DESC:A synergistic combination of beetroot and pomegranate peel extract and its method of use thereof
RELATED APPLICATION
This application is related to and takes priority from the Provisional Application 202341066542 filed with Indian Patent Office on 4/4/2024 and is incorporated herein in its entirety.

FIELD OF THE INVENTION
The present invention relates to a unique combination comprising beetroot and pomegranate peel extract with specific nitrate content and ellagic acid content and their use in improving physical performance and cardiovascular benefits. Further, the unique amounts of natural nitrate/nitrite and ellagic acid to provide synergistic health benefits is also disclosed.

BACKGROUND OF THE INVENTION
Pomegranate (Punica granatum) is a fruit cultivated in China, India and
Mediterranean countries for thousands of years (1). Pomegranate is known for their
nutritional value and medicinal properties and improve blood pressure, mood, and
increase testosterone levels in both men and women (2). Pomegranate peel extract
contains a family of molecules called ellagitannins which when ingested is hydrolyzed into ellagic acid and appears in the circulation between 30 minutes and 5 hours after consumption. Urolithin A, a derivative of ellagic acid formed by action of gut bacteria helps to rejuvenate mitochondria which is cellular powerhouses. Experimental studies conducted in animals and worms have found that Urolithin A improved muscle function and removed damaged mitochondria before they accumulate and cause cellular dysfunction in the body. The ellagic acid from pomegranate peel extract also act as antioxidants and work in different ways to improve athletic performance (3).
Pomegranates are indicated to be rich in nitrates which is converted to nitric oxide in the
body and improve athletic performance. Nitric oxide works by dilating blood vessels to
increase oxygen delivery to working muscles. This process is shown to increase
cardiorespiratory performance and improve muscle function (4).
Beetroot (Beta vulgaris) is a plant, extensively cultivated worldwide. Root of the
plant contain number of minerals including K, Cu, Mg, Zn, Ca, P, and Na, vitamins, and phytochemicals such as polyphenols and carotenoids. The red/purple color of beetroot is due to the presence of betalain pigments such as betacyanins and betaxanthins (5). Beetroot is a rich source of dietary NO3- and several studies have investigated its potential for reducing blood pressure in humans. Beetroot is an exceptionally rich source of antioxidant compounds. The powerful antioxidant, anti- inflammatory and vascular-protective effects offered by beetroot and its constituents have been clearly demonstrated by several in vitro and in vivo human and animal studies; hence its increasing popularity as a nutritional approach to help manage cardiovascular disease and cancer. In human studies beetroot supplementation has been reported to reduce blood pressure, attenuate inflammation, avert oxidative stress, and preserve endothelial function (6). Furthermore, beetroot has also been shown to increase athletic performance. Nitric oxide is known to enhance athletic performance by boosting vasodilation, which is expansion of blood vessels leading to decreased blood pressure resulting in increased blood flow allowing more nutrients and oxygen to get to working muscles mid-workout. The antioxidants and anti-inflammatory properties of beetroot can help reduce muscle soreness and inflammation, leading to faratioster recovery after high-intensity workouts. The nitrates in beetroot have also been shown to increase muscle contractility and increase strength and power output. Supplementation with beetroot can improve cardiorespiratory endurance in athletes (7).

While the role of beetroot in improving nitrate content is well known, there is little knowledge about the synergistic effect of beetroot in combination with pomegranate extracts. The present invention studies the synergy of beetroot in combination with pomegranate and its use in preventing increase in blood pressure, atherosclerosis, erectile dysfunction, memory and cognitive dysfunction, congestive heart failure and stroke.

Brief Description of Drawings
Figure 1: Graphical representation of (a) FMD mean change (%); (b) Brachial Artery
Diameter (mm); (c) Blood Flow Velocity (cm/sec) and (d) Chalder Fatigue scores among
BeepActive 500 mg (BA500), BeepActive 1000 mg (BA1000), and placebo

Description of the Invention
Physical activity in humans is associated with increased oxygen uptake (VO2) as
well as increase in the amount of carbon dioxide exhaled from the body per unit time
during physical exercise. This increased VO2 uptake and exhalation of (VO2) can be
enhanced by administering to said mammal a combination of Beetroot-
Pomegranate (BP) extract that contains specific amounts of nitrate and Ellagic acid.

