Betacryptoxanthin compositions, processes for preparation and uses thereof
Field
The compositions comprising of betacryptoxanthin extract are described herein for use of enhancing cardio-respiratory fitness for physical performance and exercise endurance and maintain healthy lung function. More particularly, betacryptoxanthin compositions as described herein are prepared by cost and time effective process and are administered in effective amounts to exercising subjects to enhance cardio-respiratory fitness. The compositions are comprised of betacryptoxanthin extract either alone or along with at least one more nutrient and suitable excipients to form compositions which can be used for pharmaceutical and nutraceutical applications. Betacryptoxanthin extract is prepared from paprika pods and paprika oleoresin, by improved process of saponification and column chromatography, in which saponification cycle time, solvent amount, column separation time and silica amount are reduced to a considerable time, thus making it cost and time effective process, which is industrially viable. Betacryptoxanthin compositions increase exercise time, endurance performance and lung capacity and maintain healthy lung and cardiovascular function during such physical activities. Betacryptoxanthin compositions as described herein enhance endurance performance by improving mitochondrial mass and muscle respiration. The compositions also improve physical performance, attenuate muscle fatigue and enhance aerobic respiration utilization capacity. The compositions are safe for consumption, prepared by cost efficient and industrially viable process and can be employed for enhancing cardio-respiratory fitness when administered to exercising subjects in effective amounts.
Background
Physical fitness of a subject is a general state of health and well-being and, more specifically the ability to perform aspects of sports, exercise or other routine physical activities. Fitness is generally achieved through correct nutrition, moderate to vigorous physical activity, exercise and rest. It is a set of attributes or characteristics seen in people and which relate to the ability to perform a given set of physical activities. Earlier, fitness was linked to the capacity to carry out the day's activities without undue fatigue. However with changes in lifestyle physical fitness is now considered a measure of the body's ability to function efficiently and effectively in work and

leisure activities, to be healthy, to resist diseases due to sedentary lifestyle and to meet emergency situations. When fitness is linked to sports activities, it has five common elements -strength, speed, endurance, flexibility, and skill. The relative contributions of each of these to the specific fitness demands of different sports are, of course, not equal. To a certain extent skill can compensate for poor fitness, but improved fitness allows skillful sportspersons to extend their performance by delaying the onset of fatigue and thus endurance plays important role to determine subject's fitness.
The fitness for exercise or sports related activities can be of two types, cardiovascular and cardio-respiratory. Cardiovascular fitness is the ability of the heart and lungs to supply oxygen-rich blood to the working muscle tissues and the ability of the muscles to use oxygen to produce energy for movement. Cardio-respiratory fitness refers to the ability of the circulatory and respiratory systems to supply oxygen to skeletal muscles during sustained physical activity. Thus cardio-respiratory fitness can be linked to endurance of a subject during prolonged exercise, sports of physical activities.
A person's ability to deliver oxygen to the working muscles is affected by many physiological parameters, including heart rate, stroke volume, cardiac output, and maximal oxygen consumption. Regular exercise improves the respiratory system by increasing the amount of oxygen that is inhaled and distributed to body tissue. Cardio-respiratory fitness can reduce the risk of heart disease, lung cancer, type 2 diabetes, stroke, and helps improve lung and heart condition, and increases feelings of wellbeing.
Although different types of aerobic and anaerobic exercises help to enhance cardio-respiratory fitness, a balanced diet, based on essential nutrients such as carbohydrates, proteins, vitamins and minerals is supposed to enhance the exercise capacity and endurance. Carotenoids such as Betacryptoxanthin [BCX], which are considered as Provitamin A are known for their effects as antioxidants and also in treatment of inflammatory arthritis. It is present in many fruits and vegetables. BCX is a carotene and helps protect cells from free radical damage. By protecting cells from free radicals and by reducing free radical activities, it may prevent many dangerous

