention, the polymolecular formulation 102 includes synergistically effective proportions of whole herbs and extracts to enhance the antidiabetic action. Further, the herb in the formulation 102 is used in the powder form and consumed in the form of capsules or tablets. In addition, the prescribed dosage of the polymolecular formulation 102 in tablet form is two tablets twice a day.

[0084] According to one embodiment of the present invention, the method for preparation of polymolecular formulation is disclosed. According to one embodiment of the present invention, the method for extraction of Guava leaf with 75% ethanol / water is provided. The Guava leaf powder at a concentration of 5 gm is mixed with 100 ml of 75% Ethanol / Water in a 250ml round bottom flask and the mixture is heated at a temperature of 80 °C for 6 hours with reflux condenser and stirring at 400 rpm. The contents of the flask are transferred into falcon tubes and centrifuged at 5000 rpm for 10 minutes. Further, supernatant obtained after centrifuge is filtered using filter paper and the residue is discarded. Ethanol from the filtrate is removed on rotavapor under vacuum at 36 °C and the remaining water is removed on a freeze dryer. The extract obtained as a light brown colored solid is stored in an airtight container at 4 °C till further use.

[0085] According to one embodiment of the present invention, the method for extraction of Jamun seed with 70% ethanol / water is provided. The Jamun seed powder at a concentration of 5 gm is mixed with 100 ml of 70% Ethanol / Water in a 250ml round bottom flask and the mixture is heated at 60 °C for 3 hours with reflux condenser under stirring at 400 rpm. The contents of the flask are transferred into falcon tubes and centrifuged at 5000 rpm for 10 minutes. Further, supernatant obtained after centrifuge is filtered using filter paper and the residue is discarded. Ethanol from thefiltrate is removed on rotavapor under vacuum at 36 °C and the remaining water is removed on a freeze dryer. The extract obtained as a light brown colored solid is stored in an airtight container at 4 °C till further use.

[0086] According to one embodiment of the present invention, the method for extraction of Tulsi leaf with water is provided. The Tulsi leaf powder at a concentration of 5gm is mixed with 100 ml of distilled water in a 250ml round bottom flask and the mixture is stirred at room temperature for 23 hours at 500 rpm. The contents of the flask were transferred into falcon tubes and centrifuged at 5000 rpm for 10 minutes. Further, supernatant obtained after centrifuge is filtered using filter paper and the residue is discarded. Solvent from the filtrate is removed on freeze dryer and the extract is obtained as a brown colored solid and stored in an airtight container at 4 °C till further use.

[0087] According to one embodiment of the present invention, the method for extraction of Daruharidra root with 70% ethanol / water is provided. 5 gm of the Daruharidra root powder at a concentration of 5gm is mixed with 100ml of 70% Ethanol / Water in a 250ml round bottom flask and the mixture is heated at 60 °C for 3 hours with reflux condenser under stirring at 400 rpm. The contents of the flask were completely transferred into falcon tubes and centrifuged at 5000 rpm for 10 minutes. Further, supernatant obtained after centrifuge is filtered using filter paper and the residue is discarded. Ethanol from the filtrate is removed on rotavapor under vacuum at 36oC and the remaining water is removed on freeze dryer till dry. The extract is obtained as a light yellow colored solid and stored in airtight container at 4 °C till further use.

[0088] Hence, for the extraction of herbs, various solvent extractions were carried out to standardize the processes and the optimal methods are chosen based on the presence of chemical ingredients and percentage yield as provided in Table 1.TABLE 1

[0089] Although various solvent extractions were carried out, as depicted in Table 1. In order to prepare a polymolecular formulation for the extraction of Guava leaves 75% ethanol / water extraction method is used. Similarly, for the extraction of Jamun seed 70% ethanol / water, for Tulsi leaves water extraction and for Daruharidra root 70% ethanol / water extraction method is used.

[0090] Subsequently, after standardizing the extract methods for various herbs, several trials with varying percentage of herb powders, extracts, and the final formulation is selected based on in- vitro results obtained as given in Table 2.TABLE 2

[0091] FIG. 2 illustrates the HPLC Chromatogram 200 based on the retention time of the active ingredients of the formulation using Liquid Chromatography Mass Spectroscopy (LCMS), according to an embodiment of the present invention. The chromatogram 200 are obtained by plotting UV Signal intensity 202 along y-axis versus time 204 along x-axis. The active ingredients from each of the herb are identified by LCMS analysis based on their mass and retention time as represented in the FIG 2. The formulation is studied to evaluate the active ingredients with respect to different retention time specific to each active ingredient of the herbs based on their elution. The results showed the presence of active ingredients, such as gallic acid, pedunculagin, catechin, ellagic acid, quercetin diglycoside, isoquercetin hyperoside, guaijaverin, rosmarinic acid and berberine. The FIG. 2 shows a UV trace of 70% ethanol extract of the polymolecular formulation at 254 nm.

[0092] Table 3 tabulates each of the herbs based on their retention time (Rt). The active ingredients from Amla fruit powder showed the presence of gallic acid and pedunculagin with a Rt of 2.30 min and 6.77 min, respectively. Similarly, the active ingredients from Guava leaves showed the presence of guaijaverin with a Rt 7.78 min, catechin with a Rt of 5.60 min, and ellagic acid derivatives with a Rt of 7.18 min. The active ingredients from Jamun fruit showed the presence of ellagic acid / quercetin derivatives with a Rt of 7.18min. The active ingredients from Tulsi leaves showed the presence of rosmarinic acid with a Rt of 8.29 min, and Daruharidra showed the presence of berberine with a Rt of 9.21 min. Hence, the active ingredients relevant to each herb in the polymolecular formulation were identified by LCMS using their mass and their retention time (Rt) is provided in Table 3.TABLE 3

[0093] Furthermore, the active ingredients relevant to each herb in the polymolecular formulation were identified by HPLC, as provided in Table 4, and four compounds have been selected for quantification by HPLC and set specifications for batch reproducibility and consistency in therapeutic efficacy as provided in Table 4.TABLE 4

[0094] Hence, the combination of herbs and extracts present in the polymolecular formulation, helps to enhance the active ingredients present in the herbs to reduce the dosage to provide optimal efficacy and to prevent insulin resistance thereby preventing diabetes progression. In order to reduce the dosage, the polymolecular formulation targets multiple mechanisms of the disease including primary and secondary complications. Primary targets of the disease include increasing insulin sensitivity of peripheral tissues, improving the function of the pancreas, reducing gut absorption,and increasing glycogen storage in the liver. The secondary adaptations can be addressed by antioxidant, anti-inflammatory, anti-lipidemic and anti-glycation properties. The polymolecular formulation 102 includes active components, which impact each of the targets, such as for instance, berberine improves insulin resistance, vitamin C and polyphenols from amla improves pancreatic function and lipid profile. Furthermore, the polymolecular formulation 102 also provides relief from the side effects, as the formulation includes an adaptogen, an antioxidant, and a stomach soother.

[0095] The invention is illustrated by the following examples which are not meant to restrict the scope of the invention in any manner.Examples Example 1: In-silico data analysis, multiple disease pathways perturbed by various chemicals of the polymolecular formulation.

[0096] The association between the pathways of the various chemical constituents of the polymolecular formulation 102 with the Insulin Sensitivity pathways is presented as ROC values. The ROC (Receiver Operating Characteristic) provides the diagnostic ability of a binary classifier system as its discrimination threshold is varied. Table 5 tabulates ROC values for various actives against multiple pathways is provided.TABLE 5

[0097] Table 5 provides that the Quercetin di glycoside with a ROC AUC of 0.6 is suggestedve to better insulin-sensitising properties. Example 2: Formulation, weightage and daily dose needed for efficacy.