The present invention provides a composition of synergistic combination comprising beetroot and pomegranate peel extract with nitrate content of 4-8% and ellagic acid content of 10-20%. The said combination is prepared by mixing beetroot extract with nitrate content of 2-18%, more preferably about 4-8% and pomegranate peel extract with ellagic acid ranging from 10-20%, more preferably, 12-18%.
The present composition is a free-flowing dry powder with or without additional excipients. Additional excipients include but not limited to solvents, bioavailability enhancers, P-glycoprotein (PGP) inhibitors, pH modifiers, emulsifiers, carriers, anticaking agents, coating agents and oils, or one or more of the combinations.
The composition may be prepared in the form of a ready-to-drink
liquid composition, or a liquid concentrate, tablet, chewable tablets, or a gummy chewable, granulate, powder, or other solid form to be added to water or other fluid to form a drinkable liquid at the time of ingestion. Preferably the composition is a free-flowing powder. The form of powder composition is more convenient because it is easy to mix with any food material and is dissolvable in water and other liquids. Herein, the term "powder composition" is intended to mean a composition in a powder form, as a whole, including materials or a mixture thereof in a form of any of powder, granules, crystalline particles, and the likes thereof.
The present invention is directed to nutraceutical compositions which support
physical performance, attenuate muscle fatigue, and enhance aerobic respiration
utilization capacity. Physical performance herein includes but not limited to the ability of body to carry out physical tasks and activities encompassing strength, endurance, speed, agility, and coordination which is crucial for both daily living and athletic endeavors.
The synergistic combination of the present invention is useful in preventing increase in blood pressure, atherosclerosis, erectile dysfunction, memory and cognitive dysfunction, congestive heart failure and stroke. The present invention is also useful for prehypertension patients in reducing the risk of developing hypertension.
Advantageously, the inventive composition enhances physiologically vital energy stores and the bio-availability of adenosine triphosphate (ATP) energy reserves. The composition also provides for regeneration of ATP in skeletal muscle, enhances the delivery and uptake of glucose in skeletal muscle, and provides essential electrolytes and other ingredients to consumers of the liquid composition. Nitric oxide (NO) is a signaling molecule involved in many cell processes, including vasodilation, or widening of the blood vessels. Wider blood vessels increase the delivery of nutrients and oxygen to the working muscles during exercise, enhancing muscle growth and recovery.

The invention is better illustrated by the examples that are ongoing studies. These examples are, however, for better understanding and not to be considered as constraining the scope of the invention.

