Methods for prevention and treatment of cardiometabolic syndrome and compositions used
therein
Field
Methods are described for the prevention and/or treatment of cardiometabolic syndrome and
associated risk factors by administering beta-cryptoxanthin compositions in an effective amount
to a subject in need thereof. More particularly, methods are described that relate to prevention
and/or treatment of cardiometabolic syndrome such as hyperlipidemia, diabetes and other
cardiovascular disorders by administering a composition comprising beta-cryptoxanthin as the
active ingredient alone or in combination with other nutrients. The compositions herein help to
improve cardiometabolic syndrome and manages associated risk factors such as body weight,
body fats, lipid profile, blood glucose and the like, when administered in an effective amount to a
subject in need thereof. The compositions herein also protect retina and liver by reducing
oxidative stress and inflammatory markers, thus improving function of vital body organs, which
are related to cardiometabolic syndrome. Beta-cryptoxanthin compositions described herein can
reduce cardiometabolic stress in a subject in need thereof. The compositions herein are safe for
human consumption and can be employed for management of cardiometabolic syndrome, when
administered in an effective amount.
Background:
Cardiometabolic syndrome is a disorder of energy intake, utilization and storage, diagnosed by a
co-occurrence of three out of five of the following medical conditions: abdominal obesity,
elevated blood pressure, elevated fasting plasma glucose, high serum triglycerides, and low highdensity
cholesterol (HDL) levels. The cardiometabolic syndrome is thus a combination of
metabolic disorders, resulting into hyperlipidemia, impaired glucose tolerance, hypertension,
oxidative stress and the tendency to develop fat around the abdomen.
Individuals with cardiometabolic syndrome are at high risk of developing heart failure and
insulin resistance, thus affecting vital organs such as the eye, liver, kidney and nervous system.
The body makes insulin to move glucose (sugar) into cells for use as energy. The prevalence of
obesity is increasing at an alarming rate in developed and developing countries (Haslam and
James, 2005). Obesity makes it more difficult for cells to respond to insulin. If the body cannot
make enough insulin to override the resistance, the blood sugar level increases and diabetes can
result. Although various risk factors such as age, high body mass index, smoking, stress,
sedentary lifestyle, and postmenopausal status are identified, high fat diet is one of the most
important risk factors leading to cardiometabolic syndrome.
Although during the last decades increasing scientific evidence has emerged that protective
health effects can be obtained from diets that are rich in fruits, vegetables, legumes and whole
grains, a significant number of the population still consumes junk food which is prevalent in
high fat and refined carbohydrates. Such diet has been a critical factor blamed for obesity,
diabetes, and a number of other cardiovascular diseases (Park et al., 2013). Rodents that are fed a
high-fat diet develop visceral obesity, insulin resistance, hyperlipidemia, endothelial dysfunction,
and hypertension (Roberts et al. 2001). Obesity is strongly associated with metabolic syndrome
and many chronic diseases that result from an imbalance between energy intake and physical
activity (Goran and Treuth 2001). It has significant health consequences such as diabetes,
hypertension, cardiovascular disease and inflammatory disorders.
Excess energy intake and reduced energy expenditure promotes cardiometabolic dysfunction
(Yang et al., 2009), oxidative stress and inflammatory pathologic factors that increase the
secretion of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-a), and C-reactive protein
(CRP) (Wang et al., 2012). Increased adipose tissue plays an important role in the development
of low-grade inflammation, which is characterized by cytokine production and stimulation of
inflammatory cytokine signaling pathways (Hotamisligil and Erbay, 2008).
However, as a result of several epidemiologic studies and some clinical trials, it has been
suggested that people with metabolic syndrome and obesity may benefit from intensive lifestyle
modifications including dietary changes and adopting a physically more active lifestyle.
(Pitsavos, Panagiotakos, et al., The Review of Diabetic Studies (2006) 3:1 18-126).
Carotenoids are known to play an important role in health and disease and the state of living
human tissue(s) based on their antioxidant function and scavenging action on singlet molecular
oxygen and peroxyl radicals (Stahl and Sies, 2003). Observational studies have proposed that
dietary carotenoid intake or circulating carotenoid serum levels are associated with a reduced
risk of mortality, cardiovascular disease, cancer, stroke, and other conditions (Krinsky and
Johnson, 2005).
Beta-cryptoxanthin (BCX) is considered a provitamin A which is present in many fruits and
vegetables and belongs to a family of carotenoids called xanthophylls. BCX is a long
hydrocarbon chain and acts as an antioxidant that 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. Studies have found that BCX may reduce the risk of
developing inflammatory disease. High intake of BCX has been shown to reduce the risk of
rheumatoid arthritis, polyarthritis and other inflammatory diseases. BCX provides anti-aging
benefits. As a powerful anti-oxidant, BCX helps keep skin cells healthy and young.
It is further observed that supplementation of highly concentrated BCX improves serum
adipocytokine profiles in obese subjects consequently reduces risk of the progression of
metabolic syndrome [Lipids Health Dis. 2012 May 14;1 1:52]. BCX also facilitates lipid
metabolism in muscle and reduced adipocyte proliferation and inflammatory response [Front
Neurol. 201 1 Nov 23;2:67].
Japanese Patent Application No. 2013 173720A relates to carotenoid agents such as
cryptoxanthin and/or an ester thereof for preventing retinopathy and is shown to exhibit superior
prevention or amelioration effect on diabetic retinopathy.
Another Japanese Patent Application No. 2010273727 relates to an oral administration
composition which exhibits the effect of reducing body fat, such as visceral fat, subcutaneous fat
and the like, and neutral fat, and relates to a food, a pharmaceutical, and a feed having the
composition; wherein the composition includes a carotenoid and a sphingolipid, or a carotenoid
and a flavonoid and/or a derivative thereof The cryptoxanthin and/or the sphingolipid and/or the
flavonoid are derived from a citrus fruit and the citrus fruit is preferably Citrus unshiu.
Japanese Patent Application No. 2014162726 relates to the use of beta-cryptoxanthin and its
derivatives to improve skin texture, to suppress chromatosis, in the retention of the skin, and in
the evaluation as moisturizing agent, whitening agent, skin beautifier, and/or for preventing
wrinkles. Thus beta-cryptoxanthin is explored for its cosmetic use in this patent application.
US Patent Application No. 200902581 11Al relates to a highly bioavailable cryptoxanthin
composition (a food or drink or a feed) for oral administration wherein the amount of dietary
fibers contained in the composition is 400 times by weight or less based on cryptoxanthin. The
reference further relates to probable use of improved absorbability of cryptoxanthin into the
living body for various effects such as its provitamin A activity and anti-oxidation action,
carcinogenesis inhibitory action, osteogenesis accelerating action, and skin whitening action.
US Patent Application No. 20120053247 relates to a nutritional supplement including betacryptoxanthin,
which may be used to maintain cardiovascular health by lowering blood pressure,
preventing high, elevated blood pressure, and/or maintaining healthy blood pressure and
reducing heart rate. The administration of beta-cryptoxanthin in combination with safflower oil
is particularly effective at the amount of 0.1 g and 20 g per day.
W.P. Kohet al (Nutrition, Metabolism and Cardiovascular Diseases 21(9), 685-690, 2011)
relates to a study of high plasma levels of beta-cryptoxanthin and lutein for decreasing the risk of
acute myocardial infarction.
Sari Voutilainen et al (Am J Clin Nutr 83:1265—71, 2006) relates to the role of main dietary
carotenoids such as lycopene, beta-carotene, alpha carotene, beta-cryptoxanthin, lutein, and
zeaxanthin in the prevention of atherosclerosis.
Further Granado-Lorencio et al. (Nutr Metab Cardiovasc Dis. 2014 Oct;24( 10): 1090-6) relates to
the effect of beta-cryptoxanthin in combination with phytosterols (PS) on cardiovascular risk and
bone turnover markers in post-menopausal women, wherein beta-cryptoxanthin improves the
cholesterol-lowering effect of PS when supplied simultaneously.
Summary
Even though the literature above discusses the effect of beta-cryptoxanthin in cosmetic
applications, body fat reduction, and diabetic retinopathy as well as for reducing risk of
myocardial infarction, they do not relate to the effect of beta-cryptoxanthin compositions on
management of metabolic syndrome and associated risk factors. Further none of the references
relate to the evaluation of beta-cryptoxanthin compositions for their effect on basic cellular
mechanism associated with diabetes, obesity and related cardiovascular risk factors in a subject,
administered with high fat diet.
Cardiometabolic syndrome is an extremely complicated issue due to the fact that it encompasses
a mesh of metabolic pathways (mainly glycemia and lipids) and involves several tissues (liver,
fat, muscle, eye and others). For example, the liver is closely involved not only in regulation of
glycemia and lipids but also in inflammation and hemostasis, which are main players in
cardiometabolic syndrome.
Patients with diabetes/pre-diabetes, concomitant cardiovascular disease (CVD), and risk factors
of hypertension, obesity, and/or dyslipidemia also have insulin resistance. The components of
cardiometabolic syndrome also include abdominal obesity, diabetes, glucose intolerance,
dyslipidemia, high blood pressure, and/or hyperuricemia. The association of diabetes and
hypertension with ocular conditions such as retinopathy, cataract, and raised intraocular pressure
(IOP) is well known. (Ref: Indian J Endocrinol Metab. 2012 Mar; 16(Supp 11): S6-S1 1, Ocular
associations of metabolic syndrome, Rupali Chopra, Ashish Chander, and Jubbin J . Jacob-
The prevalence of metabolic syndrome is rapidly increasing worldwide due for example to
sedentary lifestyles. Its association with various ocular manifestations such as non-diabetic
retinopathy, central retinal artery occlusion (CRAO), cataract, and primary open angle glaucoma
suggests that an epidemic of metabolic syndrome can have far-fetched ocular consequences as
well. Amelioration of metabolic syndrome may have a therapeutic role in preventing these ocular
conditions.
