DESC:FIELD OF THE INVENTION
[0001] The present disclosure relates generally to the field of spice compositions. Particularly, the present disclosure relates to a composition for improving glucose and lipid profile in a subject. The present disclosure further relates to a method of preparation of the composition.

BACKGROUND OF THE INVENTION
[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
[0003] Nowadays, prevalence of diabetes and cardiovascular diseases (CV) is escalating at an alarming rate and contributing to increase in mortality rates. Cost of cardiac corrective health care and/or of maintaining normal or near normal glucose profile is well beyond the budgets of majority of the population. Diet and way of life presume to play an imperative role in preventing and managing diabetes and cardiovascular diseases.
[0004] In the recent years, there has been an augmented interest globally to identify compounds isolated from natural products that are pharmacologically effective and have low or no side effects for use in preventive care and the food industry. Spices are heterogeneous collections of a wide variety of volatile and non-volatile staple dietary additives and used in culinary preparations. Spices are known to be effective as functional foods to improve health and decrease the risk of diseases. They are rich in antioxidants and comprise a diverse array of natural phyto-chemicals and many critical micronutrients that have complementary and overlapping actions. Several scientific studies suggest that spices are potent inhibitors of tissue damage and inflammation.
[0005] Though several different spices mix or masala’s are available in market for culinary purposes, however, none of them exhibits potential of improving the health of an individual and known to reduce the risk of diabetes and/or cardiac diseases, plausibly owing to disproportionate usage of different ingredients while preparing the spices mix/compositions.
[0006] Therefore, it would be desirable to develop a new and improved composition that may aid in improving glucose and lipid profile of a subject. The present invention satisfies the existing needs, as well as others, and generally overcomes the deficiencies found in the prior art.

OBJECTS
[0007] Primary object of the present invention is to provide a composition that may overcome the limitations associated with the conventional compositions.
[0008] It is an object of the present disclosure to provide a composition that may aid in improving glucose and lipid profile.
[0009] It is an object of the present disclosure to provide a composition that is substantially devoid of any side effects.
[00010] It is an object of the present disclosure to provide a composition that exhibits synergistic activity/functional reciprocity.
[00011] It is an object of the present disclosure to provide a composition that is easy to prepare and is economical.
[00012] Another object of the present disclosure is to provide a method of preparation of the composition that may aid in improving glucose and lipid profile.

SUMMARY
[00013] The present disclosure relates generally to the field of spice compositions. Particularly, the present disclosure relates to a composition for improving glucose and lipid profile in a subject. The present disclosure further relates to a method of preparation of the composition.
[00014] An aspect of the present disclosure provides a composition for improving glucose and lipid profile, said composition comprising: red chilli in an amount ranging from 5% to 30% by weight of the composition; turmeric in an amount ranging from 5% to 30% by weight of the composition; cumin in an amount ranging from 5% to 25% by weight of the composition; black pepper in an amount ranging from 5% to 25% by weight of the composition; green cardamom in an amount ranging from 2% to 20% by weight of the composition; fenugreek in an amount ranging from 5% to 20% by weight of the composition; and bay leaf in an amount ranging from 5% to 30% by weight of the composition.
[00015] In an embodiment, the composition comprises: red chilli in an amount ranging from 10% to 25% by weight of the composition; turmeric in an amount ranging from 10% to 25% by weight of the composition; cumin in an amount ranging from 5% to 20% by weight of the composition; black pepper in an amount ranging from 5% to 20% by weight of the composition; green cardamom in an amount ranging from 2% to 20% by weight of the composition; fenugreek in an amount ranging from 7% to 20% by weight of the composition; and bay leaf in an amount ranging from 15% to 25% by weight of the composition.
[00016] In an embodiment, the composition comprises: red chilli in an amount of 20% by weight of the composition; turmeric in an amount of 20% by weight of the composition; cumin in an amount of 15% by weight of the composition; black pepper in an amount of 10% by weight of the composition; green cardamom in an amount of 5% by weight of the composition; fenugreek in an amount of 10% by weight of the composition; and bay leaf in an amount of 20% by weight of the composition.
[00017] Another aspect of the present disclosure provides a process for preparing a composition comprising red chili in an amount ranging from 5 to 30% by weight of the composition; turmeric in an amount ranging from 5 to 30% by weight of the composition; cumin in an amount ranging from 5 to 25% by weight of the composition; black pepper in an amount ranging from 5% to 25% by weight of the composition; green cardamom in an amount ranging from 2 to 20% by weight of the composition; fenugreek in an amount ranging from 5% to 20% by weight of the composition; and bay leaves in an amount ranging from 5 to 30% by weight of the composition, said process comprising the steps of: (a) dry roasting each of red chili, turmeric, cumin, black pepper, green cardamom, fenugreek and bay leaves at a temperature ranging from 40°C to 100°C for a time period ranging from 1 minute to 10 minutes to obtain roasted spices; (b) dry grinding the roasted spices to obtain ground spices; (c) blending the ground spices to obtain a spice mixture; and (d) finely grinding the spice mixture to obtain the composition such that more than 75% particles have a size ranging from 1 micron to 800 microns. In an embodiment, the step of dry grinding is effected at a temperature ranging from 10°C to 50°C. In an embodiment, the ground spices have more than 75% particles with size less than 1000 microns. In an embodiment, the step of finely grinding the spice mixture is effected at a temperature ranging from 10°C to 50°C.
[00018] Other aspects, advantages, and salient features of the invention will become apparent to those skilled in the art from the following detailed description, which, taken in conjunction with the exemplary embodiments of the invention.

