Klotho nutritional supplement

Abstract

The present invention describes a nutritional supplement for increasing klotho gene expression. The nutritional supplement comprises salmon milt DNA, dried sturgeon eggs and algal oil and is formulated for oral delivery. Endogenic levels of the klotho protein have been shown to increase through the consumption of the Klotho nutritional supplement.

Description

[0001] KLOTHO NUTRITIONAL SUPPLEMENT

[0002] Technical Field

[0003] The present disclosure relates to compounds and compositions for increasing klotho gene expression, and in particular nutritional supplements for increasing endogenic klotho protein levels.

[0004] Background

[0005] Aging is an inevitable and progressive biological process At the biological level, ageing results from the impact of the accumulation of a wide variety of molecular and cellular damage over time leading to a gradual decrease in physical and mental capacity that is accompanied by a growing risk of disease and ultimately death. The aging process is considered to be regulated through an interaction between genetic, epigenetic, and external factors and is generally observed by an increase in elderliness, a quantitative and qualitative decrease in stem cells, and abnormal structure at tissue levels.

[0006] Klotho is an anti-aging single-pass membrane protein predominantly produced in the kidney, with shedding of the amino-terminal extracellular domain into the systemic circulation. Circulating levels of soluble klotho proteins in mammals, including humans, are thought to decrease with age. It was found that klotho-deficient mice exhibit accelerated aging phenotypes. On the other hand, an over-expression of the klotho gene in mice was found to extend lifespan.

[0007] In addition, klotho proteins have been implicated in a number of cellular processes related to aging. Klotho proteins suppress both oxidative stress and senescence-associated inflammation thereby functioning as both an intracellular anti-inflammatory and an anti-aging factor in the human body. Decreasing levels of the klotho protein is now known to be involved in numerous age-related pathologies, including chronic kidney disease, diabetes, cancers, cognitive decline, sarcopenia and cardiovascular diseases.

[0008] Hence, increasing klotho protein levels in the human body has diverse clinical implications. While there is ongoing research, there is a suggestion that natural expression levels of the klotho gene might be impacted by diet and exercise Furthermore, activated charcoal, probiotics and statins have demonstrated an ability to increase the production of klotho protein levels. Currently, however, there is no commercially available natural supplementation for increasing klotho protein levels through increasing klotho gene expression.

[0009] The present disclosure is directed, at least in part, to improving or overcoming one or more aspects of the prior art system.

[0010] Brief Summary of the Invention

[0011] The present disclosure describes a nutritional supplement for increasing klotho gene expression according to claim 1.

[0012] It is an object of the present disclosure to increase klotho protein levels through the increased expression of the klotho gene expression. Endogenic levels of the klotho protein are increased through the consumption of the nutritional supplement of the present disclosure.

[0013] It is a further object of the present disclosure to provide a new ingestible nutritional supplement that reduces or delays the effects of aging More particularly, the ingestible nutritional supplement replenishes or stimulates the production and release of klotho proteins to promote longevity, enhance wellness, and reduce the effects of aging at the cellular level.

[0014] Brief Description of the Drawings

[0015] The foregoing and other features and advantages of the present disclosure will be more fully understood from the following description of various embodiments, when read together with the accompanying drawings, in which:

[0016] Fig. 1 is a graph of the quantitative gene expression of the klotho and the glyceraldehyde 3-phoshate dehydrogenase genes based on real-time PCR data of blood samples collected from participants in a study relating to the effect of the kloto nutritional supplement on in vivo klotho gene expression;

[0017] Fig. 2 is a graph of the gene expression qualitative melt curves of the klotho and the glyceraldehyde 3-phoshate dehydrogenase genes based on real-time PCR data from blood samples collected from participants in the aforementioned study;

[0018] Fig. 3 is a graph representing klotho gene expression distribution in different blood samples collected from participants in the aforementioned study; Fig. 4 is a graph representing the expression of the klotho gene in comparison to the housekeeping gene GAPDH (ACt (CtKL -CtGAPDH )), for the participants receiving the klotho nutritional supplement in the aforementioned study; and

[0019] Fig. 5 is a graph presenting the relative changes in the expression of the klotho gene expression of all participants blood samples upon CAV-X intake in the aforementioned study.