Liver has an important role in cardiometabolic syndrome condition. The liver clears,
metabolizes, detoxifies, and redistributes the absorbed content of food. Its role in established
type 2 diabetes is well demonstrated, but increasing evidence implicates this organ in very early
stages of prediabetes. One major finding of the last decade has been the recognition of a
prevalence rate of 30% for hepatic steatosis in the general population, with an even higher
prevalence in obese and elderly populations. The inflammatory state of the liver in prediabetic
states is expected to impact on the cardiovascular system. There is indeed a well known interplay
between insulin resistance, inflammation, obesity, and heart disease. Insulin-resistant individuals
of normal weight have a 2.5-fold increase in risk for heart failure. The risk can be assessed by
checking various biomarkers and the compositions having desirable effect on the biomarkers can
be used effectively for managing cardometabolic syndrome. (Ref: Diabetes Metab Syndr Obes.
2013; 6: 379-388, Hepatic function and the cardiometabolic syndrome, Nicolas Wiernsperger)
Applicant has carried out rigorous experimentation and evaluation as reported herein exhibiting
the use of beta-cryptoxanthin compositions in preventing and treating cardiometabolic syndrome
by effectively reducing multiple risk factors such as hyperglycemia, hyperlipidemia and other
cardiovascular disorders, in subjects fed with high fat diet. The effect of such compositions is
described herein on biomarkers related to liver and eye. Methods for prevention and treatment of
cardiometabolic syndrome as described herein are comprised of administering betacryptoxanthin
compositions in an effective amount to a subject in need thereof and evaluating the
effect on related oxidative and inflammatory biomarkers. The compositions as described herein
are useful to improve cardiometabolic syndrome and manage associated risk factors such as body
weight, body fats, lipid profile, blood glucose and the like. The compositions herein also protect
retina by reducing oxidative stress. The compositions herein are safe for human consumption
and can be employed for management of cardiometabolic syndrome, when administered in an
effective amount.
In some embodiments, methods herein are directed to administering a beta-cryptoxanthin
composition in an effective amount(s) to treat cardiometabolic syndrome in a subject and/or are
directed to evaluating its beneficial effect on the management of cardiometabolic syndrome in a
subject. As per the methods described herein, use of compositions is directed to prevention,
treatment and improvement of associated risk factors, health conditions and vital body functions
in a subject in need thereof, thus leading to overall management of cardiometabolic syndrome.
In one embodiment, methods herein are directed to evaluating the effect(s) of beta-cryptoxanthin
composition on improvement of cardiometabolic health by administering to a subject in need
thereof, an effective amount of a composition comprising beta-cryptoxanthin alone or in
combination with other nutrients.
In one embodiment, methods described herein are comprised of administering betacryptoxanthin
compositions for improvement of cardiometabolic health by increasing antioxidant
activity and reduction in oxidative stress in retina and liver tissues.
In embodiment, methods for prevention and treatment of cardiometabolic syndrome are directed
to administering beta-cryptoxanthin compositions for reducing oxidative stress and protecting the
retina from neovascularization, when administered to a subject in need thereof, such as for
example to a subject on a high fat diet.
In another embodiment, methods herein are directed to retarding the accumulation of lipofuscin
pigment in retina and preventing the causes of retinal neovascularization, retinal vein occlusion,
and/or neovascularization in peripheral retina by administering an effective amount of betacryptoxanthin
either alone or in combination with other nutrient(s) including provitamin A to a
subject in need thereof.
In one embodiment, methods described herein are directed to use of beta-cryptoxanthin
compositions for prevention and treatment of cardiometabolic syndrome by reducing
inflammatory and oxidative stress markers on associated vital body organs such as liver and eye.
In one embodiment, methods for improvement of cardiometabolic health are comprised of
administering beta-cryptoxanthin compositions for management of a healthy lipid profile,
reduction in body fat, visceral fat, and free fatty acid levels in the body.
In one embodiment, methods described herein relate to use of beta-cryptoxanthin compositions
for management of metabolic syndrome, such as hyperglycemia, by the reduction in body
glucose levels and/or the reduction in insulin resistance, in a subject fed with a high fat diet.
The beta-cryptoxanthin composition includes an active material present including betacryptoxanthin
(BCX), which is extracted for example from paprika oleoresin by saponification
followed by purification through column chromatography. Compositions herein are enriched
with trans-beta-cryptoxanthin. In an embodiment, the extract is suspended in a suitable oil
medium to obtain 5% oil suspension. In an embodiment, the suspension was evaluated in animal
model described herein below. For human consumption, the compositions herein include final
formulations into powders, granules, beadlets, and can be administered by oral solid dosage
forms such as tablets, capsules.
In an embodiment, an effective amount herein relates to the amount of BCX present in the
composition.
In an embodiment, a daily dose duration can range from at or about 3 months to at or about 2
years, or till the desired effect is achieved in a subject. It will be appreciated that there may be
no fixed time period for the daily doses as it may be less or longer than such range. It will also
be appreciated that the dose may be given continuously daily during this period or the
administration can be stopped after obtaining a desired effect in a subject, and can also be
restarted again as needed. It is appreciated that dose periods herein include the experiment
durations or by general volunteer study period which can be extended to 12 months.
In one embodiment, methods described herein are comprised of administering betacryptoxanthin
compositions in effective daily dose of at or about 0.1 to at or about 100 mg kg
body weight, for the treatment and/or management of cardiometabolic syndrome, to improve
lipid profile, to reduce body weight, liver weight and and/or to reduce oxidative stress markers in
the retina and/or liver.
In an embodiment, an effective daily dose includes a range of at or about 250 micrograms to at
or about 30 mg/kg body weight.
In an embodiment, an effective daily dose includes a range of at or about 150 micrograms to at
or about 20 mg kg body weight.
In an embodiment, an effective daily dose includes a range of at or about 200 micrograms to at
or about 10 mg/kg body weight.
In one embodiment, methods as described herein are directed to use of beta-cryptoxanthin
compositions for reducing risk factors associated with cardiometabolic syndrome, such as for
example obesity, diabetes, hypertension, and/or hyperlipidemia, and the like.
Methods and compositions described herein are useful for the treatment and/or management of
cardiometabolic health in a subject in need thereof, such as for example a mammal fed with high
fat diet, when administered in an effective amount.
In some embodiments, daily dose range can include about 250 micrograms to about 30 mg of
beta-cryptoxanthin (BCX). In some embodiments, biological markers, genes, indicators, their
regulated pathways, and the like include, but are not limited to data which have shown decreases
in visceral fat, decreases in body weight, and decreases in liver weight. The methods as described
herein are comprised of evaluation of use of beta-cryptoxanthin compositions in high fat diet
(HFD) treated subjects, such as for example rats as an experimental model. Effects of BCX
composition are evaluated on various health parameters associated with cardiometabolic
syndrome such as total cholesterol, LDL cholesterol, triglycerides, glucose, insulin, leptin,
adiponectin and free fatty acids.
In some embodiments, compositions herein include a beta-cryptoxanthin extract administered in
the form of composition with other food grade excipients. Compositions herein can be in the
form of oil suspensions, beadlets, spray dried powders, microcapsules, tablets, capsules, caplets,
and the like. The active material is prepared by an extraction process by human intervention and
is formulated into a composition, such as with other food grade excipients and materials to obtain
the desired form.
Brief description of the drawings:
Fig.l depicts effect of beta-cryptoxanthin on body weight, visceral fat and liver weight in high
fat diet induced obese rats (n=7), (A)Final body weight, g, (B) Visceral fat, g, and (C) liver
weight.
Fig.2 depicts effect of beta-cryptoxanthin on biochemical parameters in high fat diet induced
obese rats (n=7), (A) Glucose; (B) Insulin; (C) Leptin; (D) Adiponectin; (E) Tryglyceride (TG);
(F) Total Cholesterol (TC); (G)LDL-C; (H) HDL-C; (I) Free Fatty Acids (FFA).
Fig.3 depicts effect of beta-cryptoxanthin on antioxidant status in high fat diet induced obese rats
(n=7), (A)Serum Total Antioxidant Capacity (TAC); (B) Serum malondialdehyde (MDA); (C)
Liver MDA; (D) Liver superoxide dismutase (SOD); (E) Liver catalase (CAT); (F) Liver
glutathione peroxidase (GSH-Px).
Fig.4 depicts effect of beta-cryptoxanthin for regulating CCAAT/enhancer-binding protein alpha
(C/EBPa), FAS and stearoyl-CoA desararase (SCD-1).
Fig.5 depicts effect of beta-cryptoxanthin on retina tissue vascular endothelial growth factor
(VEGF), nuclear factor erythroid derived 2-related factor 2 (Nrf-2), nuclear factor kappa light
chain enhancer of activated B cells (NFkB), Inducible nitric oxide synthase (iNOS), Intercellular
Adhesion Molecule 1 (ICAM-1); and heme-oxygenase 1 (HO-1).
Fig.6 depicts effect of beta-cryptoxanthin on HO-1 marker in retina tissue.
Fig.7 depicts effect of beta-cryptoxanthin on ICAM-1 marker in retina tissue.
