Method for inhibiting the development of Alzheimer's disease and related dementias- and for preserving cognitive function

Methods for inhibiting the development and relieving the symptoms of Alzheimer's disease and related dementias are provided. A phytoestrogenic isoflavone compound is administered to a human predisposed to developing Alzheimer's disease or a related dementia, or having Alzheimer's disease or a related dementia, in an amount effective to inhibit the development or relieve the symptoms of the disease. The phytoestrogenic isoflavone compound is selected from at least one of genistein, genistin, 6'-OMal genistin, 6'-OAc genistin, daidzein, daidzin, 6'-OMal daidzin, 6'-OAc daidzin, glycitein, glycitin, 6'-OMal glycitin, or mixtures thereof. The phytoestrogenic isoflavone compound is effective to up-regulate choline acetyltransferase mRNA and nerve growth factor mRNA.

BACKGROUND OF THE INVENTION 
The present invention relates to the discovery that phytoestrogen compounds 
are useful for up-regulating choline acetyltransferase mRNA and nerve 
growth factor mRNA in the brain, and therefore are useful for inhibiting 
the development of Alzheimer's disease and related dementias. 
Alzheimer's disease and related dementias cause marked loss in cognitive 
function, often reducing an afflicted person to an invalid state. No cure 
is known for Alzheimer's and related dementias, and the causes of these 
diseases are not well understood. 
Alzheimer's disease, however, is strongly associated with decreased choline 
acetyltransferase activity and the loss of cholinergic neurons. 
Neurochemical Studies of Early-Onset Alzheimer's Disease: Possible 
Influence on Treatment, Francis et al., Lancet, Vol. 4, pp. 7-11 (1985); 
Alzheimer's Disease: A Cell Biological Perspective, Kosik, Science, Vol. 
256, pp. 780-83 (1992), both incorporated herein by reference. Cholinergic 
neurons appear to be essential for learning and memory processes, and 
choline acetyltransferase (hereinafter "ChAT") activity and nerve growth 
factor (hereinafter "NGF") are important for the function of cholinergic 
neurons. Ovarian Steroid Deprivation Results in a Reversible Learning 
Impairment and Compromised Cholinergic Function in Female Sprague-Dawley 
Rats, Singh et al., Brain Research, Vol. 644, pp. 305-12 (1994); Effects 
of Estrogen Replacement on the Relative Levels of Choline 
Acetyltransferase, trkA, and Nerve Growth Factor Messenger RNAs in the 
Basal Forebrain and Hippocampal Formation of Adult Rats, Gibbs et al., 
Experimental Neurol., Vol. 129, pp. 70-80 (1994), both incorporated herein 
by reference. 
Postmenopausal estrogen treatment has been shown to reduce the incidence of 
Alzheimer's disease and related dementias, to relieve symptoms of 
Alzheimer's disease, and to preserve cognitive function in women. 
Estrogenic Effects on Memory in Women, Sherwin, B., Ann. N.Y. Acad. Sci., 
743, pp. 213-31 (1994); Oestrogen Replacement Therapy and Alzheimer's 
Disease, Paganini-Hill, A., Brit. J. Obstet. & Gynaecol., 103, pp. 80-86 
(1996), both incorporated herein by reference. One mechanism by which 
supplemental estrogen may provide these beneficial effects is by 
increasing ChAT, ChAT activity, and NGF messenger RNA. Gibbs et al., 
Experimental Neurol. (above), Singh et al., Brain Research (above), and 
Overiectomy Reduces ChAT Activity and NGF mRNA Levels in the Frontal 
Cortex and Hippocampus of the Female Sprague-Dawley Rat, Singh et al., 
Abstr. Soc. Neurosci., Vol. 19, p. 1254 (1993), incorporated herein by 
reference. Unfortunately, commonly used estrogens can significantly 
increase the risk of breast and uterine cancers in women, and have 
intolerable side effects for men and some women. 
It is desirable, therefore, to find compounds like estrogen which can 
maintain normal levels of ChAT and NGF in cholinergic neurons in basal 
forebrain (such as septum) and their target brain tissues (cerebral cortex 
and hippocampus) to reduce or prevent loss of cholinergic neurons to 
prevent or delay the onset on Alzheimer's disease and related dementias, 
or to relieve the symptoms of Alzheimer's disease and related dementias. 
Phytoestrogens are compounds that are structurally similar to estrogens 
which are derived from plants such as legumes, clovers, kudzu root 
(pueraria lobata), and oilseeds such as rapeseed. 
Phytoestrogens--particularly the isoflavones derived from soy and clover 
such as genistein, daidzein, glycitein, and their glucosidic 
derivatives--exhibit estrogenic properties in some mammalian and human 
tissues, and exhibit anti-estrogenic properties in other tissues by 
competitively inhibiting estrogen binding at estrogen receptor sites. 
Certain phytoestrogens, particularly genistein, are also known to have 
tyrosine kinase inhibitory activity. Unlike estrogen, however, these 
isoflavone phytoestrogens are not associated with an increased risk of 
cancer, and may actually inhibit the development of breast and uterine 
cancers. 
SUMMARY OF THE INVENTION 
A method of inhibiting the development of Alzheimer's disease and related 
dementias in a human is provided. A phytoestrogen compound selected from 
at least one of the isoflavone compounds genistein, 6-O-malonyl genistin 
(hereinafter "6'-OMal genistin"), 6-O-acetyl genistin (hereinafter "6-OAc 
genistin"), genistin, daidzein, 6-O-malonyl daidzin (hereinafter 6'-OMal 
daidzin"), 6'-O-acetyl daidzin (hereinafter "6'-OAc daidzin"), daidzin, 
glycitein, 6'O-malonyl glycitin (hereinafter "6'-OMal glycitin"), 
glycitin, or a mixture thereof, is administered to a human predisposed to 
developing Alzheimer's disease or a related dementia in an amount 
effective to inhibit the development of Alzheimer's disease or related 
dementia. 
