FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
AND
The Patent Rules, 2003
COMPLETE  SPECIFICATION
(See section 10; rule 13)
BETA-CRYPTOXANTH1N FROM PLANT SOURCE AND A PROCESS FOR ITS
PREPARATION.
OMNIACTIVE HEALTH TECHNOLOGIES LTD., an Indian
Company, registered under the Indian Companies Act, 1956 having its
registered office located at Rajan House, Appasaheb Marathe Marg,
Prabhadevi, Maharashtra, India 400025
The following specification particularly describes the invention and the manner in
which it is to be performed

Field of the invention:
The present invention relates to the natural beta-cryptoxanthin concentrates of high purity and a process for its preparation. More particularly, the present invention provides beta-cryptoxanthin concentrates containing 10-80% by weight total xanthophylls (total carotenoids) of which the trans-beta-cryptoxanthin content is 75-98% by weight and the remaining being zeaxanthin, trans-capsanthin, beta-carotene and trace amounts of other carotenoids. The concentrates are particularly useful as dietary supplements for nutrition and health promoting benefits.
The invention also provides a process for the preparation of the beta-cryptoxanthin concentrate from plant oleoresin, especially from Capsicum oleoresin. The process includes the steps of admixing the oleoresin with alcohol solvents, saponifying the xanthophyll esters, washing and purifying by eluting the crude xanthophyll viscous concentrate on a silica gel column and purifying further by washings to obtain high purity trans-beta cryptoxanthin enriched concentrate crystals.
Background of the invention
Carotenoids represent one of the most widespread groups of naturally occurring fat-soluble pigments imparting yellow, red and orange color in plants as well as in animals. These absorb light in 400-500 nm region of the visible spectrum and have a common chemical feature, a poly-isoprenoid structure, a long conjugated chain of double bonds in the central portion of the molecule and near symmetry around the central double bond. The basic structure can be modified in a number of ways such as cyclization of the end groups and introduction of oxygen functions (O-H, C=0) to yield a large family of more than 600 compounds, exclusive of cis- and trans-isomers. Mammalian species do not synthesize carotenoids and therefore these have

to be obtained from dietary sources such as fruits and vegetables and or dietary supplements.
Carotenoids are classified into hydrocarbon carotenoids, with lycopene and beta-carotene being the important members, and oxycarotenoids (xanthophylls), to which belongs mono-hydroxylated beta-cryptoxanthin while lutein, zeaxanthin and astaxanthin are dihydroxylated. In the biosynthetic pathway of enzymatic hydroxylation of symmetrical beta-carotene (beta,beta-carotene) leads to the formation of beta-cryptoxanthin (beta,beta-caroten-3-ol) whereas the same reaction starting from asymmetric alpha-carotene (beta, epsilon-carotene) gives rise to two reaction products, namely: alpha-cryptoxanthin (beta,epsilon-caroten-3"-ol) and zeinoxanthin (beta, epsilon-caroten-3-ol). Often, because of the spectral structural similarities between alpha-cryptoxanthin and zeinoxanthin, their identification is difficult and misleading unless chemical reactions are carried out such as methylation or base catalyzed isomerization. Further, the chemical structures of these are also given wrongly in many publications.

Fig.l Chemical structures of Alpha-cryptoxanthin (beta,epsilon-caroteno-3"-ol), Beta-cryptoxanthin(beta,beta-caroten-3-ol and Zeinoxanthin(beta,epsilon-caroten-3-ol).

Among the 20 of the carotenoids detected in mammalian plasma and tissues, beta-cryptoxanthin is one of the major carotenoids along with lutein, zeaxanthin, beta-carotene and lycopene accounting nearly 90%. (J. G. Bieri, E. D. Brown and J. C. Smith, Determination of individual carotenoids in human plasma by HPLC, J. Liq. Chromatogr. 8, 473-484, 1985). Beta-cryptoxanthin is a provitamin A playing an important role in the diet, finally converting in the human body into an active form of vitamin A (retinol), a nutrient important for vision, immune function, skin and bone health. Beta-cryptoxanthin has about one half the vitamin A activity of the major vitamin precursor, beta-carotene. In addition, beta-cryptoxanthin acts as an antioxidant in the body. Wingerath et. al. (1995) studied the uptake of beta-cryptoxanthin after ingestion of tangerine juice concentrate rich in beta-cryptoxanthin esters. Beta-cryptoxanthin in substantial amounts were detected both in human chylomicrons and in serum (T, Wingerath, W. Stahl and H. Sies, Beta-cryptoxanthin selectively increases in human chylomicrons upon ingestion of tangerine concentrate rich in beta-cryptoxanthin, Arch. Biochem and Biophys., 324, 385-390,1995). The bioavailability of the carotenoids from paprika oleoresin has shown the presence of beta-carotene, and beta-cryptoxanthin in higher amounts in chylomicrons compared to zeaxanthin among the volunteers (A. Perez-Galvez, H. D. Martin, H. Sies and W. Stahl, Incorporation of carotenoids from paprika oleoresin into human chylomicrons, J. Nutrition, 89, 787-793, 2003). Burri et al. (2011) have reported that beta-cryptoxanthin seems to be 7-fold greater bio-available than beta-carotene under similar conditions. Therefore, there is a strong reason to support that beta-cryptoxanthin can be a valuable and potential source of vitamin A, which needs further study and confirmation (B. J. Burri, S. Jasmine, T. Chang and T. R. Neidlinger, Beta-cryptoxanthin- and alpha-carotene-rich foods have greater apparent bioavailability than beta-carotene-rich foods in Western diets, Brit. J. Nutrition, 105, 212-219, 2011). Unlike other carotenoids, beta-cryptoxanthin is not found in most fruits or vegetables but present in certain specific foods such as capsicum species,

citrus fruits, mango, papaya, pumpkin in small amounts, 10-20 mg/l00g. Mostly, beta-cryptoxanthin is present in ester form in paprika and mandarin fruits. Breithaupt and Bamedi (2001) have analyzed a large number of fruits and vegetables and reported the beta-cryptoxanthin ester concentration levels. The highest ester concentrations were found in red chilies (17.1 mg/lOOg), tangerine and oranges (Carotenoid esters in vegetables and fruits: A screening with emphasis on beta-cryptoxanthin esters, J.Agric.49, 2064-2070, 2001). Later, Breithaupt et al., in a randomized, single- blind crossover study using a single dose of esterified or non-esterified beta-cryptoxanthin in equal amounts found no difference in the resulting plasma response among 12 volunteers suggesting a comparable bioavailability. (D. E. Breithaupt, P. Weller, M. Wolters and A. Hahn, Plasma response to a single dose of dietary Beta-cryptoxanthin ester from papaya, Carica papaya, or non-esterified beta-cryptoxanthin in adult human subjects: a comparative study, Brit. J. Nutr. 90, 795-801, 2003). Takayanagi and Mukai (2009) have developed an improved bioavailable composition of beta-cryptoxanthin derived from citrus unshiu Marc by using enzyme process and in combination with dietary fiber (US Pat. Appln. 20090258111, Highly bioavailable oral administration composition of cryptoxanthin).
In most of the Western countries and Japan, the dietary source of beta-cryptoxanthin comes from citrus fruits and their products and consequently the plasma beta-cryptoxanthin levels can be considered as a good index of the amount of fruit consumption. Similarly, fruits such as papaya widely consumed in many tropical countries (Latin America), high correlation of plasma beta-cryptoxanthin have been reported (M. S. Irwig, A. El-Sohemy, A. Baylin, N. Rifai and H. Campos, Frequent intake of tropical fruits that are rich in beta-cryptoxanthin is associated with higher plasma beta-cryptoxanthin concentrations in Costa Rican adolescents, J. Nutr. 132, 3161-3167,2002).

