Concentration of plant parts for increased bioavailability

Abstract

A method of producing high-nitrate content plant parts includes obtaining plant parts including metabolically active tissue from whole plants, roots, leaves, slices, and / or cells. The method further includes propagating the plant parts in a hydroponic medium including a nitrate salt for a period of approximately 3 hours to approximately 10 days, thus producing plant parts with a high nitrate content. The method further includes harvesting the high-nitrate content plant parts.

Description

CONCENTRATION OF PLANT PARTS FOR INCREASED BIO A VAIL ABILITYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 710,264, filed October 22, 2024 and titled “Concentration of Plant Parts for Increased Bioavailability” by Sevantilal MEHTA.SUMMARY

[0002] A method of producing high-nitrate content plant parts includes obtaining plant parts including metabolically active tissue from whole plants, roots, leaves, slices, and / or cells. The method further includes propagating the plant parts in a hydroponic medium including a nitrate salt for a period of approximately 3 hours to approximately 10 days, thus producing plant parts with a high nitrate content. The method further includes harvesting the high-nitrate content plant parts.

[0003] A nitrate-rich plant-derived composition may be prepared from the high-nitrate content plant parts described above.

[0004] A dosage form may be prepared from the composition described above, the dosage form having a single-serving mass of 1-3 g to deliver dietary nitrate in an amount suitable for support of cardiovascular health.

[0005] A method of preparing a nitrate-rich plant-derived product includes obtaining plant parts including metabolically active tissue including whole beets, beetroots, beet leaves, beetroot slices, and / or beetroot cells. The method further includes propagating the plant parts in a hydroponic medium including a nitrate salt for a period of approximately 3 hours to approximately 10 days, thus producing plant parts with a high nitrate content. The method further includes harvesting the high-nitrate content plant parts and isolating a nitrate-rich fraction from the high-nitrate content plant parts. The method further includes forming the nitrate-rich plant-derived product from the nitrate-rich fraction, the nitrate-rich plant-derived product including greater than about 10% w / w nitrate on a dry basis.

[0006] A method includes formulating a supplemental beverage, powder, chew, gummy, tablet, capsule, or other enteral dosage form, or a medical food, with or without interaction withthe oral microbiome, of 1 g to 3 g of a nitrate-rich plant-derived product including greater than 10% w / w nitrate.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] In the drawings:

[0008] Figure 1 is a flow diagram of an illustrative method of producing high-nitrate content plant parts;

[0009] Figure 2 is a flow diagram of an illustrative method of preparing a nitrate-rich plant-derived product; and

[0010] Figure 3 is an illustrative composition.

[0011] It should be understood, however, that the specific embodiments given in the drawings and detailed description thereto do not limit the disclosure. On the contrary, they provide the foundation for one of ordinary skill to discern the alternative forms, equivalents, and modifications that are encompassed together with one or more of the given embodiments in the scope of the appended claims.NOTATION AND NOMENCLATURE

[0012] Certain terms are used throughout the following description and claims to refer to particular system components and configurations. As one of ordinary skill will appreciate, companies may refer to a component by different names. This document does not intend to distinguish between components that differ in name but not function. In the following discussion and in the claims, the terms “including” and “comprising” are used in an open-ended fashion, and thus should be interpreted to mean “including, but not limited to . . . ”DETAILED DESCRIPTION

[0013] The instant disclosure generally relates to plant and plant parts and, in particular, to techniques for enhancing nutrients and phytochemicals in a plant and / or plant parts. The instant disclosure is more particularly directed to enhancing nitrate content in plants or plant parts. Although the examples given here are disclosed with regards to beets, beetroot, and the like, other plants and / or plant parts may be used in various embodiments. For example, fenugreek may beused including but not limited to the whole plant, slices, and / or its roots, shoots, stems, leaves, cells, and / or the like. The instant disclosure also relates to products, compositions, formulations, and the like made from the plant and plant parts with enhanced nutrient content, in particular enhanced nitrate content. Although beetroot powder is a specifically described product, other types of products from other plants and / or plant parts may be used in various embodiments.

[0014] The products, compositions, formulations, and the like disclosed and created via the processes herein are high in dietary nitrates and have positive effects on cardiovascular health. Nitrates may be converted to nitric oxide in the body, helping to relax and dilate blood vessels thereby improving blood flow and lowering blood pressure. These nitrates also enhance circulation, which is particularly beneficial during physical activity, making them beneficial for athletes aiming to boost endurance and oxygen flow. Their anti-inflammatory properties help reduce chronic inflammation, which is associated with diseases like heart disease, cancer, and diabetes. Furthermore, they support liver health by protecting liver cells and promoting detoxification while reducing fat accumulation in the liver.

[0015] The products, compositions, formulations, and the like disclosed and created via the processes herein are also rich in essential vitamins such as vitamin C, and they act as antioxidants that support the immune system, sleep, antioxidants, skin health, and vitamin B9 (folate) bioavailability, which is crucial for cell function, tissue growth, and fetal development during pregnancy. They also contain beta-carotene, a form of vitamin A that helps maintain healthy vision, skin, and immune function. Additionally, they provide important minerals such as potassium, which supports healthy blood pressure and heart function, magnesium for muscle function and bone health, iron to prevent anemia, and manganese and copper, which aid metabolism, antioxidant defense, and energy production. Their fiber content also promotes digestion and supports gut health.

[0016] Additionally, the products, compositions, formulations, and the like disclosed herein aid in sleep by regulating neurotransmitters and hormones involved in the sleep cycle. Specifically, they help activate the parasympathetic nervous system, which is responsible for relaxation. By binding to gamma-aminobutyric acid (GABA) receptors, a neurotransmitter that reduces brain activity and calms the nervous system, they make it easier to fall asleep. Additionally, they support the production of melatonin, the hormone responsible for regulating sleep-wake cycles. By helping to relax muscles and calm the mind, they promote deeper and morerestorative sleep, which is essential for overall health and well-being. Nitrates also improve brain function by increasing blood flow to the brain, which helps prevent cognitive decline and dementia.