DETAILED DESCRIPTION OF THE INVENTION
[00019] The embodiments herein and the various features and advantageous details thereof are explained more comprehensively with reference to the non-limiting embodiments that are detailed in the following description. Descriptions of well-known components and processing techniques are omitted so as to not unnecessarily obscure the embodiments herein. The examples used herein are intended merely to facilitate an understanding of the ways in which the embodiments herein may be practiced and to further enable those of skills in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein.
[00020] Unless otherwise specified, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions may be included to better appreciate the teaching of the present invention.
[00021] As used in the description herein, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
[00022] As used herein, the terms “comprise”, “comprises”, “comprising”, “include”, “includes”, and “including” are meant to be non- limiting, i.e., other steps and other ingredients which do not affect the end of result can be added. The above terms encompass the terms “consisting of” and “consisting essentially of”.
[00023] As used herein, the terms “composition”, “blend”, or “mixture” are all intended to be used interchangeably.
[00024] The terms “weight percent”, “vol-%”, “percent by weight”, “% by weight”, and variations thereof, as used herein, refer to the concentration of a substance as the weight of that substance divided by the total weight of the composition and multiplied by 100. It is understood that, as used here, “percent”, “%”, and the like are intended to be synonymous with “weight percent”, “vol-%”, etc.
[00025] The term, “red chilli” as used herein refers to the fruit of plants - Capsicum annuum L. belonging to the genus “Capsicum”, which is a member of the nightshade family, Solanaceae. Red chilli is widely used in many cuisines as a spice to add heat to dishes. The substances giving chili peppers their intensity when ingested or applied topically are capsaicin and related compounds known as capsaicinoids.
[00026] The term, “turmeric” as used herein refers to a flowering plant, Curcuma longa of the ginger family, Zingiberaceae, the roots of which are used in cooking. Turmeric has a warm, bitter taste and is frequently used to flavor or color curry powders, mustards, butters, and cheeses. The rhizomes are used fresh or boiled in water and dried, after which they are ground into a deep orange-yellow powder commonly used as a coloring and flavoring agent in many Asian cuisines, especially for curries, as well as for dyeing, characteristics imparted by the principal turmeric constituent, curcumin. The term Turmeric also encompasses polished, dried and cured turmeric fingers/rhizomes and comminuted forms thereof.
[00027] The term, “cumin” as used herein refers to the dried seed of the herb Cuminum cyminum, a member of the parsley family. Cumin is a flowering plant in the family Apiaceae, native to a territory including the Middle East and stretching east to India. Its seeds – each one contained within a fruit, which is dried – are used in the cuisines of many cultures in both whole and ground form. Cumin seeds contain naturally occurring substances apigenin and luteolin.
[00028] The term, “black pepper” as used herein refers to a flowering vine in the family Piperaceae, cultivated for its fruit, known as a pepper corn, which is usually dried and used as a spice and seasoning. When fresh and fully mature, the fruit is about 5 mm in diameter and dark red, and contains a single seed, like all drupes.
[00029] The term, “green cardamom” as used herein refers to a spice made from the pods of Elettaria cardamomum belonging to the family Zingiberaceae. It is native to the Indian subcontinent and Indonesia. Elettaria pods are light green and smaller.
[00030] The term, “fenugreek” as used herein refers to Trigonella foenum-graecum, which is an annual plant in the family Fabaceae, with leaves consisting of three small obovate to oblong leaflets. It is cultivated worldwide as a semiarid crop. Its seeds and leaves are common ingredients in dishes from the Indian subcontinent. Fenugreek is an herb that is similar to clover. Fenugreek is used as herb (dried or fresh leaves), spice (seeds), and vegetable (fresh leaves, sprouts, and microgreens). Fenugreek seeds smell and taste similar to maple syrup due to the chemical Sotolon.
[00031] The term, “bay leaf” and “bay leaves” as used herein refers to Indian bay leaf or malabathrum (Cinnamomum tamala, Lauraceae). The bay leaf is an aromatic leaf commonly used in cooking. It can be used whole or in a dried or ground form.
[00032] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about”. Accordingly, in some embodiments, the numerical parameters set forth in the written description are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.
[00033] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.
[00034] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
[00035] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.
[00036] The present disclosure relates generally to the field of spice compositions. Particularly, the present disclosure relates to a composition for improving glucose and lipid profile in a subject. The present disclosure further relates to a method of preparation of the composition.
[00037] The present disclosure is on the premise of a surprising discovery that when red chilli, turmeric, cumin, black pepper, green cardamom, fenugreek and bay leaves are included as part of the composition in the weight percentages as defined herein, it exhibits exceptional functional reciprocity and synergistic activity simultaneously targeting several physiological and/or patho-physiological pathways and affords dramatic improvement in glucose and lipid profile in a subject as compared to usage of individual ingredients. The observed synergistic effect is unexpected and surprising. Particularly, it could be observed that when the composition of the present disclosure is fed to Danio rerio (zebrafish models), it affords improvement in body weight, wound healing capacity, blood serum glucose level and total serum lipid while aiding in maintenance of homeostasis.
[00038] In an aspect, the present disclosure provides a spice mix powder comprising any or a combination of: red chilli (Capsicum annuum L.), turmeric (Curcuma longa), cumin (Cuminum cyminum), black pepper (Piper nigrum), green cardamom (Elettaria cardamomum), fenugreek (Trigonella foenum-graecum), and bay leaves (Cinnamomum tamala).
[00039] In an embodiment, the present disclosure provides a spice mix powder comprising a combination of: a therapeutically effective amount of red chilli (Capsicum annuum L.), a therapeutically effective amount of turmeric (Curcuma longa), a therapeutically effective amount of cumin (Cuminum cyminum), a therapeutically effective amount of black pepper (Piper nigrum), a therapeutically effective amount of green cardamom (Elettaria cardamomum), a therapeutically effective amount of fenugreek (Trigonella foenum-graecum), and a therapeutically effective amount of bay leaves (Cinnamomum tamala).
[00040] An aspect of the present disclosure provides a composition for improving glucose and lipid profile, said composition comprising: red chilli in an amount ranging from 5% to 30% by weight of the composition; turmeric in an amount ranging from 5% to 30% by weight of the composition; cumin in an amount ranging from 5% to 25% by weight of the composition; black pepper in an amount ranging from 5% to 25% by weight of the composition; green cardamom in an amount ranging from 2% to 20% by weight of the composition; fenugreek in an amount ranging from 5% to 20% by weight of the composition; and bay leaf in an amount ranging from 5% to 30% by weight of the composition.
[00041] The Capsicum annuum L. may be whole of Capsicum annuum L., part of Capsicum annuum L., an extract of Capsicum annuum L., or combinations thereof. The Curcuma longa may be whole of Curcuma longa, part of Curcuma longa, an extract of Curcuma longa, or combinations thereof. The Cuminum cyminum may be whole of Cuminum cyminum, part of Cuminum cyminum, an extract of Cuminum cyminum or combinations thereof. The Piper nigrum may be whole of Piper nigrum, part of Piper nigrum, an extract of Piper nigrum, or combinations thereof. The Elettaria cardamomum may be whole of Elettaria cardamomum, part of Elettaria cardamomum, an extract of Elettaria cardamomum or combinations thereof. The Trigonella foenum-graecum may be whole of Trigonella foenum-graecum, part of Trigonella foenum-graecum, an extract of Trigonella foenum-graecum or combinations thereof. The Cinnamomum tamala may be whole of Cinnamomum tamala, part of Cinnamomum tamala, an extract of Cinnamomum tamala or combinations thereof. The part or extract may be obtained from root, leaves, shoot, fruits, rhizome, seed, stem, pods, barks, flower, tubers, sap, bud and combinations thereof of the plant.
[00042] In a preferred embodiment, the composition comprises fruits of red chilli (Capsicum annuum L.). In a preferred embodiment, the composition comprises roots of turmeric (Curcuma longa). In a preferred embodiment, the composition comprises seeds of cumin (Cuminum cyminum). In a preferred embodiment, the composition comprises fruits of black pepper (Piper nigrum). In a preferred embodiment, the composition comprises pods of green cardamom (Elettaria cardamomum). In a preferred embodiment, the composition comprises seeds of fenugreek (Trigonella foenum-graecum). In a preferred embodiment, the composition comprises leaves of bay leaf (Cinnamomum tamala). Said parts may be dried and powdered before use.
[00043] In an embodiment, the composition comprises: red chilli in an amount ranging from 10% to 25% by weight of the composition; turmeric in an amount ranging from 10% to 25% by weight of the composition; cumin in an amount ranging from 5% to 20% by weight of the composition; black pepper in an amount ranging from 5% to 20% by weight of the composition; green cardamom in an amount ranging from 2% to 20% by weight of the composition; fenugreek in an amount ranging from 7% to 20% by weight of the composition; and bay leaf in an amount ranging from 15% to 25% by weight of the composition.
[00044] In an embodiment, the composition comprises: red chilli in an amount of 20% by weight of the composition; turmeric in an amount of 20% by weight of the composition; cumin in an amount of 15% by weight of the composition; black pepper in an amount of 10% by weight of the composition; green cardamom in an amount of 5% by weight of the composition; fenugreek in an amount of 10% by weight of the composition; and bay leaf in an amount of 20% by weight of the composition.
[00045] In an embodiment, the composition is formulated into a powder form. In an embodiment, the composition can be used as a spice mixture/seasoning. In an embodiment, the spice mix naturally contains Capsaicin, Curcumin, Rutin, Caffeic acid, Ascorbic acid, vitamins, micronutrients like Cu, Fe, Mn, K, Ca, Mg and Zn, antioxidants and the likes. The composition may further include additives including, but not limited to, edible salts, herbs, nutritional supplements, acidity modifiers, preservatives, texture additives, anti-caking agent, food grade colors and combinations thereof.
[00046] Another aspect of the present disclosure provides a process for preparing a composition comprising red chili in an amount ranging from 5 to 30% by weight of the composition; turmeric in an amount ranging from 5 to 30% by weight of the composition; cumin in an amount ranging from 5 to 25% by weight of the composition; black pepper in an amount ranging from 5% to 25% by weight of the composition; green cardamom in an amount ranging from 2 to 20% by weight of the composition; fenugreek in an amount ranging from 5% to 20% by weight of the composition; and bay leaves in an amount ranging from 5 to 30% by weight of the composition, said process comprising the steps of: (a) dry roasting each of red chili, turmeric, cumin, black pepper, green cardamom, fenugreek and bay leaves at a temperature ranging from 40°C to 100°C for a time period ranging from 1 minute to 10 minutes to obtain roasted spices; (b) dry grinding the roasted spices to obtain ground spices; (c) blending the ground spices to obtain a spice mixture; and (d) finely grinding the spice mixture to obtain the composition such that more than 75% particles have a size ranging from 1 micron to 800 microns.
[00047] In an embodiment, the step of dry grinding is effected at a temperature ranging from 10°C to 100°C. In an embodiment, the step of dry grinding is effected at a temperature ranging from 10°C to 50°C. In an embodiment, the step of dry grinding is effected at room temperature (35°C). In an embodiment, the ground spices have more than 75% particles with size less than 1000 microns. In an embodiment, the ground spices have more than 75% particles with size ranging from 1 micron to 1000 microns.
[00048] In an embodiment, the step of finely grinding the spice mixture is effected at a temperature ranging from 10°C to 100°C. In an embodiment, the step of finely grinding the spice mixture is effected at a temperature ranging from 10°C to 50°C.
[00049] In an embodiment, the composition can be used as a spice mixture that can be use in day to day cooking as a culinary mixture that improves the glucose and lipid profile of the consumer. In an embodiment, the spice mixture provides exquisite flavor to the food such that whole family can have common meals instead of making special bland food for subject having a need to improve glucose and lipid profile. In another embodiment, the spice mixture is capable of improving glucose and lipid profile of the consumer at no/minimal incremental cost. In another embodiment, the composition can be used as a seasoning/spice mixture for improving glucose and lipid profile.
[00050] The spice mixture of the present disclosure is consumed fully and no part of any spice is discarded. On the contrary, during common cooking, turmeric (usually used as a powder) and occasionally red chilli is consumed in whole whereas even if bay leaves, green cardamom and the likes are used as spices, these are used only for their volatiles that seep into the food while rest of the whole spice is discarded before consuming the food.
[00051] 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. 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 scope of the embodiments as described herein.
EXAMPLES
[00052] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.
[00053] COMPOSITION
Table 1: Composition for improving glucose and lipid profile
Ingredients Amount of Ingredients
(in grams)
Red Chilli 20
Turmeric 20
Cumin 15
Black pepper 10
Green Cardamom 5
Fenugreek 10
Bay leaf 20