The water soluble extract of marine algae extract, specifically Sargassumhorneri showed anabolic effect on bone calcification in the femoral-metaphysical tissue of young and old rats in vivo and in vitro, suggesting its role in the prevention of osteoporosis (Yamaguchi et al., Effect of marine algae extract on bone calcification in the femoral-metaphysical tissues of rats: Anabolic effect of Sargassumhorneri, J. Health Sci.,47, 533-538, 2001; Uchiyama and Yamaguchi, Anabolic effect of marine alga Sargassumhorneri, Effect on bone components in the femoral-diaphyseal and -metaphyseal tissues of young and old rats in vivo, J. Health Sci. 48, 325-330, 2002). Later, in a study of the effects of the various carotenoids, beta-cryptoxanthin showed significant increase in calcium content and alkaline phosphatase activity in the . femoral-diaphyseal (cortical bone) and femoral-metaphyseal (trabecular bone) tissues, suggesting that the beta-cryptoxanthin possesses a unique anabolic effect on bone calcification in vitro (Yamaguchi and Uchiyama, Effect of carotenoid on calcium content and alkaline phosphatase activity in rat femoral tissues in vitro: The unique anabolic effect of beta-cryptoxanthin, Biol. Pharma. Bull. 26, 1188-1191, 2003). In another study, the DNA content in bone tissues was found to increase significantly and showed inhibitory effect on bone-resorbing factors-induced bone resorption in rat bone tissues in vitro (Uchiyama et al., Anabolic effect of beta-cryptoxanthin on bone components in the femoral tissues of aged rats in vivo and in vitro, J. Health Sci. 50, 491-496, 2004; Yamaguchi and Uchiyama, Beta-cryptoxanthin stimulates bone formation and inhibits bone resorption in tissue culture in vitro, Mol. Cell. Biochem. 258, 137-144, 2004).
Thus, beta-cryptoxanthin has a potential role and effect in maintaining bone health and preventing osteoporosis. The various studies carried out by Yamaguchi et al. (2004, 2005, 2006 and 2008), have shown that regular daily intake of Satsuma mandarin juice (Citrus unshiu) and or supplemented with beta-cryptoxanthin (3-6 mg or more/day) has beneficial effects such as preventive effect on bone loss over age,

stimulatory effect on bone formation and an inhibitory effect on bone re-absorption in normal and healthy individuals and in menopausal women (Prolonged intake of juice, Citrus unshiu, reinforced with beta-cryptoxanthin has an effect on circulating bone biomarkers in normal individuals, J. Health Sci.,50,619-624, 2004; Relationship between serum beta-cryptoxanthin and circulating bone metabolic markers in healthy individuals with the intake of juice (Citrus unshiu) containing beta-cryptoxanthin. J. Health Sci.,51, 738-743, 2005; Effect of beta-cryptoxanthin on circulating bone biomarkers intake of juice (Citrus unshiu) supplemented with beta-cryptoxanthin has effect in menopausal women, J. Health Sci., 52, 758-768, 2006; Beta-Cryptoxanthin and bone metabolism: The prevention role in osteoporosis, J. Health Sci., 54, 356-369, 2008). Uchiyama and Yamaguchi (2005 & 2006) have shown that oral administration of beta-cryptoxanthin isolated from Satsuma mandarin had a preventive effect in bone loss in streptozotocin (diabetic) and ovariectomized rats in vivo studies (Oral administration of beta-cryptoxanthin prevents bone loss in streptozotocin rats in vivo, Biol. Pharm. Bull. 28, 1766-1769, 2005; Oral administration of beta-cryptoxanthin prevents bone loss in ovariectomized rats, Int. J. Mol. Med. 17, 15-20, 2006). EP Application no. 038060229, Publn. 2007/35 teaches a composition comprising of beta-cryptoxanthin and zinc for promoting osteogenesis, increasing bone mineral content thereby preventing bone diseases such as osteoarthritis. (M. Yamaguchi, Composition for promoting osteogenesis and increasing bone mineral content). US Pat No. 8,148,431B2, Apr.3, 2012 reports confirm that beta-cryptoxanthin has osteogenesis promoting effect, a bone-resorption inhibiting effect and therapeutic effect on bone diseases (M. Yamaguchi, Osteogenesis promoter containing beta-cryptoxanthin as the active ingredient).
Generally, the different bone and joint disorders such as osteoporosis, osteoarthritis and rheumatoid arthritis are common among the elderly people causing a major

health problem resulting in bone fracture. With ageing there is a decrease in bone mass and an increase in bone resorption due to various dietary reasons. In recent publications, Yamaguchi has reviewed outlining the recent advances concerning the role of beta-cryptoxanthin in the regulation of bone homeostasis and in the prevention of osteoporosis, especially the cellular and molecular mechanisms by which beta-cryptoxanthin stimulates osteoblastic bone formation and inhibits osteoclastic bone resorption (Beta-cryptoxanthin and bone metabolism: The preventive role in osteoporosis. J. Health Sci., 54, 356-369; 2008; Role of carotenoid beta-cryptoxanthin in bone homeostasis, J.Biomed.Sci.,19.1-13, 2012).
High levels of dietary intake of beta-cryptoxanthin were found to be associated with reduced risk of lung cancer among the smoking population, thereby suggesting the xanthophylls as a chemo-preventive agent for lung cancer (Yuan et al., Dietary cryptoxanlhin and reduced risk of lung cancer: the Singapore Chinese health study, Cancer Epidemiol. Biomarkers Prev. 12, 890-898, 2003). Craft et al. (2004) found beta-cryptoxanthin in the frontal cortex of brain which is considered to be associated with Alzheimer"s disease, however no exact role has been explained (N. E. Craft, T. B. Haitema, K. M. Garnett, K. A. Fitch and C. K. Dorey, Carotenoids, tocopherol and retinol concentrations in elderly human brain, J. Nutr. Health Ageing, 8, 156-162, 2004). A high plasma level of beta-cryptoxanthin has been linked to protective effect against rheumatoid arthritis. Pattison et al. attributed the incidence of inflammatory arthritis among 88 subjects to the low level of dietary intake of beta-cryptoxanthin (D. J. Pattison, D. P. Symmons, M. Lunt. A. Welch, S. A. Bingham, N. E. Day and A. J. Silman, Dietary beta-cryptoxanthin and inflammatory polyarthritis: results from a population based prospective study, Am. J. Clin. Nutri. 82,451-455, 2005). US Pat. Application 2008/0070980, describes a method of use of beta-cryptoxanthin and its esters in the manufacture of a composition for providing an increased protein formation and or prevention of loss of proteins in human and

