The present invention further relates to a method for the preparation of these compositions.

Background of the Invention Epidemiological studies strongly suggest that high intake of vegetables and fruits reduce the risk of some, but not all, cancers and vascular diseases. This is mostly attributed to natural antioxidants, such as carotene, vitamin E and vitamin C (Block and Langseth Food Technology, 48: 80 ,1994) The "French paradox", the fact that French people have a low incidence of coronary heart disease despite their high saturated fat diet, was most recently explained by the flavonoid (a class of antioxidants) content of red wine drunk by the French.

Low density lipoprotein (LDL) uptake by macrophages in the arterial intima has been implicated in the initiation of atherosclerosis. It was shown that phenolic compounds in grapes and wine inhibit the oxidation of LDL and other studies show that intake of flavonoids, mostly from onion, which contain large amounts of quercetin glucoside, is inversely associated with coronary heart disease mortality (Hertog et al. Lancet, 342: 1007, 1993).

Betalain, a natural occurring pigment, is a term that includes two classes of pigments; the betacyanins, which are red violet and the betaxanithins, which are yellow. Betalains have been detected only in red-violet, orange and yellow pigmented botanical species belonging to related families of the order caryophyllales. Of the numerous natural sources of the betalains, red beet and prickly pear are the only edibles containing these pigments.

The major betalain in beets, betanin, accounts for most of the betacyanin content of the beet.

The betacyanins are derived from two aglycons, betanidin and iso-betanidin. Betanin is a betanidin 5-o- glucoside. Betanidin contains an ortho- diphenol group, which is suspected to be a very good electron donor or hydrogen donor, working as an active antioxidant.

In addition to their antioxidative effects, betanin and betanidin are cationized molecules which may increase their protective effects on cells and organelles against oxidation. Cell and organelle membranes are negatively charged and anionic sites on the cell surface have been shown to exhibit strong affinity for cationic agents. Furthermore, successful prevention of H202 induced damage to rat jujenal mucosa and E. coli cells by cationized catalase was described (Kohen R. and Shalhoub R. Free Rad. Biol. Med. 16: 571, 1994).

There are some disorders that are reported to be associated with oxygen derived radicals (see Table 1). Also, several diseases, especially atheroschlerosis, rheumatic arthritis and inflammatory bowels are related to oxidative stress. In addition, several forms of cancer are thought to be initiated by prooxidants. Such effects and diseases could be prevented by nutritional antioxidants.

The present invention relates to compositions containing antioxidants, derived from antioxidant containing fruits and vegetable, preferably from red beets and prickly pear, and to a method for the preparation of these compositions. The compositions of the present invention are useful for prevention and treatment of any disorders and diseases that are due to oxidative stress, a partial list of which is presented in Table 1.

Summary of the invention The present invention relates to compositions containing betacyanins (betanidins and betanins) and betaxanthins from antioxidant containing fruits and vegetables, preferably red beets, useful for prevention and treatment of disorders and diseases that are due to oxidative stress, especially vascular diseases.

These compositions may be pharmaceutical compositions containing, as an active ingredient, betacyanins and betaxanthins that are extracted from antioxidant containing fruits and vegetables.

The present invention also relates to a method for the preparation of said compositions comprising heating red beet raw material; peeling it, extracting its juice with a juice extractor; mixing the juice and pulp with pectolytic or cellulytic enzymes or both, after which the red beet juice may be freeze dried or spray dried to a powder which can be packed in capsules. Said compositions are also prepared using a Luwa concentrator.

Detailed Description of the Invention The potential antioxidant activity of betacyanins (betanin and betanidin) and betaxanthins from red beet was evaluated and their bioavailability in humans was determined. it was shown that the betanin of red beet juice is absorbed quickly from the gut to the blood stream. Also it was shown that cationized betanin is strongly attached to microsomes and that the betanins and other betalains in the red beet effectively prevent lipid peroxidation by active myoglobin and, because of their cationic properties, act as membrane site specific antioxidants, increasing their protective effects on cells and organelles against oxidation.

The said invention will be further and illustrated by the following experiments. These experiments do not intend to limit the scope of the invention but to demonstrate and clarify it only.

Fractionation of betacyanins and betaxanthins bv LC on Sephadex G-25 Fresh red beets were purchased from an outdoor market.

The red beets were heated by microwave at 980c for 5 minutes, cooled to 250c and then crushed in a blender for 1 minute. The pulp was centrifUged at 10,000g for 15 minutes at 40c and the supernatant was collected for betalains estimation and further separation on Sephadex G-25 column (30cmx2cm).

