ANTIOXIDANT DERIVED FROM LENTIL AND ITS PREPARATION AND USES

Abstract: Methods of obtaining and using an extract of lentil seed coats that are enriched in polyphenolic species are presented. The extract exhibits antioxidant activities against free radicals, including superoxide. The extract can be used to stabilize and protect food, animal feeds and dietary supplements against oxidizing agents. The extract can decrease the risk of conditions associated with free radical and oxidant stress and can limit the activity of proinflammatory cells.

Description: DESCRIPTION ANTIOXIDANT DERIVED FROM LENTIL AND ITS PREPARATION AND USES Technical Field In one aspect, the invention relates to an antioxidant deπved from lentil seed husks In another aspect, the invennon relates to a process for prepaπng the antioxidant In yet another aspect, the invention relates to uses for the antioxidant Background Art Plant seed coats sometimes possess anOoxidants that protect the seed and embryo agamst oxidative damage during seed storage and germination For example, the anuoxidative components of seed coats of tamarind (Tamrmdus mdica L ) were studied by Tsuda et al (J Agric Food Chem 1994 42 2641-2674) Seed coats were extracted with ethanol, ethyl acetate, an ethyl acetate- ethanol mixture or methanol All of the solvent extracts inhibited the oxidation of linoleic acid, with the ethyl acetate extract being somewhat more active The major acove components were identified as ethyl 3,4 dihydroxybenzoate, 2-hydroxy 3,4 dihydroxyacetophenone, 3,4 dihydroxyphenyl acetate and epicatechin Essentially no antioxidant activity was detected in the germ It was reported in Tsuda et al (J Agπc Food Chem. 199442248-251 ) that pigments from red and green pea bean (Phaseola vulgaris L ) blocked the autoxidation of linoleic acid Pelargorudin glucoside, delphinidin glucoside and cyanidin glucoside were identified in an extract prepared from 05% tπfluroaceuc and 80% ethanol Later studies demonstrated that cyanidin and its glucoside block lφid peroxidation of erythrocyte membranes and posomes (Tsuda et al., J. Agπc Food Chem 1994 42 2407-10) d'Arcy and Jay (Phytochemistry 17 (4) 826-827 (1978) reported the polyphenols isolated from Lens culanans seed coats mclude tπcetin, luteolin, a diglycosyldelphmidm and two proanthocyanidms The germ contained mainly a kaempferol glycoside and 5-deoxykaempferol Masquelier, US 4,698,360, October 6, 1987 disclosed the isolation of a fraction enπched in proanthocyanidins from pine bark which possesses free radical scavenging effects The therapeutic use of the extracts is proposed for vascular pathologies, tumor promotion, hypoxia following atherosclerosis and cerebral involution associated with aging Normal cellular antioxidant defenses, consisting of enzymes (catalase, superoxide dismutases, glutathione peroxidases), antioxidant nutrients and endogenously formed antioxidants, can be impaired by environmental exposure to toxic mateπals including medications and pollutants, disease processes, as well as inadequate nutrition Free radicals and reactive oxygen species can attack DNA, leading to mutations and faulty repair mechanisms, as well as protems, thus alteπng enzyme, surface receptor and other essential functions Attack on lipids in cell membranes and in low density hpoproteins leads to the peroxidation of unsaturated fatty acid constituents with the possibility of initiating chain reactions Possible altered physiologic function include the incorporation of oxidized LDL into atherosclerotic lesions, a proposed early event in the disease process Free radicals and oxidative stress, that is the overproduction of reactive oxygen-containing molecules, has been associated with nearly 100 disorders, including certain types of cancer, retinal degeneration, cardiovascular disease, neurodegenerative diseases, ischemia-reperfusion, autoimmune conditions and other conditions associated with chronic inflammation Whether or not free radicals or reactive oxygen species are a cause or a consequence of the condition, mere are strong indications that antioxidant supplementation can at least ameliorate several of these conditions, including coronary artery disease, cataract, stroke rheumatoid arthπtos, inflammatory bowel disease, radiation injury, iron overload disease among other (Kehrer J P et al , Free radicals in biology sources, reactivities and roles in the etiology in human diseases, In Natural Antioxidants In Human Health and Disease, Academic Press 1994, 24-62), Ames et al , Proc Natl Acad Sci 1993, 90 7915-7922, Halhwell, Am J Med 1991, 91(suppl C) 14S-22S) The free radical theory of aging (Harman, D , Drugs Aging 1993, 3 60-80) proposes that the body's antioxidant and repair mechanisms are not 100% effective, so that over time, damage to DNA and other cell constituents gradually accumulates Ultimately a point is reached at which the cell can no longer function effectively, tissue and organ integπty is compromised, setting the stage for degenerative diseases and cancer Dietary antioxidants mfluence the course of these events Flavonoids possess many properties in addition to their antioxidant functions For example, flavonoids can inhibit platelet aggregation, to diminish the πsk of blood clot formation By virtue of effects on the body's detoxication systems, flavonoids are known to inhibit the initiation of certain cancers By inhibiting lntracellular signaling mechanisms, they can block cancer promotion as well (Pathak et al , Fitoterapia, 1991, 62 471-389) Antioxidants to inhibit the oxidation of water soluble nutπents, such as vitamin C, and essential fatty acids, such as linoleic acid, are desirable for improving human nutπtion Antioxidants are also desirable for their use in treating disease and in slowing the aging process Natural antioxidants which can be deπved from an inexpensive food source are especially sought after Disclosure of Invention In one embodiment of the invention, there is provided a process for producing a water-soluble antioxidant from lentil The process is earned out by first providing a quantity of lentil seed coats A soluble fraction of the lentil seed coats is extracted using water and at least one volatile organic solvent to produce a mixture of liquids containing lentil seed coat extract and solids The liquids are separated from the solids The water and at least one volatile organic solvent are then separated from the lentil seed coat extract to produce the water-soluble antioxidant Alternatively, a soluble fraction of the lentil seed coats can be extracted using hot water to produce an aqueous phase containing lentil seed coat extract and solids A portion of the lentil seed coat extract is separated as a precipitate from the aqueous phase The aqueous phase is separated from the solids and the precipitate to produce a claπfied aqueous phase The claπfied aqueous phase is extracted with a volatile organic