diseases and conditions. Many references deal with different health applications of betacryptoxanthin in humans and animals.
U.S. Patent application 20120245122A1 relates to a method for improving bone health in a subject comprising administering to the subject a carotenoid blend comprising lycopene, beta-carotene, betacryptoxanthin and combinations thereof.
PCT application WO2008023283A2 describes an esterified xanthophyll composition comprising cryptoxanthin and a method of treating breast, colon, lung, skin, cervix and ovaries cancers, as well as for treatment of cardiovascular disease, prevention of cataract and macular degeneration, as agent for the absorption of harmful ultra-violet light from the rays of the sun and quencher of photo-induced free radical and reactive oxygen species.
European patent application EP1748705 describes use of betacryptoxanthin in the manufacture of a composition for promoting an increased protein formation and/or prevention of protein loss in human or an animal, wherein the composition is for promoting an increased protein formation in sports and workout activities.
U.S. patent application US20120107380 deals with a method for producing a xanthophyll-enriched product from a xanthophyll ester source, wherein the xanthophyll ester is selected from zeaxanthin, lutein, beta-cryptoxanthin, astaxanthin, capsanthin, capsorubin, and mixtures thereof. This xanthophyll-enriched product is micro-encapsulated as a nutritional supplement and used for the treatment or the prevention of human or animal diseases including cancer-related diseases, cardiovascular diseases and inflammatory disorders.
U.S. patent 8021698B2 discloses a nutritional supplement including betacryptoxanthin which is used to maintain cardiovascular health by lowering blood pressure, preventing high, elevated blood pressure and/or maintaining healthy blood pressure.
U.S. patent 7863241 provides liquid or aerosolized composition comprising a lung surfactant polypeptide, a protease inhibitor and anti-oxidants in combination with carotenoid compounds such as leutein, zeaxanthin, cryptoxanthin, violaxanthin, carotene diol, hydroxycarotene,

hydroxylycopene, alloxanthin and dehydrocryptoxanthin, for treating pulmonary conditions and or reducing the negative effects of pulmonary inflammation.
Liu C et al (Cancer Prev Res, 2011 Aug;4(8): 1255-66) relates to evaluation of effects of betacryptoxanthin supplementation on cigarette smoke-induced squamous metaplasia, inflammation, and changes in protein levels of proinflammatory cytokine [tumor necrosis factor alpha (TNFa)] and transcription factors [nuclear factor kappa B (NF-KB) and activator protein-1 (AP-1)], as well as on smoke-induced oxidative DNA damage [8-hydroxy-2'-deoxyguanosine (8-OHdG)] in the lung tissue of ferrets. Betacryptoxanthin significantly decreased smoke-induced lung squamous metaplasia and inflammation. BCX also substantially reduced smoke-elevated TNFa levels in alveolar, bronchial, bronchiolar, and bronchial serous/mucous gland epithelial cells and in lung macrophages.
Study by Wang Xiang (NIH2014 R21 CA) suggested that increased dietary intake or higher blood levels of betacryptoxanthin (BCX) is strongly associated with a reduced risk of lung cancer in current smokers. It was hypothesized that BCX targets SIRT1 signaling pathway as its chemopreventive action. Importantly, BCX treatment restored nicotine-reduced lung SIRT1 protein to normal levels and inhibited both nicotine-induced emphysema and nicotine-promoted lung tumor development.
Iskandar AR et al (2013 Apr;6(4):309-20) describes effect of BCX as a preventive agent against emphysema and lung cancer with SIRT1 as a potential target.
Summary
Even though the patent and non-patent literature discusses the effect of betacryptoxanthin in bone health, lung cancer treatment and inflammatory conditions and in management of ill effects of smoking in lungs, there is no teaching about use of betacryptoxanthin compositions for enhancing exercise performance and endurance and maintaining lung and cardiovascular health related to exercise. Further none of the prior art references deal with evaluation of betacryptoxanthin for its protective effect on lung health through mechanism such as reduction of oxidative stress markers or improvement of lung function as well as cardiovascular function, thus resulting into improved exercise endurance and cardio-respiratory function.

Further prior processes for producing beta-cryptoxanthin have several limitations. While natural sources of beta-cryptoxanthin are available, extracts have thus far been produced only in enriched form in fruit drinks such as tangerine, Satsuma orange and persimmon. The use of a biotechnological route for producing beta-cryptoxanthin is still in preliminary development and has thus far been limited to laboratory scale production with poor yields. The synthetic approach gives a mixture of beta-cryptoxanthin and a considerable amount of impurities such as alpha-cryptoxanthin, which is most likely zeinoxanthin (a non-provitamin A), along with un-reacted anhydroluteins and zeaxanthin. Applying other processes of literature, the separation of beta-cryptoxanthin is complex, involves multiple steps and is not commercially feasible. Thus, a need exists for alternate preparation process for betacryptoxanthin composition, which is rich in trans-betacryptoxanthin, which would be cost and time effective and therefore industrially viable.
Applicant has carried out rigorous experimentation for preparation of betacryptoxanthin extract, compositions and evaluation for exploring effect of betacryptoxanthin in enhancing cardio-respiratory fitness of the exercising subjects, when administered in effective amounts. Betacryptoxanthin compositions are mainly comprised of extract enriched in trans-betacryptoxanthin which is prepared from paprika pods or oleoresin using polar and non-polar solvents. The process is comprised of two steps, saponification and column separation. The oleoresin is saponified using polar solvents and alkali at ambient to elevated temperature conditions. Saponified mass is extracted with non-polar solvent to get concentrated extract, which is purified by column chromatography to get content of at least 75% trans-betacryptoxanthin by weight of the extract. The process described herein requires significantly less saponification time and column separation time, and it is also cost effective as solvent and silica gel required are significantly less than the processes reported in prior art. According to this process, betacryptoxanthin obtained from column separation does not require further purification step, thus it further saves time of additional step. The extract thus obtained is used as such or formulated into suitable composition, by employing one or more nutrients and pharmaceutically or nutraceutically acceptable excipients.

