WO2022079539A1

WO2022079539A1 - Cinnamon based dietary supplements, and process of preparation thereof

Info

Publication number: WO2022079539A1
Application number: PCT/IB2021/059075
Authority: WO
Inventors: Bimali JAYAWARDENA; Ms. H.G Nadini DEWANGANI; Mrs. W A N M WARIYAPPERUMA
Original assignee: University Of Kelaniya
Priority date: 2020-10-16
Filing date: 2021-10-04
Publication date: 2022-04-21

Abstract

A method for preparing a cinnamon-based dietary supplement, and its composition thereof is provided. The method includes contacting cinnamon species extract with water at a pressure range of 0.07 MPa to 0.1 Mpa, for a period of 7 - 12 minutes to obtain a suspension; fractionating the suspension to obtain a phytochemical extract; incubating the phytochemical extract along with millet flour, at a temperature range of 10℃ - 20℃ for 0.5 to 2 hours at 100 – 300 rpm to form a mixture; and drying the mixture to a temperature range of 37℃ - 55℃, to obtain the dietary supplement. The dietary supplement includes cinnamon species-based polyphenols, cinnamon species-based proanthocyanidins, millet flour and optionally one or more food additives for treating a disease or disorder.

Description

CINNAMON BASED DIETARY SUPPLEMENTS, AND PROCESS OF PREPARATION THEREOF

TECHNICAL FIELD

The present disclosure relates to a dietary supplement, and more particularly, relates to cinnamon-based dietary supplements and process of preparation thereof.

BACKGROUND

Diabetes is one of the most common non-communicable diseases. It is the fourth leading cause of death in most developed countries and there is substantial evidence that it is epidemic in many developing and newly industrialized countries. Diabetes places an enormous burden on society that has increased over time, both in terms of economic costs and reduced quality of life. Diabetes is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. The chronic hyperglycemia of diabetes is associated with long-term damage, dysfunction, and failure of different organs, especially the eyes, kidneys, nerves, heart, and blood vessels (ADA, 2014). The cases of diabetes fall into two major categories as Type 1 diabetes and Type 2 diabetes.

There are many nutritional supplements, herbs, and spices that can provide benefits for diabetes management and weight loss. Cinnamon is one such spice. Cinnamomum zeylanicum (Family: Lauraceae) which is also known as Ceylon Cinnamon or true cinnamon, is an indigenous plant in Sri Lanka. Recent studies have shown many potential health effects of cinnamon such as anti-inflammatory properties, anti-microbial activity, blood glucose control, reducing cardiovascular disease and reducing risk of colonic cancer. Millets (Family: Poaceae) are small seeded species of cereal grains widely grown around the world. Millets are rich in dietary fibers, proteins, minerals, vitamins, energy and antioxidants (Lee et al., 2010). The composition of millet (per 100 g) is: moisture (11.2%- 13.1%), carbohydrates (65.5%-72%), proteins (6.2%- 12.5%), fats (0.5%- 5%), dietary fiber (11.3%- 12.5%) and minerals (1.5% - 4.4%). The millet protein has a well-balanced amino acid profile and is a good source of methionine, cystine and lysine. These essential amino acids benefit those who depend on plant food for their nourishment. The millet grain contains a high proportion of carbohydrates which are in the form of non-starchy polysaccharides and dietary fibers, which helps in prevention of constipation, lowering of cholesterol and slow release of glucose to blood stream during digestion. Millet grains are also rich in important vitamins viz. thiamine, riboflavin and niacin. The consumption of millets has been reported to protect from diabetes and other cardiovascular diseases.

Even though the health benefits of cinnamon and millets are well known through many studies overtime, a cost effective and commercially viable processing methods to extract coumarin free bioactive polyphenols containing nutraceutical and functional food ingredient applications is desirable.

SUMMARY

In one aspect of the present disclosure, a method for preparing a dietary supplement is provided. The method includes: a) contacting cinnamon species extract with water at a pressure range of 0.07 MPa to 0.1 Mpa, for a period of 7-12 minutes to obtain a suspension; b) fractionating the suspension to obtain a phytochemical extract; c) incubating the phytochemical extract along with millet flour, at a temperature range of 10°C - 20°C for 0.5 to 2 hours at 100 - 300 rpm to form a mixture; and d) drying the mixture to a temperature range of 37°C - 55°C, to obtain the dietary supplement.

In another aspect of the present disclosure, the dietary supplement composition is provided. The dietary supplement composition includes a) cinnamon species-based polyphenols having a weight percentage in the range of 5 - 50%; b) cinnamon species based proanthocyanidins having a weight percentage in the range of 5 - 50%; c) millet flour having a weight percentage in the range of 50 - 95%; and optionally one or more food additives. In some embodiments, the one or more food additives are selected from sweeteners, herbs, spices, natural ginger, natural garlic, natural salt, defatted milk proteins, natural hypoglycemic plant extracts, leavening agents, natural coloring agents, carbon dioxide, natural vitamins, minerals, preservatives, flavoring agents, gelatin or gelling agent, wetting agent, defatted milk, water or a combination thereof.