[0020] Detailed Description

[0021] This disclosure generally relates to increasing endogenic levels of the klotho proteins through an ingestible klotho nutritional supplement. The klotho nutritional supplement comprises salmon milt DNA, dried sturgeon eggs and algal oil. It has been found that the combination of salmon milt DNA, dried sturgeon eggs and algal oil act synergistically to increase the expression of klotho gene.

[0022] The term "nutritional supplement" is used in the context of the present disclosure to mean any compound or composition that can be taken by a human individual to provide, supply, or increase a nutrient(s) (e g., vitamin, mineral, essential trace element, amino acid, peptide, nucleic acid, oligonucleotide, lipid, cholesterol, steroid, carbohydrate, and the like). The nutritional supplement can comprise one or more substances which can be naturally occurring or synthetic, that provide nutritional and / or health benefits, and / or therapeutic benefits (such as polynucleotides, essential fatty acids, oils or oil-like substances of particular nutritional value, vitamins, minerals and salts, minerals / salt compounds, portions, reductions or extracts of generally consumed foodstuffs) and that are missing or are otherwise not present or consumed in sufficient quantities in the diet of certain persons.

[0023] The salmon milt DNA of the klotho nutritional supplement may be obtained from wild or reared salmon. Milt may be salmon sperm, the sperm sacs or both. The salmon milt DNA is extracted from the milt through standard extraction protocols. The salmon milt DNA may be purified through standard purification protocols. The salmon milt DNA may be stored according to standard storage protocols. The klotho nutritional supplement comprises at least 100 mg of salmon milt DNA.The amount of salmon Milt DNA in the klotho nutritional supplement is in the range of 100 mg to 600 mg. The pure DNA has a purity of 80.9 % based on 100 gram of dry matter (salmon milt) and 77.2 % based on 100 gram of crude matter (salmon milt).

[0024] The dried sturgeon eggs of the klotho nutritional supplement may be obtained from wild or farmed sturgeon. Preferably, the sturgeon eggs are obtained from farmed sturgeon. The preferred sturgeon species, for the dried sturgeon eggs, is Acipenser Baerii.

[0025] The sturgeon eggs are obtained through standard extraction protocols. The sturgeon eggs may be stored according to standard storage protocols. The sturgeon eggs are dried using standard protocols that do not destroy the various active components of the sturgeon eggs. Tn particular two methods may be used, the first is lyophilization, or freeze-drying. The second is drying the sturgeon eggs, at or near room temperature and pressure, but under reduced humidity with circulating air. The klotho nutritional supplement comprises at least 10 mg of dried sturgeon eggs. The amount of dried sturgeon eggs in the klotho nutritional supplement is in the range of 10 mg to 80 mg.

[0026] Algal oil is an oil that is obtained from marine algae. Algal oil is rich in omega-3 fatty acids, including docosahexaenoic acid (DHA) and eicosapentaenoic acid (EP A) The algal oil of the klotho nutritional supplement may be obtained from wild marine algae or cultivated marine algae. The algal oil is extracted from the marine algae through standard extraction protocols The algal oil may be purified through standard purification protocols The algal oil may be stored according to standard storage protocols. The klotho nutritional supplement comprises at least 500 mg of algal oil. The algal oil in the klotho nutritional supplement is in the range of 500 mg to 1200 mg. The effective amount of DHA is in the range of 174 mg to 415 mg.

[0027] The klotho nutritional supplement is blended using known principles and methodologies in combining the ingredients together. A preferred embodiment of the present disclosure comprises 370 mg of salmon milt DNA, 10 mg of dried sturgeon eggs and 720 mg of algal oil DHA. 720 mg of algal oil has an effective amount of 250 mg of DHA. The klotho nutritional supplement composition may contain further pharmaceutically acceptable adjutants, for example, nutritional vitamins and / or minerals, herb and / or other plant extracts, preservatives, stabilizers, colorants, flavoring agents and the like.

[0028] The klotho nutritional supplement may comprise one or more of the following ingredients selected from the group consisting of fish hydrolysates, marine collagen elastin, glycemic index reducing marine peptides, Coenzyme Q10 and selenium from anhydrous sodium selenite.

[0029] The material for preparing the fish hydrolysates may originate from any fish. Preferably, the fish hydrolysates originate from wild salmon, cod and herring and haddock. The material may be any fish material such as muscle, offcuts, heads, backbones, tail and skin that result after the filleting process or any combinations thereof. In an embodiment, the material for preparing the fish hydrolysate may be obtained from deboned and degutted fish. Grinding and hydrolysis of the material may be performed by any standard procedure.