Fig.8 depicts effect of beta-cryptoxanthin on iNOS marker in retina tissue.
Fig.9 depicts effect of beta-cryptoxanthin on nuclear factor kappa-light-chain-enhancer of
activated B cells (NFkB) marker in retina tissue.
Fig. 10 depicts effect of beta-cryptoxanthin on Nuclear factor (erythroid-derived 2) (Nrf-2)
marker in retina tissue.
Fig.l 1 depicts effect of beta-cryptoxanthin on (VEGF) marker in retina tissue.
Fig. 12 depicts effect of beta-cryptoxanthin on liver tissue beta-carotene oxygenase 2 (BC02),
tumor necrosis factor alpha (TNF-a), peroxisome proliferator-activated receptor gamma (PPAR-
), Nrf-2, NFkB, insulin receptor substrate 1 (IRS-1), HO-1.
Fig. 13 depicts effect of beta-cryptoxanthin on BC02 marker in liver tissue.
Fig. 14 depicts effect of beta-cryptoxanthin on TNF-a marker in liver tissue.
Fig. 15 depicts effect of beta-cryptoxanthin on PPAR-marker in liver tissue.
Fig. 16 depicts effect of beta-cryptoxanthin on Nrf-2 marker in liver tissue.
Fig. 17 depicts effect of beta-cryptoxanthin on NFkB marker in liver tissue.
Fig.l 8 depicts effect of beta-cryptoxanthin on HO-1 marker in liver tissue.
Fig.19 depicts effect of beta-cryptoxanthin on IRS-1 marker in liver tissue.
Detailed description:
The methods described herein are comprised of identifying a subject in need thereof,
administering beta-cryptoxanthin composition(s) in an effective amount(s), and evaluating the
effect for treatment, prevention and/or management of cardiometabolic syndrome and their
associated risk factor(s). The methods described herein can improve conditions associated with
cardiometabolic syndrome such as body weight, lipid profile, insulin resistance, blood glucose,
reduction in oxidative stress, and inflammatory markers, to protect vital organs like the eye and
liver, when administered to a subject, such as for example who is habituated for high fat diet.
Beta-cryptoxanthin for the compositions as described herein may be obtained by natural
resources and are safe for administration and thus useful for nutraceutical purposes.
Cardiometabolic syndrome, also known as syndrome X, increases the risk of developing
cardiovascular disease, particularly atherosclerosis, heart failure, dyslipidemia, diabetes, and
associated risk factors, which may be caused mainly due to imbalance of calorie intake and
energy utilization. One of the most important causes for this is a high fat diet. The syndrome also
affects vital body organs such as liver and eye. Therefore it is important to identify methods for
treating and preventing it and associated risk factors thereof by administering compositions
which are safe for administration and evaluating the effect in subjects in need thereof.
The terminology 'subject' is commonly used in the specification to refer to an individual or
mammal under test, being treated with compositions herein.
The terminology "subject in need thereof can include specific individuals or mammals who are
habituated to a diet rich in high fat and refined carbohydrates, thus lacking in fibers. Such
subjects are at high risk of developing cardiometabolic syndrome or symptoms for associated
risk factors and/or may be suffering from cardiometabolic syndrome, because of developing
abdominal obesity.
Abdominal obesity drives the progression of multiple risk factors directly, through secretion of
excess free fatty acids and inflammatory adipokines, and decreased secretion of adiponectin
(Despres JP et al, 1990; Pouliot MC, 1992; Kissebah AH et al, 1989; Carey VJ, 1997; Turkoglu
C et al, 2003). Significant effects of abdominal obesity can be dyslipidaemia and insulin
resistance, which can provide an indirect, though clinically important, link to the genesis and
progression of atherosclerosis and cardiometabolic risk. Excess abdominal obesity is
accompanied by elevated levels of C-reactive protein (CRP) and free fatty acids (FFAs), as well
as decreased levels of adiponectin. Elevated CRP is an indicator of inflammation. Abdominal
obesity may be associated with the inflammation cascade, with adipose tissue expressing a
number of inflammatory cytokines. Inflammation is now believed to play a role in the
development of atherosclerosis and type 2 diabetes. Elevated levels of CRP are considered to be
predictive of cardiovascular disease and insulin resistance.
Elevated FFA levels appear to play a significant role in the cause of insulin resistance. It has
been suggested that elevated FFAs and intracellular lipids inhibit the insulin signaling
mechanism, leading to decreased glucose transport to muscle. Adiponectin is an adipose tissuespecific
circulating protein which is involved in the regulation of lipid and glucose metabolism.
Adiponectin has been shown to be reduced in adults with obesity and type 2 diabetes. Such
components help to explain why excess abdominal obesity is considered to be a significant risk
to cardiovascular and metabolic health.
Inflammation is part of the complex biological response of vascular tissues to harmful stimuli,
such as pathogens, damaged cells, or irritants. Chronic inflammation is widely observed in
obesity. Understanding the molecular basis of inflammation has led to the identification of
markers that may also serve as new targets of therapy in the management of associated
cardiometabolic syndrome disease in obese person. The obese commonly have many elevated
markers of inflammation, including: Interlukins (IL 6, 8 and 18), TNF-a (Tumor necrosis factoralpha),
CRP (C-reactive protein), Insulin, Blood glucose, and Leptin. Inflammatory markers have
been shown to predict future cardiovascular events in subjects with and without established
cardiovascular disease (CVD).
Low-grade chronic inflammation is characterized by a two- to threefold increase in the systemic
concentrations of cytokines such as TNF-a, IL-6, and/or CRP. TNF's primary role is to regulate
the immune cells and induce inflammation. TNFa -induced reductions in insulin sensitivity in
adipocytes are partly responsible for the increased free fatty acid production and
hypertriglyceridaemia characteristic of abdominal obesity. Leptin responds specifically to
adipose-derived inflammatory cytokines. Hyperglycemia induces IL-6 production from
endothelial cells and macrophages. Meals high in saturated fat, as well as meals high in calories
have been associated with increases in inflammatory markers.
Liver plays an important role in metabolism activities and it is an important site of fat
metabolism. When this function is impaired due to a variety of reasons, fat accumulation occurs
in the liver, which may result in cirrhosis and/or increased risk of other cardiometabolic
syndromes such as for example diabetes, hypertension, disturbed lipid profile, and/or one or
more risk factors associated with these syndromes, or in combination with other associated
conditions.
As per one embodiment, the methods described herein are comprised of administering betacryptoxanthin
compositions to a subject in need thereof, in an effective amount, and evaluating
its effect on risk factors associated with cardiometabolic syndrome. Beta-cryptoxanthin
compositions herein may be administered by oral route, in combination with antioxidant or other
nutrients, using oil vehicle for suspending the composition. The oil used in the composition is
selected from the group consisting of rape seed oil, corn oil, sunflower oil and like thereof.
According to one embodiment, methods and compositions as described herein are directed to
treating macular degeneration in a subject in need thereof comprising essentially of
administering therapeutically active amounts of beta-cryptoxanthin either alone or in
combination with antioxidant or an oil.
As per one embodiment, the compositions and methods herein can improve (e.g. reduce) risk
factors associated with cardiometabolic syndrome, such as body weight, lipid profile, body
glucose, and/or insulin resistance, when administered to a subject, such as for example a subject
who is fed with a high fat diet.
In another embodiment, a method for treating dyslipidema, comprising identifying a subject
with elevated triglycerides levels, elevated serum LDL levels, or reduced HDL levels and
accordingly administering a therapeutically effective amount of a composition consisting
essentially of beta-cryptoxanthin either alone or in combination of pharmaceutically acceptable
excipients.
In further embodiment, methods described herein are comprised of administering effective
amount of a composition to a subject in need thereof for improving insulin sensitivity. The
composition may be beta-cryptoxanthin either alone or in combination of pharmaceutically
acceptable excipients.
The terminology 'high fat diet' as used in the specification includes a diet with food typically
containing about 32 to 60% of calories from fat. Such diets with 60 kcal% fat are commonly
used to induce obesity in rodents since animals tend to gain weight more quickly, thereby
allowing researchers to screen their compounds after a shorter period of time.
The type of fat is also considered when choosing a high-fat diet for an animal study. Many highfat
diets used in laboratory animal research contain more saturated fat such as lard, beef tallow,
or coconut oil and these diets are quite capable of inducing obesity in susceptible strains.
As per one embodiment, methods described herein are comprised of administering an effective
amount of beta-cryptoxanthin compositions to treat hyperlipidemia in a subject in need thereof
by lowering total cholesterol, low density lipoproteins and/or triglycerides.
According to one embodiment, methods and compositions described herein are directed to
lowering free fatty acid levels, and/or visceral fat, along with liver weight and body weight,
when administered to a subject, who may be fed with a high fat diet.
According to one embodiment, methods and beta-cryptoxanthin compositions 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 beta-cryptoxanthin compositions herein and methods of use
thereof reduce inflammatory markers.
According to one embodiment, beta-cryptoxanthin compositions and methods of use thereof can
protect organs, which may be at risk because of cardiometabolic syndrome, such as the eye and
liver by reducing oxidative stress and/or inflammatory manifestations.
In one embodiment, beta-cryptoxanthin compositions and methods herein are directed to treat
and/or be evaluate for their effect on the management of risk factors associated with
cardiometabolic syndrome, in a subject, in need thereof, when administered in an effective
amount(s).
In another embodiment, as per methods described herein, beta-cryptoxanthin compositions
described herein are evaluated for effectiveness in significantly overcoming the cardiometabolic
syndrome and associated risk factors such as body weight, body fats, lipid profile, blood glucose
and the like.