[0098] The active components relevant to each herb in the polymolecular formulation were identified, and in-silico studies were conducted. Further, the efficacy of the formulation was enhanced by combining the whole herbs and extracts of some of the herbs. Table 6 tabulates the formulation and weightage of each component, part of the plant used and the combination of whole herb or extract, mode of action relevant to the actives, and amount of each component in our formulation along with the established daily and maximum tolerated doses.TABLE 6

[0099] The weightage of each herb is allocated based on known effects on various diabetes pathways.Example 3: Effect of each component on the key diabetes pathways and its secondary complications

[0100] Table 7 tabulates the effect of each component of the polymolecular formulation on the key diabetes pathways and its associated secondary complications.TABLE 70101] In Table 7, 4 represents maximal effect and 1 represents least effect. Furthermore, collectively, the polymolecular formulation helps to improve the primary aberrations, such as plasma glucose, insulin resistance, glucose uptake, pancreas function, insulin secretion, plasma insulin levels, glucose absorption, intestinal-alpha-glucosidase activity, and glycogen storage.Subsequently, the secondary complications of diabetes, such as inflammation, antioxidant / oxidative stress, lipid lowering properties, AGEs formation, HbAlC and hepatic health.Example 4: In vitro tests

[0102] The toxicity of the polymolecular formulation was tested in an in vitro model of HepG2 cells with varying concentrations of the polymolecular formulation using the MTT assay. FIG. 3 illustrates MTT assay measured cell viability when treated with the polymolecular formulation and doxycycline (positive control) in HepG2 cells, according to an embodiment of the present invention.

[0103] FIG. 3 illustrates MTT assay measured cell viability 300 when treated with the polymolecular formulation 102 and doxycycline (positive control) in HepG2 cells at concentrations of 100, 150, 200, 300, 400, and 500 pg / ml, respectively, in accordance with an exemplary scenario. MTT asssay 300 is obtained by plotting Cell viability 302 along y-axis versus concentration 304along x-axis. The data is representative of three separate experiments, n=9. From the study, it was evident that the polymolecular formulation was found to be safe at concentrations from 100-500 pg / ml whereas, similar doses of doxycycline (positive control) induced cell death, as illustrated in FIG. 3. Hence, the study concluded that the polymolecular formulation 102 at concentrations below 300 pg / ml was found to be safe to use. As the dose-response experiments showed maximal effects at 200 pg / ml, hence efficacy studies were carried out with this concentration.

[0104] Multiple in vitro studies were conducted to test the efficacy of the polymolecular formulation in liver (HepG2) and skeletal muscle (L6) cell lines including amylase assay, glucose consumption and glucose uptake in insulin-sensitive and insulin-resistant models, and antioxidant assay. Amylase increases postprandial glucose levels by the breakdown of complex carbohydrates and inhibition of amylase enzyme helps to reduce the glucose spikes after a meal. One of the commonly used antidiabetics in the market is Acarbose, which is a potent amylase inhibitor. We measured the inhibition of amylase activity with the polymolecular formulation compared to acarbose. Briefly, amylase was incubated with varying concentrations of polymolecular formulation or acarbose (lOOpg / ml) followed by substrate (CNPG3) addition and measurement of absorbance at 405nm. Using no drug treatment, i.e., a combination of amylase and CNPG3, as 100% activity, we calculated the decrease in amylase activity with the two drug treatments.

[0105] FIG. 4 illustrates the amylase assay measured percentage inhibition 400 of amylase activity with the polymolecular formulation vs acarbose, according to an embodiment of the present invention. FIG. 4 illustrates the amylase assay measured percent inhibition 402 of amylase activity with the polymolecular formulation 102 (targlut 404) and acarbose 404. Acarbose served as the positive control. The data represented is from five separate experiments. FIG. 4 illustrates that there was a -20% reduction in amylase activity with the polymolecular formulation (targlut 404) as compared to -88% with acarbose 406. A 20% reduction in activity fits in well with our argument of mild targeting of molecular pathways, rather than complete inhibition as with acarbose -88%.

[0106] Hence, the efficacy of the polymolecular formulation 102 was evaluated by two separate glucose utilization methods. The first determined glucose consumption by the cells via an indirect method, where the amount of glucose present in the media at the end of the experiment was measured. The cells were treated with the polymolecular formulation or metformin for 24 hours or 4 hours, respectively, and the glucose in the media was measured thereafter. These treatments resulted in the enhancement of glucose uptake by the cells resulting in reduced glucose in the mediacompared to cell-free media. The greater the glucose reduction, the more effective the treatment is in utilizing the glucose. Table 8 tabulates the glucose reduction in HepG2 cells treated with four concentrations of polymolecular formulation vs Metformin for 24 hours.TABLE 8

[0107] Table 8 tabulates the Glucose reduction in HepG2 cells treated with four concentrations of the polymolecular formulation (50, 100, 200, 250 pg / ml) for 24 hours compared with metformin (0.05, 0.2 pg / ml) treatment for 4 hours or DMSO (vehicle control) or no treatment (cell control). The values represented in Table 8, are the average of two sets of experiments carried out with n = 3 / experiment.

[0108] Furthermore, HepG2 cells were incubated with media supplemented with the polymolecular formulation at four different concentrations of 50, 100, 200 & 250 pg / ml for 24 hours. The positive control for the experiment was cells treated with 0.05 and 0.2 pg / ml of metformin and the negative control was no treatment (cell control) or DMSO (vehicle) treated cells. The glucose in the media was measured and the glucose consumption was calculated using cell-free media as 100%. As tabulated in Table 8, there was a dose-dependent increase in glucose consumption (demonstrated by increased glucose reduction in the media present in the cells). This dose-dependent effect was also observed with metformin treatment. There was low glucose consumption (17%) in the cell control (no stimulation for glucose uptake). For the polymolecular formulation, maximal glucoseconsumption at 200 pg / ml (60%), which was 3.5 times higher than the cell control and 1.5 times higher than metformin (43%) was observed. Glucose consumption by the cells plateaued for drug concentrations above 300 pg / ml and a similar pattern was observed for higher doses of metformin (data not shown here).

[0109] The second method of glucose utilization measured the uptake of a fluorescent glucose analogue known as 2-NBDG, by the cells. Here, the higher the 2-NBDG uptake, the better the efficacy of the treatment. This experiment was carried out in HepG2 cells along with L6 myocytes and L6 myotubes in normal and insulin-resistant states.

[0110] FIG. 5A-5C illustrates the glucose uptake measurement 500 of a fluorescent glucose analogue 2-NBDG with glucose uptake 502 plotted along Y-xis and Dexamethasone 504 plotted along X-axis, according to an embodiment of the present invention. FIG. 5A-5C illustrates HepG2 and L6 were treated with dexamethasone for 24 hours to induce insulin resistance followed by polymolecular formulation (Trade name: Targlut) or Metformin treatment for 24 hours or 4 hours, respectively. These cells were induced with lOOnM insulin for 30min. 2-NBDG was added to the cells and 30 min later the fluorescence in the cells was measured and recorded. Normal cells with insulin serve as a positive control as is set at 100% 2-NBDG uptake. Glucose uptake in the graph corresponds to 2-NBDG uptake. The values represented here are the average of three sets of experiments carried out with n = 3 / experiment.

[0111] Moreover, the insulin resistance model was established by incubating cells with different concentrations of Dexamethasone (DXM) for 24 hours, as illustrated in FIG. 5A. Insulin treatment served as the positive control which was set to 100% for 2-NBDG uptake and the reductions in uptake due to DXM were calculated accordingly. There was a dose-dependent induction of insulin resistance (IR) in the cells evidenced by reduced glucose uptake. Insulin treatment in these conditions did not augment 2-NBDG uptake thereby establishing the insulin resistance model. Using this model, the effect of the polymolecular formulation and metformin were assessed. In FIG. 5B, insulin-resistant HepG2 cells exhibited reduced insulin response (40%) 506 which was augmented by ~1.8 fold in the presence of polymolecular formulation 102 and by -2.3 fold with Metformin. The graph 506 of FIG. 5B is obtained by plotting glucose uptake 508 along Y- axis and Insulin laong X-axis. Similarly, in L6 myoblasts and myotubes in FIG. 5C, insulin-resistant conditions resulted in a -65% reduction in 2-NBDG uptake 512. Hence, both the polymolecularformulation 102 and Metformin treatments improved the uptake by ~2-fold and ~2.8-fold, respectively.