In yet another aspect of the present disclosure, a method of treating a disease or disorder in a subject comprising administering to the subject, a therapeutically effective amount of the dietary supplement composition including: a) cinnamon species-based polyphenols having a weight percentage in the range of 5 - 50%; b) cinnamon species based proanthocyanidins having a weight percentage in the range of 5 - 50%; c) millet flour having a weight percentage in the range of 50 - 95%; and optionally one or more food additives. In some embodiments, the one or more food additives are selected from sweeteners, herbs, spices, natural ginger, natural garlic, natural salt, defatted milk proteins, natural hypoglycemic plant extracts, leavening agents, natural coloring agents, carbon dioxide, natural vitamins, minerals, preservatives, flavoring agents, gelatin or gelling agent, wetting agent, defatted milk, water, or a combination thereof.

These and other aspects and features of non-limiting embodiments of the present disclosure will now become apparent to those skilled in the art upon review of the following description of specific non-limiting embodiments of the disclosure in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF DRAWINGS

A better understanding of embodiments of the present disclosure (including alternatives and/or variations thereof) may be obtained with reference to the detailed description of the embodiments along with the following drawings, in which:

FIG. 1 shows the gas chromatography-mass spectrometry (GC-MS) profile of C. zeylanicum by pressurized water extracts, according to one embodiment of the present disclosure;

FIG. 2 shows the GC-MS profile of extract of millet flour, according to one embodiment of the present disclosure;

FIG. 3 shows the GC-MS profile of a dietary supplement, according to one embodiment of the present disclosure; and

FIG. 4 shows the Fourier-transform infrared spectroscopy (FTIR) spectra of C. zeylanicum, millet flour, and the dietary supplement, according to one embodiment of the present disclosure.

It should be appreciated by those skilled in the art that any diagram herein represents conceptual views of illustrative systems embodying the principles of the present disclosure.

DETAILED DESCRIPTION

Reference will now be made in detail to specific embodiments or features, examples of which are illustrated in the accompanying drawings. Wherever possible, corresponding or similar reference numbers will be used throughout the drawings to refer to the same or corresponding parts. Moreover, references to various elements described herein, are made collectively or individually when there may be more than one element of the same type. However, such references are merely exemplary in nature. It may be noted that any reference to elements in the singular may also be construed to relate to the plural and vice-versa without limiting the scope of the disclosure to the exact number or type of such elements unless set forth explicitly in the appended claim.

The terminologies and/or phrases used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

The term “food additives” includes substances added to food to preserve flavor or enhance taste, appearance, or other sensory qualities.

Polyphenols are chemical substances found in plants, characterized by the presence of more than one phenol group per molecule. The polyphenols include catechins, polymeric polyphenol compounds and mixtures thereof.

The present disclosure provides for a green/environmentally friendly (free of organic solvent), time and cost-effective, and an efficient process for extraction of polyphenols from an extract of cinnamon species. The high content of water-soluble bioactive polyphenols found in cinnamon bark are enriched into a hypoglycemic millet flour matrix for preparation of the dietary supplement. This dietary supplement is used as nutritional support to manage diabetes and other glycemic control problems without negative side effects or chemical interactions generally associated with pharmaceuticals or medicines.

In an exemplary embodiment, the method for preparing the dietary supplement includes the steps of obtaining the cinnamon species extract. Although the description herein refers to cinnamon species extract as an extract of Cinnamon zeylanicum, extracts of other cinnamon species may be used as well. In an example, the quills or bark of cinnamon species is used to prepare the extract, however other plant parts such as leaves, flowers, fruits and roots, may be used as well. In a preferred example, the cinnamon species extract is obtained as a powder from cinnamon quills. For this purpose, pulverized, dried cinnamon quills coarse ground and powdered to 0.1 to 25 mm particles.

The cinnamon species extract is contacted with water at a pressure range of 0.07 MPa to 0.1 Mpa for a period of 7 - 12 minutes to allow for maximum extraction of bioactive, bioavailable, stable polyphenols and proanthocyanidins or phytochemicals from the cinnamon species extract to the water, to form a suspension. In an example, the cinnamon species extract is contacted with water under the pressure of 0.098 MPa for 10 minutes for maximum extraction of phytochemicals from the cinnamon quills into water. In an example, the w/w ratio of the cinnamon species extract to water is in the range of 1:15 to 1:25. In a preferred example, the ratio of the w/w ratio of cinnamon species extract to water is 1:20.

The suspension is further fractionated to obtain a phytochemical extract. Fractionating includes centrifuging the suspension for a period of 4 - 6 minutes at 12000 - 14000 rpm to remove suspended solids. The suspended solids contain non - soluble cinnamon quill particles, while the supernatant is rich with extracted cinnamon polyphenol and proanthocyanidins, and water-soluble compounds. In an example, the suspension is centrifuged for a period of 5 minutes at 13000 rpm. The supernatant is collected and further precipitated with an organic solvent to remove polysaccharides. In an example, the organic solvent is soluble in water. The organic solvent is selected from the group consisting of ethanol, iso-propyl alcohol, propanol, and other lower alcohols. In a preferred example, the organic solvent is 75% ethanol. The precipitated polysaccharides are removed by filtration. The remaining portion of the supernatant, left behind after the filtration, is further evaporated under reduced pressure by rotary evaporation to remove the organic solvent, leaving behind the phytochemical extract rich in proanthocyanidins and polyphenols. The polyphenol concentration is in the range of 1.53 ± 0.01 mg gallic acid equivalent/g of the cinnamon species extract, and proanthocyanidins concentration is in the range of 36.08 ± 0.01 mg of catechin equivalent/g of the cinnamon species extract. In an embodiment, the phytochemical extract is substantially free of coumarins. In an example, the percentage of coumarins in the phytochemical extract is 0% or non-detectable.