In another aspect, the invention is a method of aiding the preservation of 
cognitive function in a human predisposed to loss of cognitive function. A 
phytoestrogen compound selected from genistein, 6-OMal genistin, 6-OAc 
genistin, genistin, daidzein, 6-OMal daidzin, 6-OAc daidzin, daidzin, 
glycitein, glycitin, 6-OMal glycitin, or a mixture thereof is administered 
to a human in an amount effective to aid in the preservation of the 
cognitive function of said human. 
In still another aspect, the invention is a method of up-regulating choline 
acetyltransferase mRNA in the brain of a human having, or predisposed to 
having, decreased choline acetyltransferase activity in the brain, or loss 
of cholinergic neurons. A phytoestrogen compound selected from genistein, 
6-OMal genistin, 6-OAc genistin, genistin, daidzein, 6-OMal daidzin, 6-OAc 
daidzin, daidzin, glycitein, glycitin, 6-OMal glycitin, or a mixture 
thereof, is administered to a human having, or predisposed to having, 
decreased choline acetyltransferase activity in the brain, or loss of 
cholinergic neurons, in an amount effective to up-regulate choline 
acetyltransferase mRNA in the brain of the human. 
In a further aspect, the invention is a method of up-regulating nerve 
growth factor mRNA in a human. A phytoestrogen compound selected from at 
least one of the isoflavone compounds genistein, 6-OMal genistin, 6-OAc 
genistin, genistin, daidzein, 6-OMal daidzin, 6-OAc daidzin, daidzin, 
glycitein, glycitin, 6-OMal glycitin, or a mixture thereof, is 
administered to a human in an amount effective to up-regulate nerve growth 
factor in the brain of said human. 
In a final aspect, the present invention is a method of relieving the 
symptoms of Alzheimer's disease and related dementias. A phytoestrogen 
compound selected from at least one of the isoflavone compounds genistein, 
6-OMal genistin, 6-OAc genistin, genistin, daidzein, 6-OMal daidzin, 6-OAc 
daidzin, daidzin, glycitein, 6-OMal glycitin, glycitin, or mixtures 
thereof, is administered to a human in an amount effective to relieve the 
symptoms of Alzheimer's disease or related dementia in the human. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The present invention resides in the discovery that the phytoestrogenic 
isoflavone compounds which may be derived from soy--genistein, 6-OMal 
genistin, 6-OAc genistin, genistin, daidzein, 6-OMal daidzin, 6-OAc 
daidzin, daidzin, glycitein, 6-OMal glycitin, glycitin, shown in Formulas 
1 and 2 below--are useful in up-regulating choline acetyltransferase mRNA 
and neurotrophic factors such as brain derived neurotrophic factor 
("BNDF") and nerve growth factor ("NGF") mRNA in the brain, and, 
therefore, are useful for inhibiting the development and relieving the 
symptoms of Alzheimer's disease and related dementias, and for aiding the 
preservation of cognitive function. 
______________________________________ 
Formula 1 
1 #STR1## 
Compound R.sub.1 
R.sub.2 R.sub.3 
R.sub.4 
______________________________________ 
Genistein OH H OH OH 
Daidzein OH H H OH 
Glycitein OH OCH.sub.3 H OH 
______________________________________ 
______________________________________ 
Formula 2 
2 #STR2## 
Compound R.sub.1 R.sub.2 R.sub.3 
R.sub.4 
______________________________________ 
Genistin H H OH OH 
6'-OMal genistin 
COCH.sub.2 CO.sub.2 H 
H OH OH 
6'-OAc genistin 
COCH.sub.3 H OH OH 
Daidzin H H H OH 
6'-OMal daidzin 
COCH.sub.2 CO.sub.2 H 
H H OH 
6'-OAc daidzin 
COCH.sub.3 H H OH 
Glycitin H OCH.sub.3 H OH 
6'-OMal glycitin 
COCH.sub.3 OCH.sub.3 H OH 
______________________________________ 
As used herein "inhibit" is used in accordance with its generally accepted 
meaning and includes slowing, restraining, or impeding the progression, 
development, severity, or symptoms of Alzheimer's disease and related 
dementias. The term "up-regulate" is also used in accordance with its 
generally accepted meaning and includes increasing the translational 
activity, or the concentrations of the up-regulated mRNA to increase the 
concentrations or total amounts of the moiety produced by the up-regulated 
mRNA. The term "mRNA" is used in its conventional sense and means 
messenger ribonucleic acid. 
The phytoestrogenic isoflavone compounds of Formulas 1 and 2 are naturally 
occurring substances which may be found in plants such as legumes, clover, 
and the root of the kudzu vine (pueraria root). Common legume sources of 
these phytoestrogenic isoflavone compounds include soy beans, chick peas, 
and various other types of beans and peas. Clover sources of these 
phytoestrogenic isoflavone compounds include red clover and subterranean 
clover. Soy beans are a particularly preferred source of the 
phytoestrogenic isoflavone compounds. 
The phytoestrogenic isoflavone compounds of Formulas 1 and 2 may be 
isolated from the plant sources in which they naturally occur, and several 
may be synthetically prepared by processes known in the art. For example, 
daidzein may be isolated from red clover as disclosed by Wong (J. Sci. 