[0112] Cumulatively, these experiments demonstrate that the polymolecular formulation 102 improves the glucose uptake in skeletal muscle cell line (L6)- both myoblasts and myotubes (differentiated myoblasts) as well as HepG2 cells. Most importantly, from the experiment, it was evident that the effect of the polymolecular formulation 102 in insulin- resistant cells, was to partially repairs the ability of the cells to uptake glucose. So far, Metformin is the only commercially available anti-diabetic that has displayed the ability to improve insulin resistance in an in vitro model. Insulin resistance is a hallmark of multiple chronic metabolic disorders, but its diagnosis is indirect, laborious, and inaccurate, which has diverted the focus from creating therapeutics that repair insulin resistance. Hence, the polymolecular formulation 102 exhibits tremendous antidiabetic potential to repair insulin resistance along with reducing oxidative stress, inhibiting amylase activity, repairing pancreatic tissue (in vivo study), and lowering blood glucose levels.Example 5: In vivo test

[0113] A single-dose (acute) toxicity test was conducted using 300mg / kg, 2000mg / kg and 5000mg / kg for one month in Sprague Dawley rats. The LD50 of polymolecular formulation (Trade name: Targlut) was found to be >5000 mg / kg b. wt. maximum dose based on observations such as body weight, clinical signs, mortality / morbidity and gross pathology and it was classified as a Category-5 or unclassified as per Globally Harmonized Classification System (GHS) for chemical substances and mixtures.

[0114] A 5-week efficacy study was carried out using a streptozotocin (STZ)-induced diabetes rat model with the polymolecular formulation. In the first stage of the study, Sprague Dawley rats were pre-treated with 250mg / kg of polymolecular formulation for two weeks to explore if the polymolecular formulation will impart protection against STZ-induced damage to the pancreas. As expected, post-necropsy analyses of the pancreas for these animals showed a degree of protection against STZ-induced tissue damage. During the course of pretreatment, the rats did not go into hypoglycemic shock, which established that the polymolecular formulation functions as a glucose maintenance drug rather than a glucose suppressant.

[0115] FIG. 6 illustrates an oral glucose tolerance test (OGTT) 600 in rats using polymolecular formulation 102, according to an embodiment of the present invention. FIG. 6 illustrates an OGTT test carried out in rats, wherein the rats were given glucose (2g / kg) and blood glucose 602 wasmeasured at times 604 0, 30 min, 60 min, 120 min, respectively. Group 1- Vehicle (n=6), Group 2- 250mg / kg polymolecular formulation (n=6) and Group 3- 250mg / kg polymolecular formulation (n=10). From the OGTT test, it was evident that the polymolecular formulation has the ability to increase the metabolism of blood glucose in the cells, thereby promoting a reduction in glucose concentration in the due course of time.

[0116] Furthermore, in the second stage of the study, 8-10- week-old rats were injected intraperitoneally with vehicle or 45mg / kg b.wt. STZ to induce diabetes in the rodents and 72 hours post-administration glucose levels were measured. If the glucose levels were >150mg / dl, then the animals were considered diabetic, and this was taken as day 0. After the diabetic model was established, the rats were divided into 8 groups which included vehicle, polymolecular formulation- low dose (500mg / kg), polymolecular formulation- high dose (lOOOmg / kg), metformin (200mg / kg) and the pre-treated groups. FIG. 6 illustrates the graph of blood glucose levels 600in STZ-treated rats, according to an embodiment of the present invention.

[0117] FIG. 7 illustrates the graph 700 of blood glucose levels 702 in STZ-treated rats at day 0, 7, 14, 21 with 704 vehicle, low dose polymolecular formulation (500mg / kg), high dose polymolecular formulation (lOOOmg / kg) and metformin (200mg / kg) treatment, n=10 / group. During this test, all the animals appeared to be normal during the experimental period and did not exhibit any signs of toxicity during the observation period. STZ- induced diabetic animals exhibited polyuria, polydipsia, and polyphagia, which confirmed their hyperglycemic state. Glucose measurements 702 were done on days 7, 14, and 21 respectively, and the trends were observed and plotted in graph 700 as illustrated in FIG. 7.

[0118] Hence, the animals in the four STZ-treated groups had an average glucose value of 300mg / dL at the start of the experiment and the vehicle-treated rats did not show any improvement in the glucose levels over the course of three weeks. Treatment with the polymolecular formulation (both low dose and high dose) on day 7 showed an -25% reduction in blood glucose levels followed by a gradual and sequential decrease up to the third week, similar to metformin. On day 21, glucose levels were reduced by -40% for low dose, -48% for high dose groups and -24% for metformin- treated rats. At the end of the three- week treatment period, the animals were euthanized and analyzed for various metabolic markers. The circulating insulin levels in the animals at the end of the study showed small but insignificant changes and this pattern was observed for the lipid profile as well.

[0119] Moreover, to assess the functioning of the kidney and liver, various markers specific to each organ were measured at the end of the study, as tabulated in Table 10. Creatinine and total protein did not show any significant changes, but BUN and urea increased with STZ treatment compared to vehicle control and this was ameliorated by both low and high doses of polymolecular formulation as well as metformin. The liver parameters also worsened with STZ treatment, which improved with drug treatments and these decreases were statistically significant when compared to STZ only group. Table 9 tabulates the serum kidney and liver markers in STZ rats after treatment with vehicle, low dose polymolecular formulation (500mg / kg), high dose polymolecular formulation (lOOOmg / kg) or metformin (200mg / kg) for 21 days. Kidney markers {CRE- Creatinine, Urea, BUN- blood urea nitrogen, T.PRO- total protein} and liver markers {AUT- alanine transaminase, AST- aspartate transaminase, AEP- alkaline phosphatase} are presented as an average of 10 animals. n=10 / group.

[0120] TABLE 9

[0121] Moreover, free radicals produced during metabolic reactions cause cellular damage and aggravate the pathogenesis of diabetes. Antioxidants produced endogenously such as superoxide dismutase (SOD) or glutathione peroxidase (GPx), or obtained from dietary sources such as Vitamin C, and flavonoids, counter these free radicals. As tabulated in Table 10 SOD and GPx levels were dramatically reduced in the pancreatic tissues of the diabetic rats when compared with the vehicle- only group. The lowered activity of these enzymes could be explained by the depletion of the antioxidant defense system following the increased production and accumulation of free radicals. Inthe diabetic group, glucose auto-oxidation results in the formation of hydrogen peroxide which inactivates SOD and decreases its activity. The reduced SOD values were restored in both the polymolecular formulation-treated groups, and the effect was more profound than in the metformin- treated group. GPx levels were restored in both test item and metformin-treated groups. Table 10 tabulates pancreatic oxidative stress markers in STZ rats treated with vehicle, low dose polymolecular formulation (500mg / kg), high dose polymolecular formulation (lOOOmg / kg) or metformin (200mg / kg) for 21 days. SOD- superoxide dismutase, GPx- glutathione peroxidase and malondialdehyde are presented as an average of 10 animals. n=10 / group.

[0122] TABLE 10

[0123] In addition, Malondialdehyde measurement is a determinant of the amount of lipid peroxidation in the system. Lipid peroxidation is the process of lipid oxidation as a result of oxidative stress, which leads to cell death. STZ increased malondialdehyde levels in the pancreas and the polymolecular formulation showed a dose-dependent reduction of the lipid peroxidative marker when compared to the diabetic control, which was statistically significant. STZ administration caused intense pancreatic damage evidenced by high levels of malondialdehyde, SOD, GPx and the polymolecular formulation was able to dramatically improve all three markers, which were explained by the high levels of antioxidants in the polymolecular formulation.

[0124] Furthermore, the polymolecular formulation‘s mechanism of action is an aggregate impact of selective targeting of multiple pathways responsible for the pathophysiology of diabetes. Thereby, producing efficacy as an aggregate action on several complementary mechanisms. This “soft targeting” approach enables similar or higher levels of efficacy as an additive, aggregate or synergistic mixture but enables the distribution of molecular effects to reduce toxicity in the composition. The composition therefore, enables a high degree of control on efficacy and toxicity by varying the distribution of molecular pathways selectively impacted to a higher or lower degree. Therefore, each pathway is mildly affected, referred to as "soft targeting” and the validation has been provided by in vitro and in vivo studies under example 4 and 5.