The method further includes incubating the phytochemical extract along with millet flour, at a temperature range of 10°C - 20°C for 0.5 to 2 hours at 100 - 300 rpm to form a mixture. The low temperature is used to facilitate the sorption of polyphenolic compounds into millet flour matrix. The millet flour refers to flour obtained from the millets. The millets may include one or more selected from sorghum, pearl millet, finger millet, barnyard millet, common millet, foxtail millet, kodo millet or a combination thereof. In an example, the millet flour is the flour obtained from finger millet. 10 - 30 g of millet flour (finger millet) is mixed with 50 ml of the phytochemical extract and the resultant aqueous extract is placed in a shaking incubator at 200 rpm and at a low temperature for 30 minutes. The process of shaking the cinnamon millet aqueous mixture at a low temperature is done at 15°C to facilitate the sorption of poly phenol compounds into millet flour matrix. The extract was further allowed to settle at low temperature for 1 more hour to maximize sorption. Further, the mixture was dried to a temperature range of 37°C - 55 °C, to prevent the disruption and desorption of nutrition and polyphenols. In an example, the drying can be carried out by freeze drying and spray drying or similar drying method thereof. After evaporating all the water present in the mixture, the solid mass thus obtained is homogenized by grinding the mixture into fine powder to obtain the dietary supplement.

In another exemplary embodiment, the present disclosure also provides for the dietary supplement composition. The dietary supplement composition includes a) cinnamon species-based polyphenols having a weight percentage in the range of 5- 50%; b) cinnamon species based proanthocyanidins having a weight percentage in the range of 5- 50%; c) millet flour having a weight percentage in the range of 50 - 95%; and d) optionally one or more food additives. The food additives include one or more selected from sweeteners, herbs, spices, natural ginger, natural garlic, natural salt, defatted milk proteins, natural hypoglycemic plant extracts, leavening agent, natural coloring agents, carbon dioxide, natural vitamins, minerals, preservatives, flavoring agents (coco, lemon, natural fruits to improve taste), gelatin or gelling agent, wetting agent, defatted milk, water, or a combination thereof.

In an example, the sweeteners include stevia, erythritol, or a combination thereof. In another example, the sweeteners include one or more of natural treacle, natural jaggary, and bee’s honey. In an example, the dietary supplement composition of the present invention may also use natural hypoglycemic plant extracts in combination the cinnamon species extract and the millet flour. The leavening agents include one or more selected from the group consisting of air, steam, yeast, baking powder, and baking soda. In an example, the dietary supplement composition of the present invention may also use natural hypoglycemic plant extracts or hypoglycemic drugs in combination the cinnamon species extract and the millet flour.

In yet another exemplary embodiment, the nutritional composition of the dietary supplement includes carbohydrates ranging between 60 - 72 g %; protein of concentration ranging between 7 - 8 g %; lipids of concentration ranging between 2 - 3 g %; dietary fiber ranging between 18 - 25 g %; and ash ranging between 2.4 - 2.7 g %. To achieve the nutritional composition, the dietary supplement is made with the minimum composition of combination of cinnamon pressurized water extract (phytochemical extract) and millet four. The millet flour content is in the range of 50% to 95% where more precisely 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. For better taste a natural low glycemic sweetening agent, spices, herbs, and salt or mixtures thereof is added. This may increase the attractiveness of the dietary supplement for all ages. In an example, the nutritional content of the dietary supplement may be adjusted by varying the percentage of the millet flour and the phytochemical extract depending on the age of the people to whom the dietary supplement is administered. The low-glycemic sugar includes at least one of the sweeteners selected from the group consisting of stevia and erythritol. The moisture content of the dietary supplement is kept low (to about 11% to 12%) to inhibit microbial activity level. In an example, the dietary supplement has a shelf life of 1 year when stored at room a temperature of 27 °C ± 3 °C. The dietary supplement of the present disclosure is effective for blood sugar management and cholesterol lowering effect. The dietary supplement is nontoxic and nutritious. It can be manufactured in any form such as, solid, semi-solid, powder, liquid, porridge, nutrition bar, tablet, or cracker or combination of any of these forms.

In an exemplary embodiment, the dietary supplement of the present disclosure for use as glycemic index lowering agent. In another exemplary embodiment, the dietary supplement of the present disclosure for use as weight management agent. In yet another exemplary embodiment, the dietary supplement of the present disclosure for use as satiety regulation agent.

In an exemplary embodiment, the dietary supplement of the present disclosure also concerns a method to avoid sugar drop in a subject by administering to said subject an effective amount of an extract. In an exemplary embodiment, the dietary supplement of the present disclosure also concerns a method to avoid sugar drop in a subject by administering to said subject an effective amount of the dietary supplement composition. The subject can be a human being or any other animal, preferably mammal. The animal is preferably a pet and can be chosen for example among mules or donkeys, but also cats, dogs, horses, pigs, guinea pigs, rats, mice, rabbits, gerbils, hamsters, chinchillas, and fancy rats.