[0017] The products, compositions, formulations, and the like disclosed herein include enhanced levels of secondary phytochemicals such as flavonoids, betaines, and betalains. Specifically, metabolically active plant tissues, when propagated in a defined hydroponic medium containing a nitrate salt and, optionally selected bio-enhancing agents, produce nitrate and secondary metabolites beyond levels obtainable under ordinary cultivation. Betalains have cancerfighting properties, protect cells from oxidative stress and reduce the risk of certain cancers. Other benefits include aiding in weight loss as well as offering rich antioxidant protection that defends cells from damage associated with aging and chronic diseases. Also, due to the concentration, a reduction in dosage is enabled.

[0018] The methods described herein are suitable for whole plants, excised tissues, leaves and other plant parts, slices, or cultured cells (collectively referred to generally herein as “plant parts”). In one embodiment, the plant parts, for example beetroot parts, are propagated in a hydroponic medium including a nitrate source and essential mineral nutrients by being immersed or partially submerged in the hydroponic medium. The length of exposure may vary based on the plant part being propagated and the amount of growth needed to provide a sufficient amount of the plant part used to create a final product. For example, in the case of beetroot, the exposure may be between approximately three hours and ten days in one embodiment, which results in a substantial increase in nitrate within the beetroot. The medium may include a nitrate salt compatible with plant metabolism such as ammonium nitrate, calcium nitrate, magnesium nitrate, potassium nitrate, sodium nitrate, zinc nitrate, and / or combinations thereof. Magnesium nitrate supplies both nitrate and magnesium, a cofactor for nitrate reductase, thereby facilitating rapid nitrate increase. The medium may also contain nutrients such as nitrogen, sulfur, calcium, phosphorus, potassium, and magnesium each in concentrations of at least about 100 mg / L, together with nutrients such as iodine, boron, manganese, zinc, molybdenum, copper, cobalt, and iron each in concentrations of at least about 0.025 mg / L. During propagation, conditions that sustain active metabolism without microbial contamination are maintained. For example, in any embodiment, the pH of the medium may be maintained at 5.5 to 7.0 and / or the temperature may be maintained at about 18 °C to about

[0019] The medium may also contain a bio-enhancer and / or elicitor that stimulates secondary-metabolite pathways in at least one embodiment. Bio-enhancers may include, for example, pre-killed bacterial or fungal preparations produced by autoclaving or pasteurizing a nutrient broth containing microbial biomass. A bacterial preparation may be present at about five to about fifty percent by volume of the total solution, while a fungal preparation may be included at about two to about fifty percent by volume. These materials, although no longer living, release structural polysaccharides and signaling molecules that act as elicitors of secondary metabolism. Elicitors that may also be used, include, for example, jasmonic acid, salicylic acid, chitosan, or trace-metal stressors in amounts effective to up-regulate flavonoid, betaine, or betalain biosynthesis.

[0020] Next, the plant part may be removed from the propagation medium, rinsed, and dried by air or tray drying. The plant part may then be juiced to produce a liquid material. The liquid material may be dried to a powder or, alternatively, the pre-dried plant part may be ground to a powder or the plant part may be ground before drying. Spray drying and / or freeze drying may be used to dry to a powder in various embodiments. In various embodiments, the plant part may be combined with a carrier such as maltodextrose before drying or after drying. The plant parts may be processed to a uniform nitrate-rich powder to yield a composition suitable for direct formulation or further concentration. In at least one embodiment, the nitrate-rich powder displays nitrate levels of at least 8% by weight and preferably at least 10% by weight on a dry basis and retains the natural flavor and color characteristics of the plant, e g. beetroot. In the case of beets, because beet tissue naturally contains amino acids such as arginine, glutamine, and proline, at least a portion of the nitrate may exist as amino-acid nitrate complexes. Amino-acid nitrate complexes may enhance solubility and physiological bioavailability. The nitrate-rich powder is preferably free of organic solvents and water soluble, exhibiting an appearance and palatability comparable to beet juice when reconstituted in water at one to five percent weight per weight.

[0021] The nitrate-rich powder may be incorporated into a variety of ingestible or enteral dosage forms, including beverage powders, chews, gummies, tablets, capsules, or sachets in various embodiments. Dosage forms are provided such that a serving may contain 1-5 grams, for example 1-3 grams, of the composition to deliver dietary nitrate in an amount effective to support cardiovascular health, hepatic function, and other benefits as described herein. A serving may be provided as a single dosage form or multiple dosage units. For example, a three 3 gram dosageform of the composition may be provided in a single capsule containing 3 grams of the composition, or three capsules (i.e., 3 dosage units) each containing 1 gram of the composition.

[0022] In use, ingestion as indicated above increases serum, plasma nitrate, and / or nitric oxide levels and modulates cellular signaling pathways associated with energy metabolism. Serum and / or plasma nitric oxide (NO) level may be increased thorough several mechanizms including, but not limited to, one or more of: increasing serum and / or plasma nitrite (NO2 ) levels, upregulating inducible nitric oxide synthase (iNOS), or upregulating endothelial nitric oxide synthase (eNOS).

[0023] Such composition may also down-regulate the p38 MAP kinase pathway while upregulating AMPK-ai / u.2 and PGC-la, thereby enhancing mitochondrial activity and adenosine triphosphate production in skeletal and cardiac muscle in various embodiments. The same mechanisms contribute to improved vascular function and oxygen delivery.

