DIETARY SUPPLEMENT COMPRISING LUTEIN FOR TELOMERE PROTECTION, AND METHOD FOR PRODUCTION THEREOF

Abstract

This invention discloses a dietary supplement composition. More particularly, the invention deals with the method of production of dietary supplement composition comprising natural lutein and tocopherols, optionally along with acceptable additives to a subject, wherein the dietary supplement composition reduces, ameliorates, prevents, or reverses the oxidative DNA damage and slows telomere shortening. Invention further describes a method for promoting detoxification in cells of a subject, including administering to the subject an oral formulation comprising a plurality of agents that stimulate natural stem cell production within the body; repair the stem cells fibroblasts; pro-long the replicative lifespan; slow the telomere shortening; improve the telomerase activation in stem cells; improve the telomerase activation in body cells; promote the health-span in humans; and reduce the oxidative stress. The present invention can be used for the development of functional food, dietary plan and as a nutritional supplement.

Claims

1) A method of reducing, ameliorating, preventing, or reversing oxidative DNA damage in a subject, comprising orally administering an effective dose of a dietary supplement composition comprising natural lutein and tocopherols, optionally along with acceptable additives to a subject, wherein the dietary supplement composition reduces, ameliorates, prevents, or reverses the oxidative DNA damage and slows telomere shortening.

2) The method of claim 1, wherein the dietary supplement composition comprising natural lutein is produced by a method comprising: a. saponifying Marigold Oleoresin in the presence of an alcohol or organic solvent and an alkali for about 2 hours at a preferable temperature of about 80 to 85 degrees centigrade; b. adding an aqueous solution to the reaction mass, hereby obtaining a neutralized mass; c. filtering the neutralized mass, to obtain the Lutein crystals; d. further crystalizing the Lutein crystals in the presence of alcohol at 5 degrees centigrade to obtain a purity of 75% to 80% Lutein; e. stabilizing the Lutein crystals of by adding natural tocopherols, and maintaining at room temperature; and f. vacuum drying the 75% to 80% purity Lutein crystals for about 2 hrs.

3) The method according to claim 1, wherein the natural Lutein is derived from a Marigold species of Tagetes erecta.

4) The method according to claim 1, wherein the dietary supplement composition extracts are produced/separated by a process comprising supercritical carbon dioxide extraction, ethanol/methanol extraction, filtration, centrifugation, sedimentation and/or combinations thereof.

5) The method according to claim 1, wherein the dietary supplement composition extract is administered to the subject in combination with at least one additional biologically active compound.

6) The method according to claim 5, wherein the biologically active compound is a carotenoid, an antioxidant, a vitamin, mineral salts and/or trace elements, acceptable additives or a second natural extract.

7) The method of claim 1, wherein the natural Lutein is: dispersed in oil; homogenized in an aqueous medium; dispersed in an aqueous medium; processed into emulsion; encapsulated; processed into dry material; processed into solid formulation or a combination of two or more thereof.

8) The method of claim 7 wherein the form of the dry material is stabilized beadlets, granular powder, a powder, a cold water dispersible powder, or a combination of two or more thereof.

9) The method according to claim 7, wherein the granules have a particle size distribution in the range of 20 mesh to 100 mesh (preferably in the range of 30 mesh to 60 mesh).

10) The method according to claim 6, wherein the amount of total carotenoids is in the range of 5 to 85 weight-%.

11) The method according to claim 1, wherein the dietary supplement composition is a mixture of lutein and zeaxanthin, and wherein the molar ratio of lutein to zeaxanthin is in the range of 20:1 to 1:1, preferably the molar ratio is in the range of 10:1 to 5:1.

12) The method according to claim 1, wherein the amount of the tocopherols is in the range of 0.1 to 10 weight-%, preferably in the range of 0.2 to 2 weight-%.

13) The method of claim 1, comprising orally administering an effective dose of the dietary supplement composition comprising natural lutein and tocopherols to a subject.

14) The method according to claim 1, wherein the composition does not comprise any of the following compounds: hydrolyzed lecithin products, lactose-based products, animal-based products, soy-based products or marine-based products.

15) The method according to claim 1, wherein the formulation is safe, non-toxic, gluten-free, solvent-free, pesticide-free, aflatoxin-free and free of adverse effects.

16) (canceled)

17) The method according to claim 1, wherein the subject is animal or human being.

18) The method according to claim 1, wherein the effective dose of Lutein reduces the oxidative DNA damage by at least 20% compared to a subject not administered the effective dose of Lutein.

19) The method according to claim 1, wherein the effective dose is about 6 mg to 10 mg Lutein per day.

20) The method according to claim 1, wherein the oral administration to the subject in a food or beverage product as diet plan and nutritional supplements.

21) The method according to claim 20; wherein said food products & dietary supplements are selected from the group comprising of hard-shell capsules, softgel capsules, liquid fill capsules, tablets, effervescent granules, beverages, powdered drink mixes, melt-in-mouth sachets, juices, baked goods, gummies, jelly sticks, candies or any combinations thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The foregoing and other objects and features of the present invention will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. Understanding that these drawings depict only typical embodiments of the invention and are, therefore, not to be considered limiting of its scope, the invention will be described with additional specificity and detail through use of the accompanying drawings.

[0051] FIG. 1 Illustrates a process flow diagram illustrating one presently preferred embodiment for obtaining composition comprising natural lutein and tocopherols, optionally along with acceptable additives, according to the aspects of present invention.

[0052] FIG. 2 Depicts growth curves of human adult primary fibroblasts untreated (control-DMSO), treated with Lutein (10 μg/ml, 5 μg/ml and 1 μg/ml) under standard and oxidative (10 μM H.sub.2O.sub.2) condition. Each point on the population curve represents one week.

[0053] FIG. 3 Illustrates the histogram shows the distribution of telomere lengths in sample Ctrl-PO. Bars represent the relative frequency for every particular fluorescence intensity normalized (X axis). The 20th percentile (red bars) indicates the particular length below which 20% of the telomeres have been observed. The median (MTL) and average (ATL) telomere length are also indicated in the histogram that allows the analysis of telomere length variability.

