BACKGROUND

[0001] Adenosine monophosphate-activated protein kinase (AMPK) is the primary regulator of cellular metabolism. When cells experience energy depletion, they activate AMPK which is also responsive to exercise and calorie restriction in various body tissues. Subsequently, AMPK influences the of numerous genes and proteins, thereby stimulating catabolic pathways to replenish ATP levels. The activation of AMPK primarily relies on the rations of AMP/ ATP and ADP/ATP within cells. This activation involves AMP binding to the protein and subsequent phosphorylation by a specialized kinase.

[0002] Structurally, AMPK is a heterotrimer, consisting of an a catalytic subunit and a heterodimer composed of P and y subunits. During periods of low energy within cells (characterized by depleted ATP and elevated ADP and AMP levels), ADP and AMP associate with the a subunit, activating an upstream kinase known as LKB-1. Once activated, LKB-1 phosphorylates the a subunit at Thrl72, substantially activating AMPK activity (overt OOfold). This starts a form of AMPK that engage in downstream signaling pathways that suppress anabolic processes (such as fatty acid and cholesterol synthesis) while simultaneously promoting catabolic, energy-generating pathways, including glucose uptake and fatty acid oxidation. Consequently, this activation of AMPK raises ATP levels within the cell.

[0003] Numerous compounds and natural extracts demonstrate AMPK enhancement activity by impeding mitochondrial ATP production, subsequently increasing the AMP/ ATP and ADP/ATP ratios. Among these compounds are Metformin, a medication for treating Type II diabetes, and natural substances such as berberine, arctigenin and resveratrol. It is well-established that D- ribose is an effective modulator of oxidative metabolism. D-ribose is a monosaccharide, a type of simple sugar that plays a critical role in the adenosine monophosphate (AMP) salvage pathway. The AMP salvage pathway is a metabolic pathway that allows cells to recycle and regenerate AMP, a vital nucleotide involved in various cellular processes such as energy production, signal transduction, and nucleic acid synthesis. [0004] D-ribose is a naturally occurring molecule found in all living cells. It is synthesized from glucose via the pentose phosphate pathway (PPP), ribose is essential to RNA, adenosine triphosphate (ATP), nicotinamide adenine dinucleotide (NADH), and other molecules crucial for cellular metabolism. It acts as a precursor for purine nucleotide synthesis and is also involved in insulin secretion as a messenger. While D-ribose can be obtained from foods like fruits and vegetables, it can also be found in red meat, fish, and dairy.

[0005] In the de novo synthesis pathway, D-ribose is first converted into 5-phosphoribosyl-l- pyrophosphate (PRPP) through the action of the enzyme ribose-phosphate pyrophosphokinase (RPKP). PRPP is a key intermediate used in several steps of purine nucleotide synthesis. One of the primary roles of D-ribose in purine nucleotide synthesis is to provide the ribose moiety, an essential component of the purine ring structure. The purine ring is constructed on the PRPP molecule, and the ribose moiety of PRPP provides the carbon and nitrogen atoms necessary to form the purine ring structure.

[0006] In the AMP salvage pathway, D-ribose serves as the precursor for the de novo synthesis of AMP, which occurs in the cytoplasm of cells. D-ribose is converted into phosphoribosyl pyrophosphate (PRPP), a critical intermediate molecule, by the action of an enzyme called ribosephosphate pyrophosphokinase (RPKP). PRPP then serves as a substrate for the synthesis of AMP through a series of enzymatic reactions involving other intermediates, ultimately producing AMP. AMPK is an enzyme that acts as a cellular energy sensor, monitoring the adenosine monophosphate (AMP) ratio to adenosine triphosphate (ATP), essential molecules in cellular energy production. When cellular energy levels are low, such as during periods of stress, exercise, or nutrient deprivation, the AMP to ATP ratio increases, leading to the activation of AMPK. Once activated, AMPK initiates a series of molecular events that help restore cellular energy balance.