Food Agr., Vol. 13, p. 304 (1962)) or may be isolated from the mold 
Micromonospora halophytica as provided by Ganguly and Sarre (Chem. & Ind. 
(London), p. 201 (1970)), both references of which are incorporated by 
reference herein. Daidzein may be synthetically prepared by the methods 
provided by Baker et al (J. Chem. Soc., p. 274 (1933)), Wesley et al. 
(Ber. Vol. 66, p. 685 (1933)), Mahal et al. (J. Chem. Soc., p. 1769 
(1934)), Baker et al. (J. Chem. Soc., p. 1852 (1953)), or Farkas (Ber. 
Vol. 90, p. 2940 (1957)), each reference of which is incorporated herein 
by reference. Daidzin may be synthetically prepared by the method of 
Farkas et al. (Ber., Vol. 92, p. 819 (1959)), incorporated herein by 
reference. The daidzein isoflavone conjugates 6'-OMal daidzin and 6'-OAc 
daidzin can be prepared by a conventional saponification of daidzin with a 
malonyl or an acetyl anhydride, respectively. 
Genistein may be synthetically prepared by the methods provided by Baker et 
al (J. Chem. Soc., p. 3115 (1928)); Narasimhachari et al. (J. Sci. Ind. 
Res., Vol. 12, p. 287 (1953)); Yoder et al., (Proc. Iowa Acad. Sci., Vol. 
61, p. 271 (1954); and Zemplen et al. (Acta. Chim. Acad. Sci. Hung., Vol. 
19, p. 277 (1959)), each reference of which is incorporated herein by 
reference. Genistin may be synthetically prepared by the method of Zemplen 
et al. (Ber., Vol 76B, p. 1110 (1943)), incorporated herein by reference. 
The isoflavone conjugates of genistein 6'-OMal genistein and 6'-OAc 
genistein can be prepared by a conventional saponification of genistin 
with a malonyl or an acetyl anhydride, respectively. 
A preferred method of isolating the phytoestrogenic isoflavone compounds of 
Formulas 1 and 2 from plant materials in which they naturally occur is to 
extract the plant materials with an alcohol, preferably methanol or 
ethanol, or an aqueous solution, preferably an aqueous alkaline solution, 
to remove the isoflavones from the plant material. It is preferred to 
comminute the plant material before extracting the phytoestrogenic 
isoflavone compounds to maximize recovery of the isoflavone compounds from 
the plant material. The phytoestrogenic isoflavone compounds can be 
isolated from the extract by conventional separation procedures such as 
reverse phase high performance liquid chromatography ("HPLC"). 
In a preferred embodiment, the phytoestrogenic isoflavone compounds 
genistein, genistin, 6'-OMal genistin, 6'-OAc genistin, daidzein, daidzin, 
6'-OMal daidzin, 6'-OAc daidzin, glycitein, glycitin, and 6'-OMal glycitin 
are isolated from a soy material, preferably a commercially available soy 
material. Soy materials from which the phytoestrogenic isoflavone 
compounds can be isolated include: soy beans, dehulled soy beans, soy 
meal, soy flour, soy grits, soy flakes (full fat and defatted), soy 
cotyldeons, soy molasses, soy protein concentrate, soy whey, soy whey 
protein, and soy protein isolate. In one embodiment, the phytoestrogenic 
isoflavones are extracted from soy beans, dehulled soy beans, soy meal, 
soy flour, soy grits, soy flakes, soy protein concentrate, soy whey 
protein, or soy protein isolate, preferably soy meal, soy flour, soy 
grits, or soy flakes, with a low molecular weight organic extractant, 
preferably an alcohol, ethyl acetate, acetone, or ether, and most 
preferably aqueous ethyl alcohol or methyl alcohol. Most preferably the 
extractant has a pH of about the isoelectric point of soy protein (about 
pH 4 to pH 5) to minimize the amount of soy protein extracted by the 
extractant. 
The extractant containing the phytoestrogenic isoflavones is separated from 
the insoluble soy materials to form an phytoestrogenic isoflavone enriched 
extract. If desired, a phytoestrogenic isoflavone enriched material may be 
recovered by concentrating the extract to remove the solvent and to 
produce a solid phytoestrogenic isoflavone enriched material. 
In a more preferred embodiment the phytoestrogenic isoflavone compounds are 
further purified from other soy materials soluble in the extract by 
contacting the extract with a material which adsorbs the phytoestrogenic 
isoflavones in the extract, and eluting the adsorbed phytoestrogenic 
isoflavones out of the adsorbent material with a solvent which causes the 
isoflavones to be differentially eluted from the adsorbent material. 
In a preferred embodiment, the phytoestrogenic isoflavones are separated 
from impurities in the extract by a conventional reverse phase HPLC 
separation. After extracting the isoflavones from the soy material and 
separation of the extract from the insoluble soy materials, the extract is 
filtered to remove insoluble materials that could plug an HPLC column. An 
HPLC column is prepared by packing a conventional commercially available 
HPLC column with a particulate adsorbent material which will releasably 
bind the isoflavones and impurities in the extract in a compound specific 
manner. The adsorbent material may be any reverse phase HPLC packing 
material, however, a preferred packing material may be chosen by the 
criteria of load capacity, separation effectiveness, and cost. One such 
preferred packing material is Kromasil C18 16 .mu.m 100 .ANG. beads 
available from Eka Nobel, Nobel Industries, Sweden. 
