Method of preparing and using compositions extracted from vegetable matter for the treatment of cancer

Description

This is a division of Ser. No. 09/616,150, filed Jul. 13, 2000, now U.S. Pat. No. 6,395,279 which, in turn, is a division of Ser. No. 09/162,038, filed Sep. 28, 1998 (a formal application which replaced provisional application Ser. No. 60/060,549 filed Oct. 2, 1997), now U.S. Pat. No. 6,261,565, which, in turn, is a continuation-in-part of Ser. No. 09/035,588 filed Mar. 5, 1998, now U.S. Pat. No. 6,033,714, which, in turn, is a continuation-in-part of Ser. No. 08/868,629, filed June 4, 1997, now U.S. Pat. No. 5,792,503, which, in turn, is a division of Ser. No. 08/614,545, filed Mar. 13, 1996, now U.S. Pat. No. 5,702,752.

This invention relates to compositions extracted from vegetable matter and more particularly to phytochemicals, including saponogenins and saponins, catechins, lignans, phenolic acids, and isoflavones, and especially those extracted from a family of plants including soy, flax, tea, and cocoa, and methods of using these compositions as nutritional supplements or food additives for treating neurological conditions.

As used herein, the term “isoflavone” includes malonyl, acetyl, glucoside, and aglycone forms of the isoflavones.

BACKGROUND

Currently, there is almost an epidemic of cancer; at least some of which is thought to be either caused or exacerbated by foods having a hormonal supplement derived from an animal origin. This is thought especially true for breast and prostate cancer. Other forms of cancers which are of special concern are skin cancer, colon cancer, urinary cancer, cancer of the bladder and the like.

It is thought that many of those cancers, especially breast and prostate cancers, are either preventable or treatable by a use of phytochemical fractions, especially isoflavones, as a source of supplemental hormones, and particularly if such use begins before a female reaches puberty. For males, apparently the treatments may begin at any time.

However, it is also thought that there are superior results when a plurality of such phytochemical fractions are consumed in combinations which are tailored to have a profile to treat or prevent such cancers. A proper diet should contain the desired phytochemical fractions. Many people do not have or do not like the kind of proper diet which provides the desirable effects. Hence, the challenge is to furnish the necessary phytochemical fractions in a form which is more acceptable. This is achieved in the present invention by a refining process which enables extraction, refining, isolation, and selection of specific phytochemical fractions which are combined and tailored to the needs of specific illnesses, particularly cancers.

Another object of this invention is to provide an optimized extract composition of phytochemical fractions which are present in sufficient concentration to be delivered at the proper dosage in an easy to consume form such as a pill, tablet, capsule, liquid or ingredient in a food including health bars.

This extract may be used alone or combined with one or more other plant extracts to produce the optimized composition. Further, this extract composition may be formulated with one or more other dietary nutrients, such as vitamins, minerals, amino acids, etc., to provide a nutritional supplement further optimized for a desired health effect. All of these ingredients may be combined with necessary binders, excipients, preservatives, colors and the like known to those in the industry in order to produce a suitable tablet, capsule, pill, liquid, cream, powder or food ingredient in a food including health bars.

The improved composition is obtained by fractionating a plant source high in isoflavones, lignans and other phytochemicals such as defatted soybean flakes, soy molasses, soy whey, red clover, alfalfa, flax, cocoa, tea, or kudzu root. These may be fractionated alone or in combination with these other plants known to be high in the various isoflavones, lignans, saponins and saponogenins, catechins and phenolic acids. The fractionation results in substantially removing water, carbohydrates, proteins, and lipids from the source material. The fractionation method may be preferably that disclosed in U.S. Pat. Nos. 5,702,752, 6,261,545; 6,017,555; 6,033,714 or 4,428,876, or an extraction using ethyl acetate or n-butanol may be used. U.S. Pat. Nos. 5,702,752; 6,017,555; 6,033,714; 6,261,545 are assigned to the assignee of this invention.