Betacryptoxanthin compositions are evaluated for their effect on enhancing physical performance and maintaining healthy lungs and cardiovascular functions. Betacryptoxanthin compositions described herein increase exercise performance by enhancing mitochondrial mass and muscle respiration. The compositions also exhibit positive effect on E-Cadherin (ECAD) gene expression in primary bronchial epithelial cells and manage inflammatory markers, thus protecting the lungs and improving lung capacity for exercise endurance by affecting forced expiratory volume, forced vital capacity parameters, when administered in effective amounts.
Betacryptoxanthin compositions described herein, can be administered to a subject, in the form of pharmaceutical or nutraceutical delivery systems, in the form of dietary supplement, dosage form or in suitable vehicle, convenient for administration. The compositions can be administered in the form of powders, granules, sachets, beadlets, tablets, capsules, caplets, suspensions, emulsions, solutions, energy bar, beverages, functional foods and the like. The compositions as described herein are prepared from suitable paprika source and can be administered in dosages such as about 0.001 to 10 mg/kg body weight of a subject. The dosages can also vary from about 1.0 to 9.0 mg/kg body weight or to about 2.0 to 8.0 mg/kg body weight of betacryptoxanthin. The dosage form or dietary supplement can be administered in effective amounts, as single dose for a single day, over extended time period or specific time duration, or single or multiple servings during the day, based on the requirement of the subject. Betacryptoxanthin compositions described herein may contain one or more pharmaceutically and/or nutraceutically acceptable excipients along with additive such as vitamins, lipids, carbohydrates, amino acids, trace elements, colorings, flavors, artificial sweeteners, natural antioxidants, stabilizers, preservatives and buffers.
Betacryptoxanthin compositions are comprised of extract enriched in trans-betacryptoxanthin and are prepared by economically viable process using conventional equipments. The compositions are found to be effective for enhancing physical performance during exercise, sports activities and/or routine physical activities and also enhance recovery after prolonged physical activities by enhancing cardio-respiratory fitness of a subject, when administered in effective amounts.

Detailed Description of Figures
Figure 1 depicts effect of BCX composition on ECAD genes in bronchial epithelial cells
Figure 2 depicts effect of BCX composition on Lipid profile
Figure 3 depicts effect of BCX composition on muscle oxidative stress and muscle antioxidant
enzyme
Figure 4 depicts effect of BCX on lung antioxidant enzymes
Detailed Description
Betacryptoxanthin compositions (BCX) described herein are comprised of extract with at least 75% by weight of trans-betacryptoxanthin, which are prepared by using polar and non polar solvents using industrially viable cost and time effective process. The compositions are useful to enhance performance during physical activities such as exercise and sports and to maintain healthy lungs and cardiovascular function. More particularly, betacryptoxanthin compositions are administered in suitable dosage forms to enhance cardio-respiratory fitness of a subject by increasing exercise time, endurance performance, lung capacity and maintain healthy lung and cardiovascular function during such physical activities. Betacryptoxanthin compositions enhance endurance performance by enhancing mitochondrial mass and respiration in muscles. The compositions also improve physical performance, attenuate muscle fatigue and enhance aerobic respiration utilization capacity.
Betacryptoxanthin compositions herein may be obtained by human intervention and are safe for administration and thus useful for pharmaceutical and nutraceutical applications.
The terminology 'subject' is commonly used in the specification to refer to an individual or mammal such as human being or animal under test, being treated with compositions herein.
The terminology "subject in need thereof can include specific individuals or mammals, which need to undergo sustained physical activities, exercise or sports activities for prolonged time, for which these need to have improvement in lung and cardiovascular function, to support increased energy demands to support vigorous and sustained physical activities, thus there is a need to have