The filtered extract is passed through the packed HPLC column until all the 
binding sites of the column are fully saturated with isoflavones, which is 
detected by the appearance of isoflavones in the effluent from the column. 
The HPLC column may then be eluted with a solvent to effect the 
separation. In a preferred embodiment, the eluent is a polar solvent such 
as ethanol, methanol, ethyl acetate, or acetonitrile, and preferably is an 
aqueous alcohol having an alcohol content of between about 30% and about 
90%, most preferably about 50%, and most preferably the alcohol is 
ethanol. 
The phytoestrogenic isoflavone compounds and impurities are separately 
collected from the column effluent. The isoflavone fractions of the eluent 
may be identified from other eluent fractions in accordance with 
conventional HPLC and analytical chemistry techniques. In a preferred 
embodiment the eluent fractions containing the phytoestrogenic aglucone 
isoflavones genistein, glycitein, and daidzein are collected separately 
since the aglucone isoflavones are believed to be particularly active in 
the up-regulation of ChAT and NGF mRNA, the inhibition and treatment of 
symptoms of Alzheimer's disease and related dementias, and in aiding in 
the preservation of cognitive function. Of the aglucone isoflavone 
materials, the fraction of effluent containing daidzein elutes from the 
column first, followed by a glycitein fraction, followed by the more polar 
genistein. 
The isoflavone fractions of the eluent may be collected from the column, 
and the volatile content of the solvent (e.g. alcohol) can be removed by 
evaporation. The phytoestrogenic isoflavone compounds can be recovered 
directly if the all of the solvent is removed by evaporation, or may be 
recovered by chilling the remaining solvent (e.g. water) and centrifuging 
or filtering the remaining solvent. 
In a particularly preferred embodiment the soy phytoestrogenic isoflavone 
conjugates--6'-OMal genistin, 6'-OAc genistin, 6'-OMal daidzin, 6'-OAc 
daidzin, and 6'-OMal glycitin--and the soy phytoestrogenic isoflavone 
glucosides--genistin, daidzin, and glycitin--are converted to their 
respective phytoestrogenic aglucone isoflavone forms--genistein, daidzein, 
and glycitein. The conversion of the isoflavone conjugates and isoflavone 
glucosides to the aglucone isoflavones can be effected in the soy 
substrate from which the phytoestrogenic isoflavones are to be extracted 
prior to the extraction, or may be effected in the isoflavone enriched 
extract after separation of the extract from the insoluble soy materials. 
As noted above, the aglucone isoflavone compounds are believed to be 
particularly active in the up-regulation of ChAT and NGF mRNA, the 
inhibition and treatment of symptoms of Alzheimer's disease and related 
dementias, and in aiding in the preservation of cognitive function, and 
the aglucone isoflavones are more easily separated from an extract 
containing water than their respective conjugate and glucoside forms since 
the aglucones are less water soluble. 
The isoflavone conjugates 6'-OMal genistin, 6"-OAc genistin, 6'-OMal 
daidzin, 6'-OAc daidzin, and 6'-OMal glycitin can be converted to their 
respective glucosides genistin, daidzin, and glycitin by forming an 
aqueous alkaline solution of the soy substrate containing the isoflavones 
having a pH of about 6 to about 13, preferably about pH 9 to about pH 11, 
and treating the aqueous alkaline solution at a temperature of about 
2.degree. C. to about 121.degree. C., preferably about 25.degree. C. to 
about 75.degree. C., for a period of time sufficient to effect the 
conversion, preferably about 30 minutes to about 5 hours, more preferably 
about 30 minutes to about 1.5 hours. The isoflavone glucosides genistin, 
daidzin, and glycitin can be converted to their respective aglucone forms 
genistein, daidzein, and glycitein by contacting the isoflavone glucosides 
with an enzyme capable of cleaving a 1,4-.beta.-glucoside bond--preferably 
a commercially available alpha- or beta-galactosidase enzyme, a pectinase 
enzyme, a lactase enzyme, or a gluco-amylase enzyme--at a pH at which the 
enzyme is active, typically from about pH 3 to about pH 9, and at a 
temperature of about 25.degree. C. to about 75.degree. C., more preferably 
about 45.degree. C. to about 65.degree. C., for a period of time 
sufficient to effect the conversion, typically about 1 hour to about 24 
hours, preferably about 1 hour to about 3 hours. 
The phytoestrogenic aglucone isoflavones can be separated from the soy 
substrate using conventional separation procedures. For example, the 
aglucone isoflavones may be extracted from the soy substrate with a low 
molecular weight alcohol. The aglucone isoflavones may be separated from 
the extract by conventional recrystallization processes, or by HPLC. In a 
particularly preferred embodiment, an isoflavone composition isolated from 
a soy substrate for formulation into a pharmaceutical composition or a 
dietary supplement for use in the method of the present invention includes 
at least 40% genistein, at least 15% daidzein, and at least 1% glycitein. 
In another particularly preferred embodiment of the invention, an 
isoflavone composition isolated from a soy substrate for formulation into 
a pharmaceutical composition or a dietary supplement for use in the method 
of the present invention contains at least 85% genistein, at least 5% 
daidzein, and at least 0.5% glycitein. 
Several of the isoflavone compounds of Formula 1 and Formula 2 are 
commercially available, and may be purchased for formulation into 
pharmaceutical compositions or dietary supplements useful in the method of 
the present invention. For example, genistein, daidzein, and glycitein are 
commercially available and may be purchased, for example, from Indofine 
Chemical Company Inc., P.O. Box 473, Somerville, N.J. 08876. 