The methods as described herein are comprised of evaluating effect of beta-cryptoxanthin
composition on prevention and treatment of cardiometabolic syndrome through gene expression
study on adipocyte cell system. CCAAT/enhancer binding proteins (C/EBP) are involved in
different cellular responses, such as in the control of cellular proliferation, growth and
differentiation, in metabolism, and in immunity. Their expression is regulated at multiple levels,
including hormones, mitogens, cytokines, nutrients, and other factors. The encoded protein has
been shown to bind to the promoter and modulate the expression of the gene encoding leptin, a
protein that plays an important role in body weight homeostasis. C/EBPa is involved in
adipogenesis and with normal adipocyte function. C/EBPa promotes adipogenesis by inducing
the expression of PPARy.
Fatty Acid Synthase main function is to catalyze the synthesis of palmitate from acetyl-CoA and
malonyl-CoA, in the presence of nicotinamide adenine dinucleotide (NADPH), into long-chain
saturated fatty acids. The role of fatty acid synthase is implicated in the regulation of fatty acid
synthesis and net accumulation of lipid in liver and adipose tissue. The role of fatty acid synthase
is implicated in the regulation of fatty acid synthesis and net accumulation of lipid in liver and
adipose tissue. FAS expression was controlled possibly at transcriptional level through
peroxisome proliferator- activated receptor (PPARs) and sterol regulatory element-binding
proteins (SREBPs) mediated signaling path way.
Stearoyl-CoA desaturase-1 ( SDC-1) is a key enzyme in fatty acid metabolism. The elevated
expression levels of SCDl are found to be correlated with obesity. This phenomenon depends on
increased expression of fatty acid biosynthetic enzymes that produce required fatty acids in large
quantities. Alteration in SCDl expression changes the fatty acid profile of these lipids and
produces diverse effects on cellular function. High SCDl expression is correlated with metabolic
diseases such as obesity and insulin resistance, whereas low levels are protective against these
metabolic disturbances. However, SCDl is also involved in the regulation of inflammation and
stress in distinct cell types, including -cells, adipocytes, macrophages, endothelial cells, and
myocytes.
Particularly the effect of the beta-cryptoxanthin compositions are evaluated for fat accumulation,
modulation of collagen and transforming growth factor beta (TGF-beta) signaling pathways in
high fat fed diet (HFFD) rats. The effect of beta-cryptoxanthin compositions are also evaluated
on vascular endothelial growth factor (VEGF), nuclear factor erythroid 2 (NrF2), nuclear factor
kappa-light-chain-enhancer of activated B cells (NFkB), Inducible nitric oxide synthase (INOS),
intercellular adhesion molecule 1 (ICAM-1) and heme oxygenase 1 (HO-1) pathways in retinal
tissue of HFD treated rats.
Additionally the beta-cryptoxanthin compositions are evaluated on beta-carotene oxygenase 2
(BC02), tumor necrosis factor alpha (TNF-a), peroxisome proliferator-activated receptor gamma
(PPAR-), nuclear factor erythroid 2 (Nrf-2), nuclear factor kappa-light-chain-enhancer of
activated B cells (NFkB), insulin receptor substrate 1 (IRS-1), heme oxygenase 1 (HO-1)
pathways in liver tissue of HFD treated rats.
The compositions herein include beta-cryptoxanthin concentrates of high purity. In particular,
beta-cryptoxanthin concentrates containing about 10-80% by weight total xanthophylls (total
carotenoids) of which the trans-beta-cryptoxanthin content is about 75-98% by weight and the
remaining including 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.
Processes are described 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.
In some embodiments, a process is described for the isolation of beta-cryptoxanthin crystals
containing at least or about 80% by weight of total xanthophylls (total carotenoids) in free form,
out of which the trans-beta-cryptoxanthin content is at or about or at least 98.5% by weight, the
remaining including trace amounts of zeaxanthin, trans- capsanthin, beta-carotene and other
carotenoids derived from oleoresin and extracts of plant materials such as Capsicum sources.
In some embodiments, a process is described for the preparation of beta-cryptoxanthin crystals
containing at or about or at least 40% by weight of total carotenoids, out which the trans-betacryptoxanthin
is at or about or at least 90% by weight, the remaining including trace amounts of
zeaxanthin, trans-capsanthin, beta-carotene and other carotenoids derived from oleoresin and
extracts of plant materials such as Capsicum sources.
In some embodiments, a process is described for the preparation of beta-cryptoxanthin crystals
containing at or about or at least 10% by weight of total carotenoids, out of which the trans-betacryptoxanthin
is at or about or at least 75% by weight, the remaining including zeaxanthin, transcapsanthin,
beta-carotene and traces amounts of other carotenoids derived from oleoresin and
extracts of plant materials such as Capsicum sources.
In some embodiments, a process is described for the preparation of beta-cryptoxanthin crystals
containing total carotenoids at or about 10 to at or about 80% by weight, out of which the transbeta-
cryptoxanthin content is in the range of at or about 75 to at or about 98% by weight, the rest
including zeaxanthin, trans-capsanthin, beta-carotene and trace amounts of other carotenoids
derived from a starting material like saponified Capsicum extract.
In some embodiments, a process is described for the preparation of high purity betacryptoxanthin
from capsicum oleoresin or saponified capsicum extract. In some embodiments,
residual solvent-free beta-cryptoxanthin crystals, in which trans-beta-cryptoxanthin form the
major ingredient in the total carotenoids. In one embodiment, processes herein provide recovery
of carotene hydrocarbon fractions rich in beta-carotene.
In some embodiments, the process for obtaining high purity trans-beta-cryptoxanthin includes:
saponification of esterified xanthophylls in Capsicum extract, which results in free
xanthophylls and which is purified by washing with acidified water, followed by drying to obtain
a carotenoid mass;
treating the carotenoid mass with non-polar solvent under stirring, followed by filtration and
concentration to obtain a mass;
subjecting the mass to column chromatography using silica gel and elution using non-polar
solvent to remove beta-carotene;
• eluting the column with non-polar solvent containing at or about 2% polar solvent, and
obtaining an eluent after concentration of a concentrate showing at or about 10% total
carotenoids by weight, of which trans -beta-cryptoxanthin comprises at or about 75% by weight;
treating the above concentrate with ethanol under stirring, followed by cooling to at or about 10
°C and filtering to obtain a semi -purified crystalline mass showing total xanthophylls at or about
40% by weight, of which trans-beta-cryptoxanthin comprises at or about 98%> by weight; and
washing the crystalline mass with hexane containing about 20% ethyl acetate, cooling to at or
about -10°C and filtering to obtain a high purity crystalline material showing at or about 80%
total xanthophylls by weight, of which trans- beta-cryptoxanthin comprises at or about 98.5%>
by weight.
In some embodiments, a process is described for the preparation of a beta-cryptoxanthin enriched
concentrate from plant material comprising at or about 10-80% by weight total xanthophylls, of
which at or about 75-98% by weight is trans-beta-cryptoxanthin. The process comprises: (a)
mixing an oleoresin of plant material comprising xanthophylls esters with an aliphatic alcoholic
solvent; (b) saponifying the xanthophylls esters present in the oleoresin with an alkali at an
elevated temperature; (c) removing the aliphatic alcoholic solvent followed by addition of water
to obtain a diluted resultant mixture; (d) adding a diluted organic acid to the diluted resultant
mixture to form a water layer and a precipitated xanthophylls mass; (e) removing the water layer
and washing the precipitated xanthophylls mass with a polar solvent; (f drying the precipitated
xanthophylls mass to obtain a crude xanthophylls mass; (g) washing the crude xanthophylls mass
with a non- polar solvent and concentrating the non-polar solvent washings to obtain a
concentrated crude xanthophylls mass; (h) transferring the concentrated crude xanthophylls mass
to a silica gel column and washing with a non-polar solvent; (i) eluting the column with a
mixture of non-polar and polar solvent and concentrating the elutions to obtain a trans- betacryptoxanthin-
rich xanthophylls concentrate; (j) admixing the trans-beta- cryptoxanthin-rich
xanthophylls concentrate with an aliphatic alcohol and then cooling; and (k) filtering and drying
the trans-beta-cryptoxanthin-rich xanthophylls concentrate to obtain a purified trans-betacryptoxanthin
concentrate.
In some embodiments, the xanthophylls esters in the oleoresin of plant material in step (a) are
present at or about 6-8% by weight. In some embodiments, the aliphatic alcohol of step (a) or (j)
is selected from the group consisting of ethanol, methanol, isopropyl alcohol, and mixtures
thereof.
In some embodiments, the ratio of oleoresin to alcohol in step (a) ranges from at or about 1:0.25
to at or about 1:1 weight/volume. In some embodiments, the alkali of step (b) is selected from
the group consisting of sodium hydroxide, potassium hydroxide, and mixtures thereof. In some
embodiments, the ratio of oleoresin to alkali in step (b) ranges from at or about 1:0.25 to at or
about 1:0.5 weight/weight.
In some embodiments, the elevated temperature of step (b) ranges from at or about 75 to at or
about 85 °C. In some embodiments, the addition of water in step (c) is at or about 5 times that of
the oleoresin (weight/weight).
In some embodiments, the diluted organic acid of step (d) is acetic acid or phosphoric acid. In
some embodiments, the diluted organic acid of step (d) is a solution of at or about 20% to at or
about 50% organic acid.
In some embodiments, the polar solvent of step (e) is water.