[0054] FIG. 4 Shows the representative TAT image. Both the nuclei (white) and the telomeric spots (bright white spots) are seen in the image.

[0055] FIG. 5 Illustrates the bar graphs of TAT results (Median Telomere Length) for different time points and treatments.

[0056] FIG. 6 Illustrates the bar graphs of TAT results (20th Percentile Telomere Length) for different time points and treatments.

[0057] FIG. 7 Illustrates the bar graphs of TAT results (% Short Telomeres) for different time points and treatments.

[0058] FIG. 8 Illustrates the telomere shortening rate (Median of telomere length (initial-final)/Population Doubling) at the defined time points and treatments.

[0059] FIG. 9 Illustrates the graph of telomere length vs Population doubling at different treatments. Slope of regression line in each condition, represents the telomere shortening rate. Y=aX+b; a=slope and b=y intercept.

[0060] FIG. 10 shows all grades of Lute-gen, having stability data (long term and accelerated), with a minimum self-life of two years from the date of packaging.

[0061] In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number.

DETAILED DESCRIPTION OF THE INVENTION

[0062] It is to be understood that the present invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The present invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

[0063] The use of “including”, “comprising” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. The terms “a” and “an” herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced items. Further, the use of terms “first”, “second”, and “third”, and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.

[0064] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise. By way of example, “a dosage” refers to one or more than one dosage.

[0065] The terms “comprising”, “comprises” and “comprised of” as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps.

[0066] The term “managing” or “management” includes preventing, treating and healing of a condition or disorder or ill effects or side effects. The term also encompasses maintenance of the optimum state and prevention of the further progress in the condition or disorder or ill effects or side effects.

[0067] The term “subject” is an animal or a mammal, including human beings.

[0068] The term, “plurality of agents”, “compositional elements”, and/or materials may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. In addition, no individual member of such list should be construed as an equivalent of any other member of the same list solely based on their listing in a common group without indications to the contrary.

[0069] The term “Concentrations”, “amounts”, and other “numerical data” may be expressed or presented herein in a range format. It is to be understood that such range format is used merely for convenience and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.

[0070] The term “about” means that dimensions, sizes, formulations, parameters, shapes and other quantities and characteristics are not and need not be exact, but may be approximated and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like and other factors known to those of skill. In addition, unless otherwise stated, the term “about” shall expressly include “exactly,” consistent with the specification regarding ranges and numerical data.

[0071] The term, “effective amount” refers to an amount of an ingredient which, when included in a composition, is sufficient to achieve an intended compositional or physiological effect. Furthermore, a “therapeutically effective amount” refers to a sufficient amount of an active agent, to achieve therapeutic results in treating or preventing or promoting a condition for which the active agent is known to be effective. It is understood that the “effective amount” or a “therapeutically effective amount” may be dependent in some instances on such biological factors. Further, while the achievement of therapeutic effects may be measured by a physician or other qualified medical personnel using evaluations known in the art, it is recognized that individual variation and response to treatments/therapy/conditions may make the choice of therapeutic effects a subjective decision. The determination of an effective amount is known within the ordinary skill in the art of pharmaceutical and nutritional sciences as well as medicine.

[0072] The term, “administration” or “administering” refer to the manner in which an active agent, or composition containing such, is presented or given to a subject.

[0073] The term, “oral administration” refers to a route of administration including swallowing, chewing, or sucking of an oral dosage form comprising the drug or nutritional formula.

[0074] The term, “stem cells’ refers to special human cells that have the ability to develop into many different cell types, for example, from muscle cells to brain cells. In some cases, they also have the innate ability to repair damaged tissues.

[0075] The term, “aflatoxins’ refers to a family of toxins produced by certain fungi that are found on agricultural crops such as maize (corn), peanuts, cottonseed, and tree nuts.

[0076] All documents cited in the present specification are hereby incorporated by reference in their totality. In particular, the teachings of all documents herein specifically referred to are incorporated by reference.

[0077] Example embodiments of the present invention are described with reference to the accompanying figures.

[0078] In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number.

Embodiments of the Invention

[0079] Lute-gen® is a proprietary product of company BIO-GEN EXTRACTS PRIVATE LIMITED.

[0080] In an embodiment, invention deals with a method of reducing, ameliorating, preventing, or reversing oxidative DNA damage in a subject, comprising orally administering an effective dose of a dietary supplement composition comprising natural lutein and tocopherols, optionally along with acceptable additives to a subject, wherein the dietary supplement composition reduces, ameliorates, prevents, or reverses the oxidative DNA damage and slows telomere shortening.

[0081] In an alternative embodiment, the acceptable additive is selected from group comprising gum, granulating agents, binding agents, lubricating agents, disintegrating agents, sweetening agents, coloring agents, flavoring agents, coating agents, plasticizers, preservatives, suspending agents, emulsifying agents, sweetening agents, antioxidants, surfactants, viscosity enhancers, plant cellulosic materials, additives, solvents, glidants, anti-adherents, anti-static agents, preservatives, suspending agents and spheronization agents or any combinations thereof.

[0082] In an exemplary embodiment, a method for obtaining a dietary supplement composition comprising natural lutein and tocopherols, optionally along with acceptable additives, wherein the said method comprising acts of:

a. saponifying Marigold Oleoresin in the presence of an alcohol or organic solvent and an alkali for about 2 hours at a preferable temperature of about 80 to 85 degrees centigrade;
b. adding an aqueous solution to the reaction mass, hereby obtaining a neutralized mass;
c. filtering the neutralized mass, to obtain the Lutein crystals;
d. further crystalizing the Lutein crystals in the presence of alcohol at 5 degrees centigrade to obtain a purity of 75% to 80% Lutein;
e. stabilizing the Lutein crystals of by adding natural tocopherols, and maintaining at room temperature; and
f vacuum drying the 75% to 80% purity Lutein crystals for about 2 hrs.