The phytoestrogenic isoflavone compounds of Formulas 1 and 2 may be 
administered to a human in a pharmaceutical formulation to inhibit the 
development or relieve the symptoms of Alzheimer's disease or related 
dementias, to aid in the preservation of cognitive function, or to 
up-regulate ChAT and NGF mRNA in the brain of the human. Pharmaceutical 
formulations incorporating the isoflavone compounds obtained by any of the 
methods above, or purchased from a commercial source, can be prepared by 
procedures known in the art. For example, the isoflavone compounds can be 
formulated into tablets, capsules, powders, suppositories, suspensions, 
solutions for parenteral administration including intravenous, 
intramuscular, and subcutaneous administration, and into solutions for 
application onto patches for transdermal application with common and 
conventional carriers, binders, diluents, and excipients. In a preferred 
embodiment, a pharmaceutical formulation for use in the methods of the 
present invention includes a phytoestrogenic isoflavone material 
containing at least 40% genistein, at least 15% daidzein, and at least 1% 
glycitein. In another preferred embodiment, a pharmaceutical formulation 
includes an isoflavone material containing at least 85% genistein, at 
least 5% daidzein, and at least 0.5% glycitein. 
Inert pharmaceutically acceptable carriers useful to form pharmaceutical 
formulations in accordance with the present invention include starch, 
mannitol, calcium sulfate, dicalcium phosphate, magnesium stearate, 
silicic derivatives, and/or sugars such as sucrose, lactose, and glucose. 
Binding agents include carboxymethyl cellulose and other cellulose 
derivatives, gelatin, natural and synthetic gums including alginates such 
as sodium alginate, polyethylene glycol, waxes and the like. Diluents 
useful in the invention include a suitable oil, saline, sugar solutions 
such as aqueous dextrose or aqueous glucose, and glycols such as 
polyethylene or polypropylene glycol. Other excipients include lubricants 
such as sodium oleate, sodium acetate, sodium stearate, sodium chloride, 
sodium benzoate, talc, and magnesium stearate, and the like; 
disintegrating agents including agar, calcium carbonate, sodium 
bicarbonate, starch, xanthan gum, and the like; and adsorptive carriers 
such as bentonite and kaolin. Coloring and flavoring agents may also be 
added to the pharmaceutical formulations. 
Dietary supplements incorporating the phytoestrogenic isoflavone compounds 
of Formulas 1 and/or 2 can be prepared by adding the isoflavone compounds 
to a food in the process of preparing the food, independent of the plant 
or protein material from which the isoflavone compounds are derived. The 
foods to which the isoflavone compounds may be added include almost all 
foods. For example, the isoflavone compounds can be added to foods 
including, but not limited to, meats such as ground meats, emulsified 
meats, marinated meats, and meats injected with the isoflavone compounds; 
beverages such as nutritional beverages, sports beverages, protein 
fortified beverages, juices, milk, milk alternatives, and weight loss 
beverages; cheeses such as hard and soft cheeses, cream cheese, and 
cottage cheese; frozen desserts such as ice cream, ice milk, low fat 
frozen desserts, and non-dairy frozen desserts; yogurts; soups; puddings; 
bakery products; salad dressings; and dips and spreads such as mayonnaise 
and chip dips. The isoflavone compounds are added to the food in an amount 
selected to deliver a desired dose of the isoflavone compounds to the 
consumer of the food. In a preferred embodiment, a phytoestrogenic 
isoflavone material added to a food for use as a dietary supplement in 
accordance with the methods of the present invention contains at least 40% 
genistein, at least 15% daidzein, and at least 1% glycitein. In another 
preferred embodiment, a phytoestrogenic isoflavone material added to a 
food contains at least 85% genistein, at least 5% daidzein, and at least 
0.5% glycitein. 
The phytoestrogenic isoflavone compounds of Formulas 1 and/or 2 may also be 
administered in a phytoestrogenic isoflavone enriched soy protein material 
incorporated into a dietary supplement formulation for inhibiting the 
development or relieving the symptoms of Alzheimer's disease or related 
dementias, aiding in the preservation of cognitive function, or 
up-regulating ChAT and NGF mRNA in the brain. In a preferred method of 
forming the isoflavone rich soy protein material, a phytoestrogenic 
isoflavone enriched soy protein isolate is formed. A commercially 
available defatted soy flake material is extracted with an aqueous 
alkaline solution, typically a calcium hydroxide or a sodium hydroxide 
solution having a pH of about 7.5 to about 10, to form an extract 
containing the isoflavones, protein, and other water soluble components of 
the soy flake material. The extract is then treated with an acid to lower 
the pH of the extract to about the isoelectric point of the protein, 
preferably to a pH of about 4 to about 5, and most preferably to a pH of 
about 4.4 to about 4.6, thereby precipitating a protein curd which 
captures significant amounts of the isoflavones. Preferably the conjugate 
and glucoside isoflavones are converted to aglucone isoflavones in the 
extract, as described above, to increase the amount of isoflavones 
captured in the protein curd, and to provide the benefits of the aglucone 
isoflavones. The protein curd is then separated from the extract, 
preferably by centrifugation, and dried to form the protein isolate. 
Preferably, unlike conventional processes to produce a protein isolate, 
the curd is not washed with water or is washed with a minimal amount of 
water to minimize the loss of the isoflavones from the protein isolate. 
Other phytoestrogenic isoflavone enriched soy protein materials include soy 
protein concentrates and soy whey protein materials which include 
significant amounts of the isoflavones of Formulas 1 and/or 2. 