[0007] Additionally, certain substances are investigated for their ability to mimic the effects of exercise, including AMP-activated protein kinase (AMPK) activators, such as Metformin and AICAR, and sirtuin activators, such as resveratrol, for activation of AMPK. These drugs have been shown to improve glucose uptake and insulin sensitivity, increase mitochondrial biogenesis, and reduce inflammation, among other effects. [0008] Autophagy is a cellular process that involves the degradation and recycling of cellular components, such as damaged organelles, misfolded proteins, and other cellular debris. It is an essential cellular process that helps maintain cellular health, promotes survival during nutrient deprivation, and plays a role in cellular development, differentiation, and immune responses. Various signaling pathways, including AMPK, tightly regulate autophagy. In recent years, the field of anti-aging has seen a growing interest in the metabolic demands and the updating of certain organelles in cells. In our study, the implication of D-ribose as a mediator of autophagic processes via AMPK activation has merit.

[0009] Most studies have focused on the impact of D-ribose supplementation in athletes or individuals with specific medical conditions. The doses typically range from 3 to 15 grams daily, depending on the intended purpose and the population studied. Applicant demonstrates that measurable effects may be produced utilizing much lower concentrations in healthy mammals, particularly through a previous undocumented mechanism of AMPK activation. The need to augment metabolic demands and modify specific cell organelles has gained prominence in antiaging research in recent years. Applicant’s data indicates the mechanism by which D-ribose acts to replenish energy levels by modulating AMP/ATP ratios.

DESCRIPTION OF THE DRAWINGS

[0010] Table 1 is a summary of body weight change from week 0 to week 4 of Groups 1, 2 and 3 of Experiment 1.

[0011] Table 2 is a summary of Serum ATP levels for weeks 1 to 4 of Groups 1, 2 and 3 of Experiment 1.

[0012] Table 3 is a summary of AMPk phosphorylation levels in terminal tissue of the skeletal muscle, heart, and liver of Groups 1, 2 and 3 of Experiment 1.

[0013] Table 4 is a summary of PGC-la levels in terminal tissue of the skeletal muscle, heart, and liver of Groups 1, 2 and 3 of Experiment 1.

[0014] Table 5 is a summary of citrate synthase levels in terminal tissue of the skeletal muscle, heart, and liver of Groups 1, 2 and 3 of Experiment 1. [0015] Table 6 is a summary of the mean bodyweight from week 0 to week 4 of Groups 1 to 6 of Experiment 2.

[0016] Table 7 is a summary of Serum ATP levels for weeks 1 to 4 of Groups 1 to 6 of Experiment 2.

[0017] Table 8 is a summary of ATP levels in terminal tissue of the skeletal muscle of Groups 1 to 6 of Experiment 2.

[0018] Table 9 is a summary of p-AMPk levels in terminal tissue of the skeletal muscle of Groups 1 to 6 of Experiment 2.

[0019] Table 10 is a summary of PGC-la levels in terminal tissue of the skeletal muscle of Groups 1 to 6 of Experiment 2.

[0020] Table 11 is a summary of citrate synthase activity in terminal tissue of the skeletal muscle of Groups 1 to 6 of Experiment 2.

[0021] Table 12 is a summary food consumption of the animals of Groups 1 to 6 of Experiment 2 from week 1 to 4.

[0022] Table 13 is a summary of the blood glucose levels from week 0 to week 4 of Groups 1 to 6 of Experiment 2.

[0023] Table 14 is a summary of the insulin levels at week 2 and week 4 of Groups 1 to 6 of Experiment 2.

[0024] Table 15 is a summary of the results of the endothelial nitric oxide measure of the terminal test subjects of Groups 1 to 6 of Experiment 2.

[0025] Table 16 is a summary of the superoxide dismutase measured in the serum and skeletal muscle of the terminal test subjects of Groups 1 to 6 of Experiment 2.