improved cardio-respiratory fitness in such subjects, in terms of protection and management of both vital body systems.
The terminology "cost and time effective process" means the process in which cost and time for preparation is reduced effectively by modifying the process in which saponification time is considerably reduced along with time required for separation of trans-betacryptoxanthin fraction from column chromatography. Amount of solvents such as hexane and acetone required for column separation and the silica gel used in the column is also reduced to the great extent as per the process described herein. Thus it is cost and time effective process, which is industrially viable and can be used on large scale to get a product.
The cardiovascular system is responsible for a vast set of adaptations in the body throughout exercise. It must immediately respond to changes in cardiac output, blood flow, and blood pressure. Cardiac output is defined as the product of heart rate and stroke volume which represents the volume of blood being pumped by the heart each minute. Cardiac output increases during physical activity due to an increase in both the heart rate and stroke volume. At the beginning of exercise, the cardiovascular adaptations are very rapid. Both heart rate and stroke volume vary directly with the intensity of the exercise performed and many improvements can be made through continuous training.
Another important issue is the regulation of blood flow during exercise. Blood flow must increase in order to provide the working muscle with more oxygenated blood which can be accomplished through neural and chemical regulation. Also, chemical factors such as a decrease in oxygen concentration and an increase in carbon dioxide or lactic acid concentration in the blood promote vasodilatation to increase blood flow. Although all of the described adaptations in the body to maintain homeostatic balance during exercise are very important, the most essential factor is the involvement of the respiratory system. The respiratory system allows for the proper exchange and transport of gases to and from the lungs while being able to control the ventilation rate through neural and chemical impulses. Thus fitness of both these body systems, lungs and cardiovascular systems is very important to have enhanced endurance and performance for

normal, exercise and sports activities and thus exercise performance is interlinked with this cardio-respiratory fitness.
Exercise increases the utilisation of oxygen in the body and therefore enhances the production of reactive oxygen species and impairs both enzymatic and non-enzymatic antioxidant defence systems in skeletal muscle and blood. On the other hand, it will be suggested that the activity of AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor-y coactivator la (PGC-la), nuclear respiratory factor 1 (NRF1) mitochondrial transcription factor A (TFAM), and sirtuins could play an important role in the exercise-induced adaptive response. SIRT1 is an important regulator of metabolism by controlling the activity of key transcription factors such as PGC-la, FOXOl, and p53, which play a key role in the training response. Therefore, activators of SIRT1 could have potentially beneficial effects which enhance aerobic performance, even in rats having a high endurance capacity. Recent studies have indicated that antioxidant supplementation led to the prevention of strenuous exercise induced oxidative injury in in-vivo studies. Many studies have indicated that antioxidant nutrient supplementations prevented strenuous exercise-induced oxidative injury in human subjects and rats (Khanna et al. 1999).
In some embodiments, betacryptoxanthin compositions are used in effective amount to enhance cardio-respiratory fitness in a subject and/or and beneficial effects are evaluated for exercise performance and lung capacity in a subject.
In one embodiment, compositions described herein are directed for improvement of exercise endurance and lung health by administering to a subject in need thereof, an effective amount of a composition comprising betacryptoxanthin alone or in combination with other nutrients.
As per important embodiment of this invention, the compositions are essentially comprised of extract enriched with at least 75% by weight of trans-betacryptoxanthin. The compositions comprised of trans-betacryptoxanthin extract can be either used as such for administration to the subjects and evaluation of beneficial effects.

In some embodiments, the composition of the invention can be formulated in a dosage form including, but not limited to, beadlets, microencapsulated powders, oil suspensions, liquid dispersions, capsules, pellets, ointments, soft gel capsules, tablets, chewable tablets or lotions/liquid preparations. The composition as described herein can also be provided in a food or feed (including liquid or solid) composition. Thus, it is envisioned that suitable delivery methods include, but are not limited to, oral, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, intracranial, or buccal administration. Compositions comprising trans-betacryptoxanthin may include one or more suitable pharmaceutically acceptable ingredients or food grade ingredients such as, but not limited to, carriers, binders, stabilizers, excipients, diluents, pH buffers, disintegrators, solubilizers and isotonic agents. In one more embodiment, compositions may be comprised of extract enriched with at least 75% trans-betacryptoxanthin, along with more nutrients selected from the group of, but not restricted to beta-carotene, zeaxanthin, capsanthin, carotenoids, omega 3 fatty acids, vitamins and the like alone or in the combination thereof.
The compositions of the invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of the trans-betacryptoxanthin composition. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, for example in methods of treatment or pharmaceutical compositions for use in such methods. A therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic or preventive result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease or a condition requiring treatment is identified, the prophylactically effective amount will be less than the therapeutically effective amount.