[0083] In an additional embodiment, the composition wherein the natural Lutein is derived from a Marigold species of Tagetes erecta and tocopherols from sunflowers, wherein the dietary supplement composition extracts are produced/separated by a process comprising supercritical carbon dioxide extraction, ethanol/methanol extraction, filtration, centrifugation, sedimentation and/or combinations thereof.

[0084] In an additional embodiment, solvents are aliphatic compounds selected from group comprising methanol, ethanol, anhydrous ethanol, propanol, butanol, ethyl acetate or combinations thereof, wherein the preferable solvent is ethanol.

[0085] In another embodiment, the dietary supplement composition extract is administered to the subject in combination with at least one additional biologically active compound, wherein the biologically active compound is a carotenoid, an antioxidant, a vitamin, mineral salts and/or trace elements, acceptable additives or a second natural extract.

[0086] In further embodiment, the natural Lutein extract is: dispersed in oil; homogenized in an aqueous medium; dispersed in an aqueous medium; processed into emulsion; encapsulated; processed into dry material; processed into solid formulation or a combination of two or more thereof.

[0087] In further embodiment, The method of any one of claims 1-7, wherein the natural Lutein extract is processed into dry material and/or solid formulation, wherein the form of the dry material is stabilized beadlets, granular powder, a powder, a cold water dispersible powder, or a combination of two or more thereof and the material has the particle size distribution in the range of 20 mesh to 100 mesh (preferably in the range of 30 mesh to 60 mesh).

[0088] In an additional embodiment, the solid formulation according to any one or more of the preceding claims, wherein the amount of total carotenoids is in the range of 5 to 85 weight-% and the dietary supplement composition is a mixture of lutein and zeaxanthin, and wherein the molar ratio of lutein to zeaxanthin is in the range of 20:1 to 1:1, preferably the molar ratio is in the range of 10:1 to 5:1.

[0089] In an additional embodiment the amount of the tocopherols is in the range of 0.1 to 10 weight-%, preferably in the range of 0.2 to 2 weight-%.

[0090] In an embodiment, an oral formulation for reducing, ameliorating, preventing, or reversing oxidative DNA damage in a subject, comprising orally administering an effective dose of a dietary supplement composition comprising natural lutein and tocopherols to the subject, wherein the dietary supplement composition reduces, ameliorates, prevents, or reverses the oxidative DNA damage and slower telomere shortening.

[0091] In an exemplary embodiment, an oral formulation wherein the formulation does not comprise any of the following compounds: hydrolyzed lecithin products, lactose-based products, animal-based products, soy-based products or marine-based products.

[0092] In a further embodiment the oral formulation is safe, non-toxic, gluten-free, solvent-free, pesticide-free, aflatoxin-free and free of adverse effects.

[0093] In an embodiment, a method for promoting detoxification in cells of a subject, comprising administering to a subject an oral formulation comprising a plurality of agents that stimulate natural stem cell production within the body; repair the stem cells fibroblasts; pro-long the replicative lifespan; slow the telomere shortening; improve the telomerase activation in stem cells; improve the telomerase activation in body cells; promote the health-span in humans; and reduce the oxidative stress.

[0094] In a further embodiment, the subject is animal or human being.

[0095] In an embodiment, wherein the effective dose of Lutein reduces the oxidative DNA damage by at least 20% compared to a subject not administered the effective dose of Lutein and the effective dose is about 6 mg to 10 mg Lutein per day.

[0096] In an exemplary embodiment, a method for promoting detoxification in cells of a subject according to claim 16, wherein the oral formulation is administered to the subject in a food or beverage product as diet plan and nutritional supplements.

[0097] In a further embodiment, a food product comprising the natural Lutein extract according to claim 20; wherein said food products & dietary supplements are selected from the group comprising of hard-shell capsules, softgel capsules, liquid fill capsules, tablets, effervescent granules, beverages, powdered drink mixes, melt-in-mouth sachets, juices, baked goods, gummies, jelly sticks, candies or any combinations thereof.

Detailed Process of the Invention

[0098] Lute-gen® contains Lutein, a xanthophyll found in plants. Lutein is an oxygenated carotenoid found in vegetables and fruits. It is found in macula of eye where it is believed to act as a filter and is responsible for central vision. The human body cannot synthesize lutein and its needs are to be fulfilled from dietary sources.

[0099] Lute-gen® 75% (Free) Crystals were used in the in-vitro telomere length study.

[0100] Equipment Used:

[0101] Reactor

[0102] Solvent storage tanks

[0103] External jacket for heating

[0104] Centrifuge

[0105] Filtration Unit

[0106] Blending or Homogenization Unit

[0107] Vacuum Dryer

[0108] Packing in internationally accepted packing solution

[0109] Raw Materials:

[0110] Marigold Oleoresin from Marigold flowers

[0111] Tocopherols from Sunflowers

[0112] Aliphatic Alcohol

[0113] Alkali

[0114] Water

[0115] Process:

[0116] The Marigold Oleoresin is received at the warehouse and tested for Xanthophyll content & other parameters.

[0117] Method of Obtaining Lute-Gen® Crystals:

[0118] The invention discloses the method of producing a dietary supplement composition comprising natural lutein and tocopherols, (FIG. 1) comprising acts of: [0119] a. saponifying Marigold Oleoresin in the presence of an alcohol or organic solvent and an alkali for about 2 hours at a preferable temperature of about 80 to 85 degrees centigrade; 11, 12, 13, 14, 15 [0120] b. adding an aqueous solution to the reaction mass, hereby obtaining a neutralized mass; 17, 18 [0121] c. filtering the neutralized mass, to obtain the Lutein crystals; 19 [0122] d. further crystalizing the Lutein crystals in the presence of alcohol at 5 degrees centigrade to obtain a purity of 75% to 80% Lutein; 20 [0123] e. stabilizing the Lutein crystals of by adding natural tocopherols, and maintaining at room temperature; and 20, 21 [0124] f. vacuum drying the 75% to 80% purity Lutein crystals for about 2 hrs. 22, 23

[0125] FIG. 1 shows is a process flow diagram illustrating one presently preferred embodiment for obtaining composition comprising a dietary supplement composition comprising natural lutein and tocopherols, optionally along with acceptable additives, according to the aspect of present invention.

