DESC:

FORM 2
The Patents Act 1970 (39 of 1970)
&
The Patent Rules 2003
COMPLETE SPECIFICATION
(See Section 10 and rule 13)

TITLE OF THE INVENTION:
A POLY HERBAL EXTRACT (PHE) FOR THE PREVENTION AND MANAGEMENT OF CHRONIC HYPERGLYCEMIA AND ASSOCIATED COMPLICATIONS AND ITS METHOD OF PREPARATION THEREOF

APPLICANT:
SARVOTHAM CARE LIMITED
#1-20-248, 1st Floor, Umajay Complex,
Rasoolpura, Secunderabad, Telengana, India – 500003

PREAMBLE OF THE DESCRIPTION:

THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED

CROSS- REFERENCE TO RELATED APPLICATION
This application claims the priority of the provisional application with serial number 202141049374 filed on 28th of Oct, 2021 with the title, “A POLY HERBAL EXTRACT (PHE) FOR THE PREVENTION AND MANAGEMENT OF CHRONIC HYPERGLYCEMIA AND ASSOCIATED COMPLICATIONS AND ITS METHOD OF PREPARATION THEREOF” and the contents of which is incorporated in entirety.

A) TECHNICAL FIELD OF INVENTION

[0001] The present invention generally relates to a poly herbal extract for the prevention and management of chronic hyperglycemia and associated complications.

B) BACKGROUND OF INVENTION

[0002] Hyperglycemia, a condition of blood glucose levels higher than 125 mg/dL while fasting, and greater than 180 mg/dL after 2 hours postprandial. The hyperglycemia could be due to reduced insulin secretion, either decreased utilization or increased production of glucose, excess food consumption, less physical activity, etc.
[0003] Whereas, the hyperglycemia is one of the diagnostic features of diabetes. Nonetheless the chronic diabetes confirmed through set of additional diagnostics viz. oral glucose tolerance test, HbA1C levels along with blood glucose levels. Glucose uptake by cells from the blood stream is facilitated by insulin. Inadequate insulin production by pancreas or inability of the body to use the produced insulin causes increased levels of glucose in the blood and damages to many of the body's systems, in particular to the blood vessels and the nerves.
[0004] About 422 million people worldwide have diabetes, the majority living in low-and middle-income countries, and 1.6 million deaths are directly attributed to diabetes each year. Both the number of cases and the prevalence of diabetes have been steadily increasing over the past few decades. There are two forms of diabetes, generally known as Type I diabetes and Type II diabetes. Both types of diabetes are complex diseases caused by mutations in more than one gene and by inherited and/or environmental factors
[0005] Type I diabetes or insulin-dependent diabetes mellitus (IDDM) is the form in which destruction of pancreatic beta cells results in failure to produce insulin, which is essential for survival. The etiology is unknown, but it is mostly classified as an autoimmune disorder resulting from an infection or toxic environmental insult in people whose immune system is genetically predisposed to develop a vigorous autoimmune response against pancreatic B cell antigens. This form develops most frequently in children and adolescents, and therefore was previously known as juvenile diabetes. However, it is being now increasingly noted in adults. Treatment of type I diabetes is based on daily administration of insulin.
[0006] Type II diabetes or non-insulin dependent diabetes mellitus (NIDDM) results when the pancreas produces insufficient amounts of insulin, or the body cannot properly utilize the insulin that is produced. This form is much more common and accounts for around 90% of all diabetes cases worldwide. It develops mostly in the adult age, but lately has been noted also in young people, even in adolescents. Diabetes type II is strongly familial, but environmental factors also play an important role in the development of the disease. Healthy diet, practice of physical exercise and avoidance of overweight may prevent the disease and/or control its progress. People with type II diabetes require oral agents for satisfactory blood glucose control, but about one third needs insulin for reducing their blood glucose levels.
[0007] Several complications are associated with chronic hyperglycemia which includes: (i) retinal and ophthalmic manifestations or dysfunctions such as retinopathy (damage to blood vessels of retina), glaucoma (increased fluid pressure in eye), cataract (retinal degeneration); (ii) neuropathy consisting of peripheral nerves damages, feet pains, feet low pulse and edemas, blood circulation dysfunction, walking difficulties and legs wounds; (iii) liver dysfunction consisting of hypercholesterolemia, proteinuria and hyperlipidemia; (iv) renal dysfunction or nephrotic syndrome consisting of lipiduria, hypercholesterolemia, hypoalbuminemia; edema, proteinuria, hyperlipidemia or micro albuminemia, prostate enlargement and ketones in urine; (v) hypertension; and (vi) cardiovascular disease ranging from coronary artery disease to stroke or renal vascular disease, consisting of arteriosclerosis, ischemic heart disease, valvular heart disease, disease of the heart muscle, arrythmias and noncoronary cardiovascular disease. People with diabetes, also develop neuropathy (damaged nerves) or peripheral vascular disease (blocked arteries) of the legs and either can lead to foot ulceration. Infection and foot ulceration, alone or in combination, often lead to amputation. Diabetes-related complications are a leading cause of death by the disease.
[0008] During development and progression of diabetes, many pathways are up-regulated to handle the overflow of glucose in the body. These pathways include the polyol-pathway, the glycation-pathway, the protein kinase-c pathway, the hexosamine pathway, and the enediol/alpha-ketoaldehyde pathway. In diabetes mellitus, the polyol pathway is highly active and consumes approximately 30% glucose in the body. This pathway contains two reactions catalyzed by aldose reductase (AR) and sorbitol dehydrogenase, respectively. Aldose reductase (AR) reduces glucose to sorbitol at the expense of NADPH, while sorbitol dehydrogenase converts sorbitol to fructose at the expense of NAD+, leading to NADH production. Consumption of NADPH, accumulation of sorbitol, and generation of fructose and NADH have all been implicated in the pathogenesis of diabetes and its complications.
[0009] Increased polyol pathway activity alters the redox state of the pyridine nucleotides NADP+ and NAD+, thus reducing their concentrations. Since these are important factors in many enzyme-catalyzed reactions, many other metabolic pathways may be also affected. The decreased concentration of these cofactors leads to decreased synthesis of reduced glutathione, nitric oxide, myoinositol, taurine; and the resultant oxidative stress indirectly increases formation of AGEs. Myoinositol is particularly required for the normal function of nerves. Sorbitol may also glycate nitrogen on proteins, such as collagen, producing AGE products.
[0010] The enhanced generation and accumulation of advanced glycation end-products (AGEs) have been linked to increased risk for macrovascular and microvascular complications associated with diabetes mellitus. AGEs may also activate specific receptors, like the receptor for AGEs (RAGE), which is present on the surface of all cells relevant to atherosclerotic processes, triggering oxidative stress, inflammation, and apoptosis. Such AGEs are known to affect the cells of kidney, ocular and nervous systems.
[0011] Hence there is a need to develop a composition that inhibits certain enzymes involved in polyol pathway, has anti-oxidant activity, and shows improved bioavailability in diabetic patients. There is a need to develop a poly herbal composition which provides above properties along with no side effects.
[0012] The present disclosure overcome the problems in the art and provides a poly-herbal composition for the prevention and management of chronic hyperglycemia and associated complications which shows synergistic effect and improved bioavailability in diabetic patients, without any side-effects.
[0013] The value additions and above-mentioned shortcomings, disadvantages and problems are addressed herein, as detailed below.