The dietary supplement also concerns a method for treating a disease or a disorder in a subject by administering to said subject an effective amount of the dietary supplement of the disclosure. The disclosure also concerns a method for preventing or reducing hyperglycemia in a subject comprising administering to said subject an effective amount of the dietary supplement of the disclosure. The disclosure also concerns a method for preventing obesity or diabetes in a subject comprising administering to said subject an effective amount of the dietary supplement of the disclosure. The disclosure also concerns a method for preventing or lowering cholesterol in a subject comprising administering to said subject an effective amount of the dietary supplement of the disclosure. The disclosure also concerns a method for preventing cardiovascular diseases in a subject comprising administering to said subject an effective amount of the dietary supplement of the disclosure.

The dietary supplement compositions of the present invention which when used in defined weight ratios provide positive synergistic effects over single ingredients and/or previously known combinations. One advantage of the present disclosure is that the bioactive polyphenols present in the dietary supplement composition produces synergistic and complementary beneficial effects in diabetic and pre-diabetic patients. Therefore, the composition may be readily used by those suffering with glycemic control problems and avoid further health issues caused by negative reactions and interactions that can often be debilitating for those having previously compromised general health. Another advantage of the present disclosure is that the dietary supplement composition can reduce the LDL and, triglyceride levels and increase HDL in diabetic individuals. A further advantage of the present disclosure is that the methods and ingredients used in this invention are cost effective, time saving and safe to consume.

EXAMPLES

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. The working examples depict the effect of the dietary supplement composition including cinnamon based polyphenols, cinnamon based proanthocyanidins, and millet flour, which when used in defined weight ratios is effective in lowering blood glucose, and cholesterol levels in diabetic individuals.

Example 1: Dietary supplement composition: 4 different compositions with varying weight ratios of cinnamon species extract and millet four were prepared as observed in Table 1. The cinnamon species extract as used in the present disclosure is obtained from C. zeylanicum. The millet flour is flour obtained from finger millet. The prepared formulations were evaluated for their IC50 activity against alphaglucosidase and alpha-amylase activity.

Table 1

Example 2: Preparation of the dietary supplement: The process involves two stages. The first stage involves the preparation of phytochemical extract rich in polyphenols and proanthocyanidins from cinnamon species; and the second stage involves sorption of the phytochemical extract to a flour matrix to obtain the dietary supplement. For this purpose, pulverized, dried cinnamon quills were coarse ground and powdered to 1 to 25 mm particles. Grounded cinnamon quills (10g) were extracted with pressurized water (200.0 ml) under pressure (0.098 MPa) for 10 minutes to obtain a suspension. The resultant suspension was allowed to cool to room temperature. Aqueous cinnamon suspension was centrifuged for 5min at 13000 rpm to pellet and remove suspended solids. The supernatant was collected to which was added ethanol (75%) to precipitate polysaccharides. The ethanol mixed extract was vacuum filtered through Whatman No.l paper to remove the polysaccharide precipitate; ethanol was evaporated under reduced pressure by rotary evaporation, to obtain the phytochemical extract that is rich in polyphenols and proanthocyanidins. Method of sorption of cinnamon polyphenols and proanthocyanidins to millet flour matrix was performed by mixing with 15g to 30g of millet flour in a 50 ml of cinnamon pressured water extract (phytochemical extract). A 50 ml of phytochemical extract was used to maximize solubility to allow for uniform interaction with the millet flour matrix to produce cinnamon polyphenol enriched flour matrix. Cinnamon millet aqueous extract was kept in a shaking incubator at 200 rpm and 15°C temperature for 30 minutes. Low temperature was used to obtain maximum sorption of cinnamon polyphenol and proanthocyanidins into millet flour matrix. The extract is allowed to settle at low temperature for another Ihour and subjected to drying at 37°C - 55°C. After evaporating all the water present, the solid mass thus obtained was homogenized by grinding the mixture into fine powder, to obtain the dietary supplement composition.

Example 3: Gas chromatography-mass spectrometry (GC-MS) analysis Methodology: Crude extract (4.00 mg) was dissolved in 1.00 mL of hexane and the samples were filtered using a nylon filter (0.45 pm pore size). An aliquot of the extract was injected in the split less mode into a GC/MS 7890B Gas Chromatograph (Agilent, American) equipped with a 5977B Mass Spectrometer (Agilent, American). A fused silica capillary Agilent Technology HP-5 (5% phenyl-methyl polysiloxane) column (30 m x 0.25 mm x 0.25 pm) was used for the separation. Resultant GC-MS graphs are listed in Fig. 1 - 3. The major compounds present in Fig. 1 are cinnamaldehyde, cinnamyl alcohol, eugenol, and 4-allyl-2,6-dimethoxyphenol. The major compounds present in Fig. 3 are cinnamaldehyde, cinnamyl alcohol, eugenol, and 4-allyl-2,6- dimethoxyphenol confirming the sorption of extract of cinnamon species into the millet flour. Referring to the peaks in retention times 23.50, 22.21, 25.17, 32.71 in the product (Fig. 3) and 22.21, 23.24,25.17, 32.73 in the cinnamon species extract (Fig. 1), it can be observed that these peaks are not present in the Fig. 2.