animals, resulting in enhancing performance in sports and workout activities (E. Anne. G. Resina, W. Karin and W. Adrian, Use of beta-cryptoxanthin, Mar. 20, 2008). In a recent US Pat. Application: Method of improving cardiovascular health, there is a finding that a nutritional supplement containing purified beta-cryptoxanthin (0.1 to 20 mg/day) is effective in lowering high blood pressure and also in maintaining a healthy blood pressure and cardiovascular health. However, the beta-cryptoxanthin used is purified by analytical HPLC from a mixture of alpha-cryptoxanthin. anhydroluteins, zeaxanthin, other impurities (US Pat. Application 20120053247 publication 1st March 2012, H. Showalter, Z. Defretas and L, Mortensen).
Prior Art
In view of the increasing research interest in the various health benefits of beta-cryptoxanthin as can be seen above, there have been several approaches to commercially produce this carotenoid from (1) natural sources as extracts rich in beta-cryptoxanthin, (2) biotechnology routes and (3) by total- and semi-synthesis. In fact, the various clinical studies have used either synthetic beta-cryptoxanthin or natural fruit extracts rich in beta-cryptoxanthin.
(1) Natural source: Yamaguchi (2006) prepared a high purity beta-cryptoxanthin from Satsuma orange which involved the steps of extracting the pigment hydrolyzation followed by silica gel column chromatography. The beta-cryptoxanthin fraction was further purified by octadecyl silicate silica to obtain 95% purity (M.Yamaguchi. Osteogenesis promoter containing beta-cryptoxanthin as an active ingredient, US Pat. Appin. 2006 0106115, Pub. 18lh May 2006). Takahashi and Inada (2007) have prepared Persimmon extract from pulp/juice and skin by solvent extraction and hydrolysis to liberate beta-cryptoxanthin (free). The extracts

prepared from pulp and skin showed beta-cryptoxanthin content as lmg/lOOg and 8mg/100g respectively and are useful for applications in functional foods (H. Takahashi and Y. Inada, US Pat. Appln. 20070116818 A, Publn. 24th May 2007, Extract containing beta-cryptoxanthin from Persimmon fruit). Shirakura et. al (2008) and Takayanagi and Mukai (2008) have developed commercial processes for enzyme treated Satsuma mandarin (EPSM) and emulsified mandarin extract (EME) containing 0.2 and 0.05% beta-cryptoxanthin respectively. They showed that the extracts reduced visceral fat and plasma glucose in a human comparative trial designed as placebo-controlled double blind study (Y. Shirakura, K. Takayanagi and K. Mukai, Reducing effect of beta-cryptoxanthin extracted from Satsuma mandarin on human body fat, Abstract, page 161; K. Takayanang and K. Mukai, Abstract: Beta-cryptoxanthin and Satsuma mandarin: Industrial production and health promoting benefits, page, 73, Carotenoid Science, 12. June , 2008, Abstracts of the papers presented at the 15th International Symposium on Carotenoids, Okinawa, Japan, 22nd- 27lh June, 2008).
(2) Biotechnology approach: Serrato-Joya et al. (2006) have described beta-cryptoxanthin in a laboratory scale batch production using Flavobacterium Lutescens 1TC B 008 (O. Serrato-Joya, H. Jimenez-Islas. E. Botello-Alvarez, R. Nicomartinez, and J. L. Navarrete-Bolans. Process of beta-cryptoxanthin, a provitamin A precursor by Flavobacterium Lutescens, J. Food Sci. 7LE314-E319, 2006). Louie and Fuerst disclosed a method for preparing beta-cryptoxanthin from a microorganism transformant with the beta-carotene hydroxylase gene from Arabidopsis thaliana by culturing the transformant and recovering beta-cryptoxanthin (US Pat. Appln. 20080124755, Publn. 29th May 2008, Biosynthesis of beta-cryptoxanthin in microbial hosts using Arabidopsis thaliana beta-carotene hydroxylase gene). Again, Louie and Fuerst (2009) described a method of beta-cryptoxanthin production by the use of lycopene beta-mo nocyclase and converting lycopene to beta-cryptoxanthin

through gam ma-carotene and 3-hydroxy-gamma-carotene (M. Y. Louie and E. J. Fuerst, US Pat. Appln. 2009093015, Publn. 9th April 2009, Beta-cryptoxanthin production using a novel lycopene beta-monocyclase gene). Hoshino et al. have described a process for producing zeaxanthin and beta-cryptoxanthin which comprises cultivating a recombinant microorganism expressing beta-carotene hydroxylase gene (Phaffia) under aerobic conditions in aqueous nutrient media and isolating the resulting carotenoids from the cells of recombinant microorganism or from the broth (T. Hoshino, K. Ojima and Y. Setoguchi. US. Pat. Appln. No. 20060121557 Publn. June 8th 2006, Process for producing zeaxanthin and beta-cryptoxanthin).
Unfortunately, there appears to be lack of microorganisms that can naturally produce beta-cryptoxanthin as the final product and hence fermentation technology is not feasible for commercial production. Further, in the fermentation processes the carotenoids produced are low in concentrations and as complex mixtures of products, including various added ingredients. Extensive purification steps require large amounts of solvents and the generation of considerable amounts of by-products.
(3) Synthesis: Khachik and coworkers have developed three processes for the preparation of beta-cryptoxanthin. Two methods employ lutein or lutein esters as the starting material and in the presence of acid it is converted into three forms of anhydroluteins: 3-hydroxy-3",4"-didehydro-beta-gamma-carotene (1), 3-hydroxy-2",3"-didehydro-beta-epsilon-carotene (II) and 3-hydroxy-3",-4"-didehydro-beta-beta-carotene (III). The mixture of anhydroluteins rich in anhydrolutein (III) was subjected to ionic hydrogenation in presence of an acid and chlorinated solvent to produce alpha- and beta-cryptoxanthin. The purified product showed total carotenoids 85%, of which beta-cryptoxanthin was 55 to 61% and alpha-cryptoxanthin 18 to 30% and the remaining being 3 to 8% R,R-zeaxanthin and un-