C) OBJECTIVE OF INVENTION

[0014] The primary objective of the present invention is to provide a poly herbal extract for the prevention and management of chronic hyperglycemia and associated complications.
[0015] Another objective of the present invention is to provide a poly herbal extract comprising of a hydro-alcoholic extract of dried powdered admixture of a stem or bark of Cinnamomum verum; a fruit of Emblica officinalis; a rhizome of Zingiber officinale and Curcuma longa; a root of Piper chaba or its combination thereof for treating chronic hyperglycemia and associated complications.
[0016] Yet another objective of the present invention is to provide a poly herbal extract which is absolutely free from synthetic chemicals.
[0017] Yet another objective of the present invention is to provide a poly herbal extract having synergistic effect with enhanced anti-oxidant activity and improved bioavailability in chronic hyperglycemic patients.
[0018] Yet another objective of the present invention is to provide simple and less-time consuming method of synthesizing a poly herbal extract for the prevention and management of chronic hyperglycaemia and associated complications.
[0019] These and other objectives 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 INVENTION
[0020] The various embodiments of the present invention provide a poly herbal extract for the prevention and management of chronic hyperglycemia and associated complications. The composition comprises a hydro-alcoholic extract of dried powdered admixture of a stem or bark of Cinnamomum verum; a fruit of Emblica officinalis; a rhizome of Zingiber officinale and Curcuma longa; a root of Piper chaba or its combination thereof.
[0021] According to an embodiment of the present invention, the stem or bark of Cinnamomum verum; the fruit of Emblica officinalis; the rhizome of Zingiber officinale and Curcuma longa; the root of Piper chaba is present in a ratio of 40:20:20:10:10 in the said powdered admixture.
[0022] According to another embodiment of the present invention, a method of synthesizing a poly herbal extract for the prevention and management of chronic hyperglycaemia and associated complications is provided. The method comprises: a) preparing a dried powdered admixture of a stem or a bark of Cinnamomum verum; a fruit of Emblica officinalis; a rhizome of Zingiber officinale and Curcuma longa; a root of Piper chaba or its combination thereof; b) preparing a hydro-alcoholic extract by soaking the dried powdered admixture in an aqueous alcohol; c) filtering the hydro-alcoholic extract and collecting the filtrate; d) repeating the step (b) and (c) thrice and mixing filtrates of each cycle to obtain a pooled solvent; e) concentrating the pooled solvent for removing the excess ethanol to obtain a concentrated extract; and f) lyophilizing the concentrated extract to obtain the final poly herbal extract.
[0023] According to an embodiment of the present invention, the concentration of the pooled solvent is performed at a temperature of 600C in the rota-evaporator.
[0024] According to an embodiment of the present invention, the stem or bark of Cinnamomum verum; the fruit of Emblica officinalis; the rhizome of Zingiber officinale and Curcuma longa; the root of Piper chaba is added in a ratio of 40:20:20:10:10 in the said powdered admixture.
[0025] According to an embodiment of the present invention, the dried powdered admixture is soaked in an aqueous alcohol for 24 hours in orbital shaker at 100 rpm.
[0026] According to an embodiment of the present invention, the ratio of ethanol to water is 70:30.
[0027] According to an embodiment of the present invention, the ratio of the dried powdered admixture to the solvent system is 1:8.
[0028] These and other aspects of the embodiments herein will be better appreciated and understood when considered in conjunction with the following description and 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.