The phytoestrogenic isoflavone compound can be administered to: a human 
predisposed to developing Alzheimer's disease or a related dementia for 
the purpose of inhibiting the disease; a human predisposed to loss of 
cognitive function for the purpose of aiding in the preservation of 
cognitive function; a human suffering Alzheimer's disease or a related 
dementia, particularly someone having decreased ChAT activity or 
concentrations of ChAT in the brain, or decreased cholinergic neuron 
activity or concentrations in the brain, for the purpose of relieving the 
symptoms of the disease; to a human having decreased, or predisposed to 
having decreased, ChAT activity, or concentrations of ChAT in the brain 
for the purpose of increasing ChAT activity in the brain; to a human 
having decreased, or predisposed to having decreased, cholinergic neuron 
activity or concentrations of cholinergic neurons in the brain for the 
purpose of increasing cholingeric neuron activity or concentration of 
cholinergic neurons in the brain; or to a human requiring up-regulation of 
NGF mRNA in the brain. Typically the phytoestrogenic compound should be 
administered to persons most likely to develop Alzheimer's disease or a 
related dementia, such as persons genetically predisposed to such 
diseases, or persons over 45 years of age. In a particularly preferred 
embodiment of the invention, the phytoestrogenic isoflavone compound is 
administered to a perimenopausal or postmenopausal woman. 
The particular dosage of the phytoestrogenic isoflavone compound to be 
administered to a human predisposed to developing Alzheimer's disease or a 
related dementia for the inhibition of the development of the disease will 
depend on the route of administration, and other risk factors for 
developing Alzheimer's disease or the related dementia. The dosage of the 
phytoestrogenic isoflavone compound to be administered to a human 
predisposed to a loss of cognitive function for the purpose of aiding in 
preservation of cognitive function also will depend on the route of 
administration, and the risk factors predisposing the human to loss of 
cognitive function. The dosage of the phytoestrogenic isoflavone compound 
to be administered to a human having decreased, or predisposed to having 
decreased, ChAT activity in the brain or loss of cholinergic neurons will 
depend on the route of administration and the degree of loss, or 
predisposition to loss, of ChAT activity in the brain or degree of loss, 
or predisposition to loss, of cholinergic neurons or cholinergic neuron 
activity. The dosage of the phytoestrogenic isoflavone compound to be 
administered to a human to up-regulate NGF mRNA will depend on the route 
of administration and the degree of up-regulation of the NGF mRNA desired. 
The dosage of the phytoestrogenic isoflavone compound to be administered to 
a human having Alzheimer's disease or a related dementia to relieve the 
symptoms of the disease will depend on the route of administration, the 
extent of the disease, and other risk factors. The dosage can be related 
to the degree that the human suffering the disease has decreased ChAT 
activity in the brain so the phytoestrogen isoflavone compound can be 
administered in an amount required to increase ChAT activity in the brain, 
preferably to normal levels. The dosage may also be related to the degree 
that the human suffering the disease has suffered a loss of cholinergic 
neurons, or cholinergic neuron activity, so the phytoestrogenic isoflavone 
compound can be administered in an amount required to increase the amount 
of cholinergic neurons, or cholinergic neural activity, preferably to 
normal levels. 
Generally acceptable and effective daily doses of the phytoestrogenic 
isoflavone compound for the above applications may be from about 10 mg/day 
to about 1000 mg/day. Preferably the daily dose of the phytoestrogenic 
isoflavone compound in the method of the present invention is about 20 
mg/day to about 500 mg/day, more preferably from about 30 mg/day to about 
300 mg/day, and most preferably from about 50 mg/day to about 150 mg/day. 
The phytoestrogenic isoflavone compound should be administered in an amount 
effective to increase the concentration of the isoflavones and/or their 
metabolites in the blood or urine of the human to whom the compound is 
administered. Preferably, the phytoestrogenic isoflavone compound is 
administered in an amount effective to increase the concentration of at 
least one of daidzein, equol, o-desmethylangolensin, dihydrodaidzein, 
genistein, dihydrogenistein and 6-hydroxy-o-desmethylangolensin in the 
blood or urine of the human to whom the compound is administered. 
Dihydrodaidzein, equol, and o-desmethylangolensin are metabolites produced 
by the human body in catabolism of daidzein, and dihydrogenistein and 
6'-hydroxy-o-desmethylangolensin are metabolites produced by the human 
body in the catabolism of genistein. See A Urinary Profile Study of 
Dietary Phytoestrogens. The Identification and Mode of Metabolism of New 
Isoflavanoids., Joannou et al., J. Steroid Biochem. Molec. Biol., Vol. 54, 
No. 3/4, pp. 167-84 (1995), incorporated herein by reference, including 
the structures of dihydrodaidzein, equol, o-desmethylangolensin, 
dihydrogenistein, and 6'-hydroxy-o-desmethylangolensin. 
The following non-limiting formulations illustrate pharmaceutical and 
dietary formulations including the phytoestrogenic isoflavone compounds of 
Formulas 1 and/or 2 which may be used in accordance with the methods of 
the present invention. 
FORMULATIONS 
The following Formulations 1-4 illustrate pharmaceutical formulations 
including a phytoestrogenic isoflavone compound of Formula 1 and/or 
Formula 2. In the formulations, "Active ingredient" means an isoflavone 
compound or a mixture of isoflavone compounds of Formulas 1 and/or 2. 