[0026] Table 17 is a summary of the glutathione measured in the serum and skeletal muscle of the terminal test subjects of Groups 1 to 6 of Experiment 2. Experiment 1

[0027] Eighteen mice were divided into three groups, each group containing six members. Following a seven day acclimatization phase, the treatment duration extended from the first to the fourth week. The type of treatment for each group is outlined below:

Group 1 : Control group (no exercise, no D-ribose treatment)

Group 2: No exercise, D-ribose treatment (200mg/kg B. wt.)

Group 3: Exercise, D-ribose treatment (200mg/kg B. wt.)

[0028] For the exercise regimen, the animals were subjected to a forced daily treadmill exercise running at 2.3kph for 45 minutes throughout the treatment period.

[0029] A general overview of the analysis conducted on the collected tissues of the animals of each group is summarized in the chart below:

[0030] The study of D-ribose treatment with and without exercise started on a Tuesday. Every Tuesday, body weight and ATP level in serum were assessed. In week 5 the test subjects were terminated and the tissues and serum were collected for analysis.

[0031] Referring to Table 1, no significant changes were observed in body weights in any of the treatment groups. Values are expressed in Mean ± SD; n=6. Week 0 in body weight represents body weight of animals before initiation of treatment and it is immediately after randomization. Assessment of ATP:

[0032] Blood was collected in labeled tubes after completion of week 1, week 2, and week 3 from all animals under mild anesthesia. For week 4 samples, the collection method described in the necropsy section was followed. Serum samples were isolated and preserved at -80° C until further examination.

[0033] Referring to Table 2, a progressive increase in serum ATP concentrations was observed from Week 1 to Week 4 for both Group 2 and Group 3, with the latter exhibiting the highest concentration. Values are expressed in Mean ± SD; n=6/group.

Assessment of p-AMPk:

[0034] Blood was collected in labeled tubes after completion of weeks 1, week 2, and 3 from all animals under mild anesthesia. Standard ELISA techniques, AMPK phosphorylation levels were measured in liver, heart, and skeletal muscle tissues from each group charted in Table 3.

[0035] Referring to Table 3, the administration of D-ribose alone led to a rise in phosphorylated AMPK within heart and liver tissues (Groups 2 and 3), though not significantly in skeletal muscle. The combination of D-ribose and exercise resulted in elevated AMPK phosphorylation across all examined tissues. Values are expressed in Mean ± SD; n=6/group.

Assessment of PGC-la:

[0036] Blood was collected in labeled tubes after completion of weeks 1, week 2, and 3 from all animals under mild anesthesia. Standard ELISA techniques measured PGC-la levels in liver, heart, and skeletal muscle tissues from each group charted in Table 4.

[0037] Referring to Table 4, the administration of D-ribose alone led to a rise in PGC-la within heart and liver tissues (Group 2 and 3), though not significantly in skeletal muscle. The combination of D-ribose and exercise resulted in elevated PGC-la across all examined tissues. Values are expressed in Mean ± SD; n=6/group. Assessment of Citrate Synthase:

[0038] Blood was collected in labeled tubes after completion of weeks 1, week 2, and 3 from all animals under mild anesthesia. Standard ELISA techniques measured citrate synthase levels in liver, heart, and skeletal muscle tissues from each group charted in Table 5.

[0039] Referring to Table 5, noticeable increase in citrate synthase levels was found in all examined tissues after treatment with D-ribose alone or in combination with exercise. This increase was most apparent in the liver, followed by skeletal muscle and heart. Values are expressed in Mean ± SD; n=6/group.

Experiment 2:

[0040] This study used, adult male Swiss albino mice (approximately 8 weeks old). The mice were sourced from a CPCSEA-approved laboratory, adhering to ethical protocols and animal care guidelines. The Institute Animal Ethics Committee (IAEC) sanctioned the study at the testing facility. During experimentation, the mice had unrestricted access to food and water and were kept in a pathogen-free environment with a consistent 12-hour light/dark cycle and 12-15 air exchanges per hour.