According to the embodiment of the invention, betacryptoxanthin compositions are prepared from suitable varieties of paprika, employing solvent extraction process. The plant material is derived from sources including, but not limited to, fruits and vegetables. In some embodiments of the invention, the plant material is derived from capsicums. Capsicum is a genus of flowering plants that includes several varieties of peppers, such as but not limited to red peppers, and the word "capsicum" is also used interchangeably in several parts of the world when referring to peppers. The capsicum oleoresin described herein also includes paprika oleoresin. The compositions are prepared from paprika pods or paprika oleoresin containing about 0.1-2% by weight of betacryptoxanthin. The remaining carotenes present include betacarotene, zeaxanthin, trans-capsanthin and trace amounts of other carotenoids. Preferably the compositions may contain betacryptoxanthin content of at least 75% by weight. The process of preparation includes the steps of admixing capsicum oleoresin with suitable solvent, saponifying the xanthophyll esters, extracting saponified mass with hexane and obtaining a concentrate which is loaded on silica gel column, eluting with combination of polar and non-polar solvents to obtain composition enriched with at least 75% by weight of trans-betacryptoxanthin, which can be further formulated using pharmaceutically or nutraceutically acceptable excipients and/or carriers.
In some embodiments, solvents employed in the process for preparation of betacryptoxanthin are selected from, but not limited to non-polar, semi-polar and/or polar solvents or combinations thereof. More preferably the solvents are selected from polar solvents such as acetone, ethyl acetate, acetonitrile, ether, alcohols, water and the like, either alone or in combination thereof.
In certain embodiments, the aliphatic alcohol comprises a hydrocarbon fragment derived from a fatty, non aromatic hydrocarbon and is selected from the group consisting of ethanol, methanol, isopropyl alcohol, and mixtures thereof. In some embodiments, the aliphatic alcohol is ethanol.
In some embodiments, the non-polar solvent used in the process may be selected from the group of, but not limited to pentane, hexane, cyclohexane, benzene, toluene, chloroform, diethyl ether and the like or mixtures thereof. In most preferred embodiment, the non polar solvent used herein is hexane.

In certain embodiments, the alkali is a soluble hydroxide of the alkali metals, including lithium, sodium, potassium, rubidium, or cesium, and is selected from the group consisting of sodium hydroxide, potassium hydroxide, and mixtures thereof. In some embodiments, the alkali is sodium hydroxide. In other embodiments, the alkali is potassium hydroxide.
According to still one more embodiment of the invention, the elevated temperature for saponification is above room temperature. In some embodiments, the elevated temperature ranges from about 65 to about 95° C, about 70 to about 90° C. about 75 to about 85° C, from about 75 to about 80° C, or from about 80 to about 85° C.
As per one more embodiment of the invention, suitable weight ratio of oleoresin to solvent may range from 1:0.5 to 1:20 and suitable ratio of oleoresin to alkali may range from 1:0.05 to 1: 5 by weight.
In some embodiments, the process for preparing betacryptoxanthin composition includes following steps:
1. Mixing capsicum oleoresin with ethanol in suitable ratio;
2. Saponifying the oleoresin with potassium hydroxide, wherein the ratio of oleoresin to potassium hydroxide is about 1:0.25 weight/weight; adding mixed tocopherol to the mixture
3. Applying heat to the oleoresin to elevate the temperature up to reflux at about 80-85 °C and agitating the oleoresin for about 3 to 5 hours at about 80-85 °C to get saponified mass.
4. Extracting saponified mass with hexane and concentrating the hexane washings to obtain a concentrated mass. This mass will be used as feed for column chromatorgraphy.
5. Forming slurry of hexane concentrated mass with silica and drying to get adsorbed mass on silica, which is loaded to column and eluted with hexane to obtain fractions other than betacryptoxanthin;
6. Washing the column with hexane to acetone mixture and concentrating the washings to obtain an extract composition comprising at least 75% by weight of trans-beta-cryptoxanthin.

The process described herein is cost and time effective and industrially viable in terms of scalability and reduced cycle time. The amount of solvents required in the process are very less and the time required for saponification and column separation is also significantly low. Betacryptoxanthin separated from column chromatography does not need additional step of purification, thus it saves time and still provides betacryptoxanthin having assay of about 12% w/w to comply with desired purity standard of betacryptoxanthin.
Betacryptoxanthin extract is prepared from paprika pods and paprika oleoresin, by improved process of saponification and column chromatography, in which saponification cycle time, solvent amount, column separation time and silica amount are reduced to a considerable time, thus making it cost and time effective process.
Betacryptoxanthin extract thus obtained is either used as such or formulated using suitable excipient to get betacryptoxanthin compositions.
In some embodiments, the composition of the invention can be formulated in a dosage form including, but not limited to, beadlets, microencapsulated powders, oil suspensions, liquid dispersions, capsules, pellets, ointments, soft gel capsules, tablets, chewable tablets or lotions/liquid preparations. The composition as described herein can also be provided in a food or feed (including liquid or solid) composition. Thus, it is envisioned that suitable delivery methods include, but are not limited to, oral, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, intracranial, or buccal administration. Compositions comprising trans-betacryptoxanthin may include one or more suitable pharmaceutically acceptable ingredients or food grade ingredients such as, but not limited to, carriers, binders, stabilizers, excipients, diluents, pH buffers, disintegrators, solubilizers and isotonic agents.
Betacryptoxanthin composition of the invention may be administered by oral route, in combination with antioxidant or other nutrients, using oil vehicle for suspending the composition.
In one embodiment, betacryptoxanthin compositions, process for preparation and method of using them are directed to the improvement of cardio-respiratory fitness by increasing