[0024] Because the composition is entirely plant-derived and produced without synthetic additives or solvents, it is suitable for dietary supplementation and medical-food applications. It may also be administered to companion or performance animals. Species-specific dosing guidelines may be derived from body-weight nitrate limits.

[0025] Through these methods, high-nitrate content plant parts and high-nitrate content beetroot powder may be produced at industrial scale without recourse to synthetic nitrate addition or organic-solvent extraction. The resulting compositions retain natural phytonutrients, provide a concentrated and bioavailable nitrate source, and demonstrate measurable physiological benefits.

[0026] Turning to Figure 1, a method 100 of producing high-nitrate content plant parts includes obtaining 102 plant parts including metabolically active tissue from plant parts, e.g., whole plants, roots, leaves, shoots, stems, slices, and / or cells. At 104, the plant parts are propagated in a hydroponic medium including a nitrate salt for a period of approximately 3 hours to approximately 10 days, thus producing plant parts with a high nitrate content. For example, the plant parts may be propagated in a tank including an inlet and outlet. The tank may accumulate and store the medium, and the tank inlet may be used to inlet the medium into the tank. The tank outlet may be used to discharge the medium from the tank. The plant parts may be vertically arranged, wholly or partially immersed in the medium. At 106, the high-nitrate content plant parts are harvested.

[0027] Turning to Figure 2, a method 200 of preparing a nitrate-rich plant-derived product includes obtaining 202 plant parts including metabolically active tissue including whole beets, beetroots, beet leaves, beetroot slices, and / or beetroot cells. For example, beets may be cut into slices. At 204, the plant parts are propagated in a hydroponic medium including a nitrate salt for a period of approximately 3 hours to approximately 10 days, thus producing plant parts with a high nitrate content. The nitrate salt may be ammonium nitrate, calcium nitrate, chromium(III) nitrate, copper(II) nitrate, magnesium nitrate, manganese nitrate, lithium nitrate, potassium nitrate, silver nitrate, sodium nitrate, zinc nitrate, or a combination thereof. The tolerable level of nitrate concentration may be selected based on the visible symptoms of the plant.

[0028] The medium may include nutrients such as nitrogen, sulfur, calcium, phosphorus, potassium, and magnesium in a concentration of >100 mg / L each. The medium may include nutrients such as iodine, boron, manganese, zinc, molybdenum, copper, cobalt, and iron in a concentration of >0.025 mg / L each. In at least one embodiment, the medium includes nutrients at concentrations of at least 100 mg / L of nitrogen, sulfur, calcium, phosphorus, potassium, and magnesium in an acceptable form such as salts. Nutrients at concentrations at least 0.025 mg / L of iodine, boron, manganese, zinc, molybdenum, copper, cobalt, and iron, in acceptable forms such as salts, are dissolved separately and sequentially and mixed with distilled or de-ionized water thoroughly. Some nutrient sources are water soluble and some are non-water soluble. Some exist in the form of salts, some are in chelated forms, and some have different valances. For example, chromium exists in trivalent and hexavalent form wherein the former is non-toxic and the latter is toxic.

[0029] The medium may also include a bio-enhancer including a pre-killed bacterial preparation present at approximately 5-50% by volume of the solution and / or a fungal preparation present at approximately 2-50% by volume of the solution. The bio-enhancer may be prepared by growing the fungal or bacterial culture until it sporulates. The fungal or bacterial culture along with broth may be sterilized in an autoclave, and the pre killed fungal mat may be macerated and made up to known volume with the broth in which it is grown. Dextrose may be added to the medium, and the pH of the medium may be adjusted to 6.5.

[0030] An elicitor is a component that triggers the production of phytochemicals as a defense response to stress. Plants tend to accumulate secondary metabolites in specific cells, atspecific stages, and very often in response to environmental stresses. In the event of attack of pathogens, pests, herbivores, or any other biotic and abiotic factors, plants respond by activating a range of defense mechanisms which include induction of biosynthesis of secondary metabolites as phytoalexins. The yield of the secondary metabolites are increased by subjecting the selected plant or plant parts to elicitor(s) (whether abiotic and biotic). The targeted nutrient at higher concentrations also behaves as an elicitor and contributes to the increase in secondary metabolites, thus enhancing its nutritional properties. In various embodiments, the medium may include an elicitor effective to induce secondary metabolite production or secondary phytochemicals including one or more of flavonoids, betaines, betalains, steroids, pigments, colors, gums, oleoresins, all essential oils, flavors, and the like. Furthermore, the secondary phytochemical and nutrient may develop in any part of the plant. For example, the secondary phytochemical may develop in the shoot of the plant and may move towards the root of the plant.

[0031] At 206, the high-nitrate content plant parts are harvested, and at 208 a nitrate-rich fraction is isolated from the high-nitrate content plant parts. At 210, the nitrate-rich plant-derived product is formed from the nitrate-rich fraction, the nitrate-rich plant-derived product including greater than about 10% w / w nitrate on a dry basis. For example, the juice from beetroot may be obtained, and forming the nitrate-rich plant-derived product may include drying the beetroot juice to obtain a nitrate-rich beetroot powder. Maltodextrin may be added as a carrier either prior to, during, or after the drying process, which may be performed by spray drying.

[0032] Alternatively, the step of isolating the nitrate-rich fraction may include treating the harvested high-nitrate plant parts with an aqueous ethanol solution and subsequently separating the fibrous biomass from the aqueous ethanol fraction to yield the concentrated nitrate-rich extract. In at least one embodiment, species-specific dosing guidelines for canine and equine use may be provided, based on body-weight nitrate limits.

[0033] Figure 3 illustrates a composition 300 manufactured as described above in a capsule embodiment. For example, in various embodiments, the composition 300 includes a tablet, soft gelatin capsule, hard gelatin capsule, and / or combinations thereof. In other embodiments the composition is implemented as a beverage powder, chew, gummy, tablet, sachet, lozenge, patch, spray, drop, powder, granule, syrup, elixir, food stuff, medical food, and / or combinations thereof.