[00054] PREPARATION OF COMPOSITION
[00055] All the ingredients used in preparation of the composition are of Indian origin and are readily available commercially. Particularly, each of the ingredients was procured commercially from Chennai, India. All the ingredients were weighed accurately as per Table 1 provided herein-above and dry roasted separately at 70°C for about 5 minutes to obtain roasted spices. The roasted spices were then dry grinded separately at room temperature (35°C) to obtain ground spices, such that more than 75% particles have a size below 1000 microns. The ground spices were then blended and mixed thoroughly to obtain a spice mixture. The spice mixture was then finely grinded to obtain the composition such that more than 75% particles have a size below 800 microns.
[00056] EFFICACY STUDIES
[00057] The composition of the present disclosure, prepared in accordance with Table 1 was used to assess its potential towards improving the symptoms of diabetes and/or cardiac diseases. To understand the efficacy of the composition, Type II diabetes model of zebrafish was established (referred as model group) and treated with the present composition (referred as treatment group) and the vehicle (1X PBS, referred as control group) and were screened on day 6 for any change in body weight, wound healing capacity, blood serum glucose level and total serum lipid to evaluate the therapeutic efficacy of present composition at varying dosage and its individual ingredients.
[00058] ZEBRAFISH MODEL DEVELOPMENT
Zebrafish is one of the most widely used and accepted animal for studying diabetes as they share genetic homology (at least 70%) with the humans. To evaluate the efficacy of the composition disclosed in Table 1, freshwater zebrafish of about 4 centimeters in length were selected for establishment of models for efficacy studies. Wild type strains of Zebrafish (Danio rerio) were used that were adapted and bred at the research center. All the fishes were acclimated to constant laboratory conditions (14h light and 10h dark photoperiod, normal diet and water at 28°C) for at least one week in stock aquaria before the induction of experiment. Through purposive sampling, healthy adult males with no signs of infection, equivalent body size and age (around 1-2 years) were scrutinized and used for the experiment study.
[00059] A light-dark cycle of 10h to 14h with a water temperature of 27±1 °C and pH ranging from 6.8 to 7 were adhered throughout the study. Good Animal Practice as per the Institutional Animal Ethics Committee in accordance with the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India, was followed in adherence to established protocols. The washout process was carried out once in every 4 days to keep the fishes in a clean environment and to eliminate the risk of any infection.
Obesity Induction: To increase the body weight of the fish, the fishes were fed with calorie dense high fat diet for a period of 14 days to develop adipose tissue deposits around the abdominal region. The ratio of the calorie dense feed and the mealtime is listed in Table 2 herein-below:
Table 2: Ratio of nutrients in calorie dense feed
Nutrients Percentage (%)
Protein 47.5
Fat 15.5
Fiber 2
Carbs 35