mitochondrial mass, muscle respiration, lung capacity and reduction in oxidative stress and inflammatory markers in lung tissues.
As per embodiments study is undertaken with an animal model to investigate the effects of BCX in rats after exhaustive exercise. Effect of BCX compositions is investigated on lipid profile, oxidative stress, antioxidant enzymes and muscle fatigue. The effect is also observed on promoting lung health and cardiovascular health in exercising rat models.
According to one embodiment, betacryptoxanthin compositions and methods herein are also used to treat and/or evaluate their effect on expression of inflammatory markers and/or oxidative stress markers. It is observed that betacryptoxanthin compositions herein and methods of use thereof reduce inflammatory markers.
In one embodiment, betacryptoxanthin compositions herein and methods of use thereof are directed to the improvement of cardiovascular health by management of a healthy lipid profile, reduction in body fat, visceral fat, and free fatty acid levels in the body.
In one embodiment, betacryptoxanthin compositions herein and methods of use thereof are directed to administering the composition, for example in a dose of 0.001 to 10 mg/kg body weight, for the improvement of physical performance, exercise endurance, lung health, lung capacity and cardio-respiratory fitness and/or to reduce oxidative stress markers in the lung tissue. In some embodiments, dosage ranges can include about 1 mg to 9.0 mg/kg body weight of betacryptoxanthin (BCX) administered to a subject. The dose ranges can also include about 2.0 to 8.0 mg/kg body weight of betacryptoxanthin in a subject, which are evaluated in-vitro and in-vivo for effect of betacryptoxanthin on lung and cardiovascular function as a result of sustained exercise activities, sports performance or normal physical activities.
While the compositions and methods have been described in terms of illustrative embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the compositions and methods herein. The details and
IPO M U H BAI

advantages of which are explained hereunder in greater detail in relation to non-limiting exemplary illustrations.
Examples
Example 1: Process for preparation of Betacryptoxanthin composition
Saponification: Paprika oleoresin was mixed with ethanol in a ratio of about 0.5:1 to 1:3 weight/volume and it was saponified with potassium hydroxide employing the ratio of oleoresin to potassium hydroxide to about 1:0.1 to l:0.5w/w. Mixed tocopherol (l%w/w) was added with respect to oleoresin quantity. The mixture system was heated to about 80-85 °C for about 3 to 5 hours to get saponified mass.
Column separation: Saponified mass was extracted with hexane and the extracted mass was concentrated, which was used as 'hexane extract concentrate' feed for column chromatography. Hexane concentrate was added to silica gel to form slurry and dried to get adsorbed mass on silica. This adsorbed silica was loaded in the column for separation. The column was first eluted with hexane to obtain a carotene fraction; followed by further washing with hexane to get betacarotene fraction. Hexane and acetone combination was used for separation of trans-betacryptoxanthin and the fraction was concentrated to obtain extract comprising at least about 75% by weight trans-beta-cryptoxanthin.
Paprika oleoresin having varying content of BCX is selected for experiment BCX1, BCX2 and BCX 3 as shown in table no. 1. Hexane concentrate column indicates content of BCX obtained after saponification step and the last column indicates content of BCX obtained after column separation. BCX amount was determined by routine HPLC method.

Table 1: Betacryptoxanthin (BCX) preparation steps and active contents analysis

Experiment Input
Paprika
Oleoresin BCX content in Hexane concentrate BCX content in fraction obtained from Column separation
% BCX % Yield % BCX % Yield % BCX % Area of BCX
BCX1 0.56 12.86 3.68 4.54 10.3 73.64
BCX2 0.61 11.44 4.63 4.27 11.27 74.5
BCX3 0.78 12.2 5.05 4.02 14.56 74.79
Example 2 : In vitro study in primary bronchial epithelial cell cultures (PBEC) Lung model
Normal Human Bronchial /Tracheal Epithelial Cells (NHBE, Lonza Walkersville MD) were puchased and cultured in BEGM Bulletkit Media following the supplied protocol (Lonza). Cells were cultured until they were confluent and then treated overnight with betacryptoxanthin composition (BCX). Subsequently, RNA was isolated and cDNA prepared using standard protocols and real-time PCR performed for testing E-Cadherin (ECAD).
Observation: ECAD genes showed a significant change in gene expression upon treatment with BCX. The regulation of ECAD is of note since this protein contributes to the structural and immunological function of airway epithelium through the regulation of epithelial junctions, proliferation, differentiation, and production of growth factors and proinflammatory mediators. The dose-response relationship for induction of ECAD was atypical with a peak at 4.2 ug/mL that then fell to control levels at 12.5 ug/mL ( See Fig. 1)