[0034] The composition 300 may include the nitrate-rich powder, for example beetroot powder having greater than 10% w / w nitrate. The composition may be water-soluble and exhibit appearance and palatability comparable to beetroot juice at l%-5% w / w aqueous solutions, and the composition may be free of organic solvent.

[0035] The composition 300 may be provided as a single-serving product having a mass of about one to five grams or one to three grams and configured to deliver dietary nitrate in an amount suitable to support kidney health. In another embodiment, the composition 300 may have a single-serving mass of approximately one to five grams and may be formulated to deliver dietary nitrate in an amount effective to mediate cardiovascular health issues associated with inborn errors of metabolism. Similarly, the composition 300 may have a single-serving mass of about one to five grams and may be configured to deliver dietary nitrate in an amount suitable for mediating hepatic health issues arising from inborn errors of metabolism.

[0036] In related embodiments, the composition 300 may have a single-serving mass of about one to five grams and may be configured to deliver dietary nitrate in an amount suitable for mediating kidney health issues resulting from inborn errors of metabolism.

[0037] The above methods may be used to prepare a supplemental beverage, powder, chew, gummy, tablet, capsule, or other enteral dosage form, or a medical food, with or without interaction with the oral microbiome, of 1 g to 3 g of a nitrate-rich plant-derived product including greater than 10% w / w nitrate. Specifically, in a laboratory setting, plants were subjected to thorough washing to remove soil and dust. The washed plants were surface sterilized.

[0038] In one embodiment, the grown plant component may be put into autoclavable cover. The autoclavable cover may have an opening on one of the sides and the other side may be sealed. The autoclavable cover may contain a fluid / gel, which may be a combination of formulated solution and absorbable form of solution described above. The plant may be put inside the autoclavable cover. The plant component may be partially or fully immersed in the fluid or placed on the gel. The autoclavable cover may be sealed on the other end. The amount of air in the autoclavable cover may be sufficient to grow the plant component. In one embodiment, the sealed autoclavable cover may be kept in sterile area to provide optimum condition for plant component to grow. In a second embodiment, the sterile area may have controlled temperature and humidity.

[0039] In the laboratory setting, eight hundred grams of intact roots were kept in containers immersed in formulated solutions leaving some portions of plants and plant parts above thesolution. The required temperature and light were maintained to induce growth and metabolism. In one embodiment, the magnesium nitrate used is of a higher grade than conventionally used, such as lab grade or pharmaceutical grade, as opposed to technical or fertilizer grade. This ensures higher purity and consistency in the final product.

[0040] Next, the medium was changed every 12 hours, and growth and metabolism processes were carried out for between 3 hours and 10 days. The roots of the plant thus grown in the formulated solution were then harvested, thoroughly washed, dried and powdered to suitable mesh.

[0041] The plant parts were assayed via high pressure liquid chromatography (HPLC). One gram of sample was weighed into a 100 ml flask, and 50 ml of methanol was added and refluxed for 30 minutes. The process was repeated with 3><50 ml of methanol. All the methanol extracts were combined and diluted to 250 ml with methanol, mixed and filtered to obtain a clear solution. Next, hexane, ethylacetate, and methylene chloride were mixed in the proportion of 70:20:10 and degassed.

[0042] The mobile phase was pumped at a rate of 0.8 ml / min with a back pressure of 200 psi. The injector and the detector were flushed with the mobile phase. The refractive index of the detector was set at 4x and the potentiometer chart speed was set at 0.5 cm / min. The column was equilibrated for half an hour, and the flow rate was about 1.0 ml per minute. The preparation was chromatographed and the peak response was recorded. Equal volumes of (10 pl) of the standard preparation and sample preparation were injected separately. The chromatograms were recorded and measured the responses for the peak.

[0043] The plant parts were further subj ected to detection and quantified using inductively Coupled Plasma — Optically Emission Spectrophotometer (ICP-OES) for the targeted nutrient that had accumulated several fold when compared to control. The working standard solutions were prepared in the range ofOto 1.5 ppm (0, 0.5, 1, and 1.5 ppmjby diluting appropriately the standard chromium solution with dilute nitric acid.

[0044] About 3 grams of the sample were weighed into a 30 ml silica crucible. Next, they were heated over a low Bunsen flame to volatilize the organic matter until no more smoke came out from the material. Next, the crucible was transferred to a muffle furnace having a temperature of 550° C and kept for 5-6 hours for complete ashing. The crucible was removed from the furnace and allowed to cool. The contents of the crucible were transferred into a 150 ml beaker with helpof distilled water using waterjet. Next, acid was added to the 10 ml of dilute nitric acid and 10 ml of dilute hydrochloric. The contents were boiled for about 10-15 minutes and allowed to cool. The cooled solution was made up to a known volume via distilled water. Finally, the AAS and ICP-OES were activated and allowed for stabilization. The test solutions were also aspirated.

[0045] The formulations described herein may be provided in single servings of about one to three grams, delivering dietary nitrate in amounts suitable to support liver health. In certain embodiments, they contain sufficient nitrate-rich plant-derived material to down-regulate p38 MAP kinase following administration. They may likewise up-regulate AMPK-ai / ou or PGC-la, promoting cellular energy metabolism, and may increase adenosine triphosphate levels in skeletal or cardiac muscle. Administration further elevates circulating nitric-oxide or nitrite concentrations in serum or plasma. In use, such nitrate-enriched products reduce systemic and muscular lactate accumulation, reflecting improved oxidative efficiency. They also diminish biomarkers of oxidative stress and inflammation, including malondialdehyde, C-reactive protein (CRP or hs-CRP), and interleukin-6. In some embodiments, treatment raises brain-derived neurotrophic factor (BDNF) in serum or plasma, stimulates thermogenesis within brown adipose tissue, and enhances mitochondrial biogenesis. Additional benefits include reduction of serum creatinine, an associated increase in glomerular-filtration rate, and improvements in renal function. In certain cases, the compositions lessen hepatic lipid accumulation and lower serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, consistent with improved liver integrity.