E) BRIEF DESCRIPTION OF DRAWINGS

[0029] 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. The embodiments are described in sufficient detail to enable those skilled in the art to practice the embodiments and it is to be understood that the logical, mechanical, electronic and 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.
[0030] FIG. 1 is a schematic representation showing the steps involved in the preparation of a poly herbal extract for the prevention and management of chronic hyperglycemia and associated complications, according to an embodiment of the present invention.
[0031] FIG. 2 is a schematic representation showing the detailed process steps involved in the preparation of an aqueous poly herbal extract using water as the solvent system, according to an embodiment of the present invention.
[0032] FIG. 3 is a schematic representation showing the detailed process steps involved in the preparation of a hydro-alcoholic poly herbal extract using ethanol to water in ratio 70:30 as the solvent system, according to an embodiment of the present invention.
[0033] FIG. 4 is a schematic representation showing the detailed process steps involved in the preparation of a hydro-alcoholic poly herbal extract using ethanol to water in ratio 80:20 as the solvent system, according to an embodiment of the present invention.
[0034] FIG. 5 is a schematic representation showing the detailed process steps involved in the preparation of a hydro-alcoholic poly herbal extract using ethanol to water in ratio 30:70 as the solvent system, according to an embodiment of the present invention.
[0035] FIG. 6 demonstrates the graphical representation of the free radical scavenging activity between the poly-herbal extracts prepared using aqueous alcohol as solvent system with ratio of ethanol to water as 80:20, 70:30 and 30:70, according to an embodiment of the present invention.
[0036] FIG. 7 demonstrates the graphical representation of the lipid peroxidation activity between the poly-herbal extract prepared using aqueous alcohol as solvent system with ratio of ethanol to water as 80:20, 70:30 and 30:70, according to an embodiment of the present invention.
[0037] FIG. 8 demonstrates the graphical representation of the aldose reductase inhibition activity between the poly-herbal extracts prepared using aqueous alcohol as solvent system with ratio of ethanol to water as 80:20, 70:30 and 30:70, according to an embodiment of the present invention.
[0038] FIG. 9 demonstrates AGE fluorescence at UV wavelength of 440nm of the poly-herbal extracts with ratio of ethanol to water as 80:20, 70:30 and 30:70, according to an embodiment of the present invention.

F) DETAILED DESCRIPTION OF DRAWINGS

[0039] 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. The embodiments are described in sufficient detail to enable those skilled in the art to practice the embodiments and it is to be understood that the logical, mechanical, electronic and 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.
[0040] According to an embodiment of the present invention, a poly herbal extract for the prevention and management of chronic hyperglycemia and associated complications. The composition comprises a hydro-alcoholic extract of dried powdered admixture of a stem or bark of Cinnamomum verum; a fruit of Emblica officinalis; a rhizome of Zingiber officinale and Curcuma longa; a root of Piper chaba or its combination thereof.
[0041] According to an embodiment of the present invention, the said admixture comprises the dried pulverized coarse powder (20 to 30 mesh) of the stem or bark of Cinnamomum verum; the fruit of Emblica officinalis; the rhizome of Zingiber officinale and Curcuma longa; and the root of Piper chaba in the designated ratio.
[0042] According to an embodiment of the present invention, the said admixture more particularly comprises the dried pulverized coarse powder (20 to 30 mesh) of the stem or bark of Cinnamomum verum, dried pulverized coarse powder (20 to 30 mesh) of the fruits of Emblica officinalis, dried pulverized coarse powder (20 to 30 mesh) of the rhizomes of Zingiber officinale, dried pulverized coarse powder (20 to 30 mesh) of the roots of Piper chaba, and dried pulverized coarse powder (20 to 30 mesh) of the rhizomes of Curcuma longa in the ratio of 40:20:20:10:10.
[0043] FIG. 1 is a schematic representation showing the steps involved in the preparation of a poly herbal extract for the prevention and management of chronic hyperglycemia and associated complications, according to an embodiment of the present invention. With respect to FIG. 1, the method comprises: preparing a dried powdered admixture of a stem or a bark of Cinnamomum verum; a fruit of Emblica officinalis; a rhizome of Zingiber officinale and Curcuma longa; a root of Piper chaba or its combination thereof (101); preparing a hydro-alcoholic extract by soaking the dried powdered admixture in an aqueous alcohol (102); filtering the hydro-alcoholic extract and collecting the filtrate (103); repeating the step (102) and (103) thrice and mixing filtrates of each cycle to obtain a pooled solvent (104); concentrating the pooled solvent for removing the excess ethanol to obtain a concentrated extract (105); and lyophilizing the concentrated extract to obtain the final poly herbal extract (106).
[0044] According to an embodiment of the present invention, the stem or bark of Cinnamomum verum is taken, cleaned and air-dried. The air-dried stem or bark of Cinnamomum verum are crushed and grounded to obtain a pulverized coarse powder (20 to 30 mesh).
[0045] According to an embodiment of the present invention, the fruits of Emblica officinalis were collected, graded and rinsed with water to remove any adhered soil and dirt particles. The pericarp of the fruits was removed and subjected for air-drying. The air-dried fruit pericarp of Emblica officinalis was crushed and grounded to obtain a pulverized coarse powder (20 to 30 mesh).
[0046] According to an embodiment of the present invention, the rhizomes of Zingiber officinale were taken, cleaned and air-dried. The air-dried rhizomes of Zingiber officinale are crushed and grounded to obtain a pulverized coarse powder (20 to 30 mesh).
[0047] According to an embodiment of the present invention, the roots of Piper chaba were collected, graded and rinsed with water to remove any adhered soil particles. The cleaned roots were cut into pieces and subjected for drying under sun-light. The dried roots of Piper chaba were crushed and grounded to obtain a pulverized coarse powder (20 to 30 mesh).
[0048] According to an embodiment of the present invention, the roots of Curcuma longa were collected, graded and rinsed with water to remove any adhered soil particles. The cleaned roots were cut into pieces and subjected for drying under sun-light. The dried roots of Curcuma longa were crushed and grounded to obtain a pulverized coarse powder (20 to 30 mesh).
[0049] According to an embodiment of the present invention, the said dried powdered admixture is prepared by mixing the dried pulverized coarse powder (20 to 30 mesh) of the stem or bark extract of Cinnamomum verum, dried pulverized coarse powder (20 to 30 mesh) of the fruit extract of Emblica officinalis, dried pulverized coarse powder (20 to 30 mesh) of the rhizome extract of Zingiber officinale, dried pulverized coarse powder (20 to 30 mesh) of the root extract of Piper chaba, and dried pulverized coarse powder (20 to 30 mesh) of the rhizome extract of Curcuma longa in the ratio of 40:20:20:10:10.
[0050] According to an embodiment of the present invention, the aqueous poly-herbal extract or the hydro-alcoholic extract was obtained by soaking the said powdered admixture in the suitable solvent system for 24 hours in orbital shaker at 100 rpm.
[0051] According to an embodiment of the present invention, the solvent system is either water or an aqueous alcohol.
[0052] According to an embodiment of the present invention, the aqueous alcohol is prepared by mixing ethanol to water at a varying ratio of 30:70, 70:30 and 80:20.
[0053] According to an embodiment of the present invention, the concentration of the pooled solvent is performed at a temperature of 600C in the rota-evaporator.
[0054] According to an embodiment of the present invention, the ratio of the dried powdered admixture to the solvent system is 1:8.
[0055] According to an embodiment of the present invention, an efficacy of the poly herbal extract for prevention and management of chronic hyperglycemia and associated complications is evaluated.
[0056] The invention is now illustrated by way of non-limiting example(s).