Diabetes Model Development: Alloxan, from SRL Chemicals, India was used as an inducer. Fishes were housed at an inducer concentration of 3 liters of housing water containing 2mg/ml of alloxan solution for 48 hours to induced pancreatic damage. Fishes were then housed in 0.2% glucose solution for a period of 15 days for maintaining hyperglycemia.
[00060] STUDY DESIGN
Animals were divided into 3 groups: 1) Treated groups (Diabetes + treatment with present composition/ individual ingredients), 2) Model (diseased, Diabetic), and 3) Control (treated with 1X PBS). Each group includes 12 fishes housed in 200 ml of aquarium medium. The composition was administered by means of oral gavaging for a period of 5 days. The study groups were monitored twice per day for any phenotype changes in terms of motility, visible clinical signs of redness (an indicative of inflammation) near extremities such as pelvic, pectoral and caudal region. After 5 days, screenings were carried out for the entire study group to evaluate the therapeutic efficacy for the composition.
[00061] DOSE PREPARATION
A concentrate composition was prepared using 1X Phosphate buffer (PBS) and the concentrate was diluted further using 1X PBS as mentioned herein-below in Table 3.
Table 3: Dose Concentrate preparation
Dilution Ratio 1X PBS Composition Concentrate
1:20 2000 ul 100 ul