reacted an hydro luteins (F. Khachik, US Pat. 7115,786, B2, Oct. 3, 2006, Method for production of beta-cryptoxanthin and alpha-cryptoxanthin from commercially available lutein; F. Khachik, A. N. Chang, A. Gana and E. Mazzola, Partial synthesis of (3R.6"R)-alpha-cryptoxanthin and (3R)-beta-cryptoxanthin from (3R,3"R,6"R)-lutein (J.Nat.Products70, 220-226, 2007)
In the second method, the mixture of anhydroluteins were converted to alpha- and beta-cryptoxanthin by catalytic hydrogenation using platinum supported on alumina. The final product was reddish crystals with total carotenoids 60% and the HPLC composition showing beta-cryptoxanthin and alpha-cryptoxanthin in the ratio 3:1, 7:3. or 5: land the presence of un-reacted anhydroluteins I and II and R.R-zeaxanthin. In the third method, the invention relates to a process for the synthesis of optically active 3-hydroxy-beta-ionone and transforming to beta-cryptoxanthin and using Wittig coupling reactions. The synthetic approach involves multiple step reactions and purifications leading to a mixture of beta-cryptoxanthin and R,R-zeaxanthin (F. Khachik and A. N. Chang, US Pat, Appln. No.12/48473 Publn. Date Dec.l7th 2009, Process for synthesis of 3(S)- and (3R)-3-hydroxy-beta-ionone and their transformation to zeaxanthin and beta-cryptoxanthin); Synthesis of (3S)- and (3R)-hydroxy-beta-ionone and their transformation into (3S)- and (3R)-beta-cryptoxanthin, Synthesis. 3,509-516,2011)
As explained above, natural beta-cryptoxanthin is available in enriched form in fruit drinks such as tangerine. Satsuma orange and persimmon. The biotechnological route for beta-cryptoxanthin is preliminary and limited to laboratory scale and with poor yields. The synthetic approach gives a mixture of beta-cryptoxanthin and considerable amount of impurities such as alpha-cryptoxanthin, which is most likely zeinoxanthin. a non-provitamin A, along with un-reacted anhydroluteins and

zeaxanthin. The separation of beta-cryptoxanthin is complex, involves multiple steps and is not commercially feasible.
Based on the chemical structure of anhydrolutein II (3-Hydroxy-2",3"-didehydro-beta.epsilon-carotene), one would expect hydrogenation at 2",3"- double bond to form zeinoxanthin (beta,epsilon-caroten-3-ol). In addition, one would expect no conversion of zeinoxanthin to beta-cryptoxanthin by alkali isomerization due to the absence of allylic hydroxyl group. This has been confirmed by phenyl carbinol alkali catalyzed reaction at high temperature (110 degree C) of beta-cryptoxanthin containing zeinoxanthin 10% (by HPLC), where after the resultant product showed no change in HPLC profile compared to the control. In fact, the base catalyzed reaction of so called alpha-cryptoxanthin failed to arrive at beta-cryptoxanthin (F. Khachik. A. N. Chang, A. Gana and E. Mazzola, Partial synthesis of 3(R,6"R)-alpha-cryptoxanthin and (3R)-beta-cryptoxanthin from (3R.3R",6"R)-Iutein (J.Nat.Prod.70,220-226,2007).
Bone mass decreases with increasing age. This decrease is due to increased bone resorption and reduced bone formation. The decrease in bone mass induces osteoporosis (Masayoshi Yamaguchi, Satoshi Uchiyama. Kaori Ishiyama, and Ken Hashimoto. Oral Administration in Combination with Zinc Enhances beta -Cryptoxanthin-lnduced Anabolic Effects on Bone Components in the Femoral Tissues of Rats in Vivo. Biol. Pharm. Bull. 29(2) 371—374 (2006)). Bone homeostasis is maintained through a balance between osteoblastic bone formation and osteoclastic bone resorption (Masayoshi Yamaguchi. Role Of Carotenoid Beta-Cryptoxanthin In Bone Homeostasis. Journal of Biomedical Science 2012, 19-36). Production of estrogen decreases in menopause causing imbalance in metabolism (Citrus unshiu extract. Health Ingredient for prevention of osteoporosis health ingredient for whitening and aesthetic ingredient for cosmetic.  Product monograph,

Ver.3.OHS by Oryza Oil & Fat Chemical Co Ltd.). p-cryptoxanthin has been found to have a potential anabolic effect on bone due to stimulating osteoblastic bone formation and inhibiting osteoclastic bone resorption. Oral administration of β -cryptoxanthin may have a preventive effect on bone loss with increasing age and on Osteoporosis. Role of beta-cryptoxanthin obtained from Capsicum source in strengthening bone and inhibiting bone resorption is demonstrated in the study with ovariectomized female wistar rats as described in Example 4.
From above, it is clear that presently there is lack of availability of beta-cryptoxanthin of high purity and also in appreciable amounts as a major ingredient derived from natural source for use as nutritional ingredient and in dietary supplements. The reason for this situation is low concentrations of beta-cryptoxanthin in natural sources (particularly fruits and vegetables) preventing commercialization of this molecule by traditional solvent based extraction procedures. For this purpose. Capsicum species may be considered as a first choice since it has reasonably a high content of beta-cryptoxanthin though there are many other promising options of beta-cryptoxanthin-containing materials. Commercial availability of such a material of high purity of beta-cryptoxanthin would help in establishing the potential health benefits of beta-cryptoxanthin in the various clinical trials and also as dietary supplements.
Objectives of the present invention
The main objective of the present invention is to provide natural beta-cryptoxanthin concentrates of high purity and a process for its preparation from the source material and which is safe for human consumption and useful for nutrition and bone health care.

Another objective of the present invention is to provide a process for the isolation of beta-cryptoxanthin crystals containing approximately 80% by weight of total xanthophylls (total carotenoids) in free form out of which the trans-beta-cryptoxanthin content is at least 98.5% by weight, the remaining being trace amounts of zeaxanthin, trans-capsanthin. beta-carotene and other carotenoids derived from oleoresin and extracts of plant materials such as Capsicum sources.
Yet another objective of the present invention is to provide a process for the preparation of beta-cryptoxanthin crystals containing at least 40% by weight of total carotenoids out which the trans-beta-cryptoxanthin is at least 90% by weight, the remaining being trace amounts of zeaxanthin, trans-capsanthin, beta-carotene and other carotenoids derived from Capsicum source.
Still another objective of the present invention is to provide a process for the preparation of beta-cryptoxanthin crystals containing at least 10% by weight of total carotenoids out of which the trans-beta-cryptoxanthin is at least 75% by weight, the remaining being zeaxanthin, trans-capsanthin. beta-carotene and trace amounts of other carotenoids derived from Capsicum source.
Yet another objective of the present invention is to provide a process for the preparation of beta-cryptoxanthin crystals containing total carotenoids 10 to 80% by weight out of which the trans-beta-cryptoxanthin content is in the range 75 to 98%o by weight, the rest being zeaxanthin, trans-capsanthin, beta-carotene and trace amounts of other carotenoids derived from a starting material like saponified Capsicum extract.

Still another objective of the present invention is to obtain residual solvent free beta-cryptoxanthin crystals, in which trans-beta-cryptoxanthin forms the major ingredient in the total carotenoids.
Yet another objective is to provide a simple and convenient process for the preparation of high purity beta-cryptoxanthin from capsicum oleoresin or saponified capsicum extract.
According to a feature of the present invention, there is a provision to recover carotene hydrocarbons fraction- rich in beta-carotene, from the process described herein.
Yet another feature of the present invention is to recover xanthophylls fraction
comprising mainly zeaxanthin and trans-capsanthin which has high antioxidant property.
The above objectives have been achieved by the present invention based on our following findings that
(a) the saponification process of esterified xanthophylls in Capsicum extract results in free xanthophylls, which is purified by washing with acidified water, followed by drying to obtain red colored carotenoid mass.
(b) by further treating the red mass with non polar solvent under stirring, followed
by filtration and concentration red viscous mass is obtained.
(c) by subjecting the red viscous mass through column chromatography using silica
gel and elution using non polar solvent removes the yellow colored beta-carotene.