Formulation 1 
Gelatin Capsules 
Hard gelatin capsules are prepared using the following ingredients: Active 
ingredient 0.1-1000 mg/capsule; Starch, NF 0-600 mg/capsule; Starch 
flowable powder 0-600 mg/capsule; Silicone fluid 350 centistokes 0-20 
mg/capsule. The ingredients are mixed, passed through a sieve, and filled 
into capsules. 
Formulation 2 
Tablets 
Tablets are prepared using the following ingredients: Active ingredient 
0.1-1000 mg/tablet; Microcrystalline cellulose 20-300 mg/tablet; Starch 
0-50 mg/tablet; Magnesium stearate or stearate acid 0-15 mg/tablet; 
Silicon dioxide, fumed 0-400 mg/tablet; silicon dioxide, colloidal 0-1 
mg/tablet, and lactose 0-100 mg/tablet. The ingredients are blended and 
compressed to form tablets. 
Formulation 3 
Suspensions 
Suspensions are prepared using the following ingredients: Active ingredient 
0.1-1000 mg/5 ml; Sodium carboxymethyl cellulose 50-700 mg/5 ml; Sodium 
benzoate 0-10 mg/5 ml; Purified water 5 ml; and flavor and color agents as 
needed. 
Formulation 4 
Parenteral Solutions 
A parenteral composition is prepared by stirring 1.5% by weight of active 
ingredient in 10% by volume propylene glycol and water. The solution is 
made isotonic with sodium chloride and sterilized. 
The following Formulations 5-8 illustrate dietary supplements that may be 
formed using an isolated soy protein rich in several of the 
phytoestrogenic isoflavone compounds of Formula 1 and/or Formula 2. The 
isoflavone rich isolated soy protein in the following examples typically 
contains between about 1 to about 3 milligrams of the isoflavone compounds 
per gram of soy protein. 
Formulation 5 
Ready to Drink Beverage 
A ready to drink beverage is formed of the following components: 
______________________________________ 
Ingredient Percent of composition, by weight 
______________________________________ 
Water 80-85 
Isoflavone rich isolated soy protein 
10-15 
Sucrose 5-8 
Cocoa 0.1-1 
Vitamins/Minerals 
0.1-1 
Flavor 0.1-1 
Cellulose gel 0.1-0.5 
______________________________________ 
The ready to drink beverage may be served in 8 ounce servings containing 
about 20 grams of isolated soy protein including about 20 to about 60 
milligrams of the isoflavone compounds. 
Formulation 6 
Powdered Beverage 
A powdered beverage is formed of the following components: 
______________________________________ 
Ingredient Percent of composition, by weight 
______________________________________ 
Isoflavone rich isolated soy protein 
85-90 
Sucrose 8-15 
Maltodextrin 1-5 
Vitamins/Minerals 
0.5-2 
Aspartame 0-0.5 
Flavor 0-0.5 
______________________________________ 
30 grams of the powdered beverage formulation may be added to water to form 
a serving containing about 20 grams of isolated soy protein including 
about 20 to about 60 milligrams of the isoflavone compounds. 
Formulation 7 
Food Bar 
A food bar is formed of the following components: 
______________________________________ 
Ingredients Percent of composition, by weight 
______________________________________ 
Isoflavone rich isolated soy protein 
20-30 
Corn syrup 35-45 
Rice syrup solids 
7-14 
Glycerin 1-5 
Cocoa 2-7 
Compound coating 15-25 
______________________________________ 
The food bar may be served in 70 gram portions containing about 15 grams of 
soy protein having about 15 to about 45 milligrams of the isoflavone 
compounds therein. 
Formulation 8 
Soy Yogurt 
A soy yogurt is formed of the following components: 
______________________________________ 
Ingredients Percent of composition, by weight 
______________________________________ 
Water 65-75 
Isoflavone rich isolated soy protein 
5-15 
Sucrose 3-8 
Corn starch 1-5 
Dextrin 0.3-1 
Cellulose gel 1-3 
Culture (yogurt) 0.01-0.1 
Fruit 10-20 
Vitamins/Minerals 
0.05-0.3 
______________________________________ 
The soy yogurt may be served in a 170 gram serving containing about 8 grams 
of soy protein having about 8 to about 24 milligrams of isoflavone 
compounds therein. 
The following non-limiting test examples illustrate the methods of the 
present invention.

EXAMPLE 1 
A study of the effect on ChAT and brain derived neurotrophic factor 
("BDNF") mRNAs in the brain of retired female breeder rats of diets 
supplemented with phytoestrogenic isoflavones, 17-.beta. estradiol 
(estrogen), and a control diet containing neither phytoestrogenic 
isoflavones or an estrogen-like compound is conducted. 
Fifteen retired female breeder rats weighing 300 g to 360 g are housed in 
separate cages and are maintained on a 12 hour light/12 hour dark cycle, 
with access to food and water ad libitum. The rats are randomized into 
three groups of five based on body weight. The rats are ovariectiomized, 
and are fed a casein/lactalbumin-based control diet containing no 
estrogen-like substances for a period of three days after the surgery. 
Group 1 is then fed the control diet supplemented with soy phytoestrogen 
isoflavones equivalent to a human dose of 150 mg of total isoflavones per 
day for a period of 8 weeks. Group 2 is fed the control diet supplemented 
with 17-.beta. estradiol ("E2") equivalent to a human dose of 2 mg per day 
for a period of 8 weeks. Group 3 is fed the control diet with no 
supplemental materials for the 8 week period of the experiment. 
At the end of the eight week treatments the rats are euthanized with 
phenobarbital (100 mg/kg). Blood samples are collected by cardiac puncture 
at necropsy, and serum samples are used to determine estradiol and 
phytoestrogenic isoflavone levels. The brains are removed and processed to 
determine the effect on ChAT and BDNF mRNA in the hippocampus and in the 
frontal cortex. 