Example 4: Fourier transmission-infrared (FT-IR) spectroscopy

Methodology: FTIR analysis was carried out to identify whether there is a change or modification in the functional groups of the product sample (dietary supplement) compared to the cinnamon species and binder (millet flour) samples after product preparation. FT-IR spectroscopy technique is useful for studying constituent functional groups in an analyte. The IR spectra of the cinnamon powder, binder and cinnamon polyphenol enriched dietary supplement showed common peaks with variations in intensities. In FTIR, an increase in the peak intensity usually means an increase in the amount (per unit volume) of the functional group associated with the molecular bond. Infrared spectra of samples were obtained using a FTIR spectrophotometer (PerkinElmer, L 1600300 Spectrum TWO LITA, Liantrisant, UK). The samples were analyzed by preparing potassium bromide (KBr) pellets. Anhydrous potassium bromide was used for pellet preparation. The ratio of sample and KBr was taken as 1:14 and the mixture were ground vigorously using mortar and pestle until ground into a fine powder. Small quantity of this powder was carefully put into pellet-forming mold, pressed under hydraulic pressure and then used for obtaining IR spectrum. Blank spectra of the instrument were run using prepared KBr pellet before samples were mounted on the instrument. Spectra were recorded with characteristic peaks in wave numbers from 600 to 4000 cm'¹ at 16 runs per scan at room temperature. FIG. 4 shows the FT-IR spectra of samples.

Table 2

The infrared (IR) spectra of the millet flour and polyphenol enriched matrix (dietary supplement) showed common peaks with variations in intensity. Peaks were found to occur in the range of 3600-3100 cm ¹ in all samples, which could be attributed to O-H bond stretching. It can be concluded that all three samples have hydrophilic properties. In the spectrum of dietary supplement, the intensity of this peak is quite high, and it could be correlated to increase in functional properties and better hydrophilic properties of the samples. Peak in the range of 3000-2900 cm'¹ in the spectra indicates presence of H- bonded for alcohol and phenol in millet flour and dietary supplement (product). The intensity is much higher in product spectra. The higher intensity was observed for the product sample could be correlated with higher phenolic content of the sample.

The carbonyl stretch peaks were also found in the range of 1600-1700 cm'¹ in all samples and at relatively higher intensities in the cinnamon samples. It specified the presence of amide I peaks resulting from the stretching vibration of C=O bond. Relatively high intensity peaks correspond to high levels of cinnamaldehyde, and aldehydes present in the cinnamon. In product sample, intensity of that peak shows’ intensity (medium intensity) lager than binder. It can be concluded that the additional added aldehyde compounds during product preparation. Further, medium intensity of peak suggests significant improvement of aldehyde composition because of cinnamon incorporation.

There is a peak in the range of 1550-1480 cm'¹ in the spectra of cinnamon and product sample. The peaks can be attributed to the C=C bond stretching of aromatic substance. But that peak cannot observe in binder sample, or the intensity is very low at the range. It can be concluded that the aromatic compounds in cinnamon are bound to the matrix of the binder during the product preparation process.

As observed in figure, the region of 600-1500 cm'¹ reflects similar crystallinity of the product and binder and the spectrum of cinnamon shows different pattern. These results revealed that there is no significant difference in the peaks position in binder and product in the fingerprint region which signifies limited effect of product preparation on functional groups of binder. However slight variations in peak intensities were noticed in the product. Here peak intensities of the product are higher compared to peak intensity of the millet flour indicating the functionality has been enhanced due to the incorporation of the extract into the miller flour.

Example 5: Effect of the dietary supplement composition on enzymatic activity: The prepared compositions, as provided in Example 1, were tested using alpha glucosidase and alpha amylase enzymatic assays. Alpha-glucosidase activity was assayed as follows: 50 pL of the solutions to be tested containing the inhibitors or distilled water (used as control) were added to 100 pL of the enzymatic solution consisting of 1 U/mL of Saccharomyces cerevisiae alpha-glucosidase (Sigma-Aldrich) in 50 mM phosphate buffer saline at pH 6.9 and pre-incubated during 10 min at room temperature. 50 pL of substrate solution, consisting of 5 mM p-nitrophenyl-alpha-D-glucopyranoside (PNP-G; Sigma- Aldrich) in 50 mM phosphate buffer saline at pH 6.9, was added and the mixture was incubated during 5 min at room temperature. The reaction was stopped by the addition of a solution of sodium carbonate 100 mM and the absorbance read at 405 nm. The assay was run in triplicates. The acarbose was used as positive control.

Alpha-amylase activity was assayed using the enzymatic assay of alpha-amylase inhibitor (Sigma-Aldrich) using alpha-amylase from porcine pancreas and starch from potato. The assay was run in triplicates. The acarbose was used as positive control.

Table 3

From table 3 it can be inferred those best results were obtained with composition 1 and 2. Considering both antidiabetic activity and product yield, composition 2 was used for further studies.

Example 6: In vivo Studies - Animal Study: Detailed in vivo study has been carried out on healthy and high fat diet (HFD) fed adult diabetic 60 Wistar rats (180- 280 g body weight) to evaluate the effect of cinnamon polyphenol enriched dietary supplement on blood sugar and lipid profile management. Animal experiments were designed and conducted in strict accordance with the ethical rules approved by the Ethics Committee of the Institute of Biology, Sri Lanka.