[0125] FIG. 8 illustrates a flow diagram 800 of polymolecular process standardization for the development of a polymolecular formulation, according to an embodiment of the present invention. The herbs 802 were tested for the development of the formulation, which are a blend of known 828 and unknown molecular clusters 804, in in vitro assays. The process is standardized using the work plan shown in Figure 8. Five extracts are prepared in solvents identified on the basis of their polarity and screened. Each extract is scored based on chemical and biological activity. The fractions with substantial in vitro activity are combined in different ratios and screened by in vitro assays thereby providing us with an activity distribution profile for the extracts with the unknown molecular clusters. The chemistry and molecular activity score together establish the cluster with the dominant cluster activity. The process for the development of a polymolecular formulation involves testing each herb via a multi-step process. Each herb 802 is a complex mix of molecules- both known and unknown. The first step is the identification of the known actives relevant for the disease model via literature search and computational analyses. The known chemical entities of interest are processed by a quantification-linked extraction method. Simultaneously, the herb is extracted in various solvents based on polarity 806 including water 808, ethanol (EtOH) 810, 70% ethanol (EtOH) 812, ethyl acetate (EtOAc) 814, and hexane 816. The fractions are given a score based on the chemistry including yield, cost, ease of herb acquisition, and composition. These fractions have a combination of known and unknown molecular clusters that are tested in in-vitro assays developed for the disease model. The unknown molecular clusters of interest are distinguished by the extraction method for the same. The fractions with significant in vitro activity are combined in different ratios and screened by in vitro assays and this gives us the activity distribution profile 820 for the extracts with the unknown molecular clusters. The extract combinations are given a molecular activity score. The chemistry and molecular activity score together establish the cluster with the dominant clusteractivity 836. Finally, the extract with the unknown but dominant cluster 836 is combined with the active molecules 832 and raw herb 826 or whole herb (unprocessed) to obtain dose standardized polymolecular formulation 834.In vitro validation

[0126] The components of the formulation includes guava leaf, jamun seed, tulsi and daruharidra which were extracted in five solvents selected based on polarity to study the biological activity of each fraction. Water, ethanol, 70% ethanol, ethyl acetate and hexane are the five solvents used for the extraction method. The experiments were conducted to measure biological activity which includes cell viability assay (MTT assay), antioxidant assay (FRAP assay) and glucose uptake by myoblasts (2-NBDG uptake). Two extracts for each herb were selected and tested in different ratios in glucose uptake assays to test whether the combination of the extracts had activity greater than the individual extracts.

[0127] FIG. 9 illustrates HPLC chromatograms of guava leaf extracts 900 in five separate solvents, according to an embodiment of the present invention. Guava leaf is extracted in five separate solvents selected on the basis of polarity. The five extracts includes water, ethanol, 70% ethanol, ethyl acetate and hexane. The extracts were tested for toxicity (MTT assay), antioxidant activity (FRAP assay) and glucose uptake (2-NBDG). The five guava leaf extracts were tested for toxicity and glucose uptake at concentrations of 5, 25, 50, 75, lOOpg / ml for 24h in HepG2 cells and L6 myoblasts, respectively.

[0128] FIG. 10A illustrates an impact 1000 of guava leaf extracts on cellular viability by measurement using the MTT assay, according to an embodiment of the present invention. All the extracts except ethyl acetate had cellular viability 1002 >68% at concentrations 1004 lower than 50pg / ml, as shown in Figure 10A. 70% ethanol extract had cellular viability of -68.5 ±0.01% at 50pg / ml. HepG2 cells were treated with guava leaf extracts at concentrations of 5, 25, 50, 75, 100 pg / ml for 24hours, followed by measurement of cell viability using the MTT assay.

[0129] FIG. 10B illustrates an impact 1006 of guava leaf extracts on glucose uptake by measurement using 2-NBDG, according to an embodiment of the present invention. The five guava leaf extracts shows a dose-dependent increase in glucose uptake. The 100% ethanol and 70% ethanol extracts upregulated glucose uptake by -40% at lOOpg / ml, whereas the low-toxic dose of 50pg / ml enhances glucose uptake by -20%. The water and non-polar extracts also increase glucose uptakebut the extent of increase was -10% at mid-range concentrations. Figure 10B shows the glucose uptake data 1008 for the guava leaf extracts. L6 myoblasts were incubated for 24h with guava leaf extracts at concentrations 1010 of 5, 25, 50, 75, 100 pg / ml. Glucose uptake is measured in these cells using 2-NBDG. Each colored line in the graphs corresponds to the extract listed in the legend. Data is representative of two separate experiments. n=6TABLE 11

[0130] Table 11 shows Ferric reducing antioxidant power of guava leaf extracts. It shows theFRAP activity (ferric ion reducing ability antioxidant power) for each guava leaf extract. The antioxidant ability of each extract is measured using the FRAP assay. The extracts were reconstituted in 50% DMSO and directly used in the assay. The order of antioxidant ability for these extracts were as follows: Ethanol > Water > Ethyl acetate > 70% ethanol > Hexane.TABLE 12

[0131] 70% ethanol and 100% ethanol extracts were selected based on yield, glucose uptake ability and chemical composition for further testing. Table 12 shows the percent glucose uptake values for the two guava leaf extracts when combined in three different ratios i.e., 25:75, 50:50, 75:25 and compared to 100% of each extract. There is a dose-dependent increase in glucose uptake with both extracts alone. The combination of the extracts did not have activity greater than the two alone. 70% ethanol and 100% ethanol extract alone shows almost similar glucose uptake capabilities. 70% ethanol extract is selected for the final formulation. L6 myoblasts were incubated for 24h with different combinations of the two guava leaf extracts (70% ethanol and 100% ethanol) with a final concentration of 200 pg / ml. Glucose uptake is measured in these cells using 2-NBDG. Each colored line in the graphs corresponds to the extract listed in the legend. Data is representative of two separate experiments. n=6.

[0132] FIG. 11 illustrates an effect 1100 of guava leaf extract on glucose uptake 1102 in insulin-sensitive (IR-) 1104 and insulin-resistant (IR+) L6 myoblast cells 1106, according to an embodiment of the present invention. 70% ethanol is selected for the final formulation as it has ablend of polar-soluble and non-polar soluble active molecules with antioxidant abilities, mid-range concentrations have lower toxicity, and higher glucose uptake ability in myocytes relative to the other extracts. The 70% ethanol extract is additionally tested for its impact on insulin resistant L6 myoblasts. Different concentrations of the extract were tested in insulin-sensitive (IR-, light colored bars) and insulin-resistant (IR+, dark colored bars) L6 myoblasts. Four concentrations (25, 50, 75, lOOpg / ml) of the extract were selected based on literature data on efficacious doses as well as maximum tolerated doses. There is a dose-dependent increase in glucose uptake with the extract in insulin sensitive and resistant cells. The concentration selected for the final formulation is 70pg / ml. Individually, all the herbs in the formulation positively impact glucose utilization, and the cumulative effect of higher doses of the herbs could lead to hypoglycemia, hence the mid-range doses were selected for the final formulation. The asterix marked bar demonstrates the concentration chosen for the final formulation. Cells were pretreated with 0.5 pM dexamethasone (DXM) or not for 24h, followed by treatment with the extract present in the formulation at four separate concentrations i.e., 25, 50, 75, lOOpg / ml which is shown in ascending order of the four bars. The cells were stimulated with 1 OOnM insulin followed by glucose uptake measurement using 2-NBDG. All experiments were performed twice independently, each in triplicate. The percentage of glucose uptake is shown as the mean ±SE (N = 6 in all plots).

[0133] FIG. 12 illustrates HPLC chromatogram 1200 of jamun seed extraction process in five separate solvents, according to an embodiment of the present invention. Jamun seed is extracted in five separate solvents which includes water, ethanol, 70% ethanol, ethyl acetate and hexane selected on the basis of polarity. They were tested for toxicity (MTT assay), antioxidant activity (FRAP assay) and glucose uptake (2-NBDG). The toxicity and glucose uptake studies were carried out in HepG2 cells and L6 myoblasts, respectively, at extract concentrations of 5, 25, 50, 75, I OOpg / ml for 24h.

[0134] FIG. 13A shows an impact 1300 of jamun seed extracts on cellular viability by measurement using the MTT assay, according to an embodiment of the present invention. As shown in Figure 13 A, hexane, ethyl acetate and 70% ethanol extracts had cell viability >69% at concentrations lower than 1 OOpg / ml. Cells treated with 70% ethanol extract had cellular viability 1302 of ~85 ±0.01% at concentrations 1304 below 75pg / ml. HepG2 cells were treated with jamun seed extracts at concentrations of 5, 25, 50, 75, 100 pg / ml for 24h, followed by measurement of cell viability using the MTT assay.