[0030] Fish hydrolysates are known to improve skin health, bone and cartilage health, blood lipid profile, and body-weight management. The klotho nutritional supplement can optionally include fish hydrolysate to obtain these and other desired benefits If included, an amount of fish hydrolysate sufficient to obtain these desired effects is required. Thus, the supplement can comprise at least about 500 mg of fish hydrolysate.

[0031] The material for preparing the marine collagen may originate from any fish. Preferably, the marine collagen with elastin originates from wild salmon, haddock and / or saithe. Marine collagen is generally known as a type 1 collagen that is derived from fish collagen peptides. It is generally known that collagen supplementation aids in slowing skin ageing and ameliorating joint pain. Furthermore, marine collagen may stimulate the production of new collagen in joints and skin. Marine collagen may also promote renewal and repair of skin cells and improve bone strength. The klotho nutritional supplement can optionally include marine collagen to obtain these and other desired benefits. If included, an amount of marine collagen sufficient to obtain these desired effects is required. Thus, the supplement can comprise at least about 188 mg of marine collagen. Preferably, the marine collagen is in the form of oligopeptides.

[0032] The material for preparing the marine elastin may originate from any fish. Preferably, the marine elastin originates from wild salmon, haddock and or saithe. Marine elastin enhances proliferation of human skin fibroblasts and elastin synthesis in human skin fibroblasts and improves the skin conditions. The klotho nutritional supplement can optionally include marine elastin to obtain these and other desired benefits. If included, an amount of marine elastin sufficient to obtain these desired effects is required. Thus, the supplement can comprise at least about 12 mg of marine elastin. Preferably, the marine elastin is in the form of oligopeptides.

[0033] Preferably, marine collagen and marine elastin are provided together to act synergistically for the renewal and repair of skin cells. The marine collagen and marine elastin are formed into a mixture prior to combining with the other ingredients. The mixture consists of 94% marine collagen and 6% marine elastin. The mixture may be in the amount of 200mg with 188 mg of marine collagen and 12 mg of marine elastin. In an embodiment, the marine collagen and marine elastin are extracted from the same fish.

[0034] Glycemic index reducing marine peptides are derived from fish hydrolysates. The material for preparing the peptides may originate from any fish. Preferably, the peptides originate from wild cod fish. The glycemic index reducing marine peptides may be purified and concentrated in accordance with standard practice. The glycemic index reducing marine peptides are added separately from the fish hydrolysates.

[0035] The klotho nutritional supplement comprises at least 0,121 mg of anhydrous sodium selenite which is equivalent to 55 mcg for selenium.

[0036] Coenzyme Q10 may serve to protects against congestive heart failure and improves blood circulation. Coenzyme Q10 may also act as a digestive cofactor involved in electron transport, thereby supplementing the transfer of energy in cells. The klotho nutritional supplement can optionally include Coenzyme Q10 to obtain these and other desired benefits. If included, an amount of Coenzyme Q10 sufficient to obtain these desired effects is required

[0037] The klotho nutritional supplement may further comprise one or more of the following excipients selected from the group consisting of coconut oil, refined beeswax and yellow soy lecithin.

[0038] The klotho nutritional supplement is required to be consumed orally. The preferred form of the klotho nutritional supplement for oral delivery is a soft gel capsule. The soft gel capsule is preferably composed of palm oil free glycerol, sorbitol 70% solution (noncrystalline), purified water. The soft gel capsule may comprise of colorings consisting of ferric oxide red, ferric oxide yellow and ferric oxide black.

[0039] Study on Klotho gene expression A study was conducted to test in vivo the activity of klotho nutritional supplement which was termed as CAV-X The klotho nutritional supplement contained salmon milt DNA, dried sturgeon eggs from Acipenser baerii and algal oil, marine collagen with elastin, and fish hydrolysates from salmon, cod, herring, haddock and saithe added with coenzyme Q10 (CoQl O) and selenium.