[0046] Consumption by humans, and weight adjusted doses for dogs and horses, of 1 g to 3 g or 1 g to 5 g of a nitrate-rich powder that contains not less than 10% nitrate as a beverage, powder, chew, gummy, tablet, capsule, or other enteral dosage form, or as a medical food, with or without interaction with the oral microbiome, may downregulate p38 MAPK. This mitogen-activated protein kinase (MAPK) is stress activated and is implicated in inflammation cascades. Accordingly, the products, compositions, formulations, dosage forms and the like disclosed and created via the processes herein may result in one or more of the following benefits:• Serum and / or plasma lactate level is reduced.• Skeletal muscle tissue lactate level is reduced.• Serum and / or plasma adenosine triphosphate (ATP) level is increased.• Skeletal muscle tissue adenosine triphosphate (ATP) level is increased.• Where heart muscle tissue adenosine triphosphate (ATP) level is increased.• Serum and / or plasma nitric oxide (NO) level is increased.• Serum and / or plasma nitrite (NO2") level is increased.• Inducible nitric oxide synthase (iNOS) is upregulated.• Endothelial nitric oxide synthase (eNOS) is upregulated.• Brain derived neurotrophic factor (BDNF) expression is increased as measured by BDNF level in serum and / or plasma.• Serum and / or plasma malondialdehyde level is reduced.• Serum and / or plasma cortisol is increased.• Serum and / or plasma corticosterone is increased.• C-reactive protein (CRP) and / or high-sensitivity CRP (hsCRP) is reduced.• Serum and interleukin-6 (IL-6) is reduced.• Wound healing is accelerated.• Keratinocyte migration to the site of a wound is increased.• Kidney(s) are protected from acute renal failure.• Renal atrophy and interstitial fibrosis is reduced.• The method according to claim 1 where the kidney(s) are protected against diabetic nephropathy damage.• Creatinine is reduced.• Glomerular filtration rate (GRF) is increased.• Lipid accumulation in the liver is reduced, for example in individuals with fatty liver disease, such as nonalcoholic steatohepatitis (NASH), metabolic dysfunction- associated steatohepatitis (MASH) or other etiology.• Alanine transaminase (ALT) enzyme in the liver is reduced.• The level of aspartate aminotransferase (AST) enzyme in the liver is reduced.• Systolic and / or diastolic blood pressure is lowered, for example in individuals with increased systolic and / or diastolic blood pressure.

[0047] Consumption by humans, and weight adjusted doses for dogs and horses, of 1 g to 3 g or 1 g to 5 g of a nitrate-rich powder that contains not less than 10% nitrate as a beverage, powder, chew, gummy, tablet, capsule, or other enteral dosage form, or as a medical food, with or without interaction with the oral microbiome, may upregulate AMP-activated protein kinase alpha 1 (AMPK-al) and / or AMPK-a2, a critical cellular energy switch that regulates metabolic processes. Accordingly, the products, compositions, formulations, dosage forms and the like disclosed and created via the processes herein may result in one or more of the following benefits:Serum and / or plasma lactate level is reduced.Skeletal muscle tissue lactate level is reduced.• Serum and / or plasma adenosine triphosphate (ATP) level is increased.• Skeletal muscle tissue adenosine triphosphate (ATP) level is increased.• Heart muscle tissue adenosine triphosphate (ATP) level is increased.• Serum and / or plasma nitric oxide (NO) level is increased.• Skeletal muscle tissue nitric oxide (NO) level is increased.• Serum and / or plasma nitrite (NO -) level is increased.• Inducible nitric oxide synthase (iNOS) is upregulated.• Endothelial nitric oxide synthase (eNOS) is upregulated.• Brain derived neurotrophic factor (BDNF) expression is increased as measured by BDNF level in serum and / or plasma.• Serum and / or plasma malondialdehyde level is reduced.• C-reactive protein (CRP) and / or high-sensitivity CRP (hsCRP) is reduced.• Serum and interleukin-6 (IL-6) is reduced.• Glucose uptake in skeletal muscle is increased.• Memory CD8 T cells proliferate.• Gluconeogenesis is downregulated.• Glycogenolysis is downregulated.• Lipogenesis is downregulated.

[0048] Consumption by humans, and weight adjusted doses for dogs and horses, of 1 g to 3 g or 1 g to 5 g of a nitrate-rich powder that contains not less than 10% nitrate as a beverage, powder, chew, gummy, tablet, capsule, or other enteral dosage form, or as a medical food, with or without interaction with the oral microbiome may upregulate peroxisome proliferator-activated receptor gamma coactivator 1 -alpha (PGC-la), a transcription coactivator that plays a key role in gene activation for metabolic processes. Accordingly, the products, compositions, formulations, dosage forms and the like disclosed and created via the processes herein may result in one or more of the following benefits:• Serum and / or plasma lactate level is reduced.• Skeletal muscle tissue lactate level is reduced via upregulation of lactate metabolism by muscle cells.• Serum and / or plasma adenosine triphosphate (ATP) level is increased.• Skeletal muscle tissue adenosine triphosphate (ATP) level is increased.• Heart muscle tissue adenosine triphosphate (ATP) level is increased.• Serum and / or plasma nitric oxide (NO) level is increased.• Skeletal muscle tissue nitric oxide (NO) level is increased.• Serum and / or plasma nitrite (NO?') level is increased.• Inducible nitric oxide synthase (iNOS) is upregulated.• Endothelial nitric oxide synthase (eNOS) is upregulated.• Brain derived neurotrophic factor (BDNF) expression is increased as measured by BDNFlevel in serum and / or plasma.• Serum and / or plasma malondialdehyde level is reduced.• C-reactive protein (CRP) and / or high-sensitivity CRP (hsCRP) is reduced.• Serum and interleukin-6 (IL-6) is reduced.• Thermogenesis in brown adipose tissue is upregulated.• The number of mitochondria in a cell is increased.• Hepatic fatty acid oxidation is upregulated.• A shift from fast-twitch, glycolytic (Type II) fibers to slower-twitch, fatigue-resistant Type I muscle fibers is upregulated.• Creatinine is reduced.• Where glomerular filtration rate (gfr) is increased.