(d) further eluting the column with non polar solvent containing 2% polar solvent and the eluant after concentration gives red viscous concentrate showing 10% total carotenoids by weight and 75% by weight trans-beta-cryptoxanthin.
(e) further treating the above red viscous concentrate with ethanol under stirring, followed by cooling(10 degree C) and filtering results in semi-purified red crystals mass showing total xanthophylls 40% by weight and trans-beta-cryptoxanthin 98% by weight.
f) by further washing the red crystals mass with hexane containing 20% ethyl acetate and cooling to -10 degree C and filtering gives high purity red crystalline material showing 80% total xanthophylls by weight and contains 98.5%) by weight trans-beta-cryptoxanthin.
Summary of the invention
The product and the process of the present invention is described herein below which is illustrative as shown in the examples and should not be construed to limit the scope of the present invention in any manner whatsoever.
Accordingly, the present invention provides a beta-cryptoxanthin concentrate, which contains 10-80% by weight total xanthophylls out of which 75-98% by weight being trans-beta-cryptoxanthin, the remaining being zeaxanthin. trans-capsanthin, beta-carotene and trace amounts of other carotenoids, derived from oleoresin or extract of plant material and which is useful for nutrition and health care.

The plant extract is derived from the group comprising of fruits and vegetables.
preferably capsicums.
The trans-beta-cryptoxanthin enriched concentrate is in the form selected from the group of beadlets, microencapsulated powders, oil suspensions, liquid dispersions, capsules, pellets, ointments, soft gel capsules, tablets, chewable tablets or lotions/liquid preparations.
The present invention also provides a process for the preparation of trans-beta-cryptoxanthin enriched beta-cryptoxanthin concentrate/crystals which contains 10-80% by weight total xanthophylls out of which 75-98% by weight being trans-beta-cryptoxanthin, the remaining being zeaxanthin, trans-capsanthin. beta-carotene and trace amounts of other carotenoids, suitable for human consumption as nutritional supplements which comprises:
a) Mixing the xanthophyll esters in an oleoresin with an aliphatic alcohol solvent, wherein the ratio of oleoresin to aliphatic alcohol solvent ranges from 1:1 to 1:1.5 weight/volume;
b) Saponifying the xanthophyll esters present in the oleoresin of plant material with an alkali without addition of water, wherein the ratio of oleoresin : alkali ranges from 1 : 0.25 to 1 : 0.5 weight/weight;
c) Applying heat to elevate the temperature up to reflux;
d) Allowing the saponification reaction under agitation for 3 to 5 hr;
e) Evaporating the alcohol followed by addition of water to get diluted resultant mixture, which is maintained under agitation for 1 hr;
f) Neutralizing the mixture with a diluted acid;
g) Separating the water layer from the precipitated xanthophyll mass and washing
the mass with a polar solvent 3 times to remove the soaps and the other polar soluble
materials;

h) Drying the xanthophyll crude mass under vacuum;
i) Washing the crude mass with non polar solvent and the non polar solvent washings
are concentrated to get crude viscous xanthophyll mass;
j) Transferring the viscous mass on a silica gel column and eluting with non polar
solvent to obtain non polar solvent fraction;
k) Distilling off the non polar solvent fraction to get a beta- carotene concentrate;
1) Washing with non polar : polar solvent and concentrating the washings resulting
in beta-cryptoxanthin concentrate;
m) Agitating the concentrate obtained in step (1) with ethanol, followed by cooling
and filtering results in purified beta-cryptoxanthin concentrate;
n) Washing again the purified beta-cryptoxanthin concentrate with a mixture of non
polar : polar solvents and cooling to low temperature for precipitation to obtain high
purity trans-beta-cryptoxanthin enriched beta-cryptoxanthin crystals.
The hydrocarbon solvent used is hexane or mixture of low boiling hydrocarbons. The aliphatic alcohol selected for saponification is ethanol and the alkali used is selected from sodium or potassium hydroxide.
The elevated temperature applied ranges from 80 to 85 degree C.
The acid used for neutralizing the mixture is acetic acid or phosphoric acid. The polar solvent used for washing the precipitated xanthophyll mass is water. The ratio of crude xanthophyll mass to non polar solvent is 1:10 to 1:15 weight/volume.
The silica gel column containing xanthophylls concentrate is eluted with non polar solvent to remove the carotenes to obtain beta-cryptoxanthin concentrate.

Further washing the columns with non polar : polar solvent and concentrating the washings results in beta-cryptoxanthin concentrate comprising 10% by weight total xanthophylls out of which trans-beta-cryptoxanthin content is at least 75% by weight and the remaining being beta-carotene, trans-capsanthin, zeaxanthin and traces of other carotenoids. The purified beta-cryptoxanthin concentrate comprises at least 40% by weight of total xanthophylls out of which trans-beta-cryptoxanthin content is at least 90% by weight and the high purity beta-cryptoxanthin concentrate obtained by washing the purified beta-cryptoxanthin concentrate with a mixture of non-polar : ester solvent and cooling to low temperature for precipitation results in a concentrate which comprises at least 80% by weight of total xanthophylls out of which trans-beta-cryptoxanthin content is at least 98% by weight, the rest being beta-carotene, zeaxanthin. trans-capsanthin and traces of other carotenoids.
The process by-products consist of beta-carotene, trans-capsanthin. zeaxanthin or mixtures thereof.
It may be noted that a novel feature of the present process is the preparation of high purity trans-beta-cryptoxanthin concentrate crystals from a natural source such as capsicum extract, which has not been reported in the prior art.
The following examples are given by the way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.
Example 1
A weighed quantity of 3 00g of Paprika oleoresin containing 7.72% total xanthophylls and a color value of 1.23,515 units (HPLC:- beta-carotene: 15.36%; beta-cryptoxanthin: 10%: zeaxanthin: 7.6% and trans-capsanthin: 31.50%) was mixed with  100 ml ethanol and 25g  potassium hydroxide pellet. The reaction

mixture was heated to a temperature of 80-85 degree. C with stirring. The saponification process was maintained for 3-5 hours at 80-85degree C. with gentle agitation. The reaction mixture was allowed to be cooled. Then ethanol was distilled out from the mass. A measured volume of water (700ml) was added to the reaction mixture and kept for agitation for 1 hour. The solution was neutralized with 25% acetic acid solution. The water layer from the mass was separated and washed with water thrice. The mass was collected and dried under vacuum. The concentrate obtained was 124g with total xanthophylls 3.73% by weight.
The HPLC profile of the saponified mass showed beta-carotene- 22.53%; beta-cryptoxanthin- 12.32%; zeaxanthin-l1% and t-capsanthin- 29.3%.
The concentrate was washed 2 times with 1:10 hexane wt/vol. at room temperature under stirring, filtered and the combined filtrate was concentrated to get a viscous mass. The mass obtained was 72g with total xanthophylls 3.2%. The HPLC profile of the crude xanthophylls mass showed beta-carotene- 39.01 %; beta-cryptoxanthin-21.78%: zeaxanthin- 5.70% and t-capsanthin- 9.86%.
The residue obtained after the filtration process was collected and kept separately. Yield obtained: 22g with 10% total xanthophyll content, and the HPLC profile of the residue was beta-carotene- 0.7%: beta-cryptoxanthin- 3.43%; zeaxanthin-l 5.32% and t-capsanthin- 52.84%.
The hexane concentrate was dissolved in minimum amount of hexane and subjected to column chromatographic separation. The column was packed with 1:5 Silica 100-200 mesh weight/weight. The column was washed with hexane and the separated band was collected. The fraction was concentrated. Yield obtained: 55g with total xanthophyll content 2.3%. (HPLC: beta-cryptoxanthin-99.8%).