[0135] FIG. 13B shows an impact 1306 of jamun seed extracts on glucose uptake by measurement using 2-NBDG, according to an embodiment of the present invention. Incubation ofthe myoblasts with the extracts led to a dose-dependent increase in glucose uptake 1308 with the highest glucose uptake levels displayed by 70% ethanol extract, which upregulated glucose uptake by -40% at 75pg / ml. L6 myoblasts were incubated for 24h with jamun seed extracts at concentrations 1310 of 5, 25, 50, 75, 100 pg / ml. Glucose uptake 1308 is measured in these cells using 2-NBDG. Each coloured line in the graphs corresponds to the extract listed in the legend. Data is representative of triplicates in two separate experiments. n=6TABLE 13

[0136] The antioxidant ability of each extract is measured using the FRAP assay. The extracts were reconstituted in 50% DMSO and directly used in the assay. Table 13 shows the FRAP activity (ferric ion reducing ability antioxidant power) for each jamun seed extract. The antioxidant ability of the extracts were in the following order: Ethyl acetate > Hexane > Water > Ethanol > 70% ethanol.TABLE 14

[0137] 70% ethanol and water extracts were selected based on yield, glucose uptake ability and chemical composition for further testing. Table 14 shows the percent glucose uptake values for the two jamun seed extracts when combined in three different ratios i.e., 25:75, 50:50, 75:25 and compared to 100% of each extract. Water and 70% ethanol extracts alone along with the combinations 75:25 and 25:75 shows similar glucose uptake capacity. There is a dose-dependent increase in glucose uptake with both extracts alone. As the combination of the extracts did not have activity greater than the two alone. 70% ethanol is selected for the final formulation. L6 myoblasts were incubated for 24h with different combinations of the two jamun seed extracts (70% ethanol and water) with a final concentration of 200 pg / ml. Glucose uptake is measured in these cells using 2-NBDG. Each coloured line in the graphs corresponds to the extract listed in the legend. Data is representative of two separate experiments. n=6.

[0138] From these studies, 70% ethanol is selected for the final formulation as it has both polar-soluble and non-polar soluble active molecules, and is a relatively safe extract with cellularviability of -70% at concentrations of l OOpg / ml. Glucose uptake trend for 70% ethanol extract- treated cells was greater than the other extracts. Jamun fruit is included as raw powder as well as seed extract to maximize the antidiabetic properties of the whole fruit.

[0139] FIG. 14 illustrates an effect 1400 of Jamun fruit extract and jamun seed extract on glucose uptake 1402 in insulin-sensitive (IR-) and insulin- resistant (IR+) L6 myoblast cells, according to an embodiment of the present invention. 70% ethanol extract is further tested for glucose uptake effect in insulin-sensitive and insulin-resistant myoblasts. Jamun fruit is also extracted in 70% ethanol and tested individually for its impact on glucose uptake in insulin-sensitive (IR-, light colored bars) and insulin-resistant (IR+, dark colored bars) L6 myoblasts. Four concentrations for each [(10, 30, 75, 150pg / ml- jamun fruit 1404), (5, 10, 20, 40pg / ml- jamun seed 1406)] were selected based on literature data on efficacious doses as well as maximum tolerated doses. There is a dose-dependent increase in glucose uptake with both the extracts in insulin sensitive and resistant cells. The concentration selected for the final formulation was 30pg / ml for jamun fruit and lOpg / ml for jamun seed. The concentration selected for the final formulation has been marked with asterix (*). Cells were pretreated with 0.5 pM dexamethasone (DXM) or not for 24h, followed by treatment with the jamun seed and fruit extract at four separate concentrations [(10, 30, 75, 150pg / ml- jamun fruit), (5, 10, 20, 40pg / ml- jamun seed) shown in ascending order of the four bars). The cells were stimulated with 100 nm insulin followed by glucose uptake measurement using 2- NBDG All experiments were performed twice independently, each in triplicate. The percentage of glucose uptake is shown as the mean ±SE (N = 6 in all plots).

[0140] Tulsi is extracted in five separate solvents selected on the basis of polarity which includes water, ethanol, 70% ethanol, ethyl acetate and hexane. The extracts were tested for toxicity (MTT assay), antioxidant activity (FRAP assay) and glucose uptake (2-NBDG). They were tested for toxicity and glucose uptake in HepG2 cells and L6 myoblasts, respectively, at concentrations of 5, 25, 50, 75, lOOpg / ml. The cells were incubated with the extracts for 24h followed by measurement of cell viability and glucose uptake.

[0141] FIG. 15A illustrates an impact 1500 of tulsi extracts on cellular viability 1502 by measurement using the MTT assay, according to an embodiment of the present invention. As shown in Figure 15 A, Water and ethyl acetate extracts exhibited a better toxicity profile than ethanol and hexane extracts. Water extract treated cells had cellular viability >67% at concentrations 1504 lower than lOOpg / ml. HepG2 cells were treated with tulsi extracts at concentrations of 5, 25, 50, 75, 100 pg / ml for 24h, followed by measurement of cell viability using the MTT assay.

[0142] FIG. 15B illustrates an impact 1506 of tulsi extracts on glucose uptake by measurement using 2-NBDG, according to an embodiment of the present invention. Myoblasts treated with tulsi extracts exhibited a dose-dependent increase in glucose uptake. Water extract increased the glucose uptake ability of the cells by -30% at 75pg / ml. L6 myoblasts were incubated for 24h with tulsi extracts at concentrations 1510 of 5, 25, 50, 75, 100 pg / ml. Glucose uptake 1508 is measured in these cells using 2-NBDG. Each colored line in the graphs corresponds to the extract listed in the legend. Data is representative of triplicates in two separate experiments. n=6TABLE 15

[0143] The antioxidant ability of each extract is measured using the FRAP assay. The extracts were reconstituted in 50% DMSO and directly used in the assay. Table 15 shows the FRAP activity (ferric ion reducing ability antioxidant power) for each tulsi extract.

[0144] FIG. 16 illustrates an effect 1600 of tulsi extract on glucose uptake in insulin-sensitive (IR-) and insulin-resistant (IR+) L6 myoblast cells, according to an embodiment of the present invention. Water extract of tulsi is selected for the final formulation. Tulsi extracted in water is tested for its impact on glucose uptake 1602 in insulin-sensitive (IR-, light colored bars) and insulinresistant (IR+, dark colored bars) L6 myoblasts. Four concentrations 1604 for tulsi (5, 20, 40, lOOpg / ml) were selected based on literature data on efficacious doses as well as maximum tolerated doses. There is a dose-dependent increase in glucose uptake with the extract. The asterix (*) marked bar demonstrates the concentration chosen for the final formulation. Cells were pretreated with 0.5pM dexamethasone (DXM) or not for 24h, followed by treatment with tulsi extract at four separate concentrations (5, 20, 40, lOOpg / ml- data shown in ascending order of the four bars). The cells were stimulated with 100 nm insulin followed by glucose uptake measurement using 2-NBDG. All experiments were performed twice independently, each in triplicate. The percentage of glucose uptake is shown as the mean ±SE (N = 6 in all plots).

[0145] FIG. 17 illustrates HPLC chromatograms of daruharidra root extracts 1700 in five separate solvents, according to an embodiment of the present invention. Daruharidra is extracted in five separate solvents including water, ethanol, 70% ethanol, ethyl acetate and hexane. The extracts were tested for toxicity (MTT assay), antioxidant activity (FRAP assay) and glucose uptake (2- NBDG). Daruharidra extracts were tested for toxicity and glucose uptake in HepG2 cells and L6 myoblasts, respectively, at concentrations of 5, 25, 50, 75, lOOpg / ml for 24h.

[0146] FIG. 18A illustrates an impact 1800 of daruharidra extracts on cellular viability 1802 by measurement using the MTT assay, according to an embodiment of the present invention. As shown in Figure 18 A, Cells treated with either of the extracts at concentrations 1804 lower than 50pg / ml had cell viability >75%. HepG2 cells were treated with daruharidra extracts at concentrations of 5, 25, 50, 75, 100 pg / ml for 24h, followed by measurement of cell viability using the MTT assay.