TABLE 1
Ingredients of experimental diets (grams)

	Diet 1	Diet 2	Diet 3	Diet 4	Diet 5	Diet 6
	casein	SPI	Casein/LSPC	SPI/LSPC	Casein/HSPC	SPI/HSP

SPI	0	200	0	200	0	200
Casein	200	0	200	0	200	0
DL-methionine	3	3	3	3	3	3
Corn starch	150	150	150	150	150	150
Sucrose	500	500	500	500	500	500
Cellulose, BW200	50	50	50	50	50	50
Corn oil	50	50	50	50	50	50
Mineral Mix. S10011	35	35	35	35	35	35
Vitamin Mix. V10011	10	10	10	10	10	10
Choline Bitartrate	2	2	2	2	2	2
Soy phytochemicals	0	0	2	2	10	10
Total (g)	1000	1000	1002	1002	1010	1010
(isoflavones, mg/kg diet)	0	245	341	586	705	950
1AIN formulation with minor modification by Dr. E. A. Ulman, Research Diets, Inc

TABLE 2
Final body weight, total food intake, total isoflavone intake, and tumor volume

		Food intake		Tumor volume
Treatment	Body weight	grams/m	Total isoflavone	(cm3)
Casein	22.4 ± 0.51	46.6 ± 3.1	0.00 ± 0.00	2.32 ± 0.312
SPI	23.1 ± 0.7	46.2 ± 2.8	17.00 ± 6.37	2.06 ± 0.32
Casein/LSPC	21.4 ± 0.7	41.2 ± 3.4	14.03 ± 14	1.88 ± 0.35
SPI/LSPC	22.6 ± 0.6	50.1 ± 4.7	29.36 ± 2.76	1.66 ± 0.29*
Casein/HSPC	22.2 ± 0.7	44.8 ± 6.1	76.38 ± 10.40	1.64 ± 0.22*
SPI/HSPC	22.0 ± 0.6	47.5 ± 1.7	92.53 ± 3.22	1.39 ± 0.30**
1Values are means ± SE. There are no significant differences of food intake or body weight among treatment groups.
2Compared with control group, SPI/LSPC. casein/HSPC, and SPI/HSPC had significantly smaller tumor volumes (*:p < 0.04; **:p < 0.005).

TABLE 3
Effects of treatment on apoptotic index (AI, % TUNEL),
proliferation index (PI, % PCNA Staining) and
angiogenesis (microvessel density)

Treatment	AI (% TUNEL)	PI (% PCNA)	Microvessel Density
Control (n = 2)	6.07 ± 0.88	60.1 ± 1.1	12.5 ± 3.8
Casein/HSPC	10.75 ± 0.54	51.7 ± 1.3	9.7 ± 0.7
(n = 2)
P value	<0.02	<0.01	>0.05
Values are means ± SE.

In summary, preliminary results indicate that soy products inhibit the s.c. growth of LNCaP tumor in SCID mice, possibly via induction of apoptosis, and inhibition of angiogenesis and proliferation.

Isoflavones or lignans can alleviate menopausal-related symptoms such as hot flashes and osteoporosis as well as alleviate symptoms associated with menstruation. This is further believed to be due to their estrogenic activity. It is believed that the improved composition described here will alleviate these symptoms even more effectively.

Also, isoflavones positively affect various cardiovascular-related conditions, including heart disease, cholesterol (saponins also positively affect cholesterol), angiogenesis and other vascular effects. It is believed that the improved composition will produce results for these cardiovascular conditions at least as beneficial as those hitherto known and at a reduced cost.

As explained earlier, isoflavones, lignans, and saponins are known to individually positively affect various neurological and immunological symptoms. It is believed that the improved composition will result in alleviating neurological and immunological symptoms at least as well as those compounds hitherto known and at a reduced cost. Moreover, it would be expected that some synergism would arise out of the combination described herein.

The improved composition may be administered orally, parenterally, for instance, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation or by application of an aerosol spray to mucous membranes, or to the skin by an ointment or a cream.

Administering the improved composition may be done with any suitable carrier, in solid or liquid dosage form such as tablets, capsules, powders, soft gels, solutions, suspensions, emulsions, ointments, or creams. The improved composition may also be administered as a food supplement or as a food ingredient.

The amount of the improved composition administered will vary depending on the person, the mode of administration, and the desired result. An effective amount is expected to be 10 mg to 2000 mg/per dose.