[0126] Upon completion of the extraction, the material is unloaded to produce: Lute-gen® 75% (Free) Crystals.

[0127] Lutein acts as an antioxidant, protecting cells against damaging effects of free radicals. It is thought to function as a light filter, protecting eye tissues from sunlight damage by absorbing damaging blue, violet and ultra-violet light. It reduces the amount of blue light that reaches the photoreceptors and scavenges the reactive oxygen species, thereby preventing damage to DNA and protein molecules. It reduces risk of progression of age-related macular degeneration. In general Lutein being a good antioxidant that not helps in protecting DNA and protein molecules in the eye but also in various cell types in the body.

[0128] Nutrition and lifestyle, known to modulate aging process and age-related diseases, might also affect telomerase activity. Short and dysfunctional telomeres rather than average telomere length are associated with longevity in animal models, and their rescue by telomerase maybe sufficient to restore cell and organismal viability. Improving telomerase activation in stem cells and potentially in other cells by diet and lifestyle interventions may represent an intriguing way to promote health-span in humans.

[0129] Hence, commercial production of the purified forms of dietary carotenoids particularly lutein is of great importance. These carotenoids may be used, individually or in combination, as nutritional supplements and food colorants as well as in clinical trials where their potential health benefits in the prevention of ARMD and cancer, skin care, anti-oxidant property, diabetic retinopathy, anti-wrinkle, collagen repair, anti-ageing is gaining utmost importance.

[0130] In nature lutein abundantly occur as mono and di-esters of long chain fatty acids like palmitic, stearic, myristic, oleic, linoleic, lauric and pentadecanoic acids. The lutein and zeaxanthin which are assimilated by the body in the natural ester form are biologically hydrolysed inside the human body. Only the hydrolysed free-form of lutein and zeaxanthin are transferred to the biological sites of function. High molecular weight fatty acid groups of the esters limit the dosage of free lutein and zeaxanthin consumed. Hence, the de-esterfication process is used to hydrolyze the natural carotenoid ester forms. De-esterification of a natural carotenoid ester means a process to make free the pure carotenoid from the fatty acid backbone.

[0131] Inventors have experimental evidence with the primary objective; to determine the cellular proliferation rate and telomere length in cultures of human adult primary fibroblast cells treated with Lutein under standard and oxidative stress conditions. The results obtained from this experiment has clearly drawn conclusion that lutein has a significant protective effect on telomere length erosion in vitro under oxidative stress conditions after 8 passages.

[0132] Telomere length predicts clinical outcomes and mortality, cells with shortened telomeres can remain genetically stable if the telomere maintenance system, which includes telomerase, is fully functioning and also healthy cell function.

[0133] The present invention is further elaborated with the help of the following examples. However, these examples should not be construed to limit the scope of the present invention.

Example 1: Effect of Dietary Supplement Composition Comprising Natural Lutein and Tocopherols, (Lute-Gen®) on Telomere Length; Proliferative and Telomere Length Analysis

[0134] Analysis of cellular proliferation rate and telomere length in cultures of human adult primary fibroblast cells treated with lutein under standard and oxidative stress conditions.

[0135] Primary cultures of adult human fibroblast were established. Cells are seeded at 2×103 cells/cm2 in GlutaMax™ high glucose Dulbecco's modified Eagle's medium (DMEM, Gibco®), and supplemented with 10% fetal bovine serum (FBS, HyClone, Thermo Scientific), penicillin (100 U/ml) and reptomycin (1000 U/ml). Media is renewed every 2-3 days and cells passaged at sub-confluence (70-80%) every 4-5 days.

[0136] Compounds or vehicle control are added to the cells in culture. Cell growth is monitored for each condition by counting cell numbers at each passage using a Countess™ cell counter (Invitrogen). Population doubling (PD) was calculated with the formula PD=(log (Nn/Nn-1))/log 2 where n is the passage; and N the number of cells. One PD is equivalent to one round of cell replication.

[0137] Materials and Methods

[0138] Treatments:

[0139] Eight independent condition were evaluated by proliferative Analysis:

[0140] Control: Standard cell culture condition (Ctrl)

[0141] Oxidative Stress Control: Standard cell culture condition+10 μM H.sub.2O.sub.2 (H.sub.2O.sub.2)

[0142] Standard cell culture condition+Lutein−10 μg/ml (LUT10)

[0143] Standard cell culture condition+Lutein−5 μg/ml (LUT5)

[0144] Standard cell culture condition+Lutein−1 μg/ml (LUT1)

[0145] Standard cell culture condition+Lutein−10 μg/ml+10 μM H.sub.2O.sub.2 (LUT10+H.sub.2O.sub.2)

[0146] Standard cell culture condition+Lutein−5 μg/ml+10 μM H.sub.2O.sub.2(LUT5+H.sub.2O.sub.2)

[0147] Standard cell culture condition+Lutein−1 μg/ml+10 μM H.sub.2O.sub.2 (LUT1+H.sub.2O.sub.2)

[0148] Cells were expanded during eight weeks under standard and oxidative (10 μM H.sub.2O.sub.2) cell culture condition as described previously. Fresh treatments were prepared and added to the cells at every passage and also when media was renewed. Stock solution of 10 mg/ml of the compound was prepared in DMSO and the different treatments were prepared from that stock.

[0149] Results

[0150] Tables 1, 2 and 3 show different cell passage studies.