Experiment: Five-week-old male animals were purchased from the animal center, Medical Research Institute and housed in cages (five mice to a cage) at a temperature of 25°C with 12-h light-dark cycle. Animals were allowed to acclimatize to the laboratory environment for 1 week and during that period animals were allowed free access to standard dry pellet diet and water ad libitum. After acclimatization period total of 60 rats were divided into 6 groups of 10 animals each. 30 rats were fed with high fat diet to induce diabetes while other 30 rats were fed with normal rat diet. After 6 weeks on very high fat diet, rats were randomly divided into fasting blood glucose (FBG) balanced experimental groups. Animals with FBG more than 200mg/dl were considered as diabetic rats. Groups of healthy rats were treated as follows: 1st group treated with treated with distilled water HDW, (0.25 ml per 50 g body weight), 2^nd group treated with daily dose of millet flour (HPL - heathy rats treated with placebo, 600 mg/kg) and 3^rd group treated with daily dose of cinnamon formulated millet flour (HCDS - healthy rats treated with cinnamon based dietary supplement, 600mg/kg). Groups of diabetic rats were treated as follows: 4^th group was treated with metformin (DM-300mg/kg), 5^th group treated with daily dose of millet flour (DPL - diabetic rats treated with placebo, 600 mg/kg) and 6^th group treated with daily dose of cinnamon formulated millet flour (DCDS - diabetic rats formulated with cinnamon based dietary supplement, 600mg/kg) for thirty days. In all six groups, blood for estimation of fasting blood glucose, serum total cholesterol (TC), HDL cholesterol, LDL cholesterol, triglycerides (TRI), serum creatinine, ALT and AST were drawn on day 0 and day 30 from the tail vein of the rats.

All the grouped data were statistically evaluated with Statistical Package for Social Science (SPSS) software for windows version 22.0 (IBM SPSS Inc., Chicago, Illinois, USA). Data from the experiments are presented as mean ± standard deviation. Differences between control and treated groups have tested for significance using oneway ANOVA followed by post hoc (Dennett) multiple comparison test. In all statistical analysis a P value of <0.05 was considered to indicate statistical significance.

Effect of cinnamon polyphenol enriched dietary supplement on blood glucose and lipid profile in healthy Wistar rats:

The results of the concentration of fasting blood glucose (FBG) and serum lipid profiles (mg/dl) during 30-days of treatment period is depicted in Table 4.

There is no significant reduction (p<0.05) in the mean values of FBG for healthy rats in all three treatment groups. The mean serum total cholesterol (TC), triglycerides (TRI), low density lipoprotein (LDL), and serum aspartate aminotransferase (AST) concentration in cinnamon enriched dietary supplement administrated group (group 3) shows same trend of significant decrease (p<0.05), compared to the group 1 and group 2. The mean high-density lipoprotein (HDL) in healthy rats of the 3rd group shows significant increase (P<0.05) compared with l^stand 2^nd groups. Significant differences were not observed in serum creatinine (CRE) and serum alanine amino transferase (ALT) levels for healthy rats treated with cinnamon enriched dietary supplement.

Table 4

Data are presented as mean ± SD (n=10); Different superscript letters (^{a b}) within the same row indicate that the values are significantly different (P < 0.05). Healthy rats treated with distilled water (HDW; Healthy rats treated with placebo (HPL); Healthy rats treated with cinnamon enriched natural dietary supplement (HCDS); Fasting serum glucose (FSG); Total cholesterol (TC); Triglycerides (TG); High density lipoprotein cholesterol (HDL-C); serum creatinine (CRE); Serum aspartate amino transferase (AST); Serum alanine aminotransferase (ALT).

Effect of cinnamon polyphenol enriched dietary supplement on blood glucose and lipid profile in high fat diet induced diabetic Wistar rats:

Group comparisons of FBG and serum lipid profile in treatment period for the diabetic induced rats (Day 0- Day 30 administration) shown in Table 5. There is a significant decrease (p<0.05) in the mean values of FBS in group 4 and group 6 compared to group 5.

According to the findings, supplementation of millet flour (600 mg/kg) daily dosage showed 43.26% reduction of FBG level while daily dosage of cinnamon formulated millet flour (CFMF) (600 mg/kg) resulted in 59.80% reduction FBG level on 30^th day in diabetic rats. Lipid profile in diabetic rats reflected a significant reduction (p<0.05) in serum cholesterol, LDL, triglycerides and significant increase (p<0.05) in HDL levels upon treatment with CFMF for 30 days. Also, the current findings suggested that there was no significant toxic effect (p<0.05) of the present invention on healthy rats. The mean serum total cholesterol (TC), triglycerides (TRI), and serum creatinine (CRE) concentration in cinnamon enriched dietary supplement administrated group (group 6) shows same trend of significant decrease (p<0.05), compared to the metformin administrated group and placebo group. Serum LDL and AST concentrations have significantly decreased in metformin and dietary supplement administrated groups compared to the placebo group. But the level of serum HDL cholesterol has significantly increased in group 4 and group 6 compared to the group

5. Administration of metformin for diabetic rats has significantly decreased serum ALT concentration in high fat diet induced diabetic rats compared to the group 5 and group

6.

Table 5

Data are presented as mean ± SD (n=10); Different superscript letters (^{a b}) within the same row indicate that the values are significantly different (P < 0.05). Diabetic rats treated with Metformin (DM); Diabetic rats treated with placebo (DPL); Diabetic rats treated with cinnamon enriched natural dietary supplement (DCDS); Fasting serum glucose (FSG); Total cholesterol (TC); Triglycerides (TG); High density lipoprotein cholesterol (HDL-C); serum creatinine (CRE); Serum aspartate aminotransferase (AST); Serum alanine aminotransferase (ALT).