[00062] Method of preparation of Concentrate:
i. PHF Extract Preparation:
About 15 g of the sample was weighed and transferred to a clean and dry stainless-steel container. 150 ml of distilled water was added to the sample and mixed well with a ladle. The container is then closed with a lid and preceded for steaming.
ii. Heat Steaming process:
Aluminum steam pot was used for heat steaming the sample. The lid of the steam pot was provided with a steam outlet. 1/5th of the steam pot was filled with water and pre-heated in medium flame. The container was transferred to the steam pot when the water in the vessel begins to bubble and the steam pot was closed with a lid which has a steam outlet. The steam pot was heated for about 15 minutes. After 15 minutes, the flame was switched off and the container was allowed to cool for 15 minutes (without opening the lid). After cooling, the container was removed from the steam pot.
iii. Filtration 1:
The sample in the container was filtered through a filter (large sieve) and collected in a glass bowl. The container was thoroughly rinsed with 25 ml of 2X PBS to collect the residue sticking to the wall of the container. The filtrate was transferred from the bowl to a mixer jar. The bowl was rinsed with 25ml of 2X PBS to remove the residue from the bowl and homogenized for 1 minute.
iv. Filtration 2:
The homogenized content in the jar was filtered through a filter (small sieve) and the filtrate was collected in a glass bowl. The jar was thoroughly rinsed with 50 ml PBS to collect the residue sticking to the wall of the jar. The filtrate 2 obtained is the final filtrate was used for efficacy studies. The filtrate 2 in the bowl is emptied into a clean and dry bottle, and the bowl was rinsed with 50 ml 2X PBS and transferred into the bottle.
v. Storage condition:
The final filtrate was stored at 18°C in the refrigerator. The PHF extract has a shelf life of about ten days when stored under proper storage conditions. The extract is prepared freshly every ten days considering the quality.
An intra oral dosing was pursued and a single dose volume of 15 µl of composition per day was administered through gavaging for 5 days, referred herein as treated group. The fishes dosed with only 1X PBS (devoid of any composition/ingredients), were referred herein as control group. The fishes were fed with normal dry pellet feed (Tetrabit) twice in 6 hrs light cycle usually post 3hrs of oral gavage to avoid any regurgitation. Composition dosing was optimized 1000 X lesser to the relative human doses by body weight as mentioned in Table 4 herein-below:
Table 4: Dose calculation
Body weight of Zebrafish 0.5 g
Compound Composition
Dose dilution 1:20
Dose Concentration 0.5-0.6 O.D. unit /15µl