[0049] In some aspects, methods, compositions, dosage forms, and formulations are provided according to one or more of the following examples:

[0050] Example 1: A method of producing high-nitrate content plant parts includes obtaining plant parts including metabolically active tissue from whole beets, beetroots, beet leaves, beetroot slices, and / or beetroot cells. The method further includes propagating the plant parts in a hydroponic medium including a nitrate salt for a period of approximately 3 hours to approximately 10 days, thus producing plant parts with a high nitrate content. The method further includes harvesting the high-nitrate content plant parts.

[0051] Example 2: A nitrate-rich plant-derived composition prepared from the high-nitrate content plant parts described above.

[0052] Example 3: A dosage form prepared from the composition described above, the dosage form having a single-serving mass of 1-3 g to deliver dietary nitrate in an amount suitable for support of cardiovascular health.

[0053] Example 4: A method of preparing a nitrate-rich plant-derived product includes obtaining plant parts including metabolically active tissue including whole beets, beetroots, beet leaves, beetroot slices, and / or beetroot cells. The method further includes propagating the plant parts in a hydroponic medium including a nitrate salt for a period of approximately 3 hours toapproximately 10 days, thus producing plant parts with a high nitrate content. The method further includes harvesting the high-nitrate content plant parts and isolating a nitrate-rich fraction from the high-nitrate content plant parts. The method further includes forming the nitrate-rich plant-derived product from the nitrate-rich fraction, the nitrate-rich plant-derived product including greater than about 10% w / w nitrate on a dry basis.

[0054] Example 5: A method of formulating a supplemental beverage, powder, chew, gummy, tablet, capsule, or other enteral dosage form, or a medical food, with or without interaction with the oral microbiome, of 1 g to 3 g of a nitrate-rich plant-derived product including greater than 10% w / w nitrate.

[0055] The following features may be incorporated into the various embodiments described above, such features incorporated either individually in or conjunction with one or more of the other features: high-nitrate content plant parts may be formed into a beetroot amino-acid nitrate complex powder including greater than 10% w / w nitrate. The nitrate salt may be ammonium nitrate, calcium nitrate, chromium(III) nitrate, copper(II) nitrate, magnesium nitrate, manganese nitrate, lithium nitrate, potassium nitrate, silver nitrate, sodium nitrate, zinc nitrate, or a combination thereof. The nitrate salt may be magnesium nitrate. The medium may include nitrogen, sulfur, calcium, phosphorus, potassium, and magnesium in a concentration of >100 mg / L each and iodine, boron, manganese, zinc, molybdenum, copper, cobalt, and iron in a concentration of >0.025 mg / L each. The medium may include a bio-enhancer including a pre-killed bacterial preparation present at approximately 5-50% by volume of the solution. The medium may include a bio-enhancer including a fungal preparation present at approximately 2-50% by volume of the solution. The medium may include an elicitor effective to induce secondary metabolite production. The medium may include an elicitor effective to increase secondary phytochemicals including one or more of flavonoids, betaines, and betalains. The composition may include beetroot powder having greater than 10% w / w nitrate. The nitrate may include at least one amino acid nitrate complex. The composition may be water-soluble and exhibit appearance and palatability comparable to beetroot juice at l%-5% w / w aqueous solutions. The composition may be free of organic solvent. The formulation may be a beverage powder, chew, gummy, tablet, capsule, or sachet having a single-serving mass of 1-3 g configured to deliver dietary nitrate in an amount suitable for support of liver health. The method may include providing species-specific dosing guidelines for canine and equine use, based on body-weight nitrate limits. The formulation mayinclude an amount of the nitrate-rich plant-derived product sufficient to down-regulate p38 MAPK in a subject upon administration to the subject. The formulation may include an amount of the nitrate-rich plant-derived product sufficient to up-regulate AMPK-al / a2 or PGC-la upon administration to a subject. The formulation may include an amount of the nitrate-rich plant-derived product sufficient to increases ATP in skeletal muscle or heart tissue in a subject upon administration to the subject. The formulation may include an amount of the nitrate-rich plant-derived product sufficient to increase serum, plasma nitric oxide (NO), or nitrite (NO2 ) in a subject upon administration to the subject.

[0056] The disclosure is described herein by the following representative non-limiting examples. Nothing in these examples should be considered as limiting the scope of the present disclosure.

[0057] Example 1 - Nutrient analysis

[0058] Extraction process - The nitrate-rich beetroot powder (10 g) was mixed in a conical flask containing 100 ml of pure water. The container was tightly closed to prevent any impurities and microbial contamination. The mixture was then placed in a shaker incubator and shaken overnight at room temperature for the extraction process. The extract was evaporated, and the solvent was removed under reduced pressure to obtain concentrated beetroot powder. The obtained extract powder was maintained at -20 °C.