TABLE-US-00001 TABLE 1 Population Doubling per passage Population Doubling LUT10 + LUT5 + LUT1 + Week Passage Ctrl H.sub.2O.sub.2 LUT10 LUT5 LUT1 H.sub.2O.sub.2 H.sub.2O.sub.2 H.sub.2O.sub.2 0 P0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1 P1 3.74 2.58 3.58 3.81 3.81 3.50 3.58 3.42 2 P2 3.12 2.12 3.12 2.65 3.50 3.42 3.32 2.87 3 P3 3.32 2.22 3.12 2.65 3.57 2.74 3.22 2.37 4 P4 3.42 3.66 3.50 3.42 3.50 3.42 3.42 3.12 5 P5 2.07 1.82 2.43 2.49 2.02 1.90 2.16 2.49 6 P6 2.38 2.02 2.74 1.45 3.00 2.00 2.14 2.65 7 P7 2.60 1.22 2.69 1.68 2.68 1.55 2.87 1.00 8 P8 4.06 3.12 2.74 3.12 2.74 2.30 2.34 2.30

TABLE-US-00002 TABLE 2 Cumulative Population Doubling per passage. Cumulative Population Doubling LUT10 + LUT5 + LUT1 + Week Passage Ctrl H.sub.2O.sub.2 LUT10 LUT5 LUT1 H.sub.2O.sub.2 H.sub.2O.sub.2 H.sub.2O.sub.2 0 P0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1 P1 3.74 2.58 3.58 3.81 3.81 3.50 3.58 3.42 2 P2 6.85 4.70 6.70 6.47 7.31 6.92 6.91 6.29 3 P3 10.17 6.92 9.82 9.12 10.18 9.65 10.13 9.16 4 P4 13.59 10.59 13.32 12.53 13.69 13.07 13.54 12.28 5 P5 15.66 12.41 15.75 15.02 15.71 14.97 15.70 14.76 6 P6 18.04 14.43 18.49 16.47 18.71 16.97 17.84 17.41 7 P7 20.64 15.65 21.18 18.15 21.39 18.52 20.71 18.41 8 P8 24.70 18.77 23.92 21.26 24.13 20.83 23.06 20.72

TABLE-US-00003 TABLE 3 Total number of cells per passage Total number of cells LUT10 + LUT5 + LUT1 + Week Passage Ctrl H.sub.2O.sub.2 LUT10 LUT5 LUT1 H.sub.2O.sub.2 H.sub.2O.sub.2 H.sub.2O.sub.2 0 P0 1.50E+05 1.50E+05 1.50E+05 1.50E+05 1.50E+05 1.50E+05 1.50E+05 1.50E+05 1 P1 2.00E+06 9.00E+05 1.80E+06 2.10E+06 2.10E+06 1.70E+06 1.80E+06 1.60E+06 2 P2 1.73E+07 3.90E+06 1.56E+07 1.33E+07 2.38E+07 1.81E+07 1.80E+07 1.17E+07 3 P3 1.73E+08 1.82E+07 1.35E+08 8.33E+07 1.75E+08 1.21E+08 1.68E+08 8.60E+07 4 P4 1.85E+09 2.31E+08 1.53E+09 8.89E+09 1.98E+09 1.29E+09 1.79E+09 7.46E+08 5 P5 7.77E+09 8.15E+08 8.27E+09 4.98E+09 8.04E+09 4.81E+09 8.00E+09 4.18E+09 6 P6 4.04E+10 3.31E+09 5.52E+10 1.36E+10 6.44E+10 1.93E+09 3.52E+10 2.62E+10 7 P7 2.45E+11 7.73E+09 3.57E+11 4.35E+10 4.12E+11 5.65E+10 2.58E+11 5.23E+10 8 P8 4.08E+12 6.70E+10 2.38E+12 3.77E+11 2.75E+12 2.79E+11 1.31E+12 2.58E+11

[0151] FIG. 2 shows the growth curves of human adult primary fibroblasts untreated (control-DMSO), treated. with Lutein (10 μg/ml, 5 μg/ml and 1 μg/ml) under standard and oxidative (10 μM H.sub.2O.sub.2) condition. Each point on the population curve represents one week.

[0152] After eight weeks of treatment with Lutein 10 μg/ml, 5 μg/ml and 1 μg/ml:

[0153] Under standard culture conditions, no difference was identified between the proliferations rates of the treated groups (LUT10, LUT5 and LUT1) compared to control (Ctrl).

[0154] Under oxidative culture conditions, all Lutein treated groups (LUT10+H2O2, LUT5+H.sub.2O.sub.2 and UT1+H2O2) showed an increase in proliferation rate (through oxidative protection) compared to control group (H.sub.2O.sub.2).

Example 2: Effect of Dietary Supplement Composition Comprising Natural Lutein and Tocopherols, (Lute-Gen®) on Telomere Length; Telomere Length Measurements by Telomere Analysis Technology

[0155] Methods

[0156] A high-throughput (HT) Q-FISH technique was used. This method is based on a quantitative fluorescence in-situ hybridization method modified for cells in interphase2. In brief, telomeres are hybridized with a fluorescent Peptide Nucleic Acid probe (PNA) that recognizes three telomere repeats. The images of the nuclei and telomeres are captured by a high-content screen system. The intensity of the fluorescent signal from the telomeric PNA probes that hybridize to a given telomere is proportional to the length of that telomere. The intensities of fluorescence are translated to base pairs through a standard regression curve which is generated using control cell lines with known telomere length.

[0157] Sample Preparation and HT Q-FISH: On processing day, the samples and control cell lines frozen in liquid nitrogen are thawed at 37° C. and cell counts and cellular viability are determined. Aliquots with viability lower than 60% are considered below our QC standards and are not further analyzed. Cells are seeded in clear bottom black-walled 384-well plates at 15,000 cells per well in 5 replicates for each sample and 8 replicates for each control cell line.

[0158] Cells are fixed with methanol/acetic acid (3/1, vol/vol). Once these cells have fixed onto the plate, they are treated with pepsin to digest the cytoplasm and the nuclei are processed for in situ hybridization with the PNA probe. After several washing steps following standard DAPI incubation for DNA staining, the wells are filled up with mounting medium and the plate is stored overnight at 4° C.