In some embodiments, the non-polar solvent of steps (g), (h), and (i) is selected from the group
consisting of a hexane, a pentane, a heptane, and mixtures thereof.
In some embodiments, the crude xanthophylls mass and non-polar solvent of step (g) is in a ratio
of at or about 1:10 to at or about 1:15 weight/volume. In some embodiments, the concentrated
crude xanthophylls mass of step (g) comprises beta-carotene, trans-beta-cryptoxanthin, transcapsanthin,
zeaxanthin, and trace amounts of other carotenoids, such as capsorubin or
violaxanthin.
In some embodiments, the concentrated crude xanthophylls mass and the non- polar solvent of
step (h) are in a ratio of at or about 1:5 to at or about 1:8 weight/volume. In some embodiments,
a carotene concentrate is obtained by distilling the non-polar solvent washing of step (h). In some
embodiments, the carotene concentrate is beta-carotene.
In some embodiments, the polar solvent of step (i) is selected from the group consisting of a
propanone, a pentanone, and mixtures thereof. In some embodiments, the non-polar solvent and
polar solvent of step (i) are in a ratio of at or about 95:5 to at or about 98:2. In some
embodiments, the trans-beta-cryptoxanthin-rich xanthophylls concentrate of step (i) comprises at
or about or at least 10% by weight of total xanthophylls, of which trans-beta- cryptoxanthin
content is at or about or at least 75% by weight.
In some embodiments, the cooling in step j) is performed at or about 0 °C. In some
embodiments, the purified trans-beta- cryptoxanthin concentrate of step (k) comprises at or about
or at least 40% by weight of total xanthophylls, of which trans-beta-cryptoxanthin content is at or
about or at least 90% by weight.
In some embodiments, the process further comprises a step (1): washing the purified trans-betacryptoxanthin
concentrate with a mixture of non-polar and ester solvent and cooling for
precipitation to obtain high purity trans-beta-cryptoxanthin crystals. In some embodiments, the
high purity trans-beta-cryptoxanthin crystals of step (1) comprises at or about or at least 80% by
weight of total xanthophylls, of which trans-beta- cryptoxanthin content is at or about or at least
98% by weight. In some embodiments, the ester solvent of step (1) is ethyl acetate and the nonpolar
solvent of step (1) is hexane. In some embodiments, the non-polar solvent and ester
solvent of step (1) are in a ratio of at or about 80:20 to at or about 90: 10. In some embodiments,
the temperature for cooling in step (1) is at or about - 10 °C.
In some embodiments, a process is described for the preparation of a beta-cryptoxanthin enriched
concentrate from plant material comprising at or about or at least 80% by weight total
xanthophylls, of which at or about or at least 98% by weight is trans-beta- cryptoxanthin, the
process comprising: (a) mixing an oleoresin of plant material comprising xanthophylls esters
with ethanol, wherein the ratio of oleoresin to ethanol is at or about 1:1 weight/volume; (b)
saponifying the xanthophylls esters present in the oleoresin with potassium hydroxide without
addition of water, wherein the ratio of oleoresin to potassium hydroxide is at or about 1:0.25
weight/weight; (c) applying heat to the oleoresin to elevate the temperature up to reflux at or
about 80-85 °C; (d) agitating the oleoresin for about 3 to 5 hours at or about 80-85 °C; (e)
evaporating the ethanol under vacuum followed by addition of water at or about 5 times that of
the oleoresin (weight/weight) to obtain a diluted resultant mixture and agitating for at or about 1
hour; (f) neutralizing the diluted resultant mixture with about 25% acetic acid to form a water
layer and a precipitated xanthophylls mass; (g) separating the water layer from the precipitated
xanthophylls mass and washing the mass with water to remove soaps and other polar soluble
materials; (h) drying the precipitated xanthophylls mass under vacuum to obtain a crude
xanthophylls mass; (i) washing the crude xanthophylls mass with at or about 1:10 hexane
(weight/volume) and concentrating the hexane washings to obtain a concentrated crude
xanthophylls mass; j ) transferring the concentrated crude xanthophylls mass to a silica gel
column at a ratio of at or about 1:5 (weight/weight) and eluting with hexane to obtain a carotene
fraction; (k) washing the column with at or about 98:2 hexane to acetone and concentrating the
washings to obtain a trans-beta-cryptoxanthin-rich xanthophylls concentrate; (1) admixing the
trans- beta-cryptoxanthin-rich xanthophylls concentrate with at or about 1:2 ethanol under
stirring and then cooling at or about 10 °C for about 8 hours; (m) filtering and drying the transbeta-
cryptoxanthin-rich xanthophylls concentrate under vacuum to obtain a purified trans-betacryptoxanthin
concentrate; and (n) washing the purified trans-beta-cryptoxanthin concentrate
with at or about 80:20 hexane :ethyl acetate and cooling to at or about -10 °C for about 18 hours
for precipitation to obtain high purity trans-beta-cryptoxanthin crystals.
In some embodiments, the total xanthophylls of the processes comprise byproducts selected from
zeaxanthin, trans-capsanthin, beta-carotene, trace amounts of other carotenoids, and any
combinations thereof.
In some embodiments, the plant material used in the processes or to derive the betacyrptoxanthin
concentrates is selected from at least one of the group consisting of fruits,
vegetables, and mixtures thereof. In some embodiments, the plant material is from a capsicum.
In some embodiments, beta-cryptoxanthin concentrates herein may be administered in a dosage
form selected from beadlets, microencapsulated powders, oil suspensions, liquid dispersions,
capsules, pellets, ointments, soft gel capsules, tablets, chewable tablets or lotions/liquid
preparations. In some embodiments, the beta-cryptoxanthin concentrate is added to or as part of
another composition.
In some embodiments, compositions herein includes a beta- cryptoxanthin concentrate derived
from plant material, wherein the concentrate comprises at or about or at least 10% by weight
total xanthophylls, of which at or about or at least 75% by weight is trans-beta-cryptoxanthin. In
some embodiments, the total xanthophylls comprise by-products selected from zeaxanthin, transcapsanthin,
beta-carotene, trace amounts of other carotenoids such as capsorubin or violaxanthin,
and combinations thereof. In some embodiments, the composition further comprises a
pharmaceutically acceptable ingredient or a food grade ingredient.
In some embodiments, the total xanthophylls of the beta-cryptoxanthin concentrate comprise b y
products selected from the group consisting of zeaxanthin, trans- capsanthin, beta-carotene, trace
amounts of other carotenoids such as capsorubin or violaxanthin, and combinations thereof.
In some embodiments, a beta-cryptoxanthin concentrate is provided, which contains at or about
10-80% by weight total xanthophylls, of which at or about 75-98% by weight is trans- betacryptoxanthin,
the remaining including 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. In some embodiments, the concentrate comprises at or about
or at least 10% by weight total xanthophylls, of which at or about or at least 75% by weight is
trans-beta-cryptoxanthin. In some embodiments, the concentrate comprises at or about or at least
40% by weight total xanthophylls, of which at or about or at least 90% by weight is trans-betacryptoxanthin.
In some embodiments, the concentrate comprises at or about or at least 80% by weight total
xanthophylls, of which at or about or at least 98% by weight is trans-beta-cryptoxanthin.
The plant material is derived from sources including, but not limited to, fruits and vegetables. In
some embodiments, the plant material is derived from capsicum. 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.
In some embodiments, beta-cryptoxanthin enriched concentrates herein 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 beta-cryptoxanthin enriched concentrates herein can also be
provided in a food or feed (including liquid or solid) composition. It will be appreciated that
suitable delivery methods include, but are not limited to, oral, parenteral, subcutaneous,
intravenous, intramuscular, intraperitoneal, transdermal, intracranial, or buccal administration.
Compositions herein comprising the trans-beta-cryptoxanthin enriched concentrates herein
include in some embodiments 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.
Compositions herein may include an "effective amount" of the trans-beta-cryptoxanthin enriched
concentrates. An "effective amount" refers to an amount effective, at a dose and in certain
circumstances for a period of time to achieve a desired result, for example in methods of
treatment or prevention of symptoms for use in such methods. The effective amount may vary
according to factors such as the disease state, age, sex, and weight of the individual.
The beta-cryptoxanthin compositions herein includes an active material present including betacryptoxanthin
(BCX), which is extracted for example from paprika oleoresin by saponification
followed by purification through column chromatography. Compositions herein are enriched
with trans-beta-cryptoxanthin. In an embodiment, the extract is suspended in a suitable oil
medium to obtain 5% oil suspension. In an embodiment, the suspension was evaluated in animal
model described herein below. For human consumption, the compositions herein include final
formulations into powders, granules, beadlets, and can be administered by oral solid dosage
forms such as tablets, capsules.
In an embodiment, an effective amount herein relates to the amount of BCX present in the
composition.
In an embodiment, a daily dose duration can range from at or about 3 months to at or about 2
years, or till the desired effect is achieved in a subject. It will be appreciated that there may be
no fixed time period for the daily doses as it may be less or longer than such range. It will also
be appreciated that the dose may be given continuously daily during this period or the
administration can be stopped after obtaining a desired effect in a subject, and can also be
restarted again as needed. It is appreciated that dose periods herein include the experiment
durations or by general volunteer study period which can be extended to 12 months.
In an embodiment, an effective daily dose includes a range of at or about 250 micrograms to at
or about 30 mg/kg body weight.
In an embodiment, an effective daily dose includes a range of at or about 150 micrograms to at
or about 20 mg/kg body weight.