The composition dose was administered through oral gavage. A micropipette was used to manipulate the volume of the dose administered, and the fishes were observed carefully for regurgitation or dispersion of dose through buccal or operculum opening before transferring to the study tank. The fishes were transferred from the study tank to the dampened sponge on the stage. Care was taken to ensure that the fishes were positioned rightly on the slit provided to hold the fish intact during dosing. Long tip micropipette was used to dose the treatment and Control group. Fishes that regurgitated were kept aside and were dosed again with the respective volume of the dosing. The fishes were transferred to their study tanks soon after dosing.
[00063] SCREENING
Each group was screened on day 6 of the study. Throughout the study period each group was continuously monitored twice per day for any phenotypic changes such as motility, visible clinical signs of redness (an indicative of inflammation) near extremities such as pelvic, pectoral and caudal region. Fishes exhibiting abnormal phenotypic changes were closely monitored and were reported in the observation log. Mortality was recorded on regular basis.
[00064] CHANGE IN BODY WEIGHT
The weight of the fishes was noted before initiation of dose and after administration of dose via intra oral gavage for a period of 5 days and the difference in weight of the fishes was noted that aids in further inference on loss/gain in weight of fish due to the dosage.
Table 5: Change in body weight
Study Groups Average weight of the fishes before treatment (g) Average weight of the fishes on 6th day of study
Control 0.701 0.689
Model 0.659 0.653
Present composition 0.750 0.681

The change in body weight of fish in control group was found to be moderate when compared with fishes of model group. However, in fishes treated with present composition (disclosed in Table 1) significant change in body weight was observed when compared with fishes of model group, showing the potential of the present composition in treatment of symptoms of diabetes.
[00065] WOUND HEALING BY CAUDAL FIN AMPUTATION
On 6th day fishes were amputated and were euthanized using ice cold water (4°C) and allowed to rest for 1 min. The caudal fin was amputated and placed on a clean slide without damaging the fragile caudal fin. The regenerated caudal fin was observed under Labomed CM4 stereomicroscope at 40X magnification and imaged with Labomed Camera installed with the Image view Interface for further measurement of the regenerated area. Length of regeneration was measured from the captured images of both dorsal and ventral lobes (i.e., from amputated plane to caudal fin tip) using Image J software, and the average was calculated.
Table 6: Length of regeneration of caudal fin
Study Groups Length of regeneration of caudal fin at the highest point (mm)
Control 1.231
Model 1.014
Present Composition 1.106