Examples
Objectives: The objective is to study the ergogenic potential of the novel beetroot-pomegranate (BP) extract developed by the inventors leading to improved exercise performance. BP extract being a strong anti-oxidant shall neutralize the reactive oxygen species, pro-inflammatory cytokines and excess concentration of cortisol produced during intense exercise leading to reduced sports performance, induce fatigue, and delay recovery. The purpose of the following assays are to assess the in vitro effects of BP extract on mitochondrial function, IGF-1, antioxidant activity and markers of muscle damage using C2C12 murine myoblasts and establish the synergy of combination of beetroot and pomegranate extract.
Example 1
Cell culture
C2C12 cells which is a mouse myocyte cell lines and a reliable model to study the effect of ergogenic compounds commonly used to improve exercise performance. BP extract is likely to exert positive effects on muscle strength and facilitate recovery from fatigue and attenuate exhaustive exercise-induced oxidative damage.
C2C12 mouse myocyte cell lines will be purchased from the American Type Culture Collection. C2C12 myoblasts will be cultured at 37°C in humidified 5% CO2 incubator in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% heat inactivated fetal bovine serum. For assay, C2C12 cells will be seeded in 24 well plates and incubated for 24 hours before switching to differentiation medium (DMEM supplemented with 1% horse serum) and 5 days to allow myocyte differentiation.
Cell viability assay: This assay is carried out to find out if any toxic effect of the test products on cells.
• Cytotoxicity assay will be carried out using MTT dye (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay which is based on the conversion of MTT into formazan crystals by living cells that determines mitochondrial activity.
• C2C12 cells will be seeded in 96-well plates at a density of 1 × 104 cells/mL and induced to differentiate. Cells will be treated with B, P and BP extract at various concentration and cell viability will be assessed after 24 hours by addition of MTT reagent and incubated for another 4 hours at 37ºC / 5% CO2 incubator.
• Measure absorbance at 540 nm which is directly proportional to the cell death that may be caused by the test product in which the absorbance is proportional to a cell count.
Mitochondrial density assay: During exercise the energy consumption in muscle cells increase drastically and mitochondria are the source of energy production. The mitochondrial density assay measures the effect of BP extract on muscle cells and increase the number and size of mitochondria inside the cell to cope with the additional demand of energy during exercise
• C2C12 cells will be seeded in 96-well plates at a density of 1 × 104 cells/mL, induced to differentiate, and supplemented with B, P and BP extract at various concentrations.
• After 24 hours of treatment, cells will be treated with media with Nonyl acridine orange (NAO) (100 ng/mL) dye in phosphate buffered saline and incubated for 30 minutes at 37°C.
• NAO is a metachromatic dye that specifically binds inner mitochondrial membrane. Probe fluorescence will be measured (excitation 380 nm, emission 645 nm) using microplate reader and fluorescent intensity relative to untreated control will be calculated.
Example 2
Oxygen Consumption Rate Assay: Exercise is associated with the breakdown of ATP molecules which provide energy through use of oxygen. Increased demand of energy during exercise is associated with increased demand for oxygen which can be measured in the cells in presence of BP extract
• Oxygen consumption will be assessed using an oxygen consumption rate assay kit according to the manufacturer’s instructions.
• Briefly, C2C12 cells will be grown in 96-well plates until confluence followed by induction of differentiation into myotubes.
• Subsequently cells will be treated with B, P and BP extract for 24 hours followed by treatment with 10 µL of MitoXpress-Xtra (a phosphorescent oxygen-sensitive probe whose signal increases over time when O2 is depleted in the solution) immediately before measuring the fluorescence intensity (excitation/emission, 380 nm/650 nm at 37°C, 0.5-minutes interval) using a time-resolved fluorescence plate reader.
Example 3
Antioxidant Capacity: The oxygen free radicals production increases with the rise in oxygen consumptions during high intensity exercise which in turn leads to reduced strength and increased muscle soreness. The active molecules from BP extract may interact with endogenous antioxidants to form a cooperative antioxidant network, preventing exercise-induced oxidative stress and reducing physical fatigue by scavenging the free radicals and reactive oxygen species.
• C2C12 cells grown in 24-well plates will be induced to differentiate into myotubes and treated with B, P and BP extract at various concentrations for 24 hours.
• Total antioxidant capacity will be determined in C2C12 homogenates by commercial kits. Antioxidant assay measures formation of ferryl myoglobin radical from myoglobin and hydrogen peroxide, which oxidizes the 2,2’-azino-bis 3-ethylbenzthiazoline-6-sulfonic acid (ABTS) to produce a radical cation ABTS+, a soluble green color chromogen that can be determined at 405 nm.
• In the presence of antioxidants, the radical cation is suppressed to an extent dependent on the activity of the antioxidant and the color intensity is decreased proportionally.
• Trolox, a water-soluble vitamin E analogue, serves as a standard or a control antioxidant.
Example 4
Lactate dehydrogenase and Creatine Kinase activity: Lactate dehydrogenase (LDH) and Creatine kinase (CK) are fragments of the muscle tissue and are related to muscle damage. Increased serum concentrations of these molecules are used as an indicator of damage to muscle membrane and LDH and CK are known as indicators of accumulated fatigue and exercise induced stress. The current assay explores the ability of the test products to prevent the muscle damage and hence reduce the levels of LDH and CK in the tissue culture.
• Muscle injury will be assessed by determining the released enzymes LDH and CK activity in C2C12 cells in a 24 well plate after treating the cells with B, P and BP extract at various concentrations for 24 hours.
• After the pre-treatment, cells will be exposed 2, 4-dinitrophenyl, a mitochondrial uncoupling agent for an hour to induce injury. Cell supernatant will be harvested and used for determination of LDH and CK activity using a commercial kit.
Example 5
IGF-1 Elisa assay: Insulin-like growth factor -1 (IGF-1) plays a major role in mitochondrial function and regulation of skeletal muscle growth and repair.
• C2C12 cells in 96-well plates (2 × 104 cells/well) grown until confluence and induced to differentiate into myotubes followed by addition of B, P and BP extract for 24 hours at various concentrations.
• Cell culture supernatant will be evaluated for IGF1 using commercially available ELISA kit using protocols as instructed kit manufacturer.
Examples 1-5 are underway. Example 6 was performed as follows.
Example 6
Effects of BP on flow-mediated dilation and cardiovascular health in healthy human adults: The goal of the current study is to investigate the acute effects of BP supplementation on vessel diameter, blood flow, and exercise performance. Subjects consume BP for 10 days and one hour before study. It was hypothesized that ingestion of the BP supplement would enhance vessel diameter and blood flow associated with enhanced submaximal aerobic exercise performance.
Method: This was a randomized, double-blind, placebo-controlled, cross-over clinical study initiated after obtaining written approval from an institutional ethics committee and informed consent from subjects. Healthy and physically active male/female volunteers aged between 20 to 35 years were recruited for the study and randomly assigned in 1:1:1 ratio to one of the three treatment groups i.e. BeepActive 500 mg (BA500), BeepActive 1000 mg (BA1000) or Placebo. BA was a mixture of homogenized Beetroot and pomegranate extracts in a 1:1 ratio. Subjects consumed the supplement every morning after breakfast for seven days and efficacy assessment was done at baseline and at 1 and 2 hours post-dose, Assessment included mean Flow-Mediated Dilation (FMD), brachial artery diameter, blood flow velocity, Chalder Fatigue Scale (CFQ) to assess change in subjects mental and physical energy state and biochemical parameters for safety.
Results: Twenty-one subjects completed three treatment periods. Our results showed significant improvement (p<0.05) in mean FMD% change compared to placebo regardless of BA concentration. Moreover, we have not observed any significant difference between BA500 and BA1000. The significant change was observed as early as 1-hour post dose and observed lasting over 2-hour post dose for both BA500 and BA1000 compared to placebo (Figure 1a). Similar results were observed for branchial artery diameter, and blood flow velocity (Figure 1b and Figure 1c). Significant physical fatigue scores compared to placebo against both BA500 and BA1000 were observed (Figure 1 d). No safety issues were observed throughout the study.
Conclusion: FMD is an increase in luminal blood flow and reflects vascular health that is dependent on endothelium cell function mediated by nitric oxide. Our results showed significant improvement in the mean FMD% change in subjects supplemented with BA at both concentrations as compared to placebo. A significant change was observed as early as 1-hour post dose and lasted for 2-hour post dose for both BA500 and BA1000 compared to placebo. Similar results were observed for branchial artery diameter, and blood flow velocity. Our study results also showed significant improvement in physical fatigue scores in subjects supplemented with BA at 500 and 1000 as compared to placebo. Overall, the analysis of our data revealed positive influence of BA500 and BA1000 among the subjects that could be helpful during exercise and may enhance vessel diameter, blood flow, and delay physical fatigue during exercise.