solvent to produce a solvent extract of lentil seed coat The volatile organic solvent is separated from the solvent extract to produce the water soluble antioxidant Lentil is an accepted foodstuff and the antioxidants contained therein are concentrated in the seed coat The seed coat extract also does not contain some pro-oxidant species found in the germ The recovered mateπal contains over 70% of the tannin mateπal- ke material (measured as catechin equivalents) and it possesses a range of antioxidant species A vanety of accepted food grade solvents can be used for the extraction, and the extraction is simple and direct and produces a new and unproved antioxidant herbal extract which is water soluble and enπched in the major phenolic compounds of lentil The resulting extract is characterized by a phenolics content in the range of from about 1 to about 6 milligrams of catechin equivalents per 10 milligrams of extract In the form in which it is recovered from the above processes, it contains kaempferol, quercetin, proanthocyanidins including procyanidin and prodelphinidm as glycosides, and phenolic acids including ferulic acid, protocatechuic acid and caffeic acid The extract is highly effective at reducmg organic free radicals and superoxide, and thus is a potent antioxidant In accordance with another embodiment of the invention, the extract, or a component thereof, is used to inhibit the oxidation of mateπals intended for human or animal mgestion The method is earned out simply by incorporating into the mateπals an amount of the extract, sufficient to inhibit oxidation of the matenals This aspect of the invention is believed to have good utility for inhibiting the oxidation of vitamin C and linoleic acid for use as human nutπtional supplements In accordance with another embodiment of the invention, the extract, or a component thereof, is used in the treatment of a patient having a condition of tissue inflammation The method is earned out simply by admirusteπng to the patient an amount of the extract which is sufficient to reduce the inflammation This aspect of the invention is believed to have good utility for treating conditions of chrome mflammation, such as colitis, lupus erythematoscis, rheumatoid arthπtis, ischemia, and alcoholic liver disease It is believe the invention will also be used in treating tissue inflammation resulting from an injury, such as thermal and sun trauma, toxin exposure, excessive physical activity, radiation exposure, and infections It is believed that the extract will be highly effective m the treatment of chrome intestinal inflammation when administered orally Because this condition is difficult to effectively treat by known methods, the mvention is a sigmficant advance In another embodiment of the mvention, there is provided a method for inhibiting oxidation- induced apoptosis in healthy human cells The method is earned out by mamtainmg the cells in the presence of a sufficient concentration of the extract to inhibit oxidation-induced apoptosis In a preferred embodiment, the method would be earned out by usmg the extract as a human dietary supplement to ameliorate the effects of aging or oxidation-mduced cell damage caused by diet, exposure to medications or toxins, or disease process In yet another embodiment of the mvention, there is provided a method for causing necrotic cell death in cancerous human cells The method is earned out by exposing the cells to a sufficient concentration of the extract to cause the necrotic cell death of a portion of such cells The extract has low toxicity to normal cells and high toxicity to certain cancer cells In a further embodiment of the mvention, there is provided a method for selectively inhibiting the viability of human macrophage cells without inhibiting the viability of other healthy human cells found in close association with the macrophage cells The method is earned out maintaining both the macrophage cells and the other healthy cells in the presence of a concentration of least one component found in the extract which is sufficiently high to inhibit the viability of the macrophage cells but too low to inhibit the viability of the other healthy cells which are in close association with the macrophage cells Excess macrophage numbers are involved in the pathologies of certain chronic inflammation syndromes and their selective destruction or inhibition would be beneficial to patients afflicted with such syndromes Best Mode for Carrying Out the Invention Process for producing a water-soluble antioxidant from lentil The process is earned out by first providmg a quantity of lentil seed coats A soluble fraction of the lentil seed coats is extracted usmg water and at least one volatile organic solvent to produce a mixture of liquids containing lentil seed coat extract and solids The liquids are separated from the solids The water and the at least one volatile organic solvent are then separated from the lentil seed coat extract to produce the water-soluble antioxidant Food grade lentils range in color from green to brown The literature indicates that the color change reflects storage conditions of the lentil seed, among other factors Both types are applicable to the mvention Although any vaπety of lentil can be used in the mvention, seed coat from the seeds of domestic vaπeties of Lens esculenta is preferred, smce it has been tested with good results The coats or husks can be separated from the germ or cotyledon by any means, such as manually or by sieving Soaking the whole lentils for several hours in water facilitates the separation The coats are then preferably dπed and milled to a powder, more preferably a fine powder Generally speaking, the at least one volatile organic solvent consists of carbon, oxygen and hydrogen, because such solvents have relatively low human toxicity Usually, the at least one volatile organic solvent contains four or fewer carbon atoms, for reasons of volatility and extraction efficacy, and is selected from the group consisting of an alcohol and a ketone, for reasons of efficacy and low toxicity Preferably, the at least one volatile organic solvent is selected from the group consisting of methanol, ethanol and acetone, which have been tested with good results The liquids can be separated from the solids by any convenient means, such as by filtration The water and the at least one volatile organic solvent are preferably separated from the lentil seed coat extract at a temperature which does not exceed 40° C, order to preserve the antioxidant properties of the extract Vacuum distillation can be employed with good results The final powder is preferably then dπed at low temperature, such as by vacuum drying Alternatively, a soluble fraction of the lentil seed coats can be provided usmg hot water extraction to produce an aqueous phase containing lentil seed coat extract and solids A portion of the lentil seed coat extract