Table 6 herein-above shows the length of regeneration of amputated caudal fin in Zebrafish post 5 days of treatment with present composition. It is reported in the literature that the deterioration of insulin homeostasis leads to elevated blood glucose levels that limit the reinstitution of lost tissue. A continual proliferation in the length of caudal fin was noted across the groups post amputation. In fishes of model group, wound healing was found to be reduced due to decreased in collagen deposition because of alteration in glucose homeostasis. However, fishes treated with present composition shows significant proliferation in the length of caudal fin when compared with fishes of model group.
[00066] BLOOD SERUM GLUCOSE LEVEL
On 6th day, fasting blood sugar and post prandial blood sugar level of the fishes were evaluated using a commercially handheld glucometer. The fishes were fasted for 12 hours for evaluating fasting blood sugar level. Post 12 hours of fasting, fishes were sedated in ice cold water for 30 sec and were placed laterally with minimum water. The 1µl of blood sample was collected from each fish using a syringe from the site of blood collection. Blood collection site is along the body axis and posterior to the anus in the region of the dorsal aorta. The fishes were fed, and the blood was collected for post prandial blood sugar evaluation.
Table 7: Blood Serum Glucose Level
Study Groups Fasting Blood Serum Glucose levels mg/dl Postprandial Blood Serum Glucose levels mg/dl
Control 133.75 104.25
Model 226.75 311.50
LLM Optimum 69.25 55.25

Table 7 herein-above shows the blood serum glucose level before and after dosing with present composition post 5 days treatment. It is reported in the literature that in an adult zebrafish normal blood glucose level is 50-75 mg/dl, fasting blood glucose level is 58.85±4.79 mg/dl and fasting blood glucose of the diet induced obese zebrafish is 68 ± 5 mg/dl. The post prandial blood glucose level decreased with increase in time to maintain the blood glucose homeostasis.
In the present study, control group fishes shows elevated levels of fasting blood glucose while the glucose level was found to be normal in post prandial blood. In the case of model group, glucose levels were found to be significantly high in fasting blood and post prandial blood. However, in the fishes treated with present composition, fasting blood glucose level and post prandial blood glucose level were found to be very low when compared with fishes of Control and Model group.
[00067] TOTAL SERUM LIPID
On 6th day, blood sample of zebrafish from various groups were collected for estimation of total serum lipid. The fishes were euthanized with rapid cooling method using ice cubes until it no longer responds to external stimuli. Immediately the whole blood was collected using tail cut method. A straight cut was made in between the anal fin and the caudal fin. The blood that was aspired from the cut region was collected in 10 ul of EDTA. Post blood collection the samples were centrifuged for 3 mins at 6000 RPM and the blood serum was collected. The complete serum were diluted in 10 µl of acetone, and about 2µl of DARK Liebermann-Burchard Reagent was added to each sample vial containing blood serum, and kept in dark room temperature for about 90 mins. The readings of the samples were read at 420 nm using spectrophotometer.
Table 8: Total serum lipid
Study Groups Total Serum Lipid mg/dl
Control 238.91
Model 606.36
LLM Optimum 296.18

Table 8 herein-above shows the total serum lipid level in Zebrafishes post treatment with present composition for a period of 5 days. Lipid metabolism plays a vital role in cellular signaling and acts as the source of energy. Defective lipid metabolism leads to the accumulation of lipid in the blood stream thereby widening the chances of obesity, diabetes and cardiac disease.
In the present study, fishes from the model group showcased high level of total serum lipid when compared to control. However, on treatment with present composition the lipid levels were found to be moderate when compared with fishes from Control and model group.
[00068] SYNERGISTIC ACTIVITY OF THE COMPOSITION
The compositions were prepared in accordance with Table 9 (hereinafter referred to as LLM - Low, LLM - Optimum (same as disclosed in Table 1) and LLM - High) were used for conducting experiments to assess efficacy of the composition vis-à-vis efficacy of the individual ingredients (in an amount as mentioned in column 3 (LLM - Optimum) of Table 9). The same study protocol, as detailed above was used for these studies.

Table 9: Composition LLM - Low, LLM - Optimum and LLM - High
Ingredients LLM - Low (amount in grams) LLM - Optimum (amount in grams) LLM - High (amount in grams)
Red Chilli 5 20 30
Turmeric 5 20 30
Cumin 5 15 25
Black pepper 5 10 25
Green Cardamom 2 5 20
Fenugreek 5 10 20
Bay leaf 5 20 25

[00069] Serum Blood Glucose: Table 10 herein-below shows the comparison of efficacy (Serum blood glucose level) of the composition at different dosage levels (LLM - Low, LLM - Optimum and LLM - High) vis-à-vis that of individual ingredients (in an amount as mentioned in column 3 (LLM - Optimum) of Table 9).
Table 10: Comparison of efficacy (Serum blood glucose level) of the composition at different dosage levels vis-à-vis that of individual ingredients
Study Groups Fasting Blood Serum Glucose levels mg/dl Postprandial Blood Serum Glucose levels mg/dl
Control 133.75 104.25
Model 226.75 311.50
LLM - Low 159.25 192.75
LLM - Optimum 69.25 55.25
LLM - High 63 123
Turmeric 242.75 221.00
Cumin 437.25 449.75