Then column was further eluted with hexane: acetone in the ratio of 98:2 and the eluanl was concentrated. This concentrated layer was enriched with beta-cryptoxanthin. Yield of concentrate: 5.2g with 10.26% total xanthophyll content by weight. The HPLC profile of the concentrate was beta-carotene- 1.11%: beta-cryptoxanthin- 75.56%.
Finally, the column was washed with acetone and the washings concentrated to obtain trans-capsanthin enriched residue.
Example 2
A quantity of approximately l00g of Paprika oleoresin containing 6.50% total xanlhophylls and a color value of 1.05.457 units, the HPLC profile of the oleoresin being- beta-carotene-15.73%: beta-cryptoxanthin- 9.07%; zeaxanthin-10.54% and t-capxanthin-31.38%. was mixed with .100 ml ethanol and 25g potassium hydroxide pellet. The reaction mixture was heated to a temperature of 80-85 degree C with stirring. The saponification process was maintained for 3-5 hours at 80-85 degree C. with gentle agitation.
The reaction mixture was allowed to cool. Ethanol was distilled out from the mass. A measured volume of water. 700ml was added to the reaction mixture and kept for agitation for 1 hour. The solution was neutralized with 40% acetic acid solution and the water layer from the mass was separated and washed with water three times. The mass was collected and dried under vacuum. The concentrate obtained was 126g with total xanthophylls content 3.73% by wt.
The HPLC profile of the concentrate was beta-carotene: 16.34%; beta-cryptoxanthin: 9.41%; zeaxanthin: 8.57% and t-capsanthin: 24.35%).

The concentrate was washed 2 times with 1:10 wt/vol hexane at room temperature under stirring, filtered and the combined filtrate was concentrated to get a viscous mass. The mass (hexane concentrate) obtained was 76.1 5g with total xanthophylls 3.26%. The HPLC profile of the hexane concentrate was beta-carotene:- 31.80%; beta-cryptoxanthin:-14.04%; zeaxanthin:- 4.35% and t-capsanthin:- 8.70%.
The residue obtained after the filtration was collected and kept separately. Yield obtained: 16g with 11% total xanthophylls content. The HPLC analysis of the residue showed beta-carotene:-1.22%; beta-cryptoxanthin:- 0.75%; zeaxanthin:-33.29% and t-capsanthin:-29.99%.
The hexane concentrate was dissolved in minimum amount of hexane and subjected to column chromatographic separation. The column was packed with 1:5 Silica 100-200 mesh and eluted with hexane. Separated band was collected and concentrated. Yield obtained: 54.72g with total xanthophylls content 1.08% by wt. The HPLC profile of the hexane concentrate showed beta-carotene- 85.88%).
The column was further eluted with 98:2 hexane: acetone vol/vol. the fraction solution was collected and concentrated. This fraction was enriched with beta-cryptoxanthin. Yield obtained: 4.02g with 9% total xanthophylls content. The HPLC profile of the concentrate showed 76.04% of beta-cryptoxanthin.
Finally the column was washed with acetone.
4.02g of the concentrated fraction, was stirred with 1:2 ethanol wt./vol for 1 hr and chilled for 8 hrs at 10 degree C. filtered and the precipitate was dried under vacuum. Yield obtained: 0.42g crystal with 42.45% total xanthophylls content. The HPLC profile of the crystal showed beta-cryptoxanthin:- 98.3%.

Example 3
An accurately weighed quantity of Paprika oleoresin (l00g) containing 6-8% by weight total xanthophylls and a color value of 1.00,000 units, the HPLC profile of the oleoresin was beta-carotene:- 15.36%: beta-cryptoxanlhin:-10%; zeaxanthin:-7.6% and t-eapsanthin:- 31.50%. was mixed with 100 ml ethanol and 25g potassium hydroxide pellet. The reaction mixture was heated to a temperature of 80-85 degree C. with stirring. The saponification process was maintained for 3-5 hours at 80-85 degree. C. with gentie agitation.
The reaction mixture was allowed to cool .Ethanol was distilled off from the mass. A measured volume of water (700ml) was added to the reaction mixture and kept for agitation for 1 hour. The solution was neutralized with 25% acetic acid solution. The water layer from the mass was removed followed by washing with water thrice. The mass was collected and dried under vacuum. The concentrate obtained was 121.75g with a total xanthophylls 4.92% by wt. HPLC: beta-carotene:-21.76%; beta-cryptoxanthin-: 12.74%; zeaxanthin:- 10.13% and t-capsanthin:- 38.25%.
The concentrate was washed 2 times with 1:10 hexane wt/vol at room temperature under stirring, filtered and the combined filtrate was concentrated to get a viscous mass. The mass (hexane concentrate) obtained was 85.81g with total xanthophylls 3.21%.by wt. HPLC: beta-carotene:- 35.28%; beta-cryptoxanthin:-19.65%; zeaxanthin:-3.99% and t-capsanthin:-13.88%.
The residue obtained after the filtration step was kept separately. Yield obtained: 25.65g with 10.42% by wt., total xanthophylls content. HPLC: beta-carotene-0.7%; beta-cryptoxanthin- 1.24%; zeaxanthin- 18.98% and t-capsanthin- 52.32%.

The hexane concenlrale was dissolved in minimum amount of hexane and subjected to column chromatographic separation .The column was packed with 1:5 concentrate and Silica gel 100-200 mesh wt/wl. and eluted with hexane. Separated band was eiuted and concentrated. Yield obtained: 55g with total xanthophylls content 2.29% wt..(HPLC; beta-carotene:- 99%)
The column was further eluted with 98:2 hexane : acetone vol/vol. The fraction was collected and concentrated. This concentrate was enriched with beta-cryptoxanthin. Yield obtained: 9.06g with 6.12% by wt. total xanthophylls content (HPLC: beta-cryptoxanlhin: 71.80%)
The column was finally eiuted with acetone.
Beta-Cryptoxanthin concentrate 9.06g was stirred with 1:2 ethanol wt/vol for 1 hr and chilled for 8hrs at 10 degree C, Filtered and the precipitate dried under vacuum. Yield 0.5g with 42.35% by wt.. total xanthophylls content and HPLC analysis of the crystal showed beta-cryptoxanthin content- 98.3%.
Beta-Cryptoxanthin precipitate 0.5g was dissolved in minimum amount of 80:20 hexane:ethylacctate vol/vol and chilled for 18hrs at 10 degree C filtered and the precipitate was dried under vacuum. Yield obtained: 0.03g with 80% total xanthophylls content and HPLC profile showed beta-cryptoxanthin content: 98.50%.
Example 4
Anti-resorptive property of |3- Cryptoxanthin and its effect on bone mechanical
strength
Test Materials
Description of Test materials