is separated as a precipitate from the aqueous phase The aqueous phase is separated from the solids and the precipitate to produce a claπfied aqueous phase The claπfied aqueous phase is extracted with a volatile organic solvent to produce a solvent extract of lentil seed coat The volatile organic solvent is separated from the solvent extract to produce the water soluble antioxidant The hot water extraction can be earned out at a temperature between about 25° C and 100° C but is preferably earned out at near 100° C Precipitation is made to occur by addmg salt to the aqueous phase Sodium chlonde provides good results Separation of solids from the aqueous phase, such as by filtration, is conducted before and/or after, preferably after, the precipitation step The volatile organic solvent can be of the types mentioned previous, but ethyl acetate is preferred because it has been used with good results The antioxidant composition The extraction steps yield a concentrated pigmented residue which dissolves completely in water, reduces organic free radicals, quenches superoxide, inhibits the peroxidation of linoleic acid, and inhibits the oxidation of reduced ascorbic acid The extract is characteπzed by a phenolics content in the range of from about 1 to about 6 milligrams of catechm equivalents per 10 milligrams of extract, preferably m the range of from about 2 to about 4 milligrams of catechm equivalents per 10 milligrams of extract The phenolics are predominantly in the form of polyphenols, including flavonoids and phenolic acids The flavonoids melude flavones, flavanols and condensed tannins The flavones mciude luteolin The flavanols melude quercetin and kaempferol The condensed tanmns are in the form of proanthocyanidins including procyanidin, prodelphi din and their glycosides The phenolic acids include ferulic acid, protocatechuic acid, and caffeic acid The preferred composition contains kaempferol, quercetin, proanthocyanidins including procyanidin and prodelphinidin as glycosides, and phenolic acids including ferulic acid, protocatechuic acid and caffeic acid Generally speakmg, at least 50% by weight, preferably at least 70% by weight, of the proanthocyanidin is m the form of prodelphinidin Usmg the extraction procedures employed herein, prodelphinidin accounts for at least about 15% by weight, preferably greater than about 20% by weight, of the extract This amount corresponds to about 066% to about 1 04% of the lentil hull This is on the order of 4 times greater than values previously reported by d'Arcy and Jay (1975) Phrased another way, the extraction procedures employed herem recover a much greater amount of the prodelphimdin than the pπor art techniques and produce an extract havmg a much higher level of prodelphinidin than pπor art techniques The extract is preferably further characteπzed by the substantial absence of prooxidant species Such species can be found m the germ of the lentil, but are removed by the separation process descπbed above The ability of the extract to reduce organic free radicals is descnbed in terms of its ability to reduce dipicrylhydrazyl radical Generally speakmg, the extract has an IC50 in the range of from about 1 5 to about 15 micrograms per millihter as measured by reduction of dipicrylhydrazyl radical, preferably in the range of from about 4 04 to about 14 83 micrograms per mil liter The ability of the extract to reduce superoxide is determined by following the reduction of rutro blue tetrazohum by superoxide Generally speakmg, the extract has an IC50 in the range of from about 1 to about 10 micrograms per millihter as measured by reduction of nitro blue tetrazohum by superoxide, preferably m the range of from about 3 to about 6 micrograms per millihter, and most preferably in the range of from about 4 to about 5 micrograms per millihter Use as a preservative Lentil husk extract can prevent the oxidation of foodstuffs and nutnents appropπate for human consumption and thus may be added to foods as a food-based preservative The extract stabilizes a wide range of commonly ingested nutnents In accordance with another embodiment of the invention, the extract, or a component thereof, is used to inhibit the oxidation of matenals tended for human or animal ingestion The method is earned out simply by meorporatmg to the mateπals an amount of the extract which is sufficient to inhibit oxidation of the mateπals The invention can be used with foodstuffs, beverages, livestock feed, nutπtional supplements and pharmaceutical agents, for example, but is expected to be especially useful for preventing the oxidation of foodstuffs and beverages intended for human consumption as well as nutπtional supplements such as vitamins and essential fatty acids Test results show that the extract is highly effective in stabilizing linoleic acid and in maintaining ascorbic acid (vitamin C) in a reduced state Use to relieve chrome inflammation The extract, as a potent mixture of water-soluble, plant-based antioxidants, can be used as an antioxidant supplement for humans with the overall goal of preventing or ameliorating chrome degenerative diseases in which chronic inflammation and free radical pathology has been suggested This aspect of the invention is earned out by adm isteπng to the patient an amount of the extract which is sufficient to reduce the inflammation It is believed that the invention can be used to treat coronary artery disease, stroke, lschemia-reperfusion, rheumatoid arthπhs, autoimmune disorders such as lupus erythematoscis, mflammatory bowel disease such as colitis, radiation injury, iron overload disease, alcoholic liver disease, and other conditions of inflammation such as those due to thermal insult, sun insult, excessive physical activity, toxin exposures, and infections, by administering it orally, intravenously, intramuscularly, or by any other means It is believed that the extract will be highly effective in the treatment of chrome intestinal inflammation when administered orally For example, the extract can be administered orally as tablets, capsules, powders or beverages in divided does or as a single bolus to humans as well as to animals as in a veterinary practice Generally speakmg, in the range of from about 2 to about 2,000 milligrams of the extract should be administered to a human patient daily, usually the range of from about 15 to about 1500 milligrams daily The amount administered should be sufficient to result in decreased serum or plasma hpid peroxide level or urinary secretion products of pid peroxidation such as F2 isoprostanoids, and decreased urinary excretion of 8-oxo-guanιne and other markers of DNA excision repair of oxidative damage On a cellular basis, this aspect of the mvention is descπbed as inhibiting oxidation-induced apoptosis in healthy human cells by maintaining the cells in the presence of a sufficient concentration of the extract to inhibit oxidation-induced