EXAMPLE 1
[0057] FIG. 2 is a schematic representation showing the detailed process steps involved in the preparation of an aqueous poly herbal extract using water as the solvent system, according to an embodiment of the present invention. With respect to FIG. 2, the aqueous extract is prepared by soaking the dried powdered admixture of the stem or bark of Cinnamomum verum; the fruits of Emblica officinalis; the rhizomes of Zingiber officinale and Curcuma longa; the roots of Piper chaba or its combination in water. Water is boiled and cooled prior to use as solvent system.
[0058] Example 1 illustrates formula to prepare an aqueous poly-herbal extract
Ingredients Quantity
Ratio of Dried powdered admixture of a stem or bark of Cinnamomum verum; a fruit of Emblica officinalis; a rhizome of Zingiber officinale and Curcuma longa; a root of Piper chaba or its combination thereof to water
(Powdered admixture:water) 1:8

EXAMPLE 2
[0059] FIG. 3 is a schematic representation showing the detailed process steps involved in the preparation of a hydro-alcoholic poly herbal extract using ethanol to water in ratio 70:30 as the solvent system, according to an embodiment of the present invention. With respect to FIG. 3, the hydro-alcoholic extract is prepared by soaking the dried powdered admixture of the stem or bark of Cinnamomum verum; the fruits of Emblica officinalis; the rhizomes of Zingiber officinale and Curcuma longa; the roots of Piper chaba or its combination in an aqueous alcohol solvent using ethanol to water with ratio of 70:30.
[0060] Example 2 illustrates formula to prepare a hydro-alcoholic poly-herbal extract with ratio of ethanol to water as 70:30.
Ingredients Quantity
Ratio of Dried powdered admixture of a stem or bark of Cinnamomum verum; a fruit of Emblica officinalis; a rhizome of Zingiber officinale and Curcuma longa; a root of Piper chaba or its combination thereof to aqueous alcohol
(Powdered admixture : aqueous alcohol) 1:8
Ratio of ethanol to water 70:30

EXAMPLE 3
[0061] FIG. 4 is a schematic representation showing the detailed process steps involved in the preparation of a hydro-alcoholic poly herbal extract using ethanol to water in ratio 80:20 as the solvent system, according to an embodiment of the present invention. With respect to FIG. 4, the hydro-alcoholic extract is prepared by soaking the dried powdered admixture of the stem or bark of Cinnamomum verum; the fruits of Emblica officinalis; the rhizomes of Zingiber officinale and Curcuma longa; the roots of Piper chaba or its combination in an aqueous alcohol solvent using ethanol to water at a ratio of 80:20.
[0062] Example 3 illustrates formula to prepare a hydro-alcoholic poly-herbal extract with ratio of ethanol to water as 80:20.
Ingredients Quantity
Ratio of Dried powdered admixture of a stem or bark of Cinnamomum verum; a fruit of Emblica officinalis; a rhizome of Zingiber officinale and Curcuma longa; a root of Piper chaba or its combination thereof to aqueous alcohol
(Powdered admixture : aqueous alcohol) 1:8
Ratio of ethanol to water 80:20

EXAMPLE 4
[0063] FIG. 5 is a schematic representation showing the detailed process steps involved in the preparation of a hydro-alcoholic poly herbal extract using ethanol to water in ratio 30:70 as the solvent system, according to an embodiment of the present invention. With respect to FIG. 5, the hydro-alcoholic extract is prepared by soaking the dried powdered admixture of the stem or bark of Cinnamomum verum; the fruits of Emblica officinalis; the rhizomes of Zingiber officinale and Curcuma longa; the roots of Piper chaba or its combination in an aqueous alcohol solvent using ethanol to water with ratio of 30:70.
[0064] Example 4 illustrates formula to prepare a hydro-alcoholic poly-herbal extract with ratio of ethanol to water as 30:70
Ingredients Quantity
Ratio of Dried powdered admixture of a stem or bark of Cinnamomum verum; a fruit of Emblica officinalis; a rhizome of Zingiber officinale and Curcuma longa; a root of Piper chaba or its combination thereof to aqueous alcohol
(Powdered admixture : aqueous alcohol) 1:8
Ratio of ethanol to water 30:70

[0065] The yield calculation and the bioassay studies were performed using above mention poly herbal extracts (Example 1 to Example 4) to evaluate Free Radicle Scavenging, Lipid Peroxidation, Aldose Reductase Inhibition and Advanced Glycation End (AGE) Inhibition activities.