In an embodiment, an effective daily dose includes a range of at or about 200 micrograms to at
or about 10 mg/kg body weight.
Compositions and methods of preparing beta-cryptoxanthin are disclosed in Applicant's
copending published application US 2015/0361040, which is herewith incorporated by reference.
The following examples are given by the way of illustration and therefore should not be
construed to limit the scope of the disclosures or innovations herein.
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
advantages of which are explained hereunder in greater detail in relation to non-limiting
exemplary illustrations.
Examples
Process for extraction of beta-cryptoxanthin from Paprika oleoresin
Example 1:
A weighed quantity of 100 g of Paprika oleoresin containing 7.72% total xanthophylls and a
color value of 1,23,515 units (HPLC profile of the oleoresin: beta- 15.36% carotene; 10% transbeta-
cryptoxanthin; 7.6% zeaxanthin; and 31.50% trans- capsanthin) was mixed with 100 ml
ethanol and 25 g potassium hydroxide pellet. The reaction mixture was heated to a temperature
of 80-85 °C with stirring. This saponification process was maintained for 3-5 hours at 80-85 °C
with gentle agitation. The reaction mixture was cooled, and then ethanol was distilled out from
the mass. A measured volume of water (700 ml) was added to the reaction mixture and agitated
for 1 hour. The solution was neutralized with 25% acetic acid solution. The water layer from
the mass was separated, and the mass was washed thrice with water. The mass was collected and
dried under vacuum. The saponifed mass concentrate obtained was 124 g with a total
xanthophylls content of 3.73% by weight (HPLC profile of the saponifed mass concentrate:
22.53% beta-carotene; 12.32% trans-beta-cryptoxanthin; 11% zeaxanthin; and 29.3% transcapsanthin).
T e saponified mass concentrate was washed two times with 1:10 hexane (wt/vol) at room
temperature under stirring, filtered, and the combined filtrate concentrated to obtain a
concentrated crude xanthophylls mass. The concentrated crude xanthophylls mass (hexane
concentrate) obtained was 72 g with a total xanthophylls content of 3.2% (HPLC profile of the
concentrated crude xanthophylls mass: 39.01% beta-carotene; 21.78% trans-beta-cryptoxanthin;
5.70% zeaxanthin; and 9.86% trans-capsanthin).
The residue (saponified xanthophylls) remaining after hexane wash was 22 g, which on analysis
showed a total xanthophylls content of 10% (HPLC profile of the residue: 0.7% beta-carotene;
3.43% trans-beta-cryptoxanthin; 15.32% zeaxanthin; and 52.84% trans-capsanthin).
The hexane concentrate was dissolved in a minimum amount of hexane and subjected to column
chromatographic separation. The column was packed with 1:5 concentrate to Silica 100-200
mesh (wt/wt). The column was washed with hexane, and the separated band was collected and
concentrated (yield 55 g with a total xanthophylls content of 2.3%, HPLC profile: 99.8% betacarotene).
The column was then eluted with 98:2 hexane: acetone (v/v), and the eluent collected
and concentrated. This concentrate layer was enriched with beta-cryptoxanthin (yield 5.2 g with
a total xanthophylls content of 10.26%, HPLC profile: 75.56%> trans-beta-cryptoxanthin).
Finally, the column was washed with acetone and the washings concentrated to obtain transcapsanthin
enriched residue.
Example 2
A quantity of approximately 100 g of Paprika oleoresin containing 6.50% total xanthophylls and
a color value of 1,05,457 units (HPLC profile of the oleoresin: 15.73% beta-carotene; 9.07%
trans-beta-cryptoxanthin; 10.54% zeaxanthin and 31.38% trans- capsanthin) was mixed with 100
ml ethanol and 25 g potassium hydroxide pellet. The reaction mixture was heated to a
temperature of 80-85 °C with stirring. This saponification process was maintained for 3-5 hours
at 80-85 °C with gentle agitation. The reaction mixture was cooled, and then ethanol was
distilled out from the mass. A measured volume of water (700 ml) was added to the reaction
mixture and agitated for 1 hour. The solution was neutralized with 40% acetic acid solution. The
water layer from the mass was separated, and the mass was washed thrice with water. The mass
was collected and dried under vacuum. The saponified mass concentrate obtained was 126 g
with a total xanthophylls content of 3.73% by weight (HPLC profile of the saponified mass
concentrate: 16.34% beta-carotene; 9.41% trans-beta-cryptoxanthin; 8.57% zeaxanthin; and
24.35% trans-capsanthin).
The saponified mass concentrate was washed two times with 1:10 hexane (wt/vol) at room
temperature under stirring, filtered, and the combined filtrate concentrated to obtain a
concentrated crude xanthophylls mass. The concentrated crude xanthophylls mass (hexane
concentrate) obtained was 76.15 g with a total xanthophylls content of 3.26% (HPLC profile of
the concentrated crude xanthophylls mass: 31.80% beta- carotene; 14.04% trans-betacryptoxanthin;
4.35% zeaxanthin; and 8.70% trans- capsanthin).
The residue (saponified xanthophylls) remaining after hexane wash was 16 g, which on analysis
showed a total xanthophylls content of 11% (HPLC analysis of the residue: 1.22% beta-carotene;
0.75% trans-beta-cryptoxanthin; 33.29% zeaxanthin; and 29.99% trans-capsanthin).
The hexane concentrate was dissolved in a minimum amount of hexane and subjected to column
chromatographic separation. The column was packed with 1:5 concentrate to Silica 100-200
mesh (wt/wt), eluted with hexane, and the first band separated was collected and concentrated
(yield 54.72 g with a total xanthophylls content of 1.08%, HPLC profile: 85.88% beta-carotene).
The column was then eluted with 98:2 hexane: acetone (v/v) collecting the eluent fraction and
concentrated. This fraction was enriched with beta-cryptoxanthin, yielding 4.02 g with a total
xanthophylls content of 9% (HPLC profile of the enriched beta-cryptoxanthin concentrate:
76.04% trans-beta- cryptoxanthin). Finally the column was washed with acetone.
The 4.02 g fraction concentrate was stirred with 1:2 ethanol (wt/vol) for 1 hr, chilled for 8 hrs at
10 °C, filtered, and the precipitate dried under vacuum. The yield obtained was 0.42 g
crystalline precipitate with a total xanthophylls content of 42.45%. The HPLC profile of the
crystalline precipitate showed 98.3% trans-beta-cryptoxanthin.
Example 3
A weighed quantity of Paprika oleoresin (100 g) containing 6-8% > by weight total xanthophylls
and a color value of 100,000 units (HPLC profile of the oleoresin: 15.36% beta-carotene; 10%
trans-beta-cryptoxanthin; 7.6% zeaxanthin; and 31.50% trans- capsanthin) was mixed with 100
ml ethanol and 25 g potassium hydroxide pellet. The reaction mixture was heated to a
temperature of 80-85 °C with stirring. This saponification process was maintained for 3-5 hours
at 80-85 °C with gentle agitation. The reaction mixture was cooled and then ethanol was
distilled off from the mass under vacuum. A measured volume of water (700 ml) was added to
the reaction mixture and agitated for 1 hour. The solution was neutralized with 25% acetic acid
solution. The water layer from the mass was removed, and the mass was washed thrice with
water. The mass was collected and dried under vacuum. The saponified mass concentrate
obtained was 121 .75 g with a total xanthophylls content of 4.92% by wt (HPLC profile of the
saponified mass concentrate: 21.76% beta-carotene; 12.74% trans-beta-cryptoxanthin; 10.13%
zeaxanthin; and 38.25% trans-capsanthin).
The saponified mass concentrate was washed two times with 1: 10 hexane (wt/vol) at room
temperature under stirring, filtered, and the combined filtrate concentrated to get a concentrated
crude xanthophylls mass. The concentrated crude xanthophylls mass (hexane concentrate)
obtained was 85.81 g with a total xanthophylls content of 3.21% by wt (HPLC profile of the
concentrated crude xanthophylls mass: 35.28% beta-carotene; 19.65% trans-beta-cryptoxanthin;
3.99% zeaxanthin; and 13.88% trans-capsanthin).
The residue (saponified xanthophylls) remaining after hexane wash was 25.65 g, which on
analysis showed a total xanthophylls content of 10.42% by wt (HPLC analysis of the residue:
0.7% beta-carotene; 1.24% trans-beta-cryptoxanthin; 18.98% zeaxanthin; and 52.32% transcapsanthin.
The hexane concentrate was dissolved in minimum amount of hexane and subjected to column
chromatographic separation. The column was packed with 1 :5 concentrate to Silica gel 100-200
mesh (wt/wt), eluted with 5-8 volumes of hexane, and the first band separated was eluted and
concentrated (yield 55 g with a total xanthophylls content of 2.29% wt, HPLC profile: 99% betacarotene).
The column was then eluted with 98:2 hexane: acetone (vol/vol) collecting the eluent
fraction and concentrated. This concentrate was enriched with beta-cryptoxanthin, yielding 9.06
g with a total xanthophylls content of 6.12% by wt (HPLC profile of the enriched betacryptoxanthin
concentrate: 71.80% trans-beta-cryptoxanthin). Finally the column was eluted
with acetone.
The 9.06 g beta-cryptoxanthin concentrate was stirred with 1:2 ethanol (wt/vol) for 1 hr, chilled
for 8 hours at 10 °C, filtered, and the precipitate dried under vacuum. The yield obtained was 0.5
g with a total xanthophylls content of 42.35% by wt. The HPLC profile of the crystal showed
98.3% trans-beta-cryptoxanthin content.