apoptosis The concentration of extract which is effective to accomplish this result is generally the range of from about 1 to about 100 micrograms per millihter se a§ an arrtiςariςer drug Tests demonstrate that the extract is toxic to cancer cells In yet another embodiment of the mvention, there is provided a method for causmg necrotic cell death in cancerous human cells The method is earned out by exposing the cells to a sufficient concentration of the extract to cause the necrotic cell death of a portion of such cells It appears that a concentration in the range of from about 200 to about 2,000 micrograms per millihter is adequate Use for macrophage inhibition Macrophages are an important part of the body's immune system They engulf their target cells and destroy them with oxidizing chemicals Under certain circumstances, the macrophages target healthy cells, causing a vaπety of autoimmune disorders or syndromes Rheumatoid arthπtis is an example of such a disorder In other situations, persistent inflammation, mduced m part by macrophages, causes tissue damage Inflammatory bowel disease is an example of such a disorder An effective treatment for patients suffeπng from such disorders would be desirable The extract of the invention selectively inhibits and even destroys excess macrophages The method is earned out maintaining both the macrophage cells and the other healthy cells m the presence of at a concentration of least one component found in the extract which is sufficiently high to inhibit the viability of the macrophage cells but too low to inhibit the viability of the other healthy cells which are m close association with the macrophage cells For this application, a concentration of the extract which is in the range of from about 1 to about 100 micrograms per millihter is believed suitable The mvention is further illustrated by the following examples EXAMPLE I. Preparation of the Extract a Preparation of seed coats from brown lentils Food grade brown lentils (518 g) were unmersed in deionized water for 10 hr at room temperamre then the lentil seed coats were collected manually The seed coats were air dπed and then milled to a fine powder (designated brown LH) The weight of brown LH was 36 45 g, representing a 7% yield b. Preparation of seed coats from green lentils Lentil seed coats were obtained from 1 Kg of food grade, green lentils The seeds were first moistened for 12 hr , then sieved to separate the seed coats from the cotyledons The husks were dried at 40C and then milled to a fine powder (designated green LH) The yield was 36 3 g, representing a 3 6% yield c. Solvent extraction of seed coats 1, Brown LH 3 4 g of brown LH were extracted 4 times with 1 liter of methanol-water ( 1 1 v/v) In each extraction, the solvent/husk mixture was heated to boiling for 15 minutes and then allowed to stand at room temperamre for 10 hr with constant stirring The suspension was filtered and the dark brown filtrate collected The combmed filtrates were concentrated to dryness by rotary evaporation at 40C The yield of brown residue (lentil husk methanol-water extract— LHME) was 758 mg, representing a 22 3% yield based on the initial weight of brown lentil seed coats it. Green LH 150 g of green LH were first washed with methanol (1 L, 4 times) without heating to remove chlorophyll LH was then extracted 4 times with methanol-water (1 1, v/v) with heating and stirring, as descπbed above The suspension was filtered and the dark brown filtrate was collected Upon standing a room temperature, a precipitate formed This was removed by filtration The combined filtrates were concentrated to dryness by rotary evaporation to yield 13 1 g of dark brown residue (LHME), representing a yield of 8 7 % based on the initial weight of green LH EXAMPLE II Characterization of the Extract a. Total phenolic content The percentage of total phenolic mateπal in LHME from brown seeds and green seeds was determined using the method of Same et al , J Am Oil Chemists Soc 1995, 72 1131 Solutions were prepared by dissolving dπed samples of LHME m water (5 mg/50 ml) The catechm standard ( 1 Omg/100ml) was dissolved in methanol After a reaction time of 3 minutes in 2 25% Folin-Ciocalteau reagent, sodium carbonate was added to a concentration of 2% Absorbance at 750 nm was recorded after 30 mm Results showed that LHME contains 2 33 to 3 03 mg of catechin equivalents per 10 mg of LHME Thus, 70-80% of the phenolic matenal of the LH is recovered in the LHME Recovered phenolic substances account for 3 8 to 5 2% of the dry weight ofthe LH This is a significantly greater recovery than the 2 8 to 4 2% of dry weight of seed husks reported by previous investigators b. Spectral identification of proanthocyanidins The UV-visible spectrum of lentil husk seed coat extract in water revealed a maximum absorption at 276 nm, similar to p e bark proanthocyamdms (lambda max 283 nm), and grape seed oligomenc proanthocyanidins (lamba max 279 nm) No absorption was detected between 300 and 900 run for any of these extracts in their native form Hydrolysis of the extracts was performed by heating in 5% hydrochloπc acid m butanol for 2 hr at 95 C This procedure yielded a magenta pigment m all cases This pigment is ascπbed to the flavylium cation ion acidic solution This spectral evidence for Bate-Smith reaction has been reported as charactenstic for proanthocyanidins and is confirmatory evidence for the identification of this species Furthermore, the maximum absorbance in the visible region of the spectrum appeared at 555-558 ran, and the spectrum of anthocyanidin from hydrolysis matched delphirudin c Thin laver chromatographv of LHME-Detection of phenols and flavonoids LHME was dissolved in a minimum amount of water methanol and subjected to thin layer chromatography on silica gel plates (0 2mm gel, GF254) The TLC plates were developed with butanol-acetic acid-water (4 1 2, v/v) at room temperature The TLC plates were then air dned and exposed to lodme vapor to locate species with unsaturated bonds, or sprayed with Folin- Ciocalteau reagent to visualize phenolic species, or sprayed with feme chlonde reagent containing aluminum chlonde m ethanol, in order to visualize flavonoid compounds The resulting colored complexes fluoresced in UV light at 365 nm Chromatographic analysis revealed the presence of 10 phenolic spots in LHME, 5 of which tested positive for the pyrogallol moiety ( 1 ,2,3 hydroxy derivatives similar to the gallic acid structure) Three species of LHME tested positive for flavonoids Comigration with reference glycosides, nanngin and hespeπdin, mdicated the presence of highly polar flavonoid denvatives, such as tπ and tetra glycosides Quercetin and kaempferol, and their glycosides, luteolin, ferulic acid and protocatechuic acid were identified by TLC cochromatography m vanous solvents The polyphenolic constituents acted as antioxidants as determined by the