[0159] HT Microscopy: Quantitative image acquisition and analysis is performed on a High Content Screening Opera System. Images are captured, using a 40×0.95 NA water immersion objective. UV and 488 nm excitation wavelengths are used to detect the DAPI and A488 signals respectively. With constant exposure settings, 15 independent images are captured at different positions for each well. Next, the nuclei images are used to define the region of interest for each cell, measuring telomere fluorescence intensity of the A488 image in all of them. The results of intensity for each foci are exported to the Columbus 2.4 software. Telomere length distribution and median telomere length are calculated with Life Length's proprietary program.

[0160] Validation

[0161] The TAT technology has been validated for the following parameters:

[0162] Accuracy: The establishment of a correspondence between TAT fluorescence intensity values and telomere length measurements is achieved by performing TRF (Terminal Restriction Fragmentation) in six human lymphocyte cell lines (Calibration/Method Comparison). The same set of samples is analyzed both by TAT and by the TRF reference method (Definition of TAT Systemic Error).

[0163] VALIDATION DATA shows a correlation of 0.99.

[0164] Precision: Serial analysis of the median telomere length values is performed on a human lymphocyte sample in different runs, days and plate positions in order to define TAT Random Error parameters (Standard Deviation, Variance).

[0165] VALIDATION DATA indicates that TAT has a standard deviation of 454 base pairs. Limit of Detection and Specificity: Definition of image analysis algorithms and protocol settings exist that define the lowest significant spot intensities and avoid interference by nonspecific fluorescence signals.

[0166] VALIDATION DATA defines the limit of detection at 800 base pairs and demonstrates very high specificity.

[0167] Median Reportable Range: Analysis of median telomere length of 6 cell lines is performed that covers our reportable range and defines its lower and upper limits.

[0168] VALIDATION DATA fix lower level at 4,700 base pairs and upper level at 14,400 base pairs.

[0169] Reference Range: Analysis of median telomere length have been conducted in hundreds of human samples in order to define the TAT Reference Range and its percentiles (5th, 10th, 25th, 50th 75th and 95th) for different ages.

[0170] VALIDATION DATA established population curves—normal population data base from 18 to 85 years, to extrapolate patients' data and generate reports.

[0171] Results

[0172] During the TAT protocol, the samples were assessed for:

[0173] Cell count: An automated cell counter is used to determine the total number of cells in the vials. A minimum of 100.000 cells are needed to perform TAT.

[0174] Cell viability by Tripan-Blue exclusion method. The exclusion criteria currently in place will reject aliquots with less than 60% viable cells.

[0175] Regression Curve: Internal controls are included and a regression analysis is performed for each run/plate. The plates are repeated if their regression curves have an R2 below 0.92.

[0176] Replicates: After seeding and once TAT is completed:

[0177] Samples with CV above 10% are discarded.

[0178] Samples with less than 3 valid replicates at the end of the analysis are discarded. Spot number analyzed per sample should be higher than 1,000.

[0179] Cell Count and Viability Results

[0180] All samples were analyzed by TAT.

TABLE-US-00004 TABLE 4 Concentration and viability of samples after thawing Cell conc Viability LL CODE Week (cells/mL) % Ctrl-P0 Control - Week 0 1.4  91 Ctrl-P4 Control - Week 4 0.66 91 H.sub.2O.sub.2-P4 10 μm H.sub.2O.sub.2 - Week 4 0.92 92 LUT10-P4 LUT (10 μg/ml) - Week 4 0.95 96 LUT5-P4 LUT (5 μg/ml) - Week 4 0.98 96 LUT1-P4 LUT (1 μg/ml) - Week 4 0.24 80 LUT10 + H.sub.2O.sub.2-P4 LUT (10 μg/ml) + 10 μMH.sub.2O.sub.2 - 0.21 96 Week 4 LUT5 + H.sub.2O.sub.2-P4 LUT (5 μg/ml) + 10 μMH.sub.2O.sub.2 - 0.22 81 Week 4 LUT1 + H.sub.2O.sub.2-P4 LUT (1 μg/ml) + 10 μMH.sub.2O.sub.2 - 0.31 87 Week 4 Ctrl + P8 Control - Week 8 0.25 83 H.sub.2O.sub.2-P8 10 μM H.sub.2O.sub.2 - Week 8 0.57 87 LUT10-P8 LUT (10 μg/ml) - Week 8 0.53 87 LUT5-P8 LUT (5 μg/ml) - Week 8 0.73 91 LUT1-P8 LUT (1 μg/ml) - Week 8 1.2  91 LUT10 + H.sub.2O.sub.2-P8 LUT (10 μg/ml) + 10 μMH2O2 - 0.67 92 Week 8 LUT5 + H.sub.2O.sub.2-P8 LUT (5 μg/ml) + 10 μMH2O2 - 0.28 76 Week 8 LUT1 + H.sub.2O.sub.2-P8 LUT (1 μg/ml) + 10 μMH2O2 - 0.53 87 Week 8

TABLE-US-00005 TABLE 5 Plate's Quality Control Parameters 1804048-PTAT A Coef 6.50 B Coef 7.62 R2 0.99 Control median 414 Sample median 327

[0181] Telomere Length Results from TAT Analysis

[0182] The following table (Table 6) displays the TAT parameters from the Histogram. Median telomere length, 20.sup.th percentile telomere length, percentage of short telomeres and the coefficients of variation for each set of samples. The percentage of short telomeres is defined as the percentage of those below 3 Kbp (<3 Kbp). As mentioned earlier, measurements in five replicates per sample were performed.