The 0.5 g beta-cryptoxanthin precipitate was dissolved in a minimum amount of 80:20 hexane:
ethyl acetate (vol/vol) and chilled for 18 hrs at -10 °C, filtered, and the precipitate dried under
vacuum. The yield obtained was 0.03 g with a total xanthophylls content of 80% and HPLC
profile for trans-beta-cryptoxanthin of 98.50%.
Example 4
In -vitro evaluation of beta-cryptoxanthin composition
3T3L1 murine adipocytes model system was used to understand basic cellular mechanisms
associated with diabetes, obesity and related disorders. Nutrigenomics study was carried out to
evaluate effect of beta-cryptoxanthin on adipocyte cells, particularly differentiated 3T3-L1 cells,
the study was based on real-time polymerase chain reaction (Real time PCR) with dose 12.5
ug/mL. The effect of beta-cryptoxanthin on Adipocyte differentiation and its activity for bio
markers PPARG, SCD-1, acetyl Coa carboxylase (ACC), SREBP-1, C/EBP and FAS were
evaluated.
It was observed that BCX down regulated C/EBP and fatty acid synthase (FAS) . BCX also
down regulated Stearoyl-CoA desaturase-1 (SCD-1), wherein down-regulation of SCD-1 is an
important component of leptin's metabolic actions. See Fig.4, where each set of bar graphs for
tests with BCX and without BCX (DMSO) correspond from the leftside bar respectively with the
heading legend of the factors tested (i.e. for the bar results of BCX, 0.9 for ACC alpha, 0.39 for
C/EPB alpha, 0.33 for FAS, 0.7 for PPAR gamma, and 0.38 for SCD-1).
Example 5
In -vivo evaluation of beta-cryptoxanthin composition in rat
a. Effect of beta-cryptoxanthin compositions on cardiometabolic markers, fat
accumulation, modulation of collagen and TGF-beta signaling pathways in high fat fed
diet (HFFD) rats
Animals
Male Sprague Dawley rats (7 rats/ group, 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. After acclimation for 2 weeks, the rats were divided into four
groups: Rats were randomly divided into the following groups: (1) Control, (2) High Fat Diet
(HFD) (42% of calories from fat), (3) control + beta-cryptoxanthin (2.5 mg/kg) 4) HFD (42% of
calories from fat) + beta-cryptoxanthin (2.5 mg/kg ) was administered daily as supplement for 12
weeks.
i) Effect of BCX on visceral fat:
Visceral fat decreased in HFD treated BCX rats only. HFD increased visceral fat. Similarly
decrease in body weight was observed in HFD treated BCX rats. Liver weight decreased in HFD
treated BCX rats. (Table 1, Figure 1A, B, C)
Table 1
Consumption of HF diet produced a significant (P<0.001) increase in body weight (BW)
compared to the consumption of normal diet (normal control group). Beta-cryptoxanthin
supplementation significantly reduced the body weight as compared to the HFD control group
(P<0.05). Visceral fat and liver weights were significantly higher in the HFD group as compared
to the control group (P< 0.05). In rats fed a HF diet supplemented with Beta-cryptoxanthin, a
tendency towards a decrease in visceral fat was observed (P< 0.05) (see Fig 1).
ii) Effect of BCX on Lipid and Lipoproteins:
BCX reflects its significant role in cholesterol lowering effects. According to Table 2 (Figure 2 E
to H) total cholesterol decreased in HFD treated BCX rats, whereas LDL cholesterol and TG
decreased in BCX treated rats. Amount of HDL, which is good cholesterol, was almost
unchanged, even after administering BCX supplementation.
Table 2
iii) Effect of BCX on Metabolic Markers/Hormones :
According to the study glucose, insulin and FFA (Free Fatty Acid) level decreased in HFD
treated BCX rats. On the other hand leptin was decreased significantly in HFD treated BCX and
adiponectin was increased in HFD treated BCX. It was observed BCX inhibited the glucosemediated
changes in metabolic markers and lipid profile (see Table 3, Figure 2 A-D).
Table 3 : Serum glucose, insulin, leptin, adiponectin and lipid profile in control and HFD
rats
There was a significant (P<0.001) elevation in serum glucose, insulin, and leptin levels in HFDinduced
obese rats compared with control rats.
Total cholesterol (T-C), free fatty acids (FFAs), triglycerides (TGs), high-density lipoprotein
(HDL), low-density lipoprotein (LDL) and very-low-density lipoprotein (VLDL) were checked
in serum of control and HFD-induced obese rats, respectively. The concentrations of serum lipid
profiles were significantly increased in experimental obese rats as compared to the normal rats.
Treatment with beta-cryptoxanthin significantly reduced the concentrations of serum glucose,
insulin, leptin, lipids concentrations in obese rats but decreased adiponectin concentration in
HFD rat (PO.05) (see Fig.2).
iv) Effect of BCX on oxidative stress markers:
Oxidative stress is significantly reduced in BCX treated rats in serum and liver. HFD rats had
high thiobarbituric acid reactive substance (TBARS) and BCX treated HFD rats significantly
reduced oxidative stress by reducing TBARS in retina and serum. BCX is known as provitamin
A. These results further support its antioxidant activity in reducing oxidative stress markers and
protects eyes and other associated conditions.
Table 4
b. Effect of Beta-cryptoxanthin compositions on retinal tissue in HFD treated rats
Beta-cryptoxanthin has several functions that are important for human health, including roles in
antioxidant defense and cell-to-cell communication. Beta-cryptoxanthin is a precursor of vitamin
A, which is an essential nutrient needed for eyesight, growth, development and immune
response. Increase in reactive oxygen species (ROS) is one of the major retinal metabolic
abnormalities associated with the development of diabetic retinopathy. NF-E2-related factor 2
(Nrf2), a redox sensitive factor, provides cellular defenses against the cytotoxic ROS. In stress
conditions, Nrf2 dissociates from its cytosolic inhibitor, Kelch-like ECH-associated protein 1
(Keapl), and moves to the nucleus to regulate the transcription of antioxidant genes including
the catalytic subunit of glutamylcysteine ligase (GCLC), a rate-limiting reduced glutathione
(GSH) biosynthesis enzyme.
Ocular neovascularization (NV) is a major cause of the blindness associated with ischemic
retinal disorders, such as proliferative diabetic retinopathy (PDR), retinopathy of prematurity
(ROP), and age-related macular degeneration (AMD). Studies showed that nitric oxide (NO),
produced by inducible nitric oxide synthase (iNOS), plays an important role in eye diseases such
as glaucoma, ROP and AMD.
Animals fed on HFD showed an increased upregulation of inflammatory and proangiogenic
markers. This animal model may be useful to study mechanisms of diabetic retinopathy and
therapeutic targets.
According to Figure 5 the intensity of the bands was quantified by densitometric analysis. Data
are expressed as a ratio of normal control value (set to 100 %). The bar represents the standard
error of the mean. Blots were repeated at least 3 times (n=3) and a representative blot is shown.
-actin was included to ensure equal protein loading.
HO-1 is a sensitive marker for assessing light-induced insult in the retina. Increased expression
of HO-1 is thought to be a cellular defense against oxidative damage, and its expression may
play an important role in protecting the retina against light damage (see Figure 6).
Leukocytes play a critical role in ocular diseases such as uveitis, diabetic retinopathy, and
choroidal neovascularization. Intercellular adhesion molecule (ICAM)-l is essential for the
migration of leukocytes. Control of ICAM-1 expression may lead to therapies for these diseases.
Down regulation of ICAM-1 expression to reduce retinal neovascular disease by inhibiting
leukocyte infiltration (see Figure 7).
To treat/prevent eye diseases like AMD, the effect of BCX on inhibition or induction of iNOS
can be checked in animal models. BCX decreased iNOS and may be potential for
neovascularization (see Figure 8).
It is also observed that BCX inhibited the glucose-mediated induction of NF-kB expression in
retina (see Figure 9) which suggest that selective inhibition of the NFkB pathway in glial can be
potent clinical approach for the treatment of vision loss in glaucoma.
Nrf2 is involved in the cytoprotective mechanism in the retina in response to ischemiareperfusion
injury and suggests that pharmacologic induction of Nrf2 could be a new therapeutic
strategy for retinal ischemia-reperfusion and other retinal diseases (see Figure 10).
VEGF has been considered to be a mediator of diabetic retinopathy. Inhibition of VEGF reduces
retinal neovascularization (see Figure 11).
c. Effect of Beta-cryptoxanthin compositions on liver tissue in HFD treated rats
Animals and diets
The experiment was performed using 28 male Sprague-Dawley rats (8 weeks old, weighing 180
± 20 g), purchased from the Inonu University Laboratory Animal Research Center (Malatya,
Turkey). Rats were housed in cages in a temperature and humidity controlled environment, on a
12-hr light and 12-hr dark cycle, designed for the purpose of the study. The temperature inside
the rat cages was 2 1 ± 2°C, relative humidity was 55 ±5 % and consecutive light-dark cycles
lasted 12 hours. The protocol of the study was approved by the Animal Experimentation Ethics
Committee of Inonu University (Malatya, Turkey). All procedures involving rats were conducted
in strict compliance with relevant laws, the Animal Welfare Act, Public Health Services Policy,
and guidelines established by the Institutional Animal Care and Use Committee of the Institute.