[0041] In total, 36 mice were divided into six groups, each group containing six members. Following a seven day acclimatization phase, the treatment duration extended from the first to the fourth week. The type of treatment for each group is outlined below:

Group 1 : Control Group (No exercise, no treatment)

Group 2: D-ribose treatment (75 mg/kg B.wt); dose volume: 10 mL/kg B. wt.; dose level: 75 mg/kg; concentration 7.5 mg/mL

Group 3: D-ribose + Exercise (75 mg/ kg B.wt); dose volume: 10 mL/kg B. wt.; dose level: 75 mg/kg; concentration 7.5 mg/mL

Group 4: D-ribose treatment (125) mg/ kg B.wt); dose volume: 10 mL/kg B. wt.; dose level: 125 mg/kg; concentration 12.5 mg/mL

Group 5: D-ribose + Exercise (125) mg/ kg B.wt); dose volume: 10 mL/kg B. wt.; dose level: 125 mg/kg; concentration 12.5 mg/mL

Group 6: D-ribose Treatment (800 mg/kg B.wt); dose volume: 10 mL/kg B. wt.; dose level: 800 mg/kg; concentration 80 mg/mL [0042] Test item formulations were prepared freshly before dosing, and administration was done at about the same time on each day of the experimental period. Water was used as a vehicle to formulate all test items at the specified dose levels and concentrations identified above.

[0043] For the exercise regimen, the animals were subjected to a forced treadmill exercise carried out at controlled environmental conditions with preferred lux, temperature, humidity and sound barrier. A regular treadmill (Hercules Model No. TMA21) used by humans was available for subjecting the mice to daily exercise. The treadmill was designed with an electro sensitizer (AC input: 85V-265V AC; DC output: 0V - 300V DC) in a special lane box made of acrylic Perspex partitions (4 mm thickness), which was used to fabricate the treadmill’s running platform, forming a modified three-lane rodent treadmill allowing 3 animals to run simultaneously (3 animals of a cage in one lane). The mice of the experimental groups were exercised daily for 45 minutes throughout the treatment period. Exercise comprised a treadmill running at a speed of 2.3 kph with no inclination. During the acclimatization period, as an introduction, the animals were inducted to running exercises for 15 minutes initially and for 30 minutes the next day. Subsequently the exercise duration was increase to 45 minutes, maintained from the start to the end of the in-life experimentation.

In-Life Assessments

[0044] Throughout the study period, the weekly body weight of each animal was assessed using a calibrated electronic digital weighing balance Sartorius BSA32025. Also, weekly feed intake was measured by weighing the feed offered and feed left over once weekly and recorded. The average feed consumed was calculated and expressed as grams of feed consumed/animal/day. Glucose levels in blood were measured weekly (weeks 1-4) on the starting day of every week before the day’s Exercise and dosing activities. Blood is drawn from the tail vein, and measured glucose and ketone are in strip-operated digital sensors. Glucose was measured using SD CodeFreeTM Glucometer (Glucometer strips Lot No. C038298). ATP was measured in serum weekly (week 1 to week 3) during the life phase on the starting day of every week just before the day's scheduled dosing/exercising activities and terminally in serum and skeletal muscle after week 4 post necropsy. Blood was drawn via retro-orbital plexus under mild anesthesia and immediately processed for serum separation and stored at -80 C until analysis. Additionally, ATP was also measured terminally in skeletal muscle. Enzyme-Linked Immunosorbent Assay (ELISA) Kit for Adenosine Triphosphate (ATP) was procured. Assays were performed according to the manufacturer's instructions. Serum insulin was estimated (week 2 and week 4) using a procured ready ELISA kit.

Necropsy

[0045] After completion of the fourth week of the experiment, all the animals were euthanized using an overdose of isoflurane (20% v/v in propylene glycol in a glass vacuum desiccator). Gross macroscopic observation of all internal organs was done before sampling blood and specified tissue/organs.

[0046] Blood was drawn through cardiac puncture using a 1 ml syringe with a 26G needle. The blood samples were collected into labeled tubes for further processing. The serum was separated and aliquoted to labeled tubes and used for ELISA of various biomarkers. Skeletal muscle samples were carefully excised, and flash frozen in liquid nitrogen (LN2) and stored at -80°C until further processing.