EXPERIMENTAL DETAILS
[0066] % yield of the concentrated extract:
[0067] Percentage yield of each of the concentrated extract i.e. aqueous extract and hydro-alcoholic extract with ratio 70:30, 30:70 and 80:20 was calculated for two representative batches using below formula:

[0068] BIOASSSAYS
[0069] Free Radicle Scavenging Activity
[0070] The antioxidant activity of the extracts was assessed by using a 1, 1-diphenyl 1-2-picryl-hydrazyl, (DPPH) assay. 0.1mL of extract in various concentrations was added to 3.9 ml of 6 x10-5 M methanol DPPH solution. After 30-minutes of incubation, the decrease in the absorbance was determined at 515nm using Shimadzu UV-2600 Spectrophotometer. Scavenging activity was calculated using methanol as blank and DPPH without inhibitor as control and IC50 value was determined using below formula.
[0071] The results for the antioxidant activity were compared between the poly-herbal extract prepared using aqueous alcohol as solvent system with ratio of ethanol to water as 80:20, 30:70 and 70:30.

[0072] Inhibition of Lipid Peroxidation:
[0073] The degree of lipid peroxidation was estimated by a modified thiobarbituric acid reactive substances (TBARS) assay. The procedure was performed by adhering to the guidelines approved by the Institutional Animal Ethics Committee (IAEC) of the National Institute of Nutrition. Freshly excised rat liver homogenate (10%) in phosphate buffer was used for the malondialdehyde (MDA) reaction. Initially, liver homogenate along with various concentrations of the extract was incubated after the addition of 0.07M FeSO4 for 30 minutes. To the reaction 20% acetic acid (pH 3.5), 0.8% thiobarbituric acid, and 20% trichloroacetic acid were added and vortexed, and kept for incubation for one hour in a boiling water bath. After cooling down 1-Butanol was added and centrifuged at 3000g for 10 mins. The upper organic layer containing pinkish chromogen was measured against 1-butanol at 532nm using Shimadzu UV-2600 Spectrophotometer (Ohkawa, et al., 1979). For control, distilled water was used instead of extract.
[0074] The results for the lipid peroxidation activity were compared between the poly-herbal extract prepared using aqueous alcohol as solvent system with ratio of ethanol to water as 80:20, 70:30 and 30:70.
[0075] Aldose Reductase Inhibition Activity

[0076] For inhibition studies, concentrated stocks of extracts prepared in 50 % DMSO and limiting it to 1% in the reaction volume. The assay mixture of 1 mL contained 50 µmol potassium phosphate buffer pH 6.2, 0.2 M lithium sulfate, 5µmol ß-mercapto ethanol, 10µmol DL-glyceraldehyde, 0.1µmol NADPH, and enzyme preparation (Rat Lens) along with the various concentrations of the extract as an inhibitor. The assay mixture was incubated at 37? and initiated by the addition of NADPH at 37?. The change in the absorbance at 340nm due to NADPH oxidation was followed in a Shimadzu UV-2600 Spectrophotometer. Activity in the absence of an inhibitor is considered as 100% and IC50 values were derived by non-linear regression analysis plot of percent inhibition against inhibitor concentration (Suryanarayana, et al., 2004).
[0077] The results for the aldose reductase inhibition activity were compared between the poly-herbal extract prepared using aqueous alcohol as solvent system with ratio of ethanol to water as 80:20, 70:30 and 30:70.

[0078] Advanced Glycation End-products (AGE) Inhibition Assay
[0079] For AGE inhibition studies, concentrated stocks of extracts were prepared in 50 % DMSO and limiting it to 1% in the reaction volume. One mL reaction contains BSA (10mg/ml), methylglyoxal (MGO) in 0.2M phosphate buffer pH 7.4, along with various concentrations of extract and 0.01% Sodium azide incubated at 37? in dark for 3 days. After the end of the incubation, reaction contents were filtered and unbound reactants were removed by dialysis and further protein estimation is done. The extent of inhibition of protein glycation by the extract was evaluated by non-tryptophan fluorescence by exciting at 370nm and emission at 400-500nm. For measurement 0.15mg/mL protein in 20mM phosphate buffer (pH 7.4) was used. The spectra were corrected with a phosphate buffer blank (Muthenna., et al., 2012).
[0080] The results for inhibition were compared between the poly-herbal extract prepared using aqueous alcohol as solvent system with ratio of ethanol to water as 80:20, 70:30 and 30:70.

[0081] In vitro cytotoxicity activity on Baby Hamster Kidney (BHK-21) cell line
[0082] Outline of the method
[0083] Based on out-come of above in vitro bioassays, to determine the level of cytotoxicity of poly herbal extract (PHE) of ethanol to water ratio as 70:30 was used for In Vitro MTT assay on Baby Hamster Kidney (BHK-21) cell line.
[0084] Preparation of test compounds for cytotoxicity screening
[0085] 10mg of test substance was weighed and dissolved in MEM medium supplemented with 2% inactivated FBS to obtain a stock solution of 10mg/mL. Furthermore, serial two-fold dilutions were prepared from the stock solution to prepare lower concentrations for cytotoxicity testing.
[0086] Cell line and culture medium
[0087] Baby Hamster Kidney (BHK-21) cell line was procured from NCCS, India. Stock cells were cultured in MEM supplemented with 10% inactivated Fetal Bovine Serum (FBS), penicillin (100 IU/mL), streptomycin (100 µg/mL) and amphotericin B (5 µg/mL) in an humidified atmosphere of 5% CO2 at 37? until confluent. The cells were dissociated with TPVG solution (0.2% Trypsin, 0.02% EDTA, 0.05% glucose in PBS). The stock cultures were grown in 25 cm2 culture flasks and all experiments were carried out in 96 well microtiter plates.
[0088] Determination of cell cytotoxicity by MTT Assay
[0089] The cell culture monolayer was trypsinized and the cell count was adjusted to 100,000 cells/mL using MEM containing 10% FBS. To each well of the 96 well microtiter plate, 0.1 mL of the diluted cell suspension was added. After 24 hours, on formation of partial monolayer, the supernatant was flicked off, the monolayer was washed once with DPBS and different test concentrations were added in the microtiter plates. The untreated cells were maintained as cell control for comparison. The plates were then incubated at 37? for 24 hours in 5% CO2 atmosphere, and microscopic examination was carried out and observations were noted. After 24 hours, the test solutions in the wells were discarded and 100µL of MTT diluted with DPBS added to each well. The plate was incubated for 3 hours at 37? in 5% CO2 atmosphere. The supernatant was removed and 100µL of DMSO was added and the plates were gently shaken to solubilize the formed formazan. The absorbance was measured using a micro plate reader at a wavelength of 570nm.