protection of beta carotene decomposition mduced by linoleic acid peroxidation Thm layer ehromatography of LHME and methanol extract of whole lentil was performed using silica gel plates Samples were dissolved in methanol or water Plates were developed with chloroform-methanol (12 5% methanol in chloroform, or 25% methanol in chloroform (v/v) After air drying, the plates were exposed sprayed with Folin-Ciocalteau reagent to locate phenolic compounds, or with a solution of linoleic acid and beta carotene to locate antioxidant species, according to the procedure of Mehtaetal , J Agr Food Chem (1994)42 1420-1422 Phenolic species in both LHMEs protected linoleic acid from oxidation Two prooxidant species that rapidly bleached beta carotene were detected in extract of whole lentil These were not detected LHME Consequently, major prooxidant species of whole lentils are absent from the lentil husk extracts d Fractionation of LHME 7 6 g of green LHME was dissolved in 1 liter of deiomzed water, then extracted 4 times with 700 ml of ethyl acetate, followed by extraction 4 times with 700 ml butanol The ethyl acetate extracts and butanol extracts were dπed over anhydrous sodium sulfate and concentrated m vacuo The ethyl acetate extract yielded 220 mg of yellow-brown powder, the butanol extract yielded 887 mg of brown powder The remaining aqueous extract was concentrated in vacuo and dπed to yield 6 121 g of dark brown residue, representing 80% of the starting mateπal The ethyl acetate, butanol and aqueous fractions of LHME were examined by thm layer ehromatography usmg silica gel plates The chromatograms were developed with butanol/ethanol/ water (4 1 1, v/v) and (4 1 2, v/v) and the spots were visualized usmg Folin-Ciocalteau and feme chlonde spray reagents The butanol fraction contained a mixture of dihydroxy and tπhydroxy phenols, the ethyl acetate fraction eontamed mainly dihydroxy compounds, while the aqueous fraction contain highly polar phenolics as glycosides, together with less polar polyhydroxy constituents as found in the other 2 fractions To hydrolyze flavonoid glycosides, 2 grams of the aqueous extract residue m 60 ml of 1 N HCl were refluxed for 2 hr at lOOC. The hydrofyzate was a deep magenta, indicative of a positive Bate- Smith reaction for proanthocyanidins. e Identification of phenolic species Agfycones were extracted from the hydrolyzate with butanol (4 times with 150 ml) After drying the butanol extract was concentrated in vacuo to yield 715 mg of a magenta powder. Thin layer chromatographic analysis was performed in butanol-acetic acid-water (4 1.2, v/v/v) on silica gel plates. The plates were developed with anisaldehyde reagent to reveal the presence of two procyanidin species and one predominant prodelphinidin species. EXAMPLE III FREE RADICAL QUENCHING BY LHME a. Reduction of organic free radical ta.a-diphenvl-B-pιcrvl-hvdrazvl radical. (DPPH-. (Hanto et al. Chem. Pharm. Bull. H988) 36:2090-2097. LHME was dissolved in water and the reduction of DPPH was performed as follows The 3 ml reaction mixture contained 0 05 mM DPPH in methanol and sample dissolved in water so that the final solvent concentration was methanol-water (1.1, v/v) The concentrations of antioxidant ranged from 0 98 to 500 μg ml. The decrease in absorbance at 517 nm after 1 minute was recorded. The IC50 of LHME ranged from 12.58 (+/- 0.17) to 14.83 (+/- 0.21) μg/ml. In contrast, the IC50 of the cotyledon extract was greater than 500 The ethyl acetate extract of LHME exhibited an IC50 of 6.62; the butanol extract, 4.04. For companson, the IC50s of the following reference compounds were observed: quercetin: 1.73, (+)catechin: 5 46, gallic acid: 0.63; ascorbic acid 2.56. b, Reduction of superoxide The reduction of superoxide was determined by following the reduction of mtro blue tetrazohum (NBT) by superoxide generated by three systems (I) Hypoxanthine-xanthme oxidase and NBT—The reaction mixture eontamed hypoxanthme 0 5mM, xanthine oxidase 1 67 U/ml, Tπs-HCl buffer, pH 7 4 50mM, and NBT ImM (u) Xanth e-xanthme oxidase and NBT-The reaction mixture eontamed xanthine 0 5mM, xanthme oxidase 1 67 U/ml, Tπs-HCl buffer, pH 7 4 50mM, and NBT ImM (in) Phenazme methosulfate (PMS)-NADH and NBT (Yen et al , J Agπc Food Chem (1994) 42629-632)~The reaction mixture eontamed phenazme methosulfate 60 μM, NADH 468 μM, Tπs-HCl buffer, pH 7 4, 50mM, NBT ImM LHME was dissolved in Tπs HCl at pH 7 4 and added to reaction mixtures over a concentration range from 0 7 to 333 3 μg/ml Reduction of NBT was followed by recording the absorbance at 560nm The following IC50 values were obtained For superoxide generated by (I), the IC50 was 4 52 (+/- 0 04) μg/ml, (as compared to 5 5 for ascorbic acid and I 9 for catechm) For superoxide generated by (n), the IC50 was 4 16 (+/- 0 12) μg/ml, (as compared to 7 4 for ascorbic acid and I 8 for catechin) For superoxide generated nonenzymatically by (in), the IC50 was 4 67 (+/- 0 05) μg/ml —which was the lowest of any reference compound or substance studied, including ascorbic acid (7 0), catechm (40 0), quercetin (21 1), standardized preparation of silymaπn (20 4), caffeic acid (47 25), propyl gallate (25 85), ferulic acid (201), gallic acid (19 45), pme bark proanthocyanidins ( 17 87) and green tea extract (18 8) Because all methods yielded a very low IC50, the results demonstrate an extremely effective quenching of superoxide radicals EXAMPLE IV PRESERVATION OF NUTRIENTS a. Inhibition of the oxidation of essential fattv acids LHME was evaluated according the thin layer chromatography-fluorescent method of Matsuda et al (J Agπc Food Chem 1994, 42 1850-6) Bnefly, 5 μg of sample dissolved m water was spotted on a silica gel TLC plate, which was sprayed with a 3% solution of linoleic acid m hexane The plate was dned, then irradiated continuously with UV light at 254 nm The time required for the fluorescent spots to disappear was considered the induction penod for hpid oxidation The induction penod for LHME was 183 minutes, as compared to 70 mmutes for alpha tocopherol Thus, LHME sigmficantly blocked the peroxidation of this essential fatty aαd in vitro more effectively than alpha tocopherol b Inhibition of oxidation of vitamin C Vitamin C (reduced) at a concentration of 40 μg/ml was mixed with equal volumes of antioxidant solutions over a range of 0 to 40 μg/ml in 0 1M potassium phosphate buffer, pH 7 4 (Lau et al , Analyst ( 1986) 111 665-670) The absorbance at 243 nm was followed at room temperature for 120 mmutes At a concentration of 15 μg ml, the LHME blocked approximately 90% of the degradation, while proanthocyanidin o gomeπc extract from grapeseed extract prevented 75% of degradation and catechin blocked 50% of the degradation Thus, LHME protects vitamin C in solution from oxidation, providmg a greater amount of vitamin C available to work as an antioxidant and polyphenols from the LHME can work synergistically with the vitamin