Example 3: Effect of BCX on Pulmonary Structural and Functional Changes on Skeletal Muscle against Damage induced by Exhaustive Exercise in Rats
Animals and exercise protocol
8-10 male Wistar rats per treatment arm (age: 8 week, weight: 180 ± 20 g) were housed in a controlled environment with a 12:12-h light-dark cycle at 22°C and provided with rat chow and water ad libitum. All experiments were conducted under the National Institutes of Health's Guidelines for the Care and Use of Laboratory Animals and approved by the Ethics Committee of the Veterinary Control Institute. Following a 7-day acclimatization period, rats of both the control and exercise groups were divided into groups by matched body weight. Animals were randomly divided into the following groups and all treatments were administered daily as an oral supplement per d for 8 weeks.

Group I, N=7/arm Group II, N=7/arm
Control Exercise
Control+BCX
2.5 mg/kg body weight Exercise+ BCX
2.5 mg/kg body weight
The exercise protocols were performed on a motor-driven rodent treadmill (MAY-TME, Commat Limited, Ankara, Turkey). The treadmill equipped with an electric shock grid on the rear barrier to provide exercise motivation to the animals. All exercise tests were performed during the same time period of the day to minimise diurnal effects. The animals in the chronic exercise groups were habituated by treadmill exercise over a 5-d period such as: 1st day 10 m/min, 10 min; 2nd day 20 m/min; 10 min, 3rd day 25 m/min, 10 min; 4th day 25 m/min, 20 min and 5th day 25 m/min, 30 min. Thereafter, the animals exercised at 25 m/min, 45 min/d, 5 d per week for 8 weeks (Liu et al. 2000.) To minimise diurnal effects, all animals were exercised at the same time (09.00-12.00 hours). Sample collection
The rats were killed 24 h after the last exercise in the chronic exercise group (to wean the effects of acute exercise) by cardiac puncture. Within 1 min, blood samples were transferred into EDTA-coated tubes and plasma was separated by centrifugation at 1750 g for 10 min at b48C.

Plasma samples were stored at -80°C until the time of analysis. Lung and Muscle samples were
collected and frozen at -80°C for further analyses.
Morphological study
The harvested lobes of lung were fixed in 4% formaldehyde solution for 72 hours.
Histopathologic examination of the samples were performed in all groups. Briefly, the lung
tissues from groups were embedded in paraffin and cut into a 4-um section. The lung sections
were stained with hematoxylin and eosin (H&E) solution applied to a glass slide and
photographed under a light microscope (Olympus, Japan) at a magnification of 400 for
morphological analysis.
Mean linear intercept (MLI) and mean alveolar number (MAN) were examined on two glass
slides of each group in the same size of the field of view. Using light microscopy, MLI was
determined for each region was studied on an overlay consisting of horizontal and vertical lines.
All intercepts with alveolar septal number (ASN) were counted at the intersection point of the
two lines in the central field of the view under microscope. The total length (L) of all the lines
together was divided by the number of intercepts gives the mean linear intercept for the region
studied. A formula is shown as MLI = L/ASN, which is used to estimate an average diameter of
a single alveolus in size.
MAN determined according to alveolar number (AN) in each field of view and a square area
(SA) of the field. A formula is shown as MAN = AN/SA (mm ), which is an indicator for density
of alveoli.
Laboratoary analyses
Plasma was used for the determination of glucose, lipid profile, Cortisol , serotonin, testosterone,
creatine kinase activity (CK), aspartate transaminase (AST), alanine transaminase (ALT), with
an automatic analyser (Olympus). The serum Lungs and muscle malondialdehyde (MDA) levels
were measured by HPLC (Shimadzu). The total superoxide dismutase (SOD), catalase (CAT)
and glutathione peroxidase (GPx) were measured using a commercially available assay kit
(Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer's instructions. All
proteins (NF-kB, Nrf2, HO-1) for pathways were analyzed by Western blot methods in muscle
samples.
Histological analysis