[0147] FIG. 18B illustrates an impact 1806 of daruharidra extracts on glucose uptake by measurement using 2-NBDG, according to an embodiment of the present invention. Myoblasts treated with daruharidra extracts exhibited a dose-dependent increase in glucose uptake 1808. 70% ethanol extracts increased glucose uptake by -38% at 50pg / ml. L6 myoblasts were incubated for 24h with daruharidra extracts at concentrations 1810 of 5, 25, 50, 75, 100 pg / ml. Glucose uptake is measured in these cells using 2-NBDG. Each colored line in the graphs corresponds to the extract listed in the legend. Data is representative of triplicates in two separate experiments. n=6TABLE 16

[0148] The antioxidant ability of each extract is measured using the FRAP assay. The extracts were reconstituted in 50% DMSO and directly used in the assay. Table 16 shows the FRAP activity (ferric ion reducing ability antioxidant power) for each daruharidra extract. The antioxidant ability of the extracts were in the following order: Ethyl Acetate > 70% ethanol > Hexane, Ethanol > Water.TABLE 17

[0149] 70% ethanol and 100% ethanol extracts were selected based on yield, glucose uptake ability and chemical composition for further testing. Table 17 shows the percent glucose uptake values for the two daruharidra extracts when combined in three different ratios i.e., 25:75, 50:50, 75:25 and compared to 100% of each extract. 100% ethanol and 70% ethanol extracts alone along with the combinations 75:25 and 25:75 shows similar glucose uptake capacity. There is a dosedependent increase in glucose uptake with both extracts alone. As the combination of the extracts did not have activity greater than the two alone. 70% ethanol is selected for the final formulation. L6 myoblasts were incubated for 24h with different combinations of the two daruharidra extracts (70% ethanol and 100% ethanol) with a final concentration of 200 pg / ml. Glucose uptake is measured in these cells using 2-NBDG. Each colored line in the graphs corresponds to the extract listed in the legend. Data is representative of two separate experiments. n=6.

[0150] FIG. 19 illustrates an effect 1900 of daruharidra extract / berberine on glucose uptake 1902 in insulin-sensitive (IR-) and insulin-resistant (IR+) L6 myoblast cells 1904, according to an embodiment of the present invention. From these studies, 70% ethanol is selected for the final formulation as it has both polar-soluble and non-polar soluble active molecules, and is a relatively safe extract with cellular viability of -70% at concentrations of 50pg / ml. Glucose uptake trend for 70% ethanol extract-treated cells is greater than the other extracts. Daruharidra extracted in 70% ethanol is tested for its impact on glucose uptake in insulin-sensitive (IR-, light colored bars) and insulin-resistant (IR+, dark colored bars) L6 myoblasts. Four concentrations for daruharidra (5, 20, 40, lOOpg / ml) were selected based on literature data on efficacious doses as well as maximum tolerated doses. There is a dose-dependent increase in glucose uptake with the extract. The asterix (*) marked bar demonstrates the concentration chosen for the final formulation. Cells were pretreated with 0.5 pM dexamethasone (DXM) or not for 24h, followed by treatment with daruharidra extract at four separate concentrations ((5, 20, 40, lOOpg / ml- data shown in ascending order of the four bars). The cells were stimulated with 100 nm insulin followed by glucose uptake measurement using 2-NBDG. All experiments were performed twice independently, each in triplicate. The percentage of glucose uptake is shown as the mean ±SE (N = 6 in all plots).

[0151] According to the invitro validation, guava leaf powder extracted in 75% ethanol, jamun seed extracted in 70% ethanol, tulsi extracted in water, daruharidra extracted in 70% ethanol were selected based on chemical analyses and in vitro experiments and also guava leaf powder in the raw herb form amun fruit as raw powder, bitter gourd as raw herb powder, and amla as raw herb powder were used in the final formulation.TABLE 18

[0152] Table 18 shows the ratios of botanical raw material ratios used in the trials. After finalizing the extraction methods, five combinations of the ingredients with varying ratios were prepared as shown in Tables 19 and 20 and after reviewing the chemical ingredient content and their in-vitro results one combination is selected which gave the best results. The ingredients were mixed in the ratios specified and the mixture was extracted with 70% ethanol-water for in-vitro and chemical assays.

[0153] Table 19 shows the assay results of trials for finalizing the botanical raw material ratios in the formulation.TABLE 19

[0154] FIG. 20 A illustrates a graph 2000 which represents glucose uptake 2002 measured in insulin-resistant L6 myoblasts in the presence of each of the five formulations 2004, according to an embodiment of the present invention. Metformin was used as a positive control in this study. As shown in Figure 10, both with and without insulin stimulation Tar.O and Tar.l increased glucose uptake in the insulin-resistant and insulin-sensitive cells. Tar.O was finalized for further analyses, which will be referred to as Targlut.

[0155] FIG. 20B illustrates a graph 2006 which represents glucose uptake 2008 measured in insulin-sensitive L6 myoblasts in the presence of each of the five formulations 2010, according to an embodiment of the present invention. Cells pretreated with (A) DXM (Insulin-Resistant) or not (B) (Insulin-Sensitive), followed by treatment with 200 pg / ml of the five Targlut (TAR) formulations (TAR.0, TART, TAR.2, TARA, TARA) in the presence of 100 nM insulin (solid bars) or not (shaded bars). Treatment with 0.2 pg / ml Metformin (MET) served as the positive control. All experiments were performed twice independently, each in triplicate. The percentage of glucose uptake is shown as the mean ±SE (N = 6 in A, B).

[0156] FIG. 21 illustrates a flow chart 2100 on soft targeting approach of polymolecular formulation on diabetes pathway, according to an embodiment of the present invention. FIG. 21 illustrates the soft target approach 2102 of the polymolecular formulation 102 on diabetes pathway. Gut being the first site of glucose management, where GLP-1 promotes absorption of glucose from the gut and amylase mediates carbohydrate breakdown into glucose. The polymolecular formulation exhibited an -20% inhibition in the amylase activity in an in vitro assay, whereby Acarbose 2108, a potent amylase inhibitor and commonly used antidiabetics in the market showed -90% amylaseinhibition highlighting the ‘soft targeting’ action of the polymolecular formulation 102. In addition, the polymolecular formulation 102 action on the gut 2112 shows -15% clearance of blood glucose resulting in a 15% reduction in insulin demand. Moreover, the liver also plays an instrumental role in glucose buffering and its function gets impaired in diabetics. The polymolecular formulation 102 facilitated a -30% improvement in liver function 2222 (in vivo) along with a -30% enhanced glucose uptake by liver cells (in vitro), which allows 20% glucose clearance 2224 from the system and a 35% lower insulin demand 2226. Cumulatively, better glucose clearance by the gut and liver will ameliorate the burden on the pancreas for insulin supply by -35% 2226. Pancreas in the STZ- induced rat model showed a three-fold reduction in oxidative stress 2228 with the polymolecular formulation 102 treatment for 21 days, indicating possible beta cell repair. Improved pancreatic health along with decreased insulin demand enables the phenotypic flexibility of the organ to be restored. The peripheral tissues, on the other hand, which include adipose and muscle cells experienced a two-fold improvement in insulin resistance 2236 along with a 30% increase in glucose uptake in in vitro studies 2234. Hence, the increased receptiveness of the muscle and adipose cells to insulin further contributes to a lower burden on the pancreas. This is in sharp contrast to the 85% increment 2238 in glucose uptake 2232 by metformin (in vitro), which is a commercially available glucose suppressant and can confuse the body’s glucose maintenance 2242 mechanisms and trigger the glucose production pathways. Hence, the mild effect of the polymolecular formulation 102 on the diabetic pathways exhibits glucose maintenance 2242 and homeostasis, thereby reducing stress on the glucose pathways, augmenting phenotypic flexibility and possibly preventing diabetes progression.

[0157] Therefore, the in vitro and in vivo studies demonstrated that the polymolecular formulation 102 increases glucose metabolism in the cells. The polymolecular formulation 102 has a significant effect on lowering blood glucose levels, improving liver and kidney function as well as maintaining the cellular antioxidant levels for the amelioration of the symptoms of diabetes. The polymolecular formulation treatment at both tested doses was found to be effective in promoting an anti-hyperglycemic effect with evidence of no severe degeneration in the pancreas when compared with the diabetic group. All this data supports that the polymolecular formulation 102 can be a suitable treatment option for the management and possible prevention of diabetes progression.

[0158] Further, although the present invention has been described herein with reference to particular means, materials, embodiments, techniques, and implementations, the present invention is not intended to be limited to the particulars disclosed herein; rather, the present invention extendsto all functionally equivalent structures, methods and uses, such as are within the scope of the appended claims.