[0059] Proximate analysis of nitrate-enriched beetroot powder - The individual content of macronutrients present in the nitrate-enriched beetroot sample was determined using specific methods. The energy value of total carbohydrate, protein, and fat was analyzed using Pearson’s composition and analysis of food (9thedition, 2015). The individual content of total carbohydrate, protein, and fat was analyzed using International Standard - IS 1656:2007 reaffirmed 2012, Mode of Action-Classification Schemes - MOA / CH / N, and AO AC 21stedition 2003.06-2019, respectively. The ash, moisture, and dietary fiber content were evaluated using AOAC protocols (21stedition 950.49), International Standards methods - IS 2362- 1973 and IS 11062-1984, respectively. Moreover, the content of sucrose (MOA / HPLC / 22), cholesterol (MOA / HPLC / 08), transfat (MOA / GC / 04), saturated fatty acids (SFA) (MOA / GC / 04), monounsaturated (MUFA) (MOA / GC / 04) and polyunsaturated fatty acids (PUFA) (MOA / GC / 04) were determined by following the Mode of Action-Classification Schemes.

[0060] Infra-red spectroscopy analysis of nitrate in enriched beetroot powder - The FT-TR spectra of control and nitrate-enriched beetroot samples were recorded using BRUKER system. The software model Alpha T OPUS 6.0 was used to interpret the collected data, which had a frequency range of 4000-500 cm'1and a spectral resolution of 4 cm Analysis at atomic level, such as the dipole moment, force constant, and bond strength in chemical structure were performed. The background noise from each scan was removed, adjusting for the levels of atmospheric and instrumental noise. Before the spectra for each ground sample were recorded, a blank reference was scanned and each ground sample was placed on a diamond reflection probe for three consecutive measurements. The spectra of a nitrate standard solution at concentrations ranging from 50 and 250 ppm are recorded before analyzing the samples.

[0061] ICP-MS analysis of heavy metals and minerals in nitrate enriched beetroot powder -Inductively coupled plasma mass spectrometric analysis, using the model ICP-MS, Perkin- Elmer SCIEX ELAN 6100 DRC II, was done to profile and determine the heavy metals and minerals, such as Fe, Ca, Mg, Na, K, Zn, Cu, Mn, and Se in the nitrate-enriched beetroot concentrate. Initially 1g of dried beetroot powder was digested using cone. H2SO4-H2O2. Meanwhile, inductively coupled argon plasma (ICAP 6500 Duo, Thermo-Scientific, Gloucester, UK) was used to prepare standard indium and iridium (10 g / L). This was standardized using a 1000 mg / L multi -element certified solution. The acid-digested sample was introduced into the Sturman-masters spray chamber's V-groove quartz concentric nebulizer. Concisely, a radio frequency power of 120W was kept constant as a crucial parameter, and the argon flow rates were 13, 0.8, and 0.95 L / min for the sample, cool, and auxiliary gases, respectively. The isotope intensities of Fe, Ca, Mg, Na, K, Zn, Cu, Mn, and Se were measured, using collision cell technology to remove any potential interference. Furthermore, gaseous hydrogen and helium were used in the experiment, with an average flow rate of 3.4 mL.

[0062] HPLC optimization for the detection of nitrate in enriched beetroot powder - The bioactive composition of both the standard nitrate and the nitrate enriched beet powder was profiled separately using a High-Performance Liquid Chromatography System. This system includes of a Binary Pump with 7725i Rheodyne injection port, a Solvent Degasser, Photodiode Array Detector, and C18 column having dimensions of 5 pm, 250 x 4.6 mm. Gradient elution of analytes was carried out with H2O + 0.1% TFA (trifluoroacetic acid) as solvent A and Acetonitrile + 0.1% TFA as solvent B. The optimized gradient program was followed as 20%A-80% B (0-4min), 40%A- 60%B (4-15 min), 50%A-50%B (15-25 min), and 80%A-20%B (15-25 min). The flow rate was maintained at 0.4 ml / min, with an injection volume of 5 pL. The wavelength range monitored was set between 210 and 400 nm. The Analytical data were evaluated using EMPOWER 3 data processing software.

[0063] LC-MS / MS analysis of micronutrients in nitrate-enriched beetroot powder - To profile and quantify various nutrients including, pigments and vitamins present in the nitrate-enriched beetroot powder, Liquid Chromatography-Mass Spectrometry analysis (LC- MS / MS) was conducted with PDA detector. The analysis of these nutrients of interest was performed by following the standard protocols authorized by the Association of Official Analytical Chemists (AO AC), and the Food Safety and Standards Authority of India (FSSAI) with some modification to optimize for the nature of the sample and instrument working module. Specifically, B9 (folic acid), folate (food), folate (DFE), and folate (food) were determined based on the AOAC, while the quantification of total ascorbic acid, vitamin Bl (thiamin), B2 (riboflavin), B3 (niacin), B5 (pantothenic acid), total choline, betaine, vitamin B12 (cobalamin), B6 (pyridoxin), and A (retinol) were accomplished by the methods detailed by the FSSAI. Accordingly, in the instrumentation, an asymmetry C18 column with a dimension of 150 mm x 2.1 mm, particle size 1.7 pm was used for this study because this type of columns provide a higher sensitivity in eluting phyto-based components. The mass spectrometry of the sample was performed using SYNAPT Mass Spectrometer and the peaks of the detected compounds were observed at 280 nm. By comparing the MS / MS fragmentation patterns with the library database provided, identification of the interested micronutrients such as vitamins and pigment compounds from the nitrate-enriched beetroot sample was accomplished. Results of these analyses are shown in Tables 1-5, below.Table 1: Proximate analysis of macronutrients in nitrate-rich beetrootBLQ - Below limit of quantification / LOQ - Limit of quantificationTable 2: Heavy metals and minerals content in nitrate-rich beetroot concentrateTable 3: Comparison of RT and peak area values of nitrate standard, nitrate in control and nitrate-rich beetroot concentratesTable 4: LC-MS / MS analysis of micronutrients in nitrate-rich beetroot concentrateTable 5: Comparative of overlook nutrient composition of normal and nitrate-rich beetroot

[0064] Example 2 - Cardio-performance Evaluation

[0065] The following assessments were performed to assess cardiopulmonary exercise performance in an exercising rat model:

[0066] Example 3 - Gene Expression Modulation

[0067] Expression of fatigue and metabolism-related genes including p38 MAPK, AMPK-al and a2, PGC-la and glycogen synthase (Gysi) were analyzed in heart or skeletal muscle tissues using quantitative real-time PCR in a patient or appropriate model. These markers provide insights into the molecular adaptations associated with fatigue resistance and improved cardiac performance.