C EXAMPLE V IN VITRO STUDIES a Inhibiting peroxvnrtnte-induced apoptosis Chronic inflammation is related to an overproduction of nitπc oxide, which can induce programmed cell death in epithelial cells Inflammatory mediators, including oxidants, can induce apoptosis Peroxynitnte is believed to be one of die oxidants formed from excessive superoxide and nitric oxide as produced during chrome inflammation In vitro systems have been used as models to study the pathophysiology of gut inflammation (Miller et al , Gastroenterology (1995) 109 1475-1483) Programmed cell death, apoptosis, refers to DNA directed nuclear fragmentation associated with cell death, as opposed to cell rupture (necrosis) The effect of LHME on the inhibition of apoptosis in culmred cell lines mduced by oxidative stress was studied in the following expeπment Human T84 epithelial cells, a cancer cell line deπved from gut epithelium, were grown m DMEM/F12 medium and mamtamed in a humidified, 5% carbon dioxide incubator at 37°C The T84 cells were treated with LHME steπhzed by ultrafiltration at final concentrations ranging from 10 to 30 μg/ml and incubated for 4 hr One group of cells was treated with 100 to 300 μM peroxynitnte, a sufficiently high concentration to induce apoptosis in cells incubated without antioxidants At the end of the test penod, cells were either stained for fragmentation by the TUNEL method, or cytosohc DNA fragments were quantified by a cell death detection ELISA assay Cell viability was assessed by trypan blue exclusion LHME consistently protected human cells from peroxynitπte-induced cell death b. Toxicity of LHME on established cell lines denved from human colon cancer The cell lines descπbed above were incubated m the presence of media containing 500 to 2000 μg/ml of LHME At this level, LHME extract caused necrotic cell death c. Selective inhibition of culmred normal macrophages Normal macrophages (cell line RAW 264 7) were cultured under standard conditions in the presence of 10 μg/ml of LHME Within 24 hrs , there was sigmficant loss of viability of the macrophages In contrast, other epithelial cells such as the colonic epithelial T84 cells were unaltered by the presence of LHME at a concentration of 30 μg/ml when incubated up to 5 days While certain preferred embodiments of the invention have been heretofore descnbed, the invention is not to be construed as so limited, except to the extent such limitations are found in the claims

Claims: Claims What is claimed is 1 A process for producing a water-soluble antioxidant from lentil, said process compπsmg providmg a quantity of lentil seed coats, extracting a soluble fraction of said lentil seed coats usmg water and at least one volatile organic solvent to produce a mixture of liquids containing lentil seed coat extract and solids, separating the liquids from the solids, and separating the water and the at least one volatile orgamc solvent from the lentil seed coat extract to produce the water-soluble antioxidant 2 A process as in claim 1 wherem the at least one volatile orgamc solvent consists of carbon, oxygen and hydrogen 3 A process as in claim 2 wherem the at least one volatile orgamc solvent contains four or fewer carbon atoms and is selected from the group consisting of an alcohol and a ketone 4 A process as in claim 3 wherein the at least one volatile orgamc solvent is selected from the group consisting of methanol, ethanol and acetone 5 A process as m claim 4 wherem the water and the at least one volatile orgamc solvent is separated from the lentil seed coat extract at a temperature not exceeding 40° C 6 A process as in claim 5 wherem the water and the at least one volatile orgamc solvent is separated from the lentil seed coat extract by vacuum drying 7 A process as in claim 5 wherem the lentil seed coat is from the seeds of Lens esculenta 8 A process as m claim 5 wherem the lentil seed coat extract has a prodelphinidin content of at least 15% 9 A process for producing a water-soluble antioxidant from lentil, said process compπsmg providmg a quantity of lentil seed coats, extracting a soluble fraction of said lentil seed coats usmg hot water to produce an aqueous phase containing lentil seed coat extract and solids, separating a portion of the lentil seed coat extract as a precipitate from the aqueous phase, separating the aqueous phase from the solids and the precipitate to produce a claπfied aqueous phase, extracting the claπfied aqueous phase with a volatile organic solvent to produce a solvent extract of lentil seed coat, and separating the volatile organic solvent from the solvent extract to produce the water soluble antioxidant 10 A process as in claim 9 wherein the precipitate is formed by adding salt to the aqueous phase 1 1 A process as in claim 10 wherem the salt compπses sodium chlonde 12 A process as in claim 1 1 wherem the volatile orgamc solvent compπses ethyl acetate 13 A process as in claim 12 wherein the hot water is at a temperamre of near 100 u C 14 A process as in claim 13 wherem the lentil seed coat is from the seeds of Lens esculenta 15 A process as in claim 13 wherem the lentil seed coat extract has a prodelphmidm content of at least 15% 16 An extract of lentil seed coat characteπzed by a phenolics content m the range of from about 1 to about 6 milligrams of catechm equivalents per 10 milligrams of extract 17 An extract of lentil seed coat as in claim 16 charactenzed by a phenolics content m the range of from about 2 to about 4 milligrams of catechm equivalents per 10 milligrams of extract 18 An extract of lentil seed coat as in claim 17 wherem the phenolics compπse polyphenols 19 An extract of lentil seed coat as in claim 18 wherein the polyphenols include flavonoids and phenolic acids 20 An extract of lentil seed coat as m claim 19 wherem the flavonoids are selected from the group consisting of flavones, flavanols and condensed tanmns 21 An extract of lentil seed coat as in claim 20 wherein the flavones compπse luteohn 22 An extract of lentil seed coat as in claim 21 wherein the flavanols are selected from the group consisting of quercetin and kaempferol 23 An extract of lentil seed coat as in claim 20 wherem the condensed tannins compπse proanthocyamdms 24 An extract of lentil seed coat as m claim 23 wherem the proanthocyamdms are selected from the group consisting of procyanidin, prodelphmidm, a glycoside of procyanidin, and a glycoside of prodelphmidm 25 An extract of lentil seed coat as in claim 19 wherein the phenolic acids are selected from the group consisting of ferulic acid, protocatechuic acid, and caffeic acid 26 An extract of lentil seed coat as in claim 16 further charactenzed by the substantial absence of prooxidant species 27 An extract of lentil seed coat as in claim 16 further characteπzed by an IC50 in the range of from about 1 5 to about 15 micrograms per millihter as measured by reduction of dipicrylhydrazyl radical 28 