[0159] It will be understood by those skilled in the art, having the benefit of the teachings of this specification, that the present invention is capable of modifications and other embodiments may be affected and changes may be made thereto, without departing from the scope and spirit of the present invention.G) ADVANTAGES OF THE INVENTION:

[0160] The present invention provides a polymolecular formulation and method of preparation thereof. The polymolecular formulation of the present invention serves as an effective alternative for metformin as it would function as a glucose maintenance drug rather than a suppressant. Maintenance of glucose levels in the blood would impose less burden on the pancreas to release insulin and combined with the ability of the polymolecular formulation to repair pancreatic damage due to its high antioxidant potential can provide a complementary approach towards improving diabetic symptoms. In addition, the polymolecular formulation of the present invention, adopts a similar scientific molecular development rigour as any pharmaceutical drug development. However, the polymolecular formulation of the present invention differs from conventional drugs in two ways, the first being the source and composition of the drug whereby conventional pharmaceutical drugs are synthetic molecules manufactured in the lab whereas the polymolecular formulation of the present invention is derived from a combination of herbs having same or similar in vitro and in vivo characteristics relevant to the diabetes pathways. The second distinguishing factor of the polymolecular formulation of the present invention is the mechanism of action where the molecular basis for efficacy is distributed over multiple complementary and related molecular targets, causing an additive or aggregate impact that matches and may surpass those of pharmaceutical candidates, which primarily focus on obliterating a single molecular pathway relevant to the disease. Since the activity of the polymolecular formulation of the present invention is distributed over several complementary domains, the potential for toxicity related to selective suppression of only one key molecular pathway is significantly reduced. Therefore, the polymolecular formulation of the present invention is a composite pharmacology that acts as soft targeting, wherein each of the molecular pathways targeted, will be mildly impacted without radically disrupting physiology. In addition, the synthetic molecules used in pharmaceutical drugsare substituted with molecules sourced from natural sources in the present invention. Since the polymolecular formulation of herbs is prone to tremendous variation and bio-availability, the polymolecular formulation of the present invention uses a distinct differentiated process of preparation specific to each ingredient to guarantee qualitative and quantitative molecular consistency in the formulation enabling scalability.

[0161] Furthermore, the polymolecular formulation comprising naturally occurring ingredients serves to design a drug specific for diabetes that targets multiple pathways of the disease. Besides, the polymolecular formulation acts as a precision medicine, specific for the physiological manifestations of the disease presented. The polymolecular formulation also serves as a tailored response or dose modulation, where the doses are tailored to achieve the therapeutic response that will vary from individual to individual promoting personalized medicine for the management of type 2 diabetes. Hence, the mechanism of action of the polymolecular formulation is glucose maintenance rather than glucose suppression. The polymolecular formulation of the present invention may have the ability to prevent insulin resistance and to the best of our knowledge, no other drug has not been able to achieve it.

[0162] Moreover, as diabetes causes damage to multiple organs and is a complex disease with various disruptive inflammatory and oxidative effects, the polymolecular formulation of the present invention mildly affects each pathway rather than obliterating a single pathway. When a single pathway is targeted, it becomes essential to hit it hard for efficacious results. The mild targeting approach using the polymolecular formulation ensures long-term use safety and provides a reparative rather than a disruptive effect. Besides, the polymolecular formulation provides a delicate balance in glycemic control by orchestrating glucose absorption, storage and production, as the gut is the first site of glucose management in the body. Guava leaves and bitter gourd in the polymolecular formulation have been shown to enhance gut absorption of glucose through the activation of GLP-1 (Glucagon-like peptide 1) and inhibition of amylase. GLP-1 analogues and acarbose are commonly used anti-diabetics. Taking these roles of guava leaves and bitter gourd into consideration we can assume that the polymolecular formulation facilitates an ~15% reduction in blood glucose due to increased gut absorption, resulting in a 15% reduction in insulin demand. Additionally, GLP-1 signals insulin production in the pancreas and reduces glucose production as well as increases glycogen storage in the liver, similar to insulin. The liver plays a critical role in buffering the glucose levels in the blood. The competence of the liver to handle sugar is determined by factors such as inflammation, oxidative stress, etc. Vitamin C and polyphenols from Amla, in thepolymolecular formulation, provide potent antioxidative actions and the in vivo studies of the polymolecular formulation of the present invention showed improved pancreatic and liver histology, function and oxidative markers. Hence, the improved functioning of the liver by polymolecular formulation of the present invention can further reduce the need for insulin by -20%, and ameliorate the burden on the pancreas for insulin supply by -35%. Therefore, the reduced burden on the pancreas in combination with the improved health of the pancreas with the polymolecular formulation results in strengthening of the pancreas.

[0163] Furthermore, in a diabetic state, the peripheral tissue is ineffective in glucose clearance due to insulin resistance and in turn, reduced glucose uptake. Berberine has been shown to improve insulin resistance, which is present in the polymolecular formulation of the present invention. With respect to this action, the polymolecular formulation of the present invention helps in improving glucose uptake in insulin-resistant skeletal muscle and liver cells as evidenced in in vitro studies both in the presence and absence of insulin. This, can account for -30% of glucose utilization by the body thereby reducing the signal to the pancreas for more and more insulin production. Therefore, a 2-fold improvement in insulin resistance as shown in in vitro studies of the polymolecular formulation of the present invention, a 35% reduction in insulin need, 30-40% increased glucose utilization and a constant lowering of inflammation and oxidative stress will help in better management of diabetes and as a result, might lead to a prevention of the disease.

[0164] Guava leaf has polysaccharides and polyphenols including quercetin, catechin, guaijaverin, ellagic acid, rutin, avicularin, etc, which have anti-diabetic, anti-lipidemic, antioxidant and anti-inflammatory properties. In vitro and in vivo studies have shown that catechin has anti- glycative properties and inhibits GLUT-2 mediated intestinal glucose uptake, whereas guaijaverin and quercetin inhibit DPP-IV (Dipeptidyl peptidase IV, an enzyme that blocks incretin) and a- glucosidase, a-amylase activity as well as upregulate glucose uptake in hepatocytes. Long-term administration of guava leaf extract in diabetic animals reduces fasting plasma glucose levels, improves the insulin sensitivity of target tissues along with increasing glycogen synthesis in hepatocytes. The guava leaf extract in this formulation is used to achieve intestinal absorption reduction of glucose via inhibition of carbohydrate hydrolyzing enzyme, increase glycogen synthesis and storage as well as increase glucose uptake in the liver and inhibit DPP-IV activity to block degradation of incretins including GLP-1.

[0165] Jamun seed and fruit have anti-diabetic, anti-lipidemic, antioxidant properties which are attributable to the phytochemicals including terpenoids, glycosides, saponins, flavonoids, phenols, anthocyanins, etc. Multiple in vitro and in vivo studies have shown improvement in diabetic pathways with jamun seed and jamun fruit administration. Similar to glibenclamide, jamun seed shows early signs of islet regeneration with a concomitant increase in islet number and increased insulin secretion compared to untreated diabetic rats. The hypolipidemic effects of jamun seed in STZ-treated rats could be attributable to the presence of saponins, glycosides, flavonoids, triterpenoids, which are known to activate PPARY and PPARa. Another pathway for glucose regulation by jamun seed is the inhibition of a-glucosidase and a-amylase activity, confirmed in in vitro studies. A glycoside, Jambolin in jamun has been shown to prevent conversion of starch to glucose thereby reducing the plasma glucose levels. Jamun fruit pulp have cardioprotective effects along with significantly alleviating diet-induced obesity and insulin resistance by improving diet- induced gut dysbiosis. In addition, it is a good source of antioxidants due to its high anthocyanin content. Both jamun fruit and seed extract in this formulation are used to promote pancreatic repair, beta cell regeneration and improve insulin production. They also helps in management of postprandial glucose control by inhibition of a-glucosidase and a-amylase enzymes and Hypolipidemic actions.

[0166] Bitter gourd’s antidiabetic properties have been supported and reinforced by multiple in vitro, in vivo and clinical studies. Momordicin, momordicosides, charantin, vicine, polypeptide-p (insulin-like structure) are the main constituents in bitter gourd responsible for the regulation of glucose uptake pathways. Bitter gourd has been shown to improve glucose uptake by cells and aid in lowering fasting glucose levels by reducing the levels of hepatic gluconeogenic enzymes glucose- 6-phosphatase and fructose- 1,6-bisphosphatase. Three insulin-like compounds in bitter gourd have been recorded in the literature that can increase glycogen storage in the liver by inactivating glycogen synthase (GS). Bitter gourd has high fiber content in the form of soluble fiber pectin, which aids in diabetes and lipid management. High zinc levels in bitter gourd promotes insulin secretion and glucose utilization. Bitter gourd extract has also been shown to increase GLP-1 secretion in enteroendocrine cells. Bitter gourd has been used as a whole herb in this formulation to provide fiber in addition to its other antidiabetic phytochemicals. Most of the clinical studies with bitter gourd also used it in the raw herb powder form. Bitter gourd in this formulation is used to reduce plasma glucose levels and gluconeogenesis, it will increase incretin effect and GLP-1 and also increase glycogen synthesis and storage in the liver.