Assessment of Feed Consumption Patterns:

[0047] The feed intake of animal subj ects in different experimental groups during the in-life Study. Throughout the study period, the weekly body weight of each animal was assessed using a calibrated electronic digital weighing balance Sartorius BSA32025 as charted in Table 6. Also, weekly feed intake was measured by weighing the feed offered and feed left over once weekly and recorded. Finally, the average feed consumed was calculated and expressed as grams of feed consumed/mouse/day as charted in Table 12.

[0048] Referring to Table 12 animals in the exercising groups that were treated with D-ribose at both 75 and 125 mg/kg dose levels (Group 3 and Group 5) comparatively consumed less feed initially during week 1.

Assessment of Blood Glucose:

[0049] Results of glucose and insulin levels measured in serum from week 0 to week 4 for each group are summarized in Table 13. Blood was drawn through cardiac puncture using a 1 ml syringe with a 26G needle. The blood samples were collected into labelled tubes for further processing. The serum was separated and aliquoted to labelled tubes and used for ELISA of various biomarkers.

[0050] Referring to Table 13, from Week 2 onwards, blood glucose levels were lowered in all treatment groups. Interestingly, a combination of D-ribose and regular Exercise (Group 3 and Group 5) produced a maximum effect. D-ribose improves blood glucose parameters in sedentary and exercising mammals.

Assessment of Insulin Sensitivity:

[0051] Insulin levels measured in serum (at week 2 and week 4) did not exhibit statistically significant differences between the experimental groups as charted in Table 14.

[0052] Referring to Tables 13 and 14, blood glucose lowered in a statistically significant manner without insulin elevating in parallel, indicates an improvement in the insulin sensitivity. Mechanisms such as improving insulin sensitivity in the liver and skeletal muscles to promote glycogen synthesis (which reduces blood glucose) or insulin-independent mechanisms through metabolic regulators such as AMPK etc., may be responsible for lowering circulating glucose levels in D-ribose treated animals.

Assessment of ATP:

[0053] As charted in Table 7, blood was collected in labeled tubes after completion of week 1, week 2, and week 3 from all animals under mild anesthesia. After the completion of week 1, week 2, and week 3, blood samples were gathered from all animals under mild anesthesia and placed in labeled tubes. For week 4 samples, the collection method described in the necropsy section was followed. Serum samples were isolated and preserved at -80°C until further examination.

[0054] Referring to Table 8, following a 4-week treatment period, terminal ATP levels in skeletal muscle homogenates were analyzed to assess the effects of various treatment conditions. These conditions comprised lower dose D-ribose and D-ribose with exercise. The results indicated a significant elevation in muscle ATP levels for all treatment groups relative to the respective control and maximum dose groups (Group 1 and Group 6). It was observed that the highest dosage of D- ribose (800 mg/kg in Group 6) resulted in the maximal increase in ATP levels. In contrast, administering the minimum tested dose of D-ribose (75 mg/kg in Group 2) did not yield an elevated ATP level. However, when this dosage was combined with exercise (group Group 3), a significant increase in ATP levels was observed compared to the controls. These findings corroborate the patterns discerned from serum ATP analyses, suggesting that the combination of exercise and D- ribose may synergistically enhance overall ATP reserves within the organism.

Assessment of AMPK:

[0055] After administering treatments for four weeks, the phosphorylated AMPK levels in skeletal muscle extract were quantified using ELISA. A concurrent rise in p-AMPK levels was observed in treatment groups receiving D-ribose and D-ribose with Exercise, which aligns with the increase in ATP.

[0056] Referring to Table 9, in groups treated with low doses of D-ribose at 75 mg/kg (Group 2 and Group 3) and 125 mg/kg (Group 4), mean AMPK levels appeared higher than those in control groups, although not reaching statistical significance. Notably, a significant enhancement in p- AMPK levels was detected when combining exercise and D-ribose at 125 mg/kg (Group 5) compared to the control group (Group 1). This finding underscores the importance of the D-ribose dose threshold and its synergistic effect with Exercise in inducing p-AMPK stimulation.