An extract of lentil seed coat as in claim 27 further characterized by an IC50 in the range of from about 4 04 to about 14 83 micrograms per millihter as measured by reduction of dipicrylhydrazyl radical 29 An extract of lentil seed coat as in claim 16 further characteπzed by an IC50 in the range of from about 1 to about 10 micrograms per millihter as measured by reduction of nitro blue tetrazohum by superoxide 30 An extract of lentil seed coat as in claim 29 further charactenzed by an IC50 in the range of from about 3 to about 6 micrograms per millihter as measured by reduction of nitro blue tetrazohum by superoxide 31 An extract of lentil seed coat as in claim 30 further characteπzed by an IC50 in the range of from about 4 to about 5 micrograms per millihter as measured by reduction of nitro blue tetrazohum by superoxide generated non-enzymatically in a phenazme methosulfate-NADH-NBT system 32 An extract of lentil seed coat as in claim 24 wherein at least 50% by weight of the proanthocyamdm compπses prodelphmidm 33 An extract of lentil seed coat as in claim 32 where at least 70% by weight of the proanthocya dm compπses prodelphinidin 34 An extract of lentil seed coat as in claim 33 compnsmg at least 15% by weight of prodelph idm 35 An extract of lentil seed coat as claim 34 compπsmg at least 20% by weight of prodelphinidin 36 An extract of lentil seed coat as m claim 17 consisting essentially of kaempferol, quercetin, proanthocyamdms including procyanidin and prodelphmidm as glycosides, and phenolic acids including ferulic acid, protocatechuic acid and caffeic acid 37 A method for inhibiting the oxidation of matenals mtended for human or a mal mgestion, said method compπsmg meorporatmg mto said materials an amount of an antioxidant composition deπved from lentil sufficient to inhibit oxidation of the matenals 38 A method as in claim 37 wherem the matenals are selected from the group consisting of foodstuffs, beverages, livestock feed, nutritional supplements and pharmaceutical agents 39 A method as m claim 38 wherem the mateπals are nutπtionai supplements selected from the group consisting of vitamins and essential fatty acids 40 A method as m claim 37 wherein the matenals are selected from the group consisting of foodstuffs and beverages mtended for human consumption 41 A method as m claim 39 wherein a sufficient amount of the antioxidant composition is incorporated into the foodstuff to inhibit the oxidation of linoleic acid 42 A method as in claim 39 wherem a sufficient amount of the antioxidant composition is incorporated into the foodstuff to inhibit the oxidation of ascorbic acid 43 A method as in claim 39 wherein the antioxidant composition compnses at least 15% by weight of prodelphmidm 44 A method as in claim 43 wherein the antioxidant composition consists essentially of kaempferol, quercetin, proanthocyamdms including procyanidin and prodelphmidm as glycosides, and phenolic acids including ferulic acid, protocatechuic acid and caffeic acid 45 A method for treating a patient having a condition of tissue inflammation, compnsmg administering to said patient an amount of an extract from lentil seed coat sufficient to reduce the inflammation 46 A method as m claim 45 wherem the condition of tissue inflammation is a condition of chrome inflammation selected from the group consisting of colitis, lupus erythematoscis, rheumatoid arthπtis, lschemia-reperfusion, iron overload disease, and alcoholic liver disease 47 A method as in claim 45 wherem the condition of tissue inflammation is a condition caused by injury selected from the group consisting of thermal trauma, sun trauma, toxm exposure, excessive physical activity, radiation exposure, and infection 48 A method as in claim 45 wherem the extract is administered orally, intravenously, or intramuscularly 49 A method as in claim 48 wherein the extract compnses at least 15% by weight of prodelphmidm 50 A method as in claim 49 wherein the extract consists essentially of kaempferol, quercetin, proanthocyamdms including procyanidin and prodelphmidm as glycosides, and phenolic acids including ferulic acid, protocatechuic acid and caffeic acid 51 A method as in claim 48 wherein a major portion of the proanthocyamdms are m the form of prodelphinidins 52 A method as claim 47 wherem in the range of from about 2 to about 2,000 milligrams of the extract are administered to the patient daily 53 A method as in claim 50 wherem in the range of from about 15 to about 1500 milligrams of the extract are administered to the patient daily 54 A method as claim 45 wherein a sufficient amount of the extract is administered to the patient to result in decreased serum or plasma pid peroxide level or urinary secretion products of lipid peroxidation such as F2 isoprostanoids, and decreased urinary excretion of 8-oxo-guanιne and other markers of DNA excision repair of oxidative damage 55 A method for inhibiting oxidation-mduced apoptosis in healthy human cells, said method compnsmg maintaining said cells in the presence of a sufficient concentration of an extract of lentil to inhibit oxidation-mduced apoptosis 56 A method as in claim 55 wherem the extract of lentil compnses at least 15% by weight of prodelphmidm 57 A method as claim 56 wherem the extract of lentil consists essentially of kaempferol, quercetin, proanthocyanidins including procyanidin and prodelphimdin as glycosides, and phenolic acids including ferulic acid, protocatechuic acid and caffeic acid 58 A method as m claim 56 wherem the concentration is in the range of from about 1 to about 100 micrograms per millihter 59 A method for causmg necrotic cell death in cancerous human cells, said method compnsmg exposing said cells to a sufficient concentration of an extract of lentil to cause the necrotic cell death of a portion of such cells 60 A method as m claim 59 wherem the extract of lentil compnses at least 15% by weight of prodelphmidm 61 A method as in claim 60 wherem the extract of lentil consists essentially of kaempferol, quercetin, proanthocyanidins including procyanidin and prodelphmidm as glycosides, and phenolic acids cludmg ferulic acid, protocatechuic acid and caffeic acid 62 A method as in claim 60 wherem the concentration is in the range of from about 200 to about 2,000 micrograms per millihter 63 A method for selectively inhibiting the viability of human macrophage cells without inhibiting the viability of other healthy human cells found in close association with the macrophage cells, said method compπsmg maintaining both the macrophage cells and the other healthy cells in the presence of at a concentration of least one component found m an extract of lentil which is sufficiently high to inhibit the viability of the macrophage cells but too low to inhibit the viability of the other healthy cells which are m close association with the macrophage cells 64 A method as m claim 63 wherem the extract of lentil compnses