TABLE-US-00006 TABLE 6 Telomere length results from TAT analysis. All samples meet the quality control parameters Median Length 20th Percentile Telomeres CV LL-CODE Treatment (base pairs) Length (base pairs) <3 Kbp (%) (%) Ctrl-P0 Control - Week 0 7338 3789 14.37 0.40 Ctrl-P4 Control - Week 4 6519 3381 15.83 1.37 H.sub.2O.sub.2-P4 10 μM H.sub.2O.sub.2 - Week 4 6369 3167 18.62 1.61 LUT10-P4 LUT (10 μg/ml) - Week 4 6330 3198 18.48 0.15 LUT5-P4 LUT (5 μg/ml) - Week 4 6349 3104 18.98 0.85 LUT1-P4 LUT (1 μg/ml) - Week 4 5977 3351 16.87 1.95 LUT10 + H2O2-P4 LUT (10 μg/ml) + 10 μMH.sub.2O.sub.2 - Week 4 5772 3073 19.10 5.20 LUT5 + H2O2-P4 LUT (5 μg/ml) + 10 μMH.sub.2O.sub.2 - Week 4 6077 3351 16.97 4.17 LUT1 + H2O2-P4 LUT (1 μg/ml) + 10 μMH.sub.2O.sub.2 - Week 4 5997 3381 16.72 4.70 Ctrl + P8 Control - Week 8 6330 3259 17.87 1.62 H.sub.2O.sub.2-P8 10 μM H.sub.2O.sub.2 - Week 8 5429 2782 21.93 0.40 LUT10-P8 LUT (10 μg/ml) - Week 8 5997 2977 20.21 2.58 LUT5-P8 LUT (5 μg/ml) - Week 8 6037 2945 20.51 1.64 LUT1-P8 LUT (1 μg/ml) - Week 8 6077 2848 21.29 1.00 LUT10 + H2O2-P8 LUT (10 μg/ml) + 10 μMH2O2 - Week 8 5977 2977 20.09 2.12 LUT5 + H2O2-P8 LUT (5 μg/ml) + 10 μMH2O2 - Week 8 5977 2977 20.09 2.13 LUT1 + H2O2-P8 LUT (1 μg/ml) + 10 μMH2O2 - Week 8 6037 2848 21.14 0.83

Example 3: Effect of Dietary Supplement Composition Comprising Natural Lutein and Tocopherols, (Lute-Gen®) on Telomere Length; Analysis of Telomere Length Variability

[0183] FIG. 3 Histogram shows the distribution of telomere lengths in sample Ctrl-PO. Bars represent the relative frequency for every particular fluorescence intensity normalized (X axis).

[0184] The 20th percentile (red bars) indicates the particular length below which 20% of the telomeres have been observed. The median (MTL) and average (ATL) telomere length are also indicated in the histogram. This histogram also allows for the analysis of telomere length variability. FIG. 4 shows representative TAT image, both the nuclei (white) and the telomeric spots (Bright white spots) can be seen in the image.

[0185] Data Analysis

[0186] For TAT analysis, data were grouped by condition (Ctrl, H2O2, LUT10, LUT5, LUT1, LUT10+H2O2, LUT5+H2O2, LUT1+H2O2) and time point of treatment (0, 4, and 8 weeks).

[0187] Bar graphs of TAT results (Median Telomere Length, 20th Percentile Telomere Length and % Short Telomeres) for different time points and treatments (FIGS. 5, 6 and 7).

[0188] Two-Way ANOVA Analysis was used to compare the results obtained from the TAT assay. This analysis indicates if there are significant differences among the different treatments. Significant differences are indicated in the Significance and Adj. P Value columns. From lowest to highest significance; ns: non-significant; *: p<0.05; **: p<0.01; ***p<0.001; ****p<0.0001.

[0189] Due to the fact that DNA replication during the cell cycle causes of telomere shortening, the telomere length measurements performed were normalized by the total number of population doubling (cell replication) in each condition and week.

[0190] Table 7 Summary table of cumulative population doubling and median of telomere length at different week (Week 0, 4 and 8) and treatments (Ctrl, H2O2, LUT10, LUT5, LUT1, LUT10+H2O2, LUT5+H2O2, LUT1+H2O2).

[0191] Cumulative Population Doubling

[0192] Telomere shortening rate was analysed, Median of telomere length (initial-final)/Population Doubling, at the defined time points and treatments, FIG. 8.

TABLE-US-00007 TABLE 7A Telomere shortening rate (Median of telomere length (initial-final)/Population Doubling) LUT10 + LUT5 + LUT1 + Week Ctrl H.sub.2O.sub.2 LUT10 LUT5 LUT1 H.sub.2O.sub.2 H.sub.2O.sub.2 H.sub.2O.sub.2 Cumulative Population Doubling 0 0 0 0 0 0 0 0 0 4 13.6 10.6 13.3 12.5 13.7 13.1 13.5 12.3 8 24.7 18.8 23.9 21.3 24.1 20.8 23.1 20.7 Median Telomere Length 0 7347 7347 7347 7347 7347 7347 7347 7347 4 6515 6360 6272 6340 5984 5804 6085 5980 8 6333 5636 6008 6177 6071 5972 5980 6045

[0193] Telomere Shortening Rate (bp/PD)

[0194] Telomere shortening rate (bp/PD) is shown in Table 7B.

TABLE-US-00008 TABLE 7B Telomere shortening rate (bp/PD) LUT10 + LUT5 + LUT1 + Week Passage Ctrl H.sub.2O.sub.2 LUT10 LUT5 LUT1 H.sub.2O.sub.2 H.sub.2O.sub.2 H.sub.2O.sub.2 0 P0 0 0 0 0 0 0 0 0 4 P4 61 93 81 80 100 118 93 111 8 P8 41 91 56 55 53 66 59 63

Example 4: Effect of Dietary Supplement Composition Comprising Natural Lutein and Tocopherols, (Lute-Gen®) on Telomere Length; Telomere Shortening Rate at Different Treatments

[0195] Graph of telomere length vs Population doubling at different treatments. Slope of regression line in each condition, represents the telomere shortening rate. Y=aX+b; a=slope andbb=y intercept, FIG. 9.