Dated: 27th Mar 2025

Kausalya Santhanam
Agent for the Applicant, IN/PA/1183
digitally signed for e-filing

References:
1. Morton, J. F. (1987). "Pomegranate, Punica granatum L". Fruits of Warm Climates. Purdue New Crops Profile. pp. 352–5. Archived from the original on 21 June 2012. Retrieved 14 June 2012
2. Aida Zarfeshany, Sedigheh Asgary, Shaghayegh Haghjoo Javanmard. Potent health effects of pomegranate. Adv Biomed Res. 2014; 3: 100.
3. Heber D. Pomegranate Ellagitannins. In: Benzie IFF, Wachtel-Galor S, editors. Herbal Medicine: Biomolecular and Clinical Aspects. 2nd edition. Boca Raton (FL): CRC Press/Taylor & Francis; 2011. Chapter 10. Available from: https://www.ncbi.nlm.nih.gov/books/NBK92772/.
4. Eric T. Trexler, Abbie E. Smith-Ryan, Malia N. Melvin, Erica J. Roelofs, and Hailee L. Wingfield. The effects of pomegranate extract on blood flow and running time to exhaustion. Physiol Nutr Metab. 2014 Sep; 39(9): 1038–1042.
5. Sentkowska, A.; Pyrzynska, K. Old-Fashioned, but Still a Superfood—Red Beets as a Rich Source of Bioactive Compounds. Appl. Sci. 2023, 13, 7445. https://doi.org/10.3390/app13137445
6. Tom Clifford, Glyn Howatson, Daniel J. West, Emma J. Stevenson. The Potential Benefits of Red Beetroot Supplementation in Health and Disease. Nutrients. 2015 Apr; 7(4): 2801–2822.
7. Hamid Arazi and Ehsan Eghbali. Possible Effects of Beetroot Supplementation on Physical Performance Through Metabolic, Neuroendocrine, and Antioxidant Mechanisms: A Narrative Review of the Literature. Front Nutr. 2021; 8: 660150.
,CLAIMS:Claims
I Claim:
1. A synergistic combination for enhancing oxygen uptake and vasodilation comprising beet extract with specific nitrate content and pomegranate peel extract with a measured ellagic acid content.
2. The synergistic combination as claimed in claim 1 wherein the nitrate content in beet extract is in the range of 2 to 18% and the ellagic acid content in the pomegranate peel extract is in the range of 10 to 20%.
3. The synergistic combination as claimed in claim 1 wherein the nitrate content in beet extract is in the range of 4 to 8% and the ellagic acid content in the pomegranate peel extract is in the range of 12 to 18%.
4. The synergistic combination as claimed in claim 1 wherein the said combination is formulated as a free-flowing powder.
5. The synergistic combination as claimed in claim 1 wherein the said combination is formulated as ready-to-drink liquid composition, liquid concentrate, gummy chewables, tablets, chewable tablets, granules, powder, solid form to be added to water or fluid to form a drinkable liquid.
6. The synergistic combination as claimed in claim 1 wherein the said combination is useful in enhancing physical performance wherein the said physical performance is the ability of body to carry out physical tasks and activities encompassing strength, endurance, speed, agility, coordination or athletic endeavors.
7. The synergistic combination as claimed in claim 1 wherein the said combination is useful in preventing increase in blood pressure, atherosclerosis, erectile dysfunction, memory and cognitive dysfunction, congestive heart failure and stroke.
8. The synergistic combination as claimed in claim 1 wherein the said combination is useful in reducing the risk of developing hypertension in prehypertension patients.