[0068] 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 in this disclosure have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein. Numerous other modifications, equivalents, and alternatives will become apparent once the above disclosure is fully appreciated. It is intended that thefollowing claims be interpreted to embrace all such modifications, equivalents, and alternatives where applicable.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method of producing high-nitrate content plant parts, comprising:(a) obtaining plant parts comprising metabolically active tissue selected from the group consisting of: whole plants, roots, leaves, shoots, stems, slices, and cells;(b) propagating the plant parts in a hydroponic medium comprising a nitrate salt for a period of approximately 3 hours to approximately 10 days, thus producing plant parts with a high nitrate content (“high-nitrate content plant parts”); and(c) harvesting the high-nitrate content plant parts.

2. The method of claim 1, wherein the plant parts comprise beets, the method further comprising forming the high-nitrate content plant parts into a beetroot amino-acid nitrate complex powder comprising greater than 10% w / w nitrate.

3. The method of claim 1, wherein the nitrate salt is selected from the group consisting of: ammonium nitrate, calcium nitrate, chromium(III) nitrate, copper(II) nitrate, magnesium nitrate, manganese nitrate, lithium nitrate, potassium nitrate, silver nitrate, sodium nitrate, zinc nitrate, and a combination thereof.

4. The method of claim 1, wherein the plant parts comprise fenugreek.

5. The method of claim 1, wherein the medium comprises N, S, Ca, P, K, Mg in a concentration of >100 mg / L each and I, B, Mn, Zn, Mo, Cu, Co, Fe in a concentration of >0.025 mg / L each.

6. The method of claim 1, wherein the medium comprises a bio-enhancer comprising a pre-killed bacterial preparation present at approximately 5-50% by volume of the solution.

7. The method of claim 1, wherein the medium comprises a bio-enhancer comprising a fungal preparation present at approximately 2-50% by volume of the solution.

8. The method of claim 1, wherein the medium comprises an elicitor effective to induce secondary metabolite production.

9. The method of claim 1, wherein the medium comprises an elicitor effective to increase secondary phytochemicals comprising one or more of flavonoids, betaines, and betalains.

10. A nitrate-rich plant-derived composition prepared from the high-nitrate content plant parts of claim 1.

11. The composition of claim 10, comprising a beetroot powder having greater than 10% w / w nitrate.

12. The composition of claim 11, wherein the nitrate comprises at least one amino acid nitrate complex.

13. The composition of claim 11, wherein the composition is water-soluble and exhibits appearance and palatability comparable to beetroot juice at l%-5% w / w aqueous solutions.

14. The composition of claim 11, wherein the composition is free of organic solvent.

15. The composition of claim 10, formulated as a beverage powder, chew, gummy, tablet, capsule, or sachet.

16. The composition of claim 10, having a single-serving mass of 1-3 g configured to deliver dietary nitrate in an amount suitable for support of liver health.

17. A dosage form prepared from the composition of claim 10, wherein the dosage form has a single-serving mass of 1-3 g to deliver dietary nitrate in an amount suitable for support of cardiovascular health.

18. A method of preparing a nitrate-rich plant-derived product comprising:(a) obtaining plant parts comprising metabolically active tissue selected from the group consisting of: whole beets, beetroots, beet leaves, beetroot slices, and beetroot cells;(b) propagating the plant parts in a hydroponic medium comprising a nitrate salt for a period of approximately 3 hours to approximately 10 days, thus producing plant parts with a high nitrate content (“high-nitrate content plant parts”);(c) harvesting the high-nitrate content plant parts;(d) isolating a nitrate-rich fraction from the high-nitrate content plant parts; and(e) forming the nitrate-rich plant-derived product from the nitrate-rich fraction, wherein the nitrate-rich plant-derived product comprises greater than about 10% w / w nitrate on a dry basis.

19. The method of claim 18, wherein the nitrate-rich plant-derived product is a beetroot powder.

20. A method of formulating a supplemental beverage, powder, chew, gummy, tablet, capsule, or other enteral dosage form, or a medical food, (“formulation”) with or without interaction with the oral microbiome, of 1 g to 3 g of a nitrate-rich plant-derived product comprising greater than 10% w / w nitrate.

21. The method of claim 20, further comprising providing species-specific dosing guidelines for canine and equine use, based on body-weight nitrate limits.

22. The method of claim 20, wherein the formulation comprises an amount of the nitrate-rich plant-derived product sufficient to down-regulate p38 MAPK in a subject upon administration to the subject.

23. The method of claim 20 wherein the formulation comprises an amount of the nitrate-rich plant-derived product sufficient to up-regulate AMPK-al / a2 or PGC-la upon administration to a subject.

24. The method of claim 20, wherein the formulation comprises an amount of the nitrate-rich plant-derived product sufficient to increases ATP in skeletal muscle or heart tissue in a subject upon administration to the subject.

25. The method of claim 20, wherein the formulation comprises an amount of the nitrate-rich plant-derived product sufficient to increase serum, plasma nitric oxide (NO), or nitrite (NO2") in a subject upon administration to the subject.

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