EXAMPLE 2

Tablet Manufacture

The composition provided for in this patent may be used to prepare tablets or other dosage forms. An example of a capsule preparation is provided in Example 2. The hither the concentration of the active component, the easier it is to form a tablet or emulsion. This leads to an added ability to incorporate other dietary nutrients. An example would be to prepare a phytochemical tablet which incorporates calcium and vitamin E as a supplement to maintain bone health and/or reduce post menopausal symptoms such as hot flashes. In an example of this embodiment, a 600 mg dry compression tablet was prepared containing a total of 125 mg of isoflavones concentrate (50 mg isoflavone compound). Included in the tablet formulation was a source of calcium and magnesium.

Dry compression tablets were produced by first blending the following ingredients: 4 kg of the improved composition (39.83% isoflavones), 1.91 kg sorbitol, 0.095 kg magnesium stearate, and 13.11 kg dicalcium phosphate in a 120 quart capacity Hobart mixer. This blend of ingredients was then dry compressed at 1 ton pressure with a Stokes BB2 simple press into tablets having a total weight of 600 mg containing 125.53 mg of the improved composition and therefore 50 mg of total isoflavones.

Alternatively, a photochemical concentrate may be provided in a single dosage form, a skin cream or as a food ingredient added to conventional food in amounts from 10 mg to 2000 mg/per dose, the purpose of which is to exert a positive effect on health and well being. These benefits include: cancer prevention, estrogen and sex hormone related maladies, inhibition of the pituitary-thyroid-gonadotrophic axis, alcohol dependency reduction, modulation of the cardiovascular, immune and nervous systems, antiviral effects and analgesic effects.

EXAMPLE 3

Two-piece gelatin capsules were produced by filling the receiving end of the empty size “0” capsules with 0.106 g of the improved composition (44.35% isoflavones) and closed with the capping end, providing a capsule containing 47.2 mg of total isoflavones.

EXAMPLE 4

A comparison between various sources of phytochemical preparations is given in Table 4. It is readily seen that the phytochemical components of the composition of the “Isoflavone Concentrate” of this invention is substantially higher than the corresponding amounts in the natural vegetable materials. Notably, the amount of glycone isoflavones and saponins are over 100 times more concentrated compared to the food source and over twenty times more concentrated compared to the germ of the plant which naturally concentrates these phytochemicals. Comparison of the “Isoflavone Concentrate” of this invention to other concentrates shows that the isoflavone fraction predominates in these latter products, reducing the amount of other healthful phytochemicals. Additionally, the extraction methods of these other products employ techniques which modify the components, particularly the isoflavones, so that they are not identical to the substances found in the natural vegetable material (U.S. Pat. No. 5,637,562).

One version of the improved composition was compared to other previously described compositions. The results are shown in Table 4.

TABLE 4
Comparative Products to the Invention

	Isoflavone	Isoflavone
	Glycosides in	Aglycones in	Genistein/			Phenolic
Product	Product	Product	Daidzein	Lignans	Saponins	Acids
Example	(mg/g)	(mg/g)	Ratio	(mg/g)	(mg/g)	(mg/g)
Improved	401.0	3.37	1.06 to 1	0.2	460.7	25.47
composition
Soybean	1.748-2.776a	0.044a-0.075	1.59-2.7	NA	0.9-3.2b
Soy Flour	1.969a	0.045a	3.58	0.0013		2.870c
(defatted
Soy germ	24.32d	0.85d		NA	16.7-1.98b	NA
Productc	NA	2.5-6.5c	0.5-3.5	NA	NA	NA
patent (PTI)
Producti	NA	5.1-14.7i	0.433-3.48	NA	NA	NA
patent (PTI)
Productg	NA	1.7-3.5g	0.66-2.86	NA	NA	NA
patent (PTI)
PTI	NA	970	12.8	NA	NA	NA
producth
PTI	NA	640	2.0	NA	NA	NA
producth
Soy Molasses	27.6	0.1	1.37	NA	NA	5.788
(dried)
Novogeni	0.0	550	1-1.7 to 1	NA	NA	NA
aWang II. and Murphy P. A., J. Agric. Food Chem 1994, 42, 1666-1673.
bAnderson R. L. and Wolf W. J, J. Nutr 125:58IS-588S, 1995
cSeo A. and Morr C. V., J. Agric Food Chem 1984, 32, 530-533.
dSoy Life ™ promotional literature
eWO 95/10530, PCT/US94/10697
fWO 95/10512, PCT/US94/10699
gWO 95/10529, PCT/US94/10696
hNCI paper
iNovogen promotional literature

EXAMPLE 5

The improved composition, containing the glycoside forms of isoflavones, has as one aspect an improved solubility at body temperature over the previously described compositions containing the aglycoside forms.