[0090] In vitro cytotoxicity activity on Human Retinoblastoma (Y79) cell line
[0091] Analysis of the in vitro cytotoxicity of test product against Human Retinoblastoma (Y79) cell line by WST assay:
[0092] Outline of the method: The in vitro WST assay was performed for the test product on Human Retinoblastoma (Y79) cell line to determine the level of cytotoxicity.
[0093] Preparation of test compounds for cytotoxicity screening: 10mg of test substance was weighed and dissolved in RPMI 1640 medium supplemented with 2% inactivated FBS to obtain a stock solution of 10mg/mL. Furthermore, serial two-fold dilutions were prepared from the stock solution to prepare lower concentrations for cytotoxicity testing.
[0094] Cell line and culture medium: Human Retinoblastoma (Y79) cell line procured from NCCS, India. Stock cells were cultured in RPMI 1640 supplemented with 10% inactivated Fetal Bovine Serum (FBS), penicillin (100 IU/mL), streptomycin (100 µg/mL) and amphotericin B (5 µg/mL) in an humidified atmosphere of 5% CO2 at 37°C until confluent. The cells were dissociated with TPVG solution (0.2% Trypsin, 0.02% EDTA, 0.05% glucose in PBS). The stock cultures were grown in 25 cm2 culture flasks and all experiments were carried out in 96 well microtitre plates (Tarsons India Pvt. Ltd., Kolkata, India).
[0095] Determination of cell viability by WST Assay: The monolayer cells were counted and adjusted to 2.0 x 105 cells/ml) using RPMI 1640 containing 10% FBS. To each of the wells, of the 96 well plate, 50µL of the diluted cell suspension (approximately ten thousand cells per well) was added and different concentrations of test substance (1000 - 7.8 µg/mL) in 2X concentration was added to the wells of microtitre plate. The plates were incubated at 37°C for a day in 5% CO2 atmosphere, and microscopic observations was carried out at every 24 h interval. After 24h, WST-1 was added to each well. The plates were gently shake and incubated for 3 h at 37°C in 5% CO2 atmosphere. The absorbance was measure using a microplate reader at a wavelength of 490nm. The percentage growth inhibition was calculated using the standard formula.

[0096] in vitro cytotoxicity activity on Human Hepatocellular Carcinoma (Hep G2) cell line
[0097] Analysis of the in vitro cytotoxicity of test product against Human Hepatocellular Carcinoma (Hep G2) cell line by MTT assay:
[0098] The In vitro MTT assay was performed for the test product on Human Hepatocellular Carcinoma (Hep G2) cell line to determine the level of cytotoxicity.
[0099] Preparation of test compounds for cytotoxicity screening: 10mg of test substance was weighed and dissolved in DMEM-HG medium supplemented with 2% inactivated FBS to obtain a stock solution of 10mg/mL. Furthermore, serial two-fold dilutions were prepared from the stock solution to prepare lower concentrations for cytotoxicity testing.
[00100] Cell line and culture medium: Human Hepatocellular Carcinoma (Hep G2) cell line procured from NCCS, India. Stock cells were cultured in DMEM-HG supplemented with 10% inactivated Fetal Bovine Serum (FBS), penicillin (100 IU/mL), streptomycin (100 µg/mL) and amphotericin B (5 µg/mL) in an humidified atmosphere of 5% CO2 at 37°C until confluent. The cells were dissociated with TPVG solution (0.2% Trypsin, 0.02% EDTA, 0.05% glucose in PBS). The stock cultures were grown in 25 cm2 culture flasks and all experiments were carried out in 96 well microtitre plates (Tarsons India Pvt. Ltd., Kolkata, India).
[00101] Determination of cell cytotoxicity by MTT Assay: The cell culture monolayer was trypsinized and the cell count was adjusted to 100,000 cells/mL using DMEM-HG containing 10% FBS. To each well of the 96 well microtitre plate, 0.1 mL of the diluted cell suspension was added. After 24 h, when a partial monolayer was formed, the supernatant was flicked off, the monolayer was washed once with DPBS and different test concentrations were added in the microtitre plates. The untreated cells were maintained as cell control for comparison. The plates were then incubated at 37°C for 24 h in 5% CO2 atmosphere, and microscopic examination was carried out and observations were noted. After 24 h, the test solutions in the wells were discarded and 100µL of MTT diluted with DPBS added to each well. The plate was incubated for 3 h at 37°C in 5% CO2 atmosphere. The supernatant was removed and 100µL of DMSO was added and the plates were gently shaken to solubilize the formed formazan. The absorbance was measured using a micro plate reader at a wavelength of 570 nm.