Samples of skeletal muscle (vastus lateralis) were collected from each rat in each experimental
condition and fixed with a solution of 2% glutaraldehyde in phosphate buffer at 4°C for 2 h.
Samples were treated with phosphate buffer and dehydrated in a graded series of ethanol and
embedded in Epon 812 resin (Fluka, Sigma-Aldrich). From each sample, sections of 500 nm
were obtained with ultramicrotome and subsequently stained with a solution of 1% toluidine blue
buffered with borate. They finally observed under light microscopy, and images recorded by
software.
Statistical Analyses
Data is presented as mean ± SEM. Sample size was calculated based on a power of 85% and a p
value of 0.05. Given that assumption, a sample size of seven per treatment will be calculated.
Data analysis was done between control vs exercise vs control + product X vs exercise +product
X. The data was analyzed using the GLM procedure of SAS (SAS Institute: SAS User's Guide:
Statistics). The treatments compared between control vs exercise vs control + product X vs
exercise + product X using ANOVA and student's unpaired t test; P < 0.05 was considered
statistically significant.
Results : Total cholesterol and Triglycerides were significantly decreased in Exercise treated rats
with BCX. The compositions also decreased muscle oxidative stress and improved muscle
antioxidant enzymes, in the exercising rat models.
Significant reduction in muscle oxidative stress MDA, decreased lactate and increased SOD and
GPx levels were observed in exercise treated rats with BCX and also in control rats treated with
BCX compared with control diet fed rats ( See Fig. 3)
Significant reduction in lung oxidative stress MDA, lung SOD and GPx levels were observed in
exercise treated rats with BCX and also in control rats treated with BCX compared with control
diet fed rats ( See Fig.4)
BCX compositions were found to manage lipid profile, reduce muscle oxidative stress and improve muscle antioxidant enzymes, thus reducing oxidative stress in exercising subjects, along with managing cardiovascular disease risk factors such as body lipids. The compositions also reduced lung oxidative stress and muscle oxidative stress by managing muscle and lung antioxidant enzymes. Betacryptoxanthin compositions support healthy lung and cardiovascular

function during such physical activities such as exercise, sports or related strenuous activities. The compositions attenuate muscle fatigue by reducing lactate content and enhance cardio-respiratory fitness for physical performance and exercise endurance and maintain healthy lung function.

Claims:
1. Betacryptoxanthin composition comprising extract enriched with trans-betacryptoxanthin, prepared using cost and time effective process, which exhibits activity including improving lung health, physical performance and cardio-respiratory fitness, when administered to exercising subjects in effective amounts.
2. Betacryptoxanthin composition of claim 1, which is comprised of at least 75% by weight of trans-betacryptoxanthin, prepared by industrially viable cost effective process of saponification and column separation.
3. Betacryptoxanthin composition of claim 1, which is administered in effective amounts of about 0.001 to 10 mg/kg body weight to exercising subjects for improving lung health, physical performance and cardio-respiratory fitness.
4. Betacrytoxanthin composition of claim 1, which improves lung health through reduction in oxidative stress and enhancement of antioxidant enzymes, when administered to exercising subjects in effective amounts.
5. Betacryptoxanthin composition of claim 1, which improves physical performance through enhancement of exercise endurance, reduction in oxidative muscle stress, reduction in muscle lactate and improvement in antioxidant muscle enzymes, when administered to exercising subjects in effective amounts.
6. Betacryptoxanthin composition of claim 1, which improves cardio-respiratory fitness by reducing oxidative stress markers and supporting healthy cardiovascular, muscle and lung function, when administered to exercising subjects in effective amounts.
7. Process for preparation of betacryptoxanthin composition comprising trans-betacryptoxanthin extract, which is cost and time effective and is comprised of;
i. mixing capsicum oleoresin with aliphatic alcohol in a ratio of about 1:0.25 to 1:5 ; ii. saponifying the oleoresin with potassium hydroxide, wherein the ratio of oleoresin to potassium hydroxide is about 1: 0.1 to 1: 1 w/w and adding mixed tocopherol to the
mixture

iii. applying heat to the oleoresin to reflux at about 80-85 °C for about 3 to 5 hours to get
saponified mass. iv. extracting saponified mass with non polar solvent and concentrating for feeding to
column chromatography. v. washing the column with mixture of polar and non-polar solvent and concentrating the washings to obtain an extract composition comprising at least 75% by weight of trans-beta- cryptoxanthin.
8. The process of claim 7, wherein aliphatic alcohol used is selected from the group of
ethanol, methanol, isopropyl alcohol, butanol and the like, either alone or in combination
thereof.
9. The process of claim 7, wherein polar solvent used is selected from the group of acetone, ethyl acetate, acetonitrile, ether, alcohols, water and the like, either alone or in combination thereof; while non polar solvent used is selected from the group of pentane, hexane, cyclohexane, benzene, toluene, chloroform, diethyl ether and the like or mixtures thereof.
10. The process of claim 7, wherein composition rich in trans-betacryptoxanthin is prepared using saponification and column separation by cost and time effective process, which can be administered to the exercising subjects in effective amounts for improving lung health, physical performance and cardio-respiratory fitness.