TABLE-US-00009 TABLE 8 Telomere shortening rate at different treatments. Condition Slope (Telomere Shortening Rate) R.sup.2 Ctrl y = −41.782x + 7264.9 R.sup.2 = 0.914 H.sub.2O.sub.2 y = −91.272x + 7340.7 R.sup.2 = 0.999 LUT10 y = −57.039x + 7250 R.sup.2 = 0.928 LUT5 y = −56.796x + 7261.1 R.sup.2 = 0.917 LUT1 y = −55.261x + 7163.8 R.sup.2 = 0.768 LUT10 + H2O2 y = −71.479x + 7181.9 R.sup.2 = 0.999 LUT5 + H2O2 y = −61.587x + 7222 R.sup.2 = 0.879 LUT1 + H2O2 y = −66.365x + 7187.3 R.sup.2 = 0.804

[0196] Observations and Conclusions

[0197] The results obtained during the initial analysis of the samples indicated cell viabilities within acceptable range for all samples. The standard regression curve's coefficient of determination (R2) for the plate analyzed, met the quality control standards (R2>0.98).

[0198] Summarized Version of the Results:

[0199] Telomere analysis following 8 weeks of treatments:

[0200] Oxidative Stress decreased proliferation capacity and increased telomere shortening rate in cultures of human primary fibroblast.

[0201] For control in standard culture condition (Crtl):

[0202] The median telomere length decreased by 1014 bp (ie: from 7347 bp to 6333 bp).

[0203] The 20th percentile median telomere length shortened by 530 bp (ie: from 3789 bp to 3259 bp).

[0204] The percentage of short telomeres (<3 kpb) increased by 4% (ie: from 14% to 18%).

[0205] For control in oxidative culture condition (H.sub.2O.sub.2):

[0206] The median telomere length decreased by 1712 bp (ie: from 7347 bp to 5636 bp).

[0207] The 20th percentile median telomere length shortened by 1007 bp (ie: from 3789 bp to 2782 bp).

[0208] The percentage of short telomeres (<3 kpb) increased by 7% (ie: from 14% to 21%).

[0209] For treated groups in standard conditions (LUT10, LUT5, LUT1):

[0210] A significant increase in telomere shortening rate was observed after LUT1 treatment.

[0211] No other significant differences were observed under these conditions

[0212] For treated groups in oxidative culture condition (LUT10+H.sub.2O.sub.2, LUT5+H.sub.2O.sub.2, LUT1+H.sub.2O.sub.2):

[0213] Oxidative Stress decreased proliferation capacity and increased telomere shortening rate.

[0214] A significant difference in the telomere shortening rate was identified between LUT10+H.sub.2O.sub.2, LUT5+H.sub.2O.sub.2 and LUT1+H.sub.2O.sub.2 compared to control (H.sub.2O.sub.2) after 8 passages.

[0215] According to the data obtained from this assay, Lutein has a significant protective effect on telomere length erosion in vitro under oxidative stress conditions after 8 passages.

[0216] According to non-limiting exemplary aspect of the present invention, it describes a method of increasing telomere length in humans comprising administering an effective dietary composition comprising Lutein crystal.

[0217] According to non-limiting exemplary aspect of the present invention, it describes a particularly effective dietary supplement in stimulating natural stem cell production within the body.

[0218] According to embodiments of the present invention, a new approach to slowing aging and increasing longevity by increasing the number of healthy cells in the human body and also to increase the length of the telomeres in the DNA of these healthy cells. Telomeres in existing cells can be lengthened and the length of telomeres in newly produced stem cells can be increased with the daily administration of this dietary formulation. The nutritional supplements may repair telomere shortening and decrease the rate of telomere shortening.

[0219] According to yet another embodiment of the present invention, the dietary nutritional supplements disclosed below support telomere lengthening to aid in attaining a maximum life span, as well as repair telomere shortening and decrease the rate of telomere shortening.

[0220] According to a non-limiting exemplary aspect of the present invention describes a novel Lute-gen® dietary supplement for its antioxidant's activity to telomeric damage, pro-longing the replicative lifespan and slowing telomere shortening. Improving telomerase activation in stem cells and potentially in other cells by diet and lifestyle interventions may represent an intriguing way to promote health-span in humans.

[0221] According to further non-limiting exemplary aspect of the present invention, dietary supplement for its antioxidant's activity to telomeric damage.

[0222] Inventive step involved in the present invention deals with the method of administering the composition containing lutein and tocopherol crystals to subjects in the form of dietary supplement so that the oxidative stress is reduced, reduction of telomerase shortening occurs and in turn resulting in increase in the health span of humans.

[0223] The invention of Lute-gen® has enormous potential as the crystals of lutein+tocopherol can be used as dietary supplement to increase the health span of humans and at the same time it can be produced on a large scale in the industry.

[0224] In an embodiment, all grades of Lute-gen®, having stability data (long term and accelerated), with a minimum shelf-life of two years from the date of packaging.

[0225] Tablets, effervescent tablets, hard-shell capsules, softgel capsules, liquid filled capsules, stick packs and sachets, beverages, dairy (butter and cheese), bakery, gummies, jelly sticks, droppers and cosmetics (FIG. 10).

[0226] According to a non-limiting exemplary aspect of the present invention can be used for the development of diet plan and nutritional supplements.

[0227] Merely for illustration, only representative number/type of graph, chart, block, and sub-block diagrams were shown. Many environments often contain many more block and sub-block diagrams or systems and sub-systems, both in number and type, depending on the purpose for which the environment is designed.

[0228] While specific embodiments of the invention have been shown and described in detail to illustrate the inventive principles, it will be understood that the invention may be embodied otherwise without departing from such principles.

[0229] Reference throughout this specification to “one embodiment”, “an embodiment”, or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment”, “in an embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

[0230] It should be understood that the figures and/or screen shots illustrated in the attachments highlighting the functionality and advantages of the present invention are presented for example purposes only. The present invention is sufficiently flexible and configurable, such that it may be utilized in ways other than that shown in the accompanying figures.

[0231] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

REFERENCES

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