RESULTS
[00102] Table 1 demonstrates the result of % yield for the concentrated extract i.e. aqueous extract and hydro-alcoholic extract with ethanol to water ratio 30:70, 70:30, and 80:20. With respect to Table 1, highest percentage yield was obtained for the hydro-alcoholic extract with ratio of ethanol to water as 70:30.
Batch Particulars % yield of an aqueous extract % yield of hydro-alcoholic extract with ratio of ethanol to water as
30:70 70:30 80:20
Batch – 1 18.00% 14.47% 24.01 % 18.52%
Batch – 2 -- -- 21.50% 18.19%

[00103] Free Radicle Scavenging Activity
[00104] FIG. 6 demonstrates the graphical representation of the free radical scavenging activity between the poly-herbal extracts prepared using aqueous alcohol as solvent system with ratio of ethanol to water as 80:20, 70:30 and 30:70, according to an embodiment of the present invention. With respect to FIG. 6, free radical scavenging activity or anti-oxidant was relatively higher for the extract with ratio of ethanol to water 80:20 in comparison to the other two extracts i.e. the extract with ratio of ethanol to water 70:30 and 30:70. However, the anti-oxidant activity is almost similar for the poly-herbal extract with ratio of ethanol to water 80:20 and 70:30 extracts.

[00105] Lipid Peroxidation Inhibition Activity:
[00106] FIG. 7 demonstrates the graphical representation of the lipid peroxidation activity between the poly-herbal extract prepared using aqueous alcohol as solvent system with ratio of ethanol to water as 80:20, 70:30 and 30:70, according to an embodiment of the present invention. With respect to FIG. 7, the lipid peroxidation inhibition activity was relatively higher for the extract with ratio of ethanol to water 70:30 in comparison to the other two extracts i.e., the extract with ratio of ethanol to water 30:70 and 80:20.
[00107] Aldose Reductase Inhibition Activity
[00108] Table 2 illustrates IC50 value of the poly-herbal extracts with ratio of ethanol to water as 80:20, 70:30 and 30:70, according to an embodiment of the present invention. With respect to Table 2, the IC50 value was found higher for the poly-herbal extract with ratio of ethanol to water as 70:30 in comparison to extracts with ratios 30:70 and 80:20.
Table 2: IC50 value of the poly-herbal extracts with ratio of ethanol to water as 80:20, 70:30 and 30:70.
Poly-herbal extract with ratio of Ethanol:Water as IC50 (µg)
80:20 8.25
70:30 7.43
30:70 12.99

[00109] FIG. 8 demonstrates the graphical representation of the aldose reductase inhibition activity between the poly-herbal extracts prepared using aqueous alcohol as solvent system with ratio of ethanol to water as 80:20, 70:30 and 30:70, according to an embodiment of the present invention. With respect to FIG. 8, the extract with ratio of ethanol to water 70:30 exhibited better inhibition activity in comparison to the other two extracts i.e., the extract with ratio of ethanol to water 30:70 and 80:20.

[00110] Advanced Glycation End-products (AGE) Inhibition Activity:
[00111] Table 3 illustrates AGE fluorescence at UV wavelength of 440nm of the poly-herbal extracts with ratio of ethanol to water as 80:20, 70:30 and 30:70, according to an embodiment of the present invention. FIG. 9 demonstrates AGE fluorescence at UV wavelength of 440nm of the poly-herbal extracts with ratio of ethanol to water as 80:20, 70:30 and 30:70, according to an embodiment of the present invention. With respect to Table 3 and FIG. 9, percentage inhibition of non-tryptophan AGE fluorescence at 440 nm was relatively higher with the extract with ratio of ethanol to water 70:30 in comparison to the extract with ratio of ethanol to water 30:70 and 80:20. Lower the fluorescence, higher is the AGE inhibition activity.
Table 3: AGE fluorescence at UV wavelength of 440nm of the poly-herbal extracts with ratio of ethanol to water as 80:20, 70:30 and 30:70.
Concentration (µg) Poly-herbal extract with ratio of Ethanol:Water as
80:20 70:30 30:70
20 44.20 30.67 54.137
100 37.90 22.82 45.046
200 10.86 21.96 32.945

[00112] Based on the result of the bioassay studies it is concluded that the poly herbal extracts with ratio of ethanol to water as 70:30 showed better free radicle scavenging, lipid peroxidation, aldose reductase inhibition and advanced glycation end-products (AGE) inhibition activities.
[00113] Also, the poly herbal extracts with ratio of ethanol to water as 70:30 showed highest % yield.

[00114] In vitro cytotoxicity activity on Baby Hamster Kidney (BHK-21) cell line
[00115] Table 4 illustrates analysis of the In vitro cytotoxicity of PHE of ethanol to water ratio as 70:30 against Baby Hamster Kidney (BHK-21) cell line by exposing the cells to different concentrations ranging from 1000µg/mL to 7.8µg/mL using MTT assay method, according to an embodiment of the present invention. MTT assay was employed to test the cytotoxic effect of selected concentrations by measuring the metabolic activity through a colorimetric determination. With respect to Table 4, the results indicates that the test product (RR222192) on the tested cell line exhibited higher toxicity at 1000µg/mL. The CTC50 values obtained from MTT assay for the given test product (RR222192) on Baby Hamster Kidney (BHK-21) cells was found to be 583.838µg/mL.
Test product
Concentration
(µg/mL) Percentage cytotoxicity after treatment CTC50
(µg/mL)

RR222192 1000 68.34±2.83

583.838

500 41.56±0.12
250 36.54±1.20
125 39.05±0.94
62.5 32.98±0.97
31.25 28.63±1.30
15.625 20.18±1.19
7.8 18.47±2.48