In summary, a detailed in vivo animal study was conducted on normal and high- fat diet fed adult diabetic Wistar rats to assess the effect of cinnamon polyphenol rich natural dietary supplement on blood sugar management, lipid profile and other hematological parameters. From table 5, treatment of dietary supplement has shown, a significant reduction in FBS (59.80%), total cholesterol (23.35%), triglycerides, (13.19%), low density cholesterol (46.47%) and an elevation of high-density cholesterol (63.44%) in diabetic rats after 30 days’ supplementation, when compared to the initial levels (p<0.05). Toxicity was not observed in healthy after treatment with the dietary supplement.

Example 7: Effect of the dietary supplement composition in diabetic and non-diabetic individuals - Human trials: A detailed human study has been carried out on adult, type II diabetic patients to evaluate the effect of cinnamon polyphenol enriched natural dietary supplement on blood sugar and lipid profile management. This clinical trial was designed and conducted in strict accordance with the ethical rules approved by the Ethics Review Committee of University of Kelaniya. Following ethical committee approval (reference no 40) type II diabetic adults with ages between 40 and 65 years were selected from the local community to participate in this study. Inclusion criteria included subjects aged 18 or more, both genders with type II diabetic condition. Exclusion criteria comprised healthy, non-diabetic individuals and have gastrointestinal symptoms or diseases. The study also excluded subjects with pregnancy, lactation, and allergy to cinnamon.

A written informed consent was obtained from each volunteer after explaining the aim and experiment risk procedures. Forty, type II diabetic adults were selected and randomly allocated in to 2 groups (n = 20): test group and placebo group. At baseline (before interventions), demographic and clinical characteristic data, such as anthropometric data, medical condition, physical activity (based on International Physical Activity Questionnaire (IPAQ) short version (Craig, et al., 2003) and dietary intake (based on Food Frequency Questionnaire (FFQ) were collected from all participants. The participants were asked not to ingest any cinnamon on the day before the intervention. The test sample group received 10g of cinnamon enriched dietary supplement daily for 14 days. The second group assigned as placebo group received placebo sample containing millet flour used to make the formulation without cinnamon for the same length of time. They were instructed not to take any supplements or traditional herbs during the study. At the end of 14th day biochemical and anthropometric outcomes were re-evaluated. Confidentiality of participant’s data was maintained throughout the procedure.

Outcome measures

Biochemical and anthropometric outcomes were measured at the baseline and after two weeks of the intervention. As biochemical measures fasting blood sugar (FBS), glycated Hemoglobin (HbAlC), fasting serum lipid levels (Total cholesterol, Triglyceride, HDL and LDL, total cholesterol and HDL ratio, serum C- reactive protein concentration, serum key hepatic enzyme levels (AST, ALT) and renal function (Serum creatinine level).

Statistical analysis.

Statistical analyses were performed using Statistical Package for the Social Sciences software (version 23; IBM SPSS Inc, Chicago, USA). The normal distribution of the variable was checked by Kolmogorov-Smirnov test. The paired t test was used to compare all blood parameters between pre- and post-treatment. Significant differences between groups were determined by using an independent t test. A value of 5% was used as the a error to define the significance. Results are tabulated in table 6. Parameters of the patients at baseline and post-treatment. * Significantly different from baseline (P<0.05).

Table 6

The efficacy and safety of the present invention was evaluated for a two-week period, double-blinded group study. Subjects were randomized and were administered either the dietary supplement or a placebo for a two-week period. The study showed that subjects receiving the cinnamon formulated dietary supplement experienced the significant reduction (p<0.05) in fasting blood glucose (10%), total cholesterol (11%), triglycerides (9.8%), low density cholesterol (7.6%) and glycated hemoglobin (6,88%). An elevation of high-density cholesterol (15.09%) was also observed in patients compared to the initial levels. There was no evidence of toxicity in any of the subjects.

Example 8: Stability determination: A stability chamber was used to assess the stability of the dietary supplement at room temperature. Freshly prepared sample was stored in a stability chamber for six months. After six month of storage period, activity was checked using alpha glycosidase and alpha amylase assays, and the results are demonstrated in table 7.

Table 7

From table 7, it can be observed that no significant change in alpha amylase inhibition activity or alpha glycosidase inhibition activity was observed even after the product was stored for a year. According to the outcomes it can be concluded that the said dietary composition can be stored at room temperature for at least one year without affecting to its biological activity, physical and chemical properties.

INDUSTRIAL APPLICABILITY

The present disclosure aims to overcome the limitations in all above-mentioned prior arts. In an exemplary embodiment, the present disclosure provides an easy protocol for extraction of bioactive polyphenols and proanthocyanidins from cinnamon bark using waterbased solvent, while selectively excludes the extraction of potentially toxic coumarin. Coumarin has strong anticoagulant properties and potentially toxic effects on the liver.

The method used to extract cinnamon compounds is an environmentally friendly technique also it is a cost effective and time saving method. This is achieved by direct extraction of bioactive polyphenol and proanthocyanidins from grounded cinnamon bark powder using water, without further purification methods.

The phytochemical extract obtained through the method of the present invention is rich with polyphenol (1.53 ± 0.01 mg gallic acid equivalent/g extract) and proanthocyanidins (36.08 ± 0.01 mg of catechin equivalent/ g extract).

The dietary supplement is safe and non- toxic as proved by the studies on healthy rats.