The E2 and phytoestrogenic isoflavones are found to significantly 
up-regulate both ChAT and BDNF mRNA in the frontal cortex portion of the 
brain of the rats compared to the control. 
EXAMPLE 2 
A study of the effect on NGF mRNAs in the brain of young female rats of 
diets supplemented with phytoestrogenic isoflavones, 17-.beta. estradiol 
("E2"), and a control diet containing neither phytoestrogenic isoflavones 
or an estrogen-like compound is conducted. 
Fifteen young adult female rats weighing 180 g to 250 g are housed in 
separate cages and are maintained on a 12 hour light/12 hour dark cycle, 
with access to food and water ad libitum. The rats are randomized into 
three groups of five based on body weight. The rats are ovariectiomized, 
and are fed a casein/lactalbumin-based control diet containing no 
estrogen-like substances for a period of three days after the surgery. 
Group 1 is then fed the control diet supplemented with soy phytoestrogen 
isoflavones equivalent to a human dose of 150 mg of total isoflavones per 
day for a period of 8 weeks. Group 2 is fed the control diet supplemented 
with 17-.beta. estradiol ("E2") equivalent to a human dose of 2 mg per day 
for a period of 8 weeks. Group 3 is fed the control diet with no 
supplemental materials for the 8 week period of the experiment. 
At the end of the eight week treatments the rats are euthanized with 
phenobarbital (100 mg/kg). Blood samples are collected by cardiac puncture 
at necropsy, and serum samples are used to determine estradiol and 
phytoestrogenic isoflavone levels. The brains are removed and processed to 
determine the effect on NGF mRNA in the hippocampus and in the frontal 
cortex. 
The phytoestrogenic isoflavones are found to marginally up-regulate NGF 
mRNA in the hippocampus portion of the brain of the young rats relative to 
the control, and E2 is found to significantly up-regulate NGF mRNA in the 
hippocampus portion of the brain of the young rats. 
EXAMPLE 3 
Five to fifty men having mild to moderate Alzheimer's disease are selected 
for clinical study. The men are divided into two groups, one of which 
receives a soy protein dietary supplement containing between 30 mg to 500 
mg per day of phytoestrogenic isoflavones, the other of which receives a 
soy protein dietary supplement from which the phytoestrogenic isoflavones 
have been removed by alcohol extraction. The diets of the two groups are 
selected to contain no further source of isoflavones, and no source of 
estrogen or androgen is administered to either of the two groups. The 
diets are continued for 6 to 12 months. 
Prior to beginning the diets, the patients are benchmarked as to cognitive 
ability. The benchmarked symptoms are measured again for each group after 
the groups have been on the diets for the prescribed period of the study. 
Activity of the phytoestrogenic isoflavone compounds to aid in the 
preservation of cognitive function and to inhibit the development of 
Alzheimer's disease is shown by significant retention of cognitive 
function in the patients on the diet containing the phytoestrogenic 
isoflavone compounds relative to the patients on the diet containing no 
phytoestrogenic isoflavone compounds. Activity of the phytoestrogenic 
isoflavone compounds to relieve the symptoms of Alzheimer's disease is 
shown by improved cognitive function in the patients on the diet 
containing the phytoestrogenic isoflavone compounds relative to the 
cognitive function of the same patients at the start of the study. 
EXAMPLE 4 
Five to fifty perimenopausal or postmenopausal women having mild to 
moderate Alzheimer's disease are selected for clinical study. The women 
are divided into two groups, one of which receives a soy protein dietary 
supplement containing between 30 mg to 500 mg per day of phytoestrogenic 
isoflavones, the other of which receives a soy protein dietary supplement 
from which the phytoestrogenic isoflavones have been removed by alcohol 
extraction. The diets of the two groups are selected to contain no further 
source of isoflavones, and no source of estrogen or androgen is 
administered to either of the two groups. The diets are continued for 6 to 
12 months. 
Prior to beginning the diets, the patients are benchmarked as to cognitive 
ability. The benchmarked symptoms are measured again for each group after 
the groups have been on the diets for the prescribed period of the study. 
Activity of the phytoestrogenic isoflavone compounds to aid in the 
preservation of cognitive function and to inhibit the development of 
Alzheimer's disease is shown by significant retention of cognitive 
function in the patients on the diet containing the phytoestrogenic 
isoflavone compounds relative to the diet containing no phytoestrogenic 
isoflavone compounds. Activity of the phytoestrogenic isoflavone compounds 
to relieve the symptoms of Alzheimer's disease is shown by improved 
cognitive function in the patients on the diet containing the 
phytoestrogenic isoflavone compounds relative to the cognitive function of 
the same patients at the start of the study. 
Utility of the phytoestrogenic isoflavone compounds of Formula 1 or 2 for: 
inhibiting the development of Alzheimer's disease or a related dementia in 
a human; relieving the symptoms of Alzheimer's disease or a related 
dementia in a human; aiding the preservation of cognitive function in a 
human; up-regulating ChAT mRNA in the brain of a human; or up-regulating 
NGF mRNA in the brain of a human is evidenced by activity in at least one 
of the above examples. 
It is to be understood that the foregoing are merely preferred embodiments 
of the invention and that various changes and alterations can be made 
without departing from the spirit and broader aspects thereof as set forth 
in the appended claims, which are to be interpreted in accordance with the 
principles of patent law including the Doctrine of Equivalents.