One ml of the supernatant was elected with acetic acid (1%). Three ml fractions were collected and tested for betanins at 536nm, betanidins at 542nm and betaxanthins at 480nm. The concentration of betacyanins was estimated using £ value of 65,000 for betanin, 54,000 for betanidin and 60,000 for betaxanthin (figure 1).

HPLC identification of betacyanins Red beet juice was run on HPLC at ambient temperature. The HPLC separation was done by a column of Water Novo-pack C-18, 3.9x150mm and a Merk Hitachi pump L6200A and a UV-Vis detector. The eluents were: solvent A - CH3OH/0.05M KH2PO4 18:82; solvent B - CH3OH gradient: 100% solvent A to 50% solvent B for 15 minutes.

The chromatograms were monitored at 535nm. The betanin and betanidin fractions were further separated by HPLC using the method described in Schwartz S.J. and von Elbe J.H.J. Agric Food Chem., 28:540, 1980.

Betanin, the main pigment in red-beet roots, attains a concentration of 600 mg/l in several varieties (Schwartz S.J. and von Elbe J.H.J. Agric Food Chem., 28:540, 1980).

The results brought here, which are based on HPLC separation, show that fresh red beet juice contains betanin at a concentration of about 400 mg/l and about 40 mg/l of iso- betanin.

Antioxidation activity Betanin and betanidin (and betaxanthins results not shown) were found to act as antioxidants in a model system containing linoleate, which its peroxidation is catalyzed by myoglobin or cytochrome C (figure 2).

The assay of diene conjugation was carried out according to the method described by Ben Aziz et al. Anal. Biochem. 84:38, 1970. The reaction mixture contained linoleic acid (0.05%), EDTA (0.5my1) in 0. it phosphate buffer at pH 7. Both assays employed a double beam recording.

carotene - linoleate oxidation activity was assayed colorimetrically as described in Kanner J. Am. Oil. Chem. Doc. 16:24,1979. The technique consists of following the decrease in absorbance at 460nm in the cuvette of a double beam recording spectrophotometer. The test sample contained 1.5 ml of buffered carotene - linoleate mixture at pH 7, 0.1 - 0.4 ml active fraction and distilled water, in a mixture as follows: ,8 carotene 14 I1M, linoleate 2mM, linoleate acid hydroperoxydes 2ism, Tween 20, 0.05% phosphate buffer pH 7 0. iM, EDTA 0.5M (except in the model catalyzed by Fe- ascorbic acid). The blank sample contained all the reagents except carotene.

Low density lipoproteins (LDL) were prepared from blood collected in EDTA by venipuncture (1.5mg/ml) from four nonsmoking healthy male volunteers and was centrifuged at 150 g at 40c to prepare plasma. Plasma LDL was prepared as described in Frankel et al. Lancet 341:434, 1993. Prior to the oxidation experiments LDL were exhaustively dialyzed with deoxygenated phosphate buffered (lOmM, pH 7.4) saline (100mM) for 24 hours. The final concentration of each sample was diluted to an equal protein content (0.25mg/ml LDL) with phosphate buffered saline (lOmM). The oxidative susceptibility of LDL was evaluated. LDL oxidation was determined by oxygen absorption during lipid peroxidation of LDL in a reaction system containing activated metmyoblobin (30to), 30 ,uM H202, using oxygen monitor with a Clark electrode.

Spectral changes showing the reduction of ferryl myoglobin radical with betanin and betanidin were determined spectrophotometrically at 545nm and 575 nm after interaction of equimolar concentration of metmyoglobin with H202 (50KlM).

It was shown that betanin and betanidin inhibit linoleate peroxidation 3 and 1.3 fold respectively, better than catechin, a well known antioxidant found in large concentrations in many fruits and vegetables, especially in wines and tea. In the same model system betanin was found to be 15 fold more active than cc tocopherol, the natural membrane antioxidant.

Betanin antioxidant activity was also examined in a model system containing activated myoglobin which initiate microsomal lipid peroxidation. In this model system the Iso of betanin was less than 2.5 I1M, and much lower than that of several flavonoids and phenolic acids such as catechin, quercetin, caffeic and gallic acids (table 2).

Finally, betanin was found to inhibit human LDL oxidation much better than catechin, the most active flavonoid in wines (figure 3).

Nutritional studv One of the most important questions concerning the in vivo activity of many of many natural antioxidants found in fruits and vegetables is their bioavailability.

To answer this important question three healthy volunteers drank 300ml red beet juice which contain about 120 mg betanin. Urine was collected from these volunteers at 0 and every 2 hours for a period of 10 hours. Urine was collected in plastic bottles marked with a subject code number and the data of collection. The urine was run on HPLC as described in HPLC identification of betacyanins.