Assessment of PGC-la:

[0057] ELISA assays were conducted to determine the terminal PGC-la levels in skeletal muscle homogenates after four weeks of treatment in various experimental groups.

[0058] Referring to Table 10, PGC-la protein levels are predominantly regulated by the impact of exercise, as only the groups subjected to exercise (Group 3 and Group 5) displayed significantly elevated PGC-la levels compared to the control levels observed in Group 1.

Assessment of Citrate Synthase:

[0059] Citrate synthase activity in skeletal muscle homogenate was assessed using a standard colorimetric kit-based approach among all experimental groups following a 4-week treatment period.

[0060] Referring to Table 11, a considerable augmentation in citrate synthase activity was detected in all treatment groups, excluding the D-ribose treated Group at 75 mg/kg (Group 2). Intriguingly, the combination of exercise with the same dose of D-ribose (Group 3) exhibited a significantly elevated citrate synthase activity compared to the control values, highlighting the vital role of exercise in regulating metabolic markers linked with energy homeostasis and mitochondrial biogenesis in skeletal muscle. This finding demonstrates the importance of D-ribose and exercise in eliciting the desired modulatory impacts on these biomarkers when applied at comparatively low doses.

Assessment of Endothelial Nitric Oxide in Skeletal Muscle:

[0061] Exercise maintains a redox state (by upregulation of the antioxidant systems) via increased coupling and phosphorylation of eNOS. D-ribose enhancement of available high-energy phosphate pool maximizes the effect. Therefore, the eNOS profile in skeletal muscle homogenate was studied in different treatment groups, including the exercised animals that received D-ribose.

[0062] Referring to Table 15, eNOS activation was observed to be elevated over control in animals that received a combination of D-ribose + Exercise (Group 3 and Group 5).

Antioxidant Markers (Reduced Glutathione (GSH) and Superoxide Dismutase (SOD)) in Skeletal muscle and Serum:

[0063] The antioxidant status was measured in different treatment groups after the termination of the treatment period by estimating SOD and GSH levels in serum and skeletal muscle homogenate using kit-based methods.

[0064] Table 16 shows SOD levels in serum and skeletal muscle homogenate. Table 17 shows GSH levels in serum and skeletal muscle homogenate. Both SOD and GSH levels were significantly enhanced over control in the Exercise + D-ribose supplemented groups (Group 3 and Group 5).

Conclusion

[0065] In conclusion, the combined studies examined the efficacy of oral D-ribose supplementation in enhancing energy levels with or without exercise in a mammalian model. The results demonstrated that administration of D-ribose quickly increased levels of AMPK and citrate synthase, indicating immediate enhancement of oxidative metabolism, a reduction in circulating blood glucose without additional insulin input, and increasing high-energy nucleotides. Subsequently, the enhanced metabolic status led to enhanced features of antioxidant status in skeletal muscle and serum ATP levels in sedentary and exercising animals at ingestion levels previously not considered active. Due to the dual -experiment nature of the studies, heightened ATP, the phosphorylated AMPK, and citrate synthase levels also increased not only in skeletal muscle and serum, but in critical organs such as the liver and heart as well, indicating a fundamental modification in mitochondrial activity, specifically altering citrate synthase across multiple tissues and confirming the phenomena in skeletal muscle. Additionally, increased levels of AMPK activity, elevated high energy phosphates, and PGC-la in skeletal muscle supports the notion that D-ribose is active in the signaling pathway for mitochondrial biogenesis. The research found no significant changes in feed intake or body weight gain throughout the study, although trends support a body weight reduction. Therefore, it can be deduced that oral D-ribose effectively augments serum ATP levels and contributes to maintaining energy levels in exercise and sedentary mammals by acting through the AMP-mediated AMPK pathway, broadly enhancing parameters of oxidative metabolism while improving insulin sensitivity, thus mimicking, and enhancing the health advantages of regular physical activity.