Separate solutions (0.02% in distilled water) were made for genistein, genistin, daidzein, daidzin, and isoflavone concentrate in volumetric flasks. Samples were then placed in a 37° C. water bath for 17 hours, followed by rapid filtration through a 0.2 micron syringe-type filter to remove particulates. Filtered samples were then analyzed for isoflavone concentration by HPLC. Results are tabulated as shown in Table 5.

TABLE 5
Differential Solubility of Isoflavone Glycosides vs. Aglycones

Isoflavone	Genistein		Daidzein
sample	(ppm)	Genistin (ppm)	(ppm)	Daidzin (ppm)

Genistein	7.42
Genistin		33.89
Daidzein			3.64
Daidzin				48.51
Isoflavone	0.492	30.075	0.672	37.69
Concentrate

The glycoside forms, genistin and daidzin, are at least 4.57 and 13.32 fold higher in concentration at 37° C. than their corresponding aglycone forms, respectively.

The modifications made to the isoflavones are to remove the carbohydrate attached to the isoflavone moiety. This modification renders the isoflavone less soluble in water. Maintenance of the natural modification, glycosylation, enhances solubility. This fact is shown in the comparative solubility chart of Table 5. This chart shows that the genistin isoflavone is 4.6 times higher and the daidzin isoflavone is 13.3 times higher than the corresponding non-glycosylated form. Higher solubility can lead to better bioavailability to intestinal organisms. The glycosylation does not inhibit absorption in the gut because the intestinal microflora convert the glycone form to the aglycone form before absorption occurs.

EXAMPLE 6

Extraction of Lignans from Flax

Lignans can be readily extracted from flax using this following method.

978 g of defatted flax meal (F1) was extracted with 2000 g of 85% ethanol at 40° C. for 10 minutes, forming a slurry. The resulting slurry was filtered and extraction was repeated twice with a total of 6000 g of ethanol.

The ethanolic fraction was then evaporated under vacuum at 70° C., resulting in an aqueous fraction of 1186 g. The aqueous fraction was combined with 1000 g of water and mixed.

The mixed sample was then ultra-filtered through a 5000 molecular weight cutoff membrane, resulting in a 767 g permeate fraction and a retentate action of 1283 g.

The retentate fraction was freeze-dried, resulting in a 27.84 g sample (F2).

The 767 g permeate fraction at 50° C. was fed to a 35 ml bed volume, XAD-4 resin column at a rate of 10 ml/min. The column effluent was collected and dried, resulting in a 14.8 g sample (F3). XAD-4 is a trademark for an absorbent resin, available from Rohm & Haas.

The column was then eluted with four bed volumes (140 ml) of 70% ethanol at 50° C. The eluent sample was evaporated under vacuum at 70° C. and dried, resulting in a 1.79 g sample (F4). The four fractions were then analyzed for their lignan content, measured as the concentration by weight of secoisolariciresinol. As Table 6 shows, this extraction method enriches lignan concentration.

TABLE 6
LIGNAN CONCENTRATIONS AS SECOISOLARICIRESINOL

	FRACTION	F1	F2	F3	F4
	SECO. CONC. (mg/g)	2.3	1.9	4.8	13.4
	PHENOLIC ACID

While the present invention has been disclosed in terms of the preferred embodiment in order to facilitate a better understanding of the invention, it should be appreciated that the invention can be embodied in various ways without departing from the principles of the invention. Therefore, the invention should be understood to include all possible embodiments, modifications, and equivalents to the described embodiment which do not depart form the principles of the inventions as set out in the appended claims.