[0040] The study investigated in vivo the potential effects of the klotho nutritional supplement (CAV-X) in the expression of the Klotho gene which is associated with aging and longevity. Blood samples drawn from 14 apparently healthy males and females from all ages taking CAV-X (treated group) or taking no supplementation (control group) were analyzed for changes in the expression of the klotho gene. Participants’ blood samples were collected before CAV-X intake, as well as after 30 or after 60 days on CAV-X intake. In addition, blood samples were collected for some participants after 30 and 60 days upon CAV-X intake discontinuation. Treatment with CAV-X for 60 days dramatically increased the expression of the anti-aging klotho gene by up to 250-fold; p < 0.0001.

[0041] The dramatic effect on gene expression of Klotho was evident even 30 days after CAV-X discontinuation albeit with a 10-15% reduction. However, the effect on the Klotho gene expression was lost after 60 days of CAV-X discontinuation. No significant changes in all analyzed genes expression were observed in the control group. Hence, the klotho nutritional supplement enhances dramatically the expression of the anti-aging gene Klotho which demonstrates a powerful effect in aging prevention

[0042] Materials and Methods i. Participants and Study Design

[0043] Overall, 14 persons apparently healthy male and female participants from all ages were recruited and treated for two months with CAV-X. The treatment consisted of one nutritional supplement in the form of a soft gel capsule each day of the treatment. Accordingly, 5 individuals were evaluated in parallel without receiving any supplementation of CAV-X (control group). Blood samples were collected along with clinical evaluation of overall functions at the beginning and at the end of the treatment period (day 0 and day 60). The measurements were also repeated two months after treatment discontinuation. Inclusion criteria were an age between 18 and 85 years, non-smoking status, normal blood pressure values, body mass index values below 30 kg / m2, no clinical cardiovascular disease, no history of any other chronic disease. ii. Blood Sampling

[0044] Three samples of whole peripheral blood were collected from each participant: before treatment (day 0), during treatment (day 30), at treatment conclusion (day 60), as well as after treatment discontinuation (day 90 and day 120). The whole blood samples were collected in 10 mL EDTA tubes and mixed immediately with RNAzol-RT for RNA isolation. iii. RNA Isolation

[0045] Total RNA was isolated using a RNAzol-RT (MRC) according to the manufacturer’s instructions. RNA was quantified by spectrophotometry, and cDNA was synthesized from 1 pg of the total RNA using the High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol. iv. Quantitative Real-Time PCR (qPCR)

[0046] The expression of target genes in the tested samples was performed using Klotho gene specific primers. The housekeeping gene glyceraldehyde 3-phoshate dehydrogenase (GAPDH) was used as an endogenous control. Described briefly, qPCR was performed using the Rotor Gene Q (Qiagen). Individual qPCR reactions were carried out in 10 pL reaction mix with 2x Maxima SybrGreen Universal PCR Master Mix (Applied Biosystems). Each sample was analyzed in triplicate. Fold-differences in each gene expression were calculated using the comparative Ct method, where data were normalized to day 0 for each participant. v. Statistical analysis

[0047] All statistical analyses were performed using the GraphPad Prism 8 software. Data were presented as mean ± SD, and the paired or unpaired Student's t-test or Analysis of variance were chosen to analyze the statistical significance between two groups. P-values < 0.05 was considered statistically significant.

[0048] Results

[0049] The expression of the Klotho gene was monitored in the CAV-X treated group, as well as in control group (no CAV-X intake). Reference is made to Figs. 1 and 2. In Fig. 1, the x axis represents the number of cycles in RT-PCR and the y axis represents the normalized fluorescence. In Fig. 2, the x axis represents the temperature in degrees Celsius and the y axis represents the dF / dT change in fluorescence level with respect to per unit increase in temperature. In Figs. 1 and 2, the dotted lines marked with “0” denotes the expression of the corresponding genes (Klotho [“KL”] and the reference GAPDH gene) before CAV-X intake. The solid lines marked with “60” denotes the expression of these genes after 60 days on CAV-X (black characters) The DNA synthesis and quantitation curves that correspond to each gene’s expression are shown.

[0050] With reference to Fig. 3, diamonds represent the relative levels of gene expression of each participant’s blood sample drawn after 60 days on CAV-X (time 60 DAYS). Circles represent the relative levels of KL gene expression of each participant’s blood sample drawn the day before CAV-X intake (time 0 DAYS). ACt on the X-axis represents the expression difference CtKL-CtGAPDH With reference to Fig. 4 dots represent KL expression values of participants before CAV-X intake (0 DAYS), while triangles represent KL gene expression values of the same participants after 60 days on CAV-X. All the graphs of Figs. 1 to 4 indicate an increased expression of the KL gene relative to the GAPDH gene.