Production of bioactive coumarin-free polyphenols and proanthocyanidins enriched dietary supplement is proved efficacious to control the blood sugar levels in high fat diet induced diabetic rats.

The dietary supplement can reduce the LDL and, triglyceride levels and increase HDL in diabetic rats. Also, this dietary supplement can maintain the liver health of diabetic rats according to the observation of liver function markers AST and ALT.

The dietary supplement is stable, having an extended shelf life of about twelve months or greater at room temperature.

The dietary supplement can be used in any form such as tablet, nutrition bar, cracker or porridge for consumption.

The dietary supplement is not only for controlling glycemic index in diabetic individuals, but this also provide beneficial nutrition which help to manage day to day nutrition requirement with general diet.

The dietary supplement of the present disclosure is suitable for patients having prediabetes, diabetes and healthy people as well. It provides solution for individuals with high blood sugar and related complications and is also an essential nutrition source for the people without causing any negative side effects or chemical interactions normally associated with other pharmaceuticals or medicines. Synergistic effects of the ingredients of the combination provide greater beneficial effects than single compound and/or other known combinations. While aspects of the present disclosure have been particularly shown and described with reference to the embodiments above, it will be understood by those skilled in the art that various additional embodiments may be contemplated by the modification of the disclosed methods without departing from the spirit and scope of what is disclosed. Such embodiments should be understood to fall within the scope of the present disclosure as determined based upon the claims and any equivalents thereof.

Claims

23 CLAIMS What is claimed is:

1. A method for preparing a dietary supplement, the method comprising: a) contacting cinnamon species extract with water at a pressure range of 0.07 MPa to 0.1 Mpa, for a period of 7-12 minutes to obtain a suspension; b) fractionating the suspension to obtain a phytochemical extract; c) incubating the phytochemical extract along with millet flour, at a temperature range of 10°C - 20°C for 0.5 to 2 hours at 100 - 300 rpm to form a mixture; and d) drying the mixture to a temperature range of 37°C - 55 °C, to obtain the dietary supplement.

2. The method according to claim 1, wherein the phytochemical extract comprises proanthocyanidins and polyphenols.

3. The method according to claim 2, wherein the polyphenol concentration is in the range of 1.53 ± 0.01 mg gallic acid equivalent/g of the cinnamon species extract, and proanthocyanidins concentration is in the range of 36.08 ± 0.01 mg of catechin equivalent/g of the cinnamon species extract.

4. The method according to claim 1, wherein the cinnamon species extract is an extract of Cinnamon zeylanicum.

5. The method according to claim 1, wherein the cinnamon species extract is in the form of powder, and wherein the powder has a particle size in the range of 0.1 - 25mm.

6. The method according to claim 1, wherein the w/w ratio of the cinnamon species extract to the millet flour is in the range of 1:1 to 1:5.

7. The method according to claim 1, wherein fractionating further comprises: a) centrifuging the suspension for a period of 4 - 6 minutes at 12000 - 14000 rpm to remove suspended solids and collect supernatant; b) precipitating the supernatant with an organic solvent to remove polysaccharides; and c) evaporating the organic solvent under reduced pressure, to obtain the phytochemical extract in the form of dry powder.

8. The method according to claim 1, wherein the dietary supplement obtained has coumarin having a weight percentage in the range of 0 - or non-detectable

9. A dietary supplement composition comprising: a) Cinnamon species-based polyphenols having a weight percentage in the range of 5-50%; b) Cinnamon species based proanthocyanidins having a weight percentage in the range of 5-50%; c) millet flour having a weight percentage in the range of 50 - 95%; and d) and optionally one or more food additives.

10. The dietary supplement according to claim 9, wherein the polyphenol concentration is in the range of 1.53 ± 0.01 mg gallic acid equivalent/g of the cinnamon, and proanthocyanidins concentration is in the range of 36.08 ± 0.01 mg of catechin equivalent/g of the cinnamon.

11. The dietary supplement composition according to claim 9, wherein the cinnamon comprises Cinnamon zeylanicum.

12. The dietary supplement composition according to claim 9, wherein the millet of the millet flour is selected from a group comprising sorghum, pearl millet, finger millet, barnyard millet, common millet, foxtail millet, kodo millet or a combination thereof.

13. The dietary supplement composition according to claim 9, wherein the food additives are selected from a group comprising sweeteners, herbs, spices, natural ginger, natural garlic, natural salt, defatted milk proteins, natural hypoglycemic plant extracts, levering agent, natural coloring agents, carbon dioxide, natural vitamins, minerals, preservatives, flavoring agents (coco, lemon, natural fruits, etc.), gelatin or gelling agent, wetting agent, (defatted milk, water ) or a combination thereof.

14. The dietary supplement composition according to claim 13, wherein the sweeteners include stevia, erythritol, or a combination thereof.

15. The dietary supplement composition according to claim 9, is a solid, semi-solid, powder, liquid, porridge, nutrition bar, cracker or a combination thereof.

16. The dietary supplement composition according to claim 9, having a shelf life of 1 year when stored at a room temperature of 27 °C ± 3 °C.

17. A method of treating a disease or disorder in a subject comprising administering to the subject, a therapeutically effective amount of the composition according to claim 9.

18. The method according to claim 17, wherein the disease or disorder comprises diabetes, cholesterol and other cardiovascular diseases, or a combination thereof.

19. The method according to claim 17, wherein the subject is a mammal.