[00070] Total Serum lipid: Table 11 herein-below shows the comparison of efficacy (Total Serum lipid) of the composition at different dosage levels (LLM - Low, LLM - Optimum and LLM - High) vis-à-vis that of individual ingredients (in an amount as mentioned in column 3 (LLM - Optimum) of Table 9).
Table 11: Comparison of efficacy (Total Serum lipid) of the composition at different dosage levels vis-à-vis that of individual ingredients
Study Groups Total Serum Lipid mg/dl
Control 238.91
Model 606.36
LLM Low 343.34
LLM Optimum 296.18
LLM High 247.36
Bay leaf 435.37
Fenugreek 481.88

The aforesaid efficacy studies establish that the composition of the present disclosure affords improved therapeutic efficacy in adult Zebrafish treated with the present composition and that to at varying concentration/dosage levels of the ingredients such as Turmeric, Cumin, Bay leaf and Fenugreek disclosed hereinabove in Table 10 and Table 11. Accordingly, it could be concluded that composition of the present disclosure, exhibiting synergistic effect at each of the low, optimum and high dosage, offers reduction in the risk of diabetes and/or cardiac diseases in a consumer as compared to usage of the individual ingredients.

ADVANTAGES
[00071] The present disclosure provides a composition that overcomes the limitations associated with the conventional compositions.
[00072] The present disclosure provides a composition that improves the glucose and lipid profile of a subject.
[00073] The present disclosure provides a composition that is substantially devoid of any side effects.
[00074] The present disclosure provides a composition that exhibits synergistic activity/functional reciprocity.

[00075] The present disclosure provides a composition that is easy to prepare and is economical.
[00076] The present disclosure provides a method of preparation of the composition.

,CLAIMS:1. A composition for improving glucose and lipid profile, said composition comprising:
red chilli in an amount ranging from 5% to 30% by weight of the composition;
turmeric in an amount ranging from 5% to 30% by weight of the composition;
cumin in an amount ranging from 5% to 25% by weight of the composition;
black pepper in an amount ranging from 5% to 25% by weight of the composition;
green cardamom in an amount ranging from 2% to 20% by weight of the composition;
fenugreek in an amount ranging from 5% to 20% by weight of the composition; and
bay leaf in an amount ranging from 5% to 30% by weight of the composition.
2. The composition as claimed in claim 1, wherein the composition comprises:
red chilli in an amount ranging from 10% to 25% by weight of the composition;
turmeric in an amount ranging from 10% to 25% by weight of the composition;
cumin in an amount ranging from 5% to 20% by weight of the composition;
black pepper in an amount ranging from 5% to 20% by weight of the composition;
green cardamom in an amount ranging from 2% to 20% by weight of the composition;
fenugreek in an amount ranging from 7% to 20% by weight of the composition; and
bay leaf in an amount ranging from 15% to 25% by weight of the composition.
3. The composition as claimed in claim 1, wherein the composition comprises:
red chilli in an amount of 20% by weight of the composition;
turmeric in an amount of 20% by weight of the composition;
cumin in an amount of 15% by weight of the composition;
black pepper in an amount of 10% by weight of the composition;
green cardamom in an amount of 5% by weight of the composition;
fenugreek in an amount of 10% by weight of the composition; and
bay leaf in an amount of 20% by weight of the composition.
4. A process for preparing a composition comprising red chili in an amount ranging from 5 to 30% by weight of the composition; turmeric in an amount ranging from 5 to 30% by weight of the composition; cumin in an amount ranging from 5 to 25% by weight of the composition; black pepper in an amount ranging from 5% to 25% by weight of the composition; green cardamom in an amount ranging from 2 to 20% by weight of the composition; fenugreek in an amount ranging from 5% to 20% by weight of the composition; and bay leaves in an amount ranging from 5 to 30% by weight of the composition, said process comprising the steps of:
a) dry roasting each of red chili, turmeric, cumin, black pepper, green cardamom, fenugreek and bay leaves at a temperature ranging from 40°C to 100°C for a time period ranging from 1 minute to 10 minutes to obtain roasted spices;
b) dry grinding the roasted spices to obtain ground spices;
c) blending the ground spices to obtain a spice mixture; and
d) finely grinding the spice mixture to obtain the composition such that more than 75% particles have a size ranging from 1 micron to 800 microns.
5. The process as claimed in claim 4, wherein the step of dry grinding is effected at a temperature ranging from 10°C to 50°C.
6. The process as claimed in claim 4, wherein the ground spices have more than 75% particles with size less than 1000 microns.
7. The process as claimed in claim 4, wherein the step of fine grinding the spice mixture is effected at a temperature ranging from 10°C to 50°C.