[0051] As can be noted from the above graphs, significant differences in the expression of the KL gene between the groups was observed after 60 days of treatment. Two months after treatment discontinuation, the expression of the Klotho gene in the treatment group decreased to initial values (i.e. values before CAV-X treatment). In contrast, in the control group, expression of the KL gene did not change during this time period.

[0052] Conclusion

[0053] The study shows that the klotho nutritional supplement (CAV-X) intake increased the expression of the KL gene after 30 daysof treatment up to 4-fold (p <0.001) in all participants, while after 60 days this increase was dramatic, up to 250-fold (p<0.0001) compared to 0 days treatment, as well as to KL gene expression of the control group, where no significant changes observed.

[0054] Characteristic example of a case of 83 years-old male with no detectable expression of KL gene at 0 days, presented with high levels of KL gene expression after 60 days on CAV- X. In addition, 30 days after CAV-X intake discontinuation, the expression of the KL gene was still at dramatically high levels, albeit 10-15% lower compared to the 60 days treatment levels. However, two months after CAV-X intake discontinuation, KLOTHO gene expression levels were back to the initial levels observed at 0 days time point. With reference to Fig 5, the values for each time point are presented as means +SEM of gene expression fold induction relative to 0 time values.

[0055] The skilled person would appreciate that foregoing embodiments may be modified or combined to obtain the klotho nutritional supplement of the present disclosure.

[0056] Industrial Applicability

[0057] This disclosure describes a nutritional supplement that is intended for increasing klotho protein levels in humans. The nutritional supplement has an oral mode of delivery.

[0058] Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein.

[0059] Where technical features mentioned in any claim are followed by reference signs, the reference signs have been included for the sole purpose of increasing the intelligibility of the claims and accordingly, neither the reference signs nor their absence have any limiting effect on the technical features as described above or on the scope of any claim elements.

[0060] One skilled in the art will realise the disclosure may be embodied in other specific forms without departing from the disclosure or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting of the disclosure described herein. Scope of the invention is thus indicated by the appended claims, rather than the foregoing description, and all changes that come within the meaning and range of equivalence of the claims are therefore intended to be embraced therein.

Claims

Claims1. A klotho nutritional supplement comprising salmon milt DNA, dried sturgeon eggs and algal oil.

2. The klotho nutritional supplement of claim 1 wherein the salmon milt DNA is in the range of 100 mg to 600 mg, the dried sturgeon eggs is in the range of 10 mg to 80 mg and the agal oil is in the range of 500 mg to 1200 mg.

3. The klotho nutritional supplement of claim 1 or 2 comprising at least 100 mg of salmon milt DNA.

4. The klotho nutritional supplement of claim 1, 2 or 3 comprising at least 10 mg of dried sturgeon eggs.

5. The klotho nutritional supplement of claim 1, 2, 3 or 4 comprising at least 500 mg of algal oil.

6. The klotho nutritional supplement of any one of preceding claims comprising 370 mg of salmon milt DNA, 10 mg of dried sturgeon eggs and 720 mg of algal oil.

7. The klotho nutritional supplement of any one of the preceding claims further comprising one or more of the following ingredients selected from the group consisting of fish hydrolysates, marine collagen, marine elastin, glycemic index reducing marine peptides, Coenzyme Q10 and selenium.

8. The klotho nutritional supplement of any one of the preceding claims further comprising one or more of the following excipients selected from the group consisting of coconut oil, refined beeswax and yellow soy lecithin.

9. The klotho nutritional supplement of any one of the preceding claims, wherein thenutritional supplement is in the form of a soft gel capsule.

10. The klotho nutritional supplement of claim 9 wherein the soft gel capsule is composed of palm oil free glycerol, sorbitol 70% solution (non-crystalline), purified water and colorings consisting of ferric oxide red, ferric oxide yellow and ferric oxide black.

11. The klotho nutritional supplement of any one of the preceding claims wherein the dried sturgeon eggs are obtained from Acipenser Baerii.

12. The klotho nutritional supplement of claim 7 wherein the marine collagen and marine elastin are combined in a mixture.

13. The klotho nutritional supplement of claim 12 wherein the mixture consists of 94% marine collagen and 6% marine elastin.

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