at least 15% by weight of prodelphimdin 65 A method as in claim 64 wherein the extract of lentil consists essentially of kaempferol, quercetin, proanthocyanidins mcludmg procyamdm and prodelphmidm as glycosides, and phenolic acids mcludmg ferulic acid, protocatechuic acid and caffeic acid 66 A method as m claim 63 wherein the concentration is m the range of from about 1 to about 100 micrograms per millihter AMENDED CLAIMS [received by the International Bureau on 12 January 1998 (12.01.98); original claims 1-66 replaced by new claims 1-25 (4 pages)] 1 A process for producing a water-soluble antioxidant from lentil, said process compnsmg providmg a quantity of lentil seed coats, extracting a soluble fraction of said lentil seed coats usmg water and at least one volatile orgamc solvent to produce a mixture of liquids containing lentil seed coat extract and solids, separating the liquids from the solids, and separating the water and the at least one volatile orgamc solvent from the lentil seed coat extract to produce the water-soluble antioxidant 2 A process as m claim 1 wherein the at least one volatile orgamc solvent is selected from the group consisting of methanol, ethanol and acetone 3 A process as m claim 2 wherein the lentil seed coat is from the seeds of Lens esculenta and the lentil seed coat extract has a prodelphmidm content of at least 15% 4 A process for producmg a water-soluble antioxidant from lentil, said process compπsmg providmg a quantity of lentil seed coats, extracting a soluble fraction of said lentil seed coats usmg hot water to produce an aqueous phase containing lentil seed coat extract and solids, separating a portion of the lentil seed coat extract as a precipitate from the aqueous phase, separating the aqueous phase from the solids and the precipitate to produce a claπfied aqueous phase, extracting the claπfied aqueous phase with a volatile organic solvent to produce a solvent extract of lentil seed coat, and separating the volatile orgamc solvent from the solvent extract to produce the water soluble antioxidant 5 A process as m claim 4 wherein the volatile orgamc solvent compnses ethyl acetate 6 A process as m claim 5 wherein the lentil seed coat is from the seeds of Lens esculenta and the lentil seed coat extract has a prodelphmidm content of at least 15% 7 An extract of lentil seed coat charactenzed by a phenolics content m the range of from about 1 to about 6 milligrams of catechm equivalents per 10 milligrams of extract 8 An extract of lentil seed coat as m claim 7 charactenzed by a phenolics content m the range of from about 2 to about 4 milligrams of catechin equivalents per 10 milligrams of extract and wherem the lentil seed coat is from the seeds of Lens esculenta 9 An extract of lentil seed coat as m claim 8 further charactenzed by the substantial absence of prooxidant species 10 An extract of lentil seed coat as claim 9 further characteπzed by an IC50 in the range of from about 1 5 to about 15 micrograms per millihter as measured by reduction of dipicrylhydrazyl radical 11 An extract of lentil seed coat as m claim 9 wherem the polypheonols compπses condensed tanmns mcludmg proanthocyamdm and at least 50% by weight of the proanthocyamdm compπses prodelphmidm 12 An extract of lentil seed coat as in claim 11 compπsmg at least 15% by weight of prodelphmidm 13 An extract of lentil seed coat as claim 8 consisting essentially of kaempferol, quercetin, proanthocyamdms mcludmg procyamdm and prodelphmidm as glycosides, and phenolic acids mcludmg ferulic acid, protocatechuic acid and caffeic acid 14 A method for inhibiting the oxidation of mateπals mtended for human or ammal mgestion, said method compnsmg meorporatmg mto said matenals an amount of an antioxidant composition deπved from lentil sufficient to inhibit oxidation of the mateπals 15 A method as in claim 14 wherein the mateπals are selected from the group consisting of foodstuffs, beverages, livestock feed, nutritional supplements and pharmaceutical agents 16 A method as m claim 15 wherein the antioxidant composition compπses at least 15% by weight of prodelphmidm 17 A method for treating a patient having a condition of tissue inflammation, compπsmg administering to said patient an amount of an extract from lentil seed coat sufficient to reduce the inflammation 18 A method as m claim 17 wherem the extract compnses at least 15% by weight of prodelphimdin and wherein in the range of from about 2 to about 2,000 milligrams of the extract are administered to the patient daily 19 A method as m claim 17 wherein a sufficient amount of the extract is administered to die patient to result m decreased serum or plasma hpid peroxide level or urinary secretion products of hpid peroxidation such as F2 isoprostanoids, and decreased urinary excretion of 8-oxo- guanine and other markers of DNA excision repair of oxidative damage 20 A method for inhibiting oxidation-mduced apoptosis in healthy human cells, said method compπsmg mamtammg said cells m the presence of a sufficient concentration of an extract of lentil to inhibit oxidation-mduced apoptosis 21 A method as m claim 20 wherem the extract of lentil compπses at least 15% by weight of prodelphmidm and wherem the concentration is in the range of from about 1 to about 100 micrograms per millihter 22 A method for causmg necrotic cell death in cancerous human cells, said method compπsmg exposmg said cells to a sufficient concentration of an extract of lentil to cause the necrotic cell death of a portion of such cells 23 A method as m claim 22 wherem the extract of lentil compπses at least 15% by weight of prodelphmidm and wherein the concentration is m the range of from about 200 to about 2,000 micrograms per millihter 24 A method for selectively inhibiting the viability of human macrophage cells without inhibiting the viability of other healthy human cells found in close association with the macrophage cells, said method compπsmg mamtammg both the macrophage cells and the other healthy cells in the presence of at a concentration of least one component found m an extract of lentil which is sufficiently high to inhibit the viability of the macrophage cells but too low to inhibit the viability of the other healthy cells which are close association with the macrophage cells 25 A method as m claim 24 wherem the extract of lentil compπses at least 15% by weight of prodelphmidm and the concentration is in the range of from about 1 to about 100 micrograms per millihter

Inventors: MUANZA DAVID N, RONZIO ROBERT A, SPARKS WILLIAM S, MUANZA, DAVID, N., RONZIO, ROBERT, A., SPARKS, WILLIAM, S.
Applicants: BIOTICS RESEARCH CORP, MUANZA DAVID N, RONZIO ROBERT A, SPARKS WILLIAM S, BIOTICS RESEARCH CORPORATION, MUANZA, DAVID, N., RONZIO, ROBERT, A., SPARKS, WILLIAM, S.
Application year: 1997
Main classification: A01N65
IPC codes: A23L1, A23L3, A61K31, A61K36, A61P3, A61P29, A61P35, A61P37, A61P39, C09K15, C12N5