A peak of betanin was identified 3 hours after drinking the juice and 60g/50mi of urine were found (figure 4). The total betanin collected in urine during 10 hours was about 10011g, which is about 0.1% of what was ingested.

Figure 5 shows that red beet juice is absorbed quickly from the gut, most probably from the stomach and only after 8 - 9 hours from the intestine. Red beet slices are absorbed only partially from the stomach and mostly from the intestine.

This study demonstrates that betanin, which is the 5-o- -glucose of betanidin, is absorbed from the gut to the blood stream and could be identified in urine at relatively high concentrations.

These are a few preferred embodiments ofthe present invention: Red beet juice enriched with betalains after enzvmatic treatment Red beet juice is produced from heated raw material at 980c for 30 minutes and peeled with a carborodium peeler. The beets are brought to room temperature by immersion in tap water. Juice is extracted from the beets by a "Santos" juice extractor. The juice is mixed for 30 minutes at room temperature with the pulp and pectolytic and cellulytic enzymes Pectinex Ultra JP-L and Celluclast 1.5L respectively at concentrations of 0. 1% each.

After the enzymatic treatment the juice is filtered through a Brawn machine separator and is pasteurized by a plate pasteurizer at 980c for 10 seconds, to achieve the high temperature needed for hot filing glass bottles of 250 ml. the bottles are cooled by tap water and brought to room temperature.

This treatment decreases the pulp from 35% to 7% and enriches the extraction of the juice from 64 % to 92%.

Concentrated red beet juice Concentrated red beet juice is prepared by using a Luwa concentrator. By this method the juice is concentrated to 60 Brix and 80% dry matter. The concentrated red beet juice contains about 3 mg/gr betacyanins. This concentrate could be used as an additive for foods, cosmetics and nutraceuticals.

Capsules of red beet betanins antioxidants Red beet juice, after enzymatic treatment, is freeze dried or spray dried to a powder.

This powder is packed in capsules of 0.5 gr which contain 2mg betacyanin each. Two capsules contain 4 mg of betacyanins which is the equivalent of one glass of red beet juice produced by the above mentioned methods. The dried powder may also be used as an antioxidant in food products Table 1 Some disorders reported to be associated with oxygen-derived radicals Inflammatory-Immune Injury Glomerulonepirritis, vasculitis, autoimmune disease, adult respiratory distress syndrome, rheumatoid arthritis, inflammatory bowel disease, pancreatitis Cancer Radiation induced cancer, cervical carcinoma, hepatocellular carcinoma, promotors of carcinogenesis, cancer in inflammatory bowel disease Ischemia/reoxygenation Stroke, myocardial infraction, organ transplantation (heart, lung, skin, cornea, kidney), organ preservation, reattachment of severed limbs, frostbite, Dupuytren's contracture, hemorrhagic shock, endotoxic shock, crush injury Metal overload Hemochromatosis, thalassemia, kwashlorkor, chemotherapy for leukemias, fUlminant hepatic failure, Wilson's disease, alcohol induced iron overload, nickel induced carcinogenesis, lead poisoning Toxins Hemolytic drugs, lead, halogenated hydrocarbons, ozone, oxides of nitrogen, asbestos, other mineral dusts, sulfUr dioxid, paraquat, aluminum, cigarette smoke, diabetogenic drugs, fava beans (hemolytic agents), anthracyclines (cardiotoxicity), heavy metals (nephrotoxicity), photosensitizing drugs, contact dermatitis Eye disorders Cataract development, deterioration after ocular hemorrhage, photochemical retinal damage, retinopathy of prematurity (retrolental fibroplasia) Inborn disease Porphyrias, sickle cell anemia, Fanconi's anemia, neuronal ceroid lipofUscinoses, thalassemia Insufficient antioxidant protection Keshan disease (severe selenuim deficiency), hemolytic disease of prematurity, retinopathy of prematurity, bronchopulmonary dysplasia, intracranial hemorrhage, neurological degeneration due to severe vitamin E deficiency (in inborn errors affecting intestinal fat absorption), acquired immunodeficiency syndrome Brain and central nervous system disorders Stroke, trauma, neurotoxicities (eg, of aluminum), effects of hyperbaric oxygen, Parkinson's disease, potentiation of traumatic injury, cerebral malaria TABLE 2 Inhibition of Ferryl Microsomal Lipid Peroxidation by Betanin* Betanin, µM TBARS, A 336 nm Inactivation, % Control, 0 0.335 0 2.5 0.158 50 5.0 00.20 95 125 0015 95 25.0 00 15 95 <BR> <BR> <BR> <BR> <BR> Microsomes were incubated 1 h at 370C with buffer phosphate pH 7.0, 0.05 M.