[0167] Amla has the highest levels of naturally occurring heat stable Vitamin C of any ripe fruit in the world. It exerts antidiabetic effects through its antioxidant and free radical scavenging properties. The polyphenol compounds and fiber content in amla contributes to its lipid lowering and anti-glycation properties. Ellagic acid derivatives have the ability to lower a-glucosidase and a- amylase activity. Amla is used in the whole herb form in the formulation to achieve Lipid lowering and to reduce inflammation, advanced glycated end products (AGEs) and oxidative stress.

[0168] Tulsi has antioxidant, immunomodulatory, cardioprotective, anti- inflammatory, antidiabetic and hepatoprotective properties which are attributable to eugenol, rosmarinic acid, and ursolic acid. Ursolic acid and rosmarinic acid have been shown to reduce cortisol levels. Ursolic acid reduces cortisol levels by blocking the CRHR1 receptor in the anterior pituitary that is responsible for the synthesis and release of cortisol. In the formulation, tulsi serves as an adaptogen to reduce stress and related cortisol levels, to reduce inflammation and oxidative stress.

[0169] Berberine is a stable quaternary benzylisoquinoline alkaloid present in multiple commonly used medicinal plants including Coptis chinesis, Daruharidra or Berberis aristata. Berberine displays anti-diabetic properties in in vitro and in vivo studies in addition to its antiobesity, hepatoprotective, anti-inflammatory and anti-microbial properties. The anti-diabetic actions of berberine include increasing insulin sensitivity by increasing the expression of GLUTs, alleviation of insulin resistance, inhibiting gluconeogenesis in the liver, modulating the gut microbiota (by increasing the ratio of Bacteroidetes: Firmicutes and proportion of SCFA-producing bacteria), promoting intestinal GLP-1 secretion, reducing intestinal glucose absorption and lowering inflammation and oxidative stress. Berberine also exhibits antihyperlipidemic properties and has been shown to reduce total cholesterol, total triglycerides and LDL by increasing the expression of hepatic LDL receptors. Daruharidra is used in the formulation to achieve improved insulin resistance and augmented glucose utilization via GLUT1 and GLUT4, increased GLP-1 secretion and inhibition of DPP4 and modulation of the gutmicrobiota.

[0170] The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and / or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments.

[0171] It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within scope of the appended claims.

[0172] Although the embodiments herein are described with various specific embodiments, it will be obvious for a person skilled in the art to practice the invention with modifications. However, all such modifications are deemed to be within the scope of the claims. It is also to be understood that the following claims are intended to cover all of the generic and specific features of the embodiments described herein and all the statements of the scope of the embodiments which as a matter of language might be said to fall there between. The scope of the embodiments of the present invention is ascertained by the claims to be submitted at the time of filing the complete specification.

Claims

CLAIMSWe Claim:

1. A polymolecular formulation for preventing diabetes progression, the polymolecular formulation comprising synergistically effective proportions of:(i) a whole herb, and(ii) extracts comprising guava leaf powder at a concentration of 175 mg, guava leaf extract at a concentration of 28 mg, jamun fruit powder at a concentration of 1 OOmg, jamun seed extract at a concentration of 20mg, bitter gourd fruit powder at a concentration of 150 mg, amla fruit powder at a concentration of 100 mg, tulsi leaf extract at a concentration of 20 mg, and Daruharidra root extract at a concentration of 4 mg.

2. The formulation of claim 1, wherein the guava leaf extract is obtained by 75% ethanol / water extraction method.

3. The formulation of claim 1, wherein jamun seed extract is obtained by 70% ethanol / water extraction method.

4. The formulation of claim 1, wherein Tulsi leaf extract is obtained by water extraction method.

5. The formulation of claim 1 , wherein Daruharidra root extract is obtained by 70% ethanol / water extraction method.

6. The formulation of claim 1 , wherein the whole herb and the extracts are in the ratio of 1 : 1 at the raw material stage.

7. The formulation of claim 1, further comprising one or more pharmaceutical excipients comprising at least one of: povidone K 30, microcrystalline cellulose powder, dicalcium phosphate, cross carmellose sodium, magnesium stearate, silicon dioxide and coating materials comprising opadry white and opadry maroon.

8. The formulation of claim 1, wherein the polymolecular formulation is prepared in the form of a tablet or a capsule.

9. The formulation of claim 1, wherein the active ingredients of the polymolecular formulation are effective against multiple diabetic pathways.

10. The formulation of claim 1 , wherein the polymolecular formulation helps in amelioration of the disease phenotype and additionally a prevention of insulin resistance / diabetes progression.

11. A method of preparation of a polymolecular formulation for preventing diabetes progression, the method comprising: combining synergistically effective proportions of a whole herb and extracts comprising guava leaf powder at a concentration of 175 mg, guava leaf extract at a concentration of 28 mg, jamun fruit powder at a concentration of lOOmg, jamun seed extract at a concentration of 20mg, bitter gourd fruit powder at a concentration of 150 mg, amla fruit powder at a concentration of 100 mg, tulsi leaf extract at a concentration of 20 mg, and Daruharidra root extract at a concentration of 4 mg.

12. The method of claim 10, comprising extracting of guava leaf powder for preparation of the polymolecular formulation by: mixing Guava leaf powder at a concentration of 5 gm with 100 ml of 75% Ethanol / Water in a 250ml round bottom flask; heating the mixture at a temperature of 80o C for 6 hours with reflux condenser and stirring the mixture at 400 rpm; transferring the mixture into falcon tubes and centrifuging at 5000 rpm for 10 minutes; filtering the supernatant obtained after centrifuge using filter paper and discarding the residue; removing the ethanol from the filtrate on rotavapor under vacuum at 36 degree Celsius and removing the remaining water on a freeze dryer; and storing the extract obtained in an airtight container at 4 degree Celsius till further use.

13. The method of claim 10, comprising extracting of Jamun seed for preparation of the polymolecular formulation by: mixing Jamun seed powder at a concentration of 5 gm with 100 ml of 70% ethanol / water in a 250ml round bottom flask and heating the mixture at 60 degree Celsius for 3 hours with reflux condenser under stirring at 400 rpm; transferring the contents of the flask into falcon tubes and centrifuged at 5000 rpm for 10 minutes; obtaining supernatant after centrifuge is filtered using filter paper and discarding the residue; removing Ethanol from the filtrate on rotavapor under vacuum at 36 degree Celsius removing remaining water on a freeze dryer; storing the extract obtained in an airtight container at 4degree Celsius for subsequent use.

14. The method of claim 10, comprising extracting of Tulsi leaf for preparation of the polymolecular formulation by: mixing Tulsi leaf powder at a concentration of 5gm with 100 ml of distilled water in a 250ml round bottom flask and stirring the mixture at room temperature for 23 hours at 500 rpm; transferring the contents of the flask into falcon tubes and centrifuging at 5000 rpm for 10 minutes; filtering supernatant obtained after centrifuge using filter paper and discarding the residue; and removing the solvent from the filtrate on freeze dryer, obtaining the extract and storing in an airtight container at 40 degree Celsius for subsequent use.

15. The method of claim 10, comprising extracting of Daruharidra root for preparation of the polymolecular formulation by: mixing about 5 gm of the Daruharidra root powder at a concentration of 5gm with 100ml of 70% Ethanol / Water in a 250ml round bottom flask and heating the mixture at 60 degree Celsius for 3 hours with reflux condenser under stirring at 400 rpm; transferring the heated mixture into falcon tubes and centrifuging at 5000 rpm for 10 minutes;filtering the supernatant obtained after centrifuge using filter paper and discarding the residue. removing Ethanol from the filtrate on rotavapor under vacuum at 36 degree Celsius and removing remaining water on freeze dryer till dry; and obtaining extract and storing in airtight container at 40 degree Celsius for subsequent use.

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