Prior the starting the experiment, animals were assigned to either a regular diet (control; 12% of
calories from fat) or a high-fat diet (HFD, 42% of calories from fat).Control or HFD composed
according to the American Institute of Nutrition AIN-93 (Reeves et al., 1993) recommendations
of casein (20%), soybean oil (7%), wheat starch (53.2%), sucrose (10%), potato starch (5%), 1-
cysteine (0.3%), vitamin mix AIN-93M (1%) and mineral mix AIN-93M (3.5%). The high-fat
diets (42% calories from fat) were obtained from the basal AIN-93 diet, by replacement of wheat
starch with fat (tallow 15% and soybean oil 10%).For induction of obesity (insulin resistance),
the rats were fed with HFD for 1 weeks
Experimental protocol
After a one week period of adaptation to laboratory conditions, the animals were randomly
divided into four equal groups: 1) Control (n=7): untreated rats were allowed free access to a
standard diet; 2) BCX Group, rats were fed a standard diet supplemented with beta-cryptoxanthin
(n=7) 2.5 mg/kg; 3) HFD Group (high-fat diet; 42% of calories from fat; n =7 ) : rats were fed a
high-fat diet 4) HFD+BCX Group, rats were fed a high-fat diet (42% of calories from fat; n =7)
supplemented with beta-cryptoxanthin 2.5 mg/kg. 5% suspension of beta-cryptoxanthin was
dissolved in corn oil. At the end of the experiment, all rats were sacrificed by cervical
dislocation. Blood samples were taken from rats in the morning upon overnight fasting for
biochemical analyses and their visceral fat and liver samples were removed and weighed after
sacrificing the animals.
Laboratory measurements
Blood was collected by cardiac puncture using an anticoagulant-free vacutainer tube, later
centrifuged at 3,000 x g for 10 min to obtain serum and kept frozen at -80°C until it was assayed
for biochemical parameters and malondialdehyde (MDA). Serum biochemical parameters were
estimated using an automatic analyzer (Samsung LABGEO PT10, Samsung Electronics Co,
Suwon, Korea). Repeatability and device/method precision of LABGEOPX10 was established
according to the IVR-PT06 guideline. Serum insulin, leptin and adiponectin levels were
measured with the Rat Insulin Kit (Linco Research Inc, St. Charles, MO, USA) by ELISA (Elx-
800; Bio-Tek Instruments Inc, Vermont, USA).
The total antioxidant capacity (TAC) was measured using an antioxidant assay kit (Sigma, St
Louis, MO, USA). Trolox was used as an antioxidant standard to calculate Trolox equivalent
antioxidant capacity; absorbance readings were taken at 520 nm. Lipid peroxidation was
measured in terms of MDA formation, which is the major product of membrane lipid
peroxidation done by a previously described method (Karatepe, 2004) with slight modification.
The liver MDA content was measured by high performance liquid chromatography (HPLC,
Shimadzu, Tokyo, Japan) using a Shimadzu UV-vis SPD-10 AVP detector and a CTO-10 AS
VP column in a mobile phase consisting of 30 mM KH2P04 and methanol (82.5+17.5, v/v; pH
3.6) at a flow rate of 1.2 ml/min. Column effluents were monitored at 250 nm and the volume
was 20 ΐ . The liver homogenate (10%, w/v) was prepared in 10 mM phosphate buffer (pH 7.4),
centrifuged at 13,000 xg for lOmin at 4 °C, and the supernatant was collected and stored at -80
°C for MDA analysis (Karatepe, 2004). Activities of superoxide dismutase (SOD), catalase
(CAT) and glutathione peroxidase (GPx) of homogenized liver were measured using a
commercial kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer's
instructions.
Western blots Analysis
For Western blot analyses protein extraction was performed by homogenizing the liver in 1ml
ice-cold hypotonic buffer A, containing 10 mM2-[4-(2-Hydroxyethyl)-l-piperazinyl]
ethanesulfonic acid [HEPES] (pH 7.8), 10 mMKCl, 2 mM MgC12, 1mM DTT, O.lmM EDTA,
and 0.1 mMphenylmethylsulfonyl-fluoride (PMSF). The homogenates were added with 80 ΐ of
10% Nonidet P-40 (NP-40) solution and then centrifuged at 14,000 xg for 2 min. The
precipitates were washed once with 500 ΐ of buffer A plus 40 of 10% NP-40, centrifuged, resuspended
in 200 ΐ of buffer C [50 mM HEPES [pH 7.8], 50 mMKCl, 300 mM NaCl, O.lmM
EDTA, 1mM dihiothreitol [DTT], O.lmM PMSF, 20% glycerol], and recentrifugedat 14,800 xg
for 5 min. The supernatants were collected for determinations of NF-, VEGF, iNOS, ICAM,
Nrf2, and HO-1 according to the method described by Sahin et al. [2012]. Equal amounts of
protein (50 g) were electrophoresed and subsequently transferred onto a nitrocellulose
membrane (Schleicher and Schuell Inc., Keene, NH, USA).
Antibodies against NF-, TNF-a, Nrf2, HO-1, PPAR-, and p-IRSl, (Abeam (Cambridge, UK)
were diluted ( 1:1000) in the same buffer containing 0.05% Tween-20. Protein loading was
controlled sing a monoclonal mouse antibody against -actin (A5316; Sigma). Bands were
analyzed densitometrically using an image analysis system (Image J; National Institute of
Health, Bethesda, USA). (Figure 12)
Statistical analysis
Sample size was calculated based on a power of 85% and a P-value of 0.05. Data are expressed
as mean ± standard deviation. Differences among the groups were evaluated using the General
Linear Model (GLM) procedure of SAS at baseline. If ANOVA indicated significance, a Fisher's
multiple comparison test was performed. The alpha level of significance was set at P < 0.05.
Effect of beta-cryptoxanthin compositions on oxidative metabolites and antioxidant
capacity in liver tissue
Antioxidant capacity, catalase, superoxide dismutase (SOD) increased in BCX treated rats and
decreased TBARS in liver. These results suggest BCX protects liver from oxidative stress due to
hyperglycemia.
Table 5
GSHPx: (Glutathione peroxidase); CAT: (catalase); SOD: (superoxide dismutase); TAC:
(total antioxidant capacity)
Rats fed a HFD had lower levels of total antioxidant capacity (TAC) activities of superoxide
dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) and higher
malondialdehyde (MDA) concentration than rats fed a standard diet (P < 0.001 for all). Betacryptoxanthin
administration increased activities of these enzymes and decreased MDA
concentration in rats fed a HFD (P < 0.05) (See Fig 3)
BCX compositions upregulate BC02 expression. BC02 acts as a protective antioxidant and
plays a crucial role in protection against oxidative damage (see Figure 13).
It is also observed that decrease in TNF alpha decreases oxidative stress in liver tissue (see
Figure 14).
BCX activated PPAR gamma in HFD treated rats shows that BCX had a significant role in CMS
and antioxidant pathways (see Figure: 15).
BCX compositions decrease Nrf2 expression, which improves glucose homeostasis, possibly
through its effects on fibroblast growth factor 2 1 (Fgf 21) and/or insulin signaling in liver tissue
of HFD rats (see Figure 16).
Further NFkB decreased in HFD treated with BCX. These results show BCX as antioxidant anti
inflammatory provitamin A (see Figure 17).
According to Figure- 18 increased HO-1 is associated with diabetes and may contribute to the
progression of insulin resistance in obese patients by promoting chronic inflammation. HFD rats
treated with BCX decreased HOI.
Decreased IRS-1 was also associated with a decrease in glucokinase expression and a trend
toward increased blood glucose. HFD rats treated BCX increased IRS-1 in liver so there is a
potential decrease in blood glucose and glucose management (See Figure 19).
It is observed that beta-cryptoxanthin inhibited liver NFkB and TNF-a expression by 22% and
14% and enhanced liver Nrf2, HO-1, PPAR-a, andp-IRSl levels were enhanced by 1.1.43, 1.41,
3.53, and 1.33, fold, respectively (P< 0.001).
We claim:
1. A method for prevention and treatment of cardiometabolic syndrome, comprising administering
a beta-cryptoxanthin composition in an effective amount to a subject fed with high fat diet; and
evaluating effect of composition on cardiometabolic markers, oxidative stress markers and
inflammatory makers, to assess overall management of cardiometabolic syndrome.
2. The method of claim 1, wherein the beta-cryptoxanthin composition is administered to the
subject fed with high fat diet in a daily dose of about 0.1 to 100 mg/kg body weight and
evaluated to assess overall management of cardiometabolic syndrome.
3. The method of claim 1, wherein the beta-cryptoxanthin composition is administered in an
effective amount to the subject fed with high fat diet and is evaluated on cardiometabolic
markers including one or more of body weight, body fat, lipid profile, blood glucose, blood
insulin, leptin, and liver weight.
4. The method of claim 3, wherein the lipid profile is evaluated through assessment of one or more
of triglycerides levels, serum LDL levels, serum HDL levels and total cholesterol levels, when
administered to the subject fed with high fat diet.
5. The method of claim 1, wherein the beta-cryptoxanthin composition is administered in an
effective amount to the subject fed with high fat diet and is evaluated on oxidative stress markers
and inflammatory markers related to vital body organs such as eye and liver.
6. The method of claim 4, wherein the evaluating includes checking an effect on oxidative stress
markers including one or more of SOD, CAT, GPx, and TBARS to assess liver function and
indications of cardiometabolic syndrome.
7. The method of claim 4, wherein the evaluating includes checking effect on inflammatory stress
markers including one or more of HO-1, ICAM, INOS, NfkB, NrF2, and VEGF to assess eye
function and indications of cardiometabolic syndrome.