Samples Total Xanthophyll content Source
BCX-A 80.3% Obtained through Marigold oleoresin
BCX-B 0.793% Obtained through Orange fruits
BCX-C 10%* Obtained through Paprika oleoresin
For experimental purpose the sample was diluted to 1%.
Test system: Wistar rats of age between 8 to 10 weeks and weighing between 180-230 gm.
Housing of Animals
Animals were divided into 5 groups of 6 animals in each group. Each cage was labeled with the name of group, protocol no., species /strain and sex of animals.
The total no. of cages used was 10 for 30 animals. Each cage housed three animals at the temperature (25°C ± 2) and 50-70 % relative humidity with 12 h light/dark cycle. All the animals had free access to water. Rice husk was used as bedding in the cages. The cages were cleaned on daily basis.
Bilateral ovariectomy procedure:
All surgical instruments were sterilized before use. The dorsal skin of rat was shaved and disinfected using Povidone iodine solution. Ovariectomy was performed by two dorso-lateral incisions, approximately 1 cm long above the ovaries. With the use of a sharp dissecting scissors, skin cut was made almost together with the dorsal muscles to access peritoneal cavity. The ovary was found surrounded by a variable amount of fat. Blood vessels were ligated to prevent blood loss. The connection between the fallopian tube and the uterine horn was cut and the ovary was moved out and three single catgut stitches were placed on the skin.

Grouping

GROUP TREATMENT Dose No. of Animals
Group-1 Sham Control (Corn oil) 0.5 ml/100 g 6
Group-2 Ovariectomized control (OVX control) - 6
Group-3 OVX + β Cryptoxanthin (BCX-A) 20μg/100g 6
Group-4 OVX + β Cryptoxanthin (BCX-B) 20μg/100g 6
Group-5 OVX + β Cryptoxanthin (BCX-C) 20μg/100g 6
Study Procedure
Rats from group 1 were operated for sham surgery under Ketamine (70mg/kg) + Xylazine (lOmg/kg) (intra peritoneal) anesthesia. Rats from group 2. 3, 4 & 5 were operated for bilateral ovariectomy (OVX) under Ketamine + Xylazine (i.p.) anesthesia. The OVX-operated animals were fed with standard commercial laboratory chow amounts matched with Sham operated group. The operated animals were housed individually and were allowed to recover for 2 weeks.
Test compounds were dissolved in corn oil. Concentrations of 20μg/100 g of body weights were administered orally to rats in respective groups through oral gavage needle once daily for 3 weeks. Control rats received corn oil (0.5 ml/100 g of body
weight) orally.
On last day of treatment, urine samples were collected by micturation induced by manual pressure from overnight fasted animals and preserved at -20°C till further
analysis.

Statistical Analysis: All results were analyzed using One Way ANOVA followed by Dunnetf s multiple comparison test. Considering confidence interval of P <0.05.
Estimation of Bone Collagen metabolite (Pyridinoline Crosslinks) in urine:
Pyridinoline Crosslinks are the bone collagen metabolites which appear in urine when bone resorption process is accelerated hence considered as an early important marker of osteoporosis.
OVX control animals showed significant increase in Pyridinoline crosslinks in urine which confirms the successful induction of osteoporosis after ovariectomy procedure. BCX-C treatment moderately reduced the urinary excretion of Pyridinoline Crosslinks to a considerable extent as shown below in Table No. 1. This confirms the therapeutic utility of BCX treatment in estrogen deficiency related osteoporosis.
Table No. 1: Pyridinoline Crosslinks Determination

Pyridinoline Crosslinks (nmol/mmol of Creatinine)
Sham Control OVX
control BCX-A BCX-B BCX-C
17.15 17.64 6.71 10.27 14.48
1.89 21.69 9.80 9.64 15.64
29.71 22.31 10.52 10.04 16.87
33.50 14.68 12.15 10.80 10.26
4.40 14.79 34.37 24.17 13.77
1.50 13.97 12.19 23.12 24.62
Mean= 14.69 17.51 14.29 14.68 15.94

Determination of bone (femur) density:
Cessation of the ovarian function in humans (eads to increase in bone turnover, a negative bone balance, and a net decrease in bone density; these changes are also evident in surgically ovariectomized rats.
A significant OVX induced decrease in bone density was observed in group-2. BCX-A, BCX-B and BCX-C treatment prevented OVX associated decrease in bone density. BCX-C showed better activity than BCX-A and BCX-B in preventing OVX induced loss of bone density as shown below in Table No. 2.
Table No. 2: Bone Density Determination

Bone Density (g/cm3)
Sham Control OVX control BCX-A BCX-B BCX-C
1.253 1.121 1.173 1.1858 1.1902
1.186 1.131 1.178 1.0669 1.2437
1.169 1.167 1.175 1.1785 1.2065
1.208 1.131 1.186 1.1893 1.1593
1.230 1.162 1.141 1.0782 1.1965
1.205 1.130 1.171 1.2490 1.1978
Mean ± SD= 1.209 ±0.030 1.140 ±0.019 1.171 ±0.015 1.157 ±0.070 1.198 ±0.027
The results obtained from the above experiments confirm that p- Cryptoxanthin obtained from Paprika source (BCX-C) shows better anti-resorptive property than (3-Cryptoxanthin obtained from Marigold and Orange source.
Determination of ultimate failure load of bones (Tibiae)
Bone fragility can be defined broadly as the susceptibility to fracture. One function of bones is to carry loads. Fractures occur when loads exceed the bone strength, so

weakened bones should be considered fragile. During traumatic loading, such as falling on the ground, fracture will occur if the energy from the fall exceeds the mechanical energy that the bone can absorb. Osteoporotic bones absorb very little energy before breaking (failure load) and are therefore more susceptible to fracture resulting from trauma. In this study, the failure load was measured using Three Point Bending test.
In ovariectomized animals there was significant decrease in maximal load values for tibial mid shaft indicating the significant loss of cancellous bone. From the results given below in Table No. 3 it is evident that, BCX-C treatment significantly-prevented the loss of mechanical strength of cancellous bone.
Table No. 3 Determination of Failure Load of Bone

Failure load (N)
Sham Control OVX control BCX-A BCX-B BCX-C
77.14 45.47 51.9 59.63 66.66
69.89 43.22 65.25 56.91 57.00
58.33 54.11 53.41 59.77 65.72
61.22 45.63 49.71 62.38 54.31
58.33 42 73.71 75.23 65.66
55.76 45.62 63.28 57.65 48.90
Mean ±
SD= 63.445
±8.31 46.00 ± 4.24 59.54 ± 9.38 61.92 ±6.79 59.70 ± 7.39
The data represented in Table 1, 2 and 3 are plotted in Graphs 1, 2 and 3 respectively, as shown in the drawings accompanied with the specification.

Conclusion
p-cryptoxanthin possess significant anti-osteoporotic activity in OVX rat model. BCX-C (Paprika source) significantly improved mass and mechanical strength of bones in ovariectomized rats than BCX-A (Marigold source) & B (Orange source).