[00116] in vitro cytotoxicity activity on Human Retinoblastoma (Y79) cell line:
[00117] Table 5 shows the analysis of the in vitro cytotoxicity of test product against Human Retinoblastoma (Y79) cell line by WST assay
Test Product Concentration (µg/mL) Percentage cytotoxicity after
treatment CTC50
(?g/mL)
RR222192
1000 72.16± 0.57
438.53
500 55.28± 12.63
250 36.81± 1.40
125 39.18± 1.62
62.5 32.98± 0.90
31.25 29.02± 1.14
15.625 20.71± 1.06
7.8 18.21± 2.37
Table 5: Analysis of the in vitro cytotoxicity of test product against Human Retinoblastoma (Y79) cell line by WST assay
[00118] Test product was assayed for in vitro cytotoxicity study against Human Retinoblastoma (Y79) cell line by exposing the cells to different concentrations ranging from 1000µg/mL to 7.8µg/mL. WST assay was employed to test the cytotoxic effect of selected concentrations by measuring the metabolic activity through a colorimetric determination. In the present study, test product (RR222192) on the tested cell line exhibited higher toxicity at 1000µg/mL. The CTC50 values obtained from WST assay for the given test product (RR222192) on Human Human Retinoblastoma (Y79) cell line were found to be 438.53µg/mL.

[00119] in vitro cytotoxicity activity on Human Hepatocellular Carcinoma (Hep G2) cell line:
[00120] Table 6 shows the analysis of the in vitro cytotoxicity of test product against Human Hepatocellular Carcinoma (HepG2) cell line by MTT assay
[00121] Table 6: Analysis of the in vitro cytotoxicity of test product against Human Hepatocellular Carcinoma (HepG2) cell line by MTT assay
Test product Concentration (µg/mL) Percentage cytotoxicity
after treatment CTC50
(?g/mL)

RR222192 1000 69.95 ± 3.00

509.13
500 44.57 ± 0.43
250 39.52 ± 1.24
125 42.05 ± 1.97
62.5 36.24 ± 0.83
31.25 32.07 ± 1.04
15.625 24.12 ± 1.14
7.8 22.22 ± 2.27

[00122] Test product was assayed for in vitro cytotoxicity study against Human Hepatocellular Carcinoma (Hep G2) cell line by exposing the cells to different concentrations ranging from 1000µg/mL to 7.8µg/mL. MTT assay was employed to test the cytotoxic effect of selected concentrations by measuring the metabolic activity through a colorimetric determination. In the present study, test product (RR222192) on the tested cell line exhibited higher toxicity at 1000µg/mL. The CTC50 values obtained from MTT assay for the given test product (RR222192) on Human Hepatocellular Carcinoma (Hep G2) cells were found to be 509.13µg/mL.
G) ADVANTAGES OF INVENTION
[00123] The present invention provides a poly herbal extract for the prevention and management of chronic hyperglycemia and associated complications. The additive effect of ‘admixture’ of Cinnamomum verum, Emblica officinalis, Curcuma longa, Zingiber officinale and Piper chaba shows enhanced anti-oxidant activity and improved bioavailability in diabetic patients. The said poly herbal extract which is absolutely free from synthetic chemicals.
[00124] 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 the spirit and scope of the claims presented in the complete specification or non-provisional application.

Dated this 26th day of October, 2022

For SARVOTHAM CARE LIMITED
BY THEIR AGENT

(DR. BABITHA THARAPPAN)
IN/PA-1614

,CLAIMS:Claims:
We claim:
1. A poly herbal extract for the prevention and management of chronic hyperglycemia and associated complications, comprises:
a hydro-alcoholic extract of dried powdered admixture of a stem or bark of Cinnamomum verum; a fruit of Emblica officinalis; a rhizome of Zingiber officinale and Curcuma longa; a root of Piper chaba or its combination thereof.
2. The poly herbal extract as claimed in claim 1, wherein the stem or bark of Cinnamomum verum; the fruit of Emblica officinalis; the rhizome of Zingiber officinale and Curcuma longa; the root of Piper chaba is present in a ratio of 40:20:20:10:10 in the said powdered admixture.
3. The poly herbal extract as claimed in claim 1, wherein the ratio of ethanol to water is 70:30.
4. A method of synthesizing a poly herbal extract for the prevention and management of chronic hyperglycaemia and associated complications, comprises:
preparing a dried powdered admixture of a stem or a bark of Cinnamomum verum; a fruit of Emblica officinalis; a rhizome of Zingiber officinale and Curcuma longa; a root of Piper chaba or its combination thereof (101);
preparing a hydro-alcoholic extract by soaking the dried powdered admixture in an aqueous alcohol (102);
filtering the hydro-alcoholic extract and collecting the filtrate (103);
repeating the step (102) and (103) thrice and mixing filtrates of each cycle to obtain a pooled solvent (104);
concentrating the pooled solvent for removing the excess ethanol to obtain a concentrated extract (105); wherein the concentration of the pooled solvent is performed at a temperature of 600C in the rota-evaporator; and
lyophilizing the concentrated extract to obtain the final poly herbal extract (106).
5. The method as claimed in claim 4, wherein the stem or bark of Cinnamomum verum; the fruit of Emblica officinalis; the rhizome of Zingiber officinale and Curcuma longa; the root of Piper chaba is added in a ratio of 40:20:20:10:10 in the said powdered admixture.
6. The method as claimed in claim 4, wherein the dried powdered admixture is soaked in an aqueous alcohol for 24 hours at 100 rpm in orbital shaker.
7. The method as claimed in claim 4, wherein the ratio of ethanol to water is 70:30.
8. The method as claimed in claim 4, wherein the ratio of the dried powdered admixture to the solvent system is 1:8.

Dated this 26th day of October, 2022

For SARVOTHAM CARE LIMITED
BY THEIR AGENT

(DR. BABITHA THARAPPAN)
IN/PA-1614