We claim:
1) A beta-cryptoxanthin concentrate, which contains approximately 10-80% by
weight total xanthophylls out of which approximately 75-98% by weight being trans-
beta-cryptoxanthin, the remaining being zeaxanthin, trans-capsanthin, beta-carotene
and trace amounts of other carotenoids, derived from plant carotenoid source and
which is useful for human nutrition and health care.
2) The beta-cryptoxanthin concentrate as claimed in claim 1. wherein the concentrate comprises at least 80% by weight total xanthophylls, out of which the trans-beta-cryptoxanthin content is at least 98% by weight, the remaining being zeaxanthin. trans-capsanthin and other carotenoids.
3) The beta-cryptoxanthin concentrate as claimed in claims 1 & 2, wherein the concentrate comprises at least 40% by weight total xanthophylls, out of which the trans-beta-cryptoxanthin content is at least 90% by weight, the remaining being zeaxanthin. trans-capsanthin and other carotenoids.

4) The beta-cryptoxanthin concentrate as claimed in claims 1 to 3, wherein the concentrate comprises at least 10% by weight total xanthophylls, out of which the trans-beta-cryptoxanthin content is at least 75% by weight, the remaining being zeaxanthin, trans-capsanthin and other carotenoids.
5) The beta-cryptoxanthin concentrate as claimed in claims 1 to 4, wherein the plant extract is derived from the group comprising of fruits and vegetables, preferably capsicum species.

6) The beta-cryptoxanthin concentrate as claimed in claims 1 to 5, wherein the trans-beta-cryptoxanthin concentrate is in the form selected from the group of beadlets, microencapsulated powders, oil suspensions, liquid dispersions, capsules, pellets. ointments, soft gel capsules, tablets, chewable tablets or lotions/ liquid preparations.
7) A process for the preparation of trans-beta-cryptoxanthin enriched concentrate which contains approximately 10-80% by weight total xanthophylls out of which approximately 75-98% by weight being trans-beta-cryptoxanthin, the remaining being zeaxanthin, trans-capsanthin, beta-carotene and trace amounts of other carotenoids. comprising the steps:

a) Mixing xanthophylls esters in an oleoresin with an aliphatic alcoholic solvent. wherein the ratio of oleoresin to alcohol ranges from 1:0.25 to 1: 0.5 weight/volume;
b) Saponifying the xanthophylls esters present in the oleoresin of plant material with an aliphatic alcoholic alkali solution without addition of water, wherein the ratio of oleoresin : alkali ranges from 1: 0.25 to 1 : 0.5 weight/weight;
c) Applying heat to elevate the temperature up to reflux;
d) Allowing the saponification reaction under agitation for 3 to 5 hr;
e) Evaporating the alcohol under vacuum followed by addition of water to get diluted resultant mixture, which is maintained under agitation for 1 hr;
f) Neutralizing the mixture with a diluted acid;

g) Separating the water layer from the precipitated xanthophylls mass and washing the mass with a polar solvent to remove the soaps and the other polar soluble materials;
h) Drying the resultant xanthophylls crude mass under vacuum;
i) Washing the crude mass with non polar solvent and the non polar solvent washings are concentrated to get crude viscous xanthophylls mass;
j) Transferring the viscous mass on a silica gel column and eluting with non polar solvent to obtain carotene fraction;
k) Distilling off the non polar solvent fraction to get a beta- carotene concentrate;
1) Washing the column with non-polar : polar solvent mixture and concentrating the washings resulting in beta-cryptoxanthin rich xanthophylls concentrate;
m) Admixing the xanthophylls concentrate with aliphatic alcohol under stirring and cooling at low temperature;
n) Filtering and drying under vacuum to obtain purified beta-cryptoxanthin concentrate;
o) Washing again the purified beta-cryptoxanthin concentrate with a mixture of non-polar : ester solvent and cooling to low temperature for precipitation to obtain high purity trans-beta-cryptoxanthin crystals.

8) The process of claim 7 step (a) & (b), wherein the aliphatic alcohol is ethanol. methanol, iso-propyl alcohol or mixture of thereof.
9) The process of claim 7 step (b), wherein the alkali used is selected from sodium or potassium hydroxide.

10) The process of claim 7 step (c), wherein the elevated temperature ranges from 80 to 85 degree C.
11) The process of claim 7 step (e). wherein the water added is 5 times that of the oleoresin weight/weight.
12) The process of claim 7 step (f), wherein the acid used is acetic acid or phosphoric acid.
13) The process of claim 7 step (g), wherein the polar solvent used is water.
15) The process of claim 7 step (i) & (1). wherein the non polar solvent used selected from hexane, pentane, heptanes or mixtures thereof.
15) The process of claim 7 step (i), wherein the ratio of crude xanthophylls mass to non polar solvent is 1:10 to 1: 15 weight/volume.
16) The process of claim 7 step (i), wherein the viscous xanthophylls mass comprises beta-carotene, trans-beta-cryptoxanthin, trans-capsanthin and zeaxanthin along with trace amounts of other carotenoids.

17) The process of claim 7 step (j), wherein the ratio of xanthophylls concentrate to non polar solvent is 1:5 to 1:8 weight/volume.
18) The process of claim 7 step (j), wherein the non polar solvent is selected from hexane, heptane, pentane and mixtures thereof.
19) The process of claim 7 step (k). wherein the carotene concentrate is beta-carotene.
20) The process of claim 7 step (1). wherein the polar solvent selected from the group consisting of propanone. pentanone or mixtures thereof.
21) The process of claim 7 step (1). wherein the ratio of non-polar : polar solvent ratio is in the range of 95:5 to 98:2.
22) The process of claim 7 step (o), wherein the ester solvent is ethyl acetate.
23) The process of claim 7 step (o), wherein the ratio of non polar: ester solvent is in the range of 80:20 to 90:10.
24) The process of claim 7 step (1), wherein the resulting xanthophylls comprises 10% by weight total xanthophylls out of which trans-beta-cryptoxanthin content is at least 75% by weight and the remaining being beta-carotene, trans-capsanthin. zeaxanthin and traces of other carotenoids.
25) The process of claim 7 step (n), wherein the purified beta-cryptoxanthin
concentrate comprises at least 40% by weight of total xanthophylls out of which

trans-beta-cryptoxanthin content is at least 90% by weight, the rest being beta-carotene, trans-capsanthin. zeaxanthin and traces of other carotenoids.
26) The process of claim 7 step (o). wherein the high purity beta-cryptoxanthin concentrate comprises at least 80% by weight of total xanthophylls out of which trans-beta-cryptoxanthin content is at least 98% by weight, the rest being beta-carotene, zeaxanthin. trans-capsanthin and traces of other carotenoids.
27) The beta-cryptoxanthin concentrate as claimed in claims 1 to 6, wherein the trans-beta-cryptoxanthin enriched concentrate obtained from capsicum source is useful for strengthening bone and inhibiting bone resorption.
28) The process of claim 7. wherein the by-products consist of beta-carotene, trans-capsanthin, zeaxanthin or mixtures thereof.