WO2004084832A2

WO2004084832A2 - Measurement of estrogen activity in plants and a composition therefrom

Info

Publication number: WO2004084832A2
Application number: PCT/US2004/008689
Authority: WO
Inventors: Karen O. Klein; R. Jeffrey Chang
Original assignee: The Regents Of The University Of California
Priority date: 2003-03-21
Filing date: 2004-03-18
Publication date: 2004-10-07
Also published as: WO2004084818A3; WO2004084832A3; WO2004084818A2

Abstract

Detection and measurement of estrogen like bioactivity in plants is provided. An ultra sensitive recombinant cell bioassay is utilized in an aspect of the invention. Also provided are previously undetermined measurements of estrogen like bioactivity in named herbs, including measurements of significant estrogen like bioactivity in fo-ti. In an aspect, a glycone group is removed from soy, providing an increase in estrogen bioactivity. The measurements provided may be utilized for various medical purposes, including estrogen replacement therapy (ERT). A composition is also provided that may be utilized for ERT, therapeutics and nutriceutics.

Description

MEASUREMENT OF ESTROGEN ACTIVITY IN PLANTS AND A COMPOSITION THEREFROM

CROSS REFERENCE TO RELATED APPLICATION

This application is based on and claims priority to U.S. provisional patent application 60/456,909, filed March 21, 2003, attorney docket number 0027.0001P, entitled Measurement Of Estrogen Activity And Development Of Herbal Products, and assigned to the Assignee of this application and incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates generally to estrogen activity, and more particularly to detection and measurement of estrogen like activity in plants, and the development of products for estrogen replacement therapy (ERT).

BACKGROUND OF THE INVENTION

A current and continued topic in women's health is the matter of risks and benefits of estrogen replacement therapy. Scientific uncertainty and lack of consensus of standard ERT has driven many women to seek alternative sources of estrogen, including herbal remedies. Consequently, the natural hormone alternatives industry is presently active and flourishing.

Since the initial identification of estrogen activity in plant extracts in 1926, numerous plants have been described with estrogenic properties. In the early 1980's, detection of plant estrogens was demonstrated in human urine using gas chromatography - mass spectrometry and NMR, which showed that diet may be a source of estrogen. Despite the considerable history of herbs used for their estrogen-like effects, only recently have there been reports of measurement of estrogenic activity in some herbs. However, as described below, the certainty of the conclusions of these reports is unclear.

Competitive binding for estrogen receptor in an endometrial cell line has been exhibited by extracts of red clover, hops, and chaste tree berry. Further, dong quai and licorice have been shown to have weak binding affinity, and black cohosh has been shown to have no binding affinity. Glabridin, the major isoflavone in licorice, has been shown to have estrogenic properties as measured by competition for estrogen receptor in human breast cancer cells (T47D). Licorice and red clover extracts have been shown to act like estrogen agonists by stimulating cell proliferation in breast cancer cell lines (MCF-7 and T47D). Dong quai and black cohosh had little estrogen receptor binding in this system. Estrogenic activity in hops has been described using a human endometrial cell line (Ishikawa Var I), and using a radioreceptor assay with MCF-7 cells. A similar yeast system was used to show estrogen activity in red clover extracts. By comparison, it has been suggested that growth of MCF-7 cells may be independent of estrogenic activity in dong quai and ginseng. These herbs were shown to exhibit no estrogenic activity in a gene expression system using HeLa cells co-transfected with an estrogen-dependent reporter plasmid and human estrogen receptor ERα or ERβ. In addition, exposure to black cohosh or licorice failed to stimulate growth of MCF-7 cells, and were shown to have no effect on HeLa cells. Although these and many other conflicting and variable estrogen activity studies exist, several herbal preparations are available to consumers, including preparations having red clover, dong quai, cohosh, soy, licorice, and chaste tree berry.

Efforts to quantify estrogen bioactivity in plants and herbal preparation have been limited by inadequate methodological techniques, variation in biopotency among plant products, and different measures of relative estrogen bioactivity. Manufacturers of herbal products currently use high pressure liquid chromatography (HPLC) to determine whether herbs contain isoflavones and similar chemical structures. HPLC is a process that seeks out characteristic peaks of known chemical structures, but does not seek out estrogen like bioactivities in subject herbs. What is needed is a method to effectively and accurately quantify estrogen like bioactivity in plants and herbal preparations.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein: Fig. 1 is a standard curve constructed in assay media with 17- β estradiol standards, interpolation of an unknown represented, used in an embodiment of the invention;

Fig. 2 is a bar graph representation of estrogen activity in various herbs obtained utilizing an embodiment of the invention; and

Fig. 3 is an illustration of a molecular structure of soy extract with glycoside and without the glucose module (aglycone), used in an embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Exemplary embodiments are described with reference to specific configurations. Those of ordinary skill in the art will appreciate that various changes and modifications can be made while remaining within the scope of the appended claims. Additionally, well- known elements, materials, components, methods, process steps, specific formulations, process parameters and the like may not be set forth in detail in order to avoid obscuring the invention.

A method to detect and measure estrogen like bioactivity in plants, including herbs, is described herein. Also presented are previously unknown measurements of estrogen activity in named herbs. The detection and measurement methods as described herein may be utilized for various medical purposes, including estrogen replacement therapy (ERT). Further described is a composition that may be utilized for ERT. Features of the invention are achieved in part by utilizing an ultra-sensitive recombinant cell bioassay. Recombinant cell bioassay is a technique that has previously been employed to assess very low levels of estradiol in the serum of children and post-menopausal women. A recombinant cell bioassay is described in "Estrogen Levels in Childhood Determined by an Ultrasensitive Recombinant Cell Bioassay" by Karen Oerter Klein, Jeffrey Baron, Michael J. Colli, Donald P. McDonnell, and Gordon B. Cutler, Jr., The Journal of Clinical Investigation, Inc., Volume 94, December 1994, p. 2475-2480. In an embodiment, the present invention makes use of, adapts, and adds method steps to the recombinant cell bioassay for use with detecting and measuring estrogen like activity in plants. 100 In an embodiment, the present invention provides a method to detect and measure estrogen bioactivity in plants, plant extracts, herbs and herb extracts including red clover (trifolium pretense L.), dong quai (angelica sinensis), black cohosh (cimiciuga racemosa), soy, licorice (glycyrrhiza glabra), chaste tree berry (vitex agnus-castus L.) , fo-ti (polygonum multiflorum), hops (humulus lupulus L.), and any combinations thereof It is

105 to be appreciated that plants and herbs other than those named herein can be applied to the methods of the present invention. In another embodiment, features of the discussion and claims may be applied to measurement of estrogen bioactivity in substances other than plant or herb extracts.

110 The method of measurement as described herein shows fo-ti as having significant estrogen like bioactivity. Further, utilizing the measurement method as discussed herein, it is determined that removing a glycone group from soy (as compared to glycosylated soy) results in a significant increase in the estrogen bioactivity of soy. In an embodiment, the present invention removes a glycone group from soy (a-glycosylated soy, also

115 referred to as Soy A) to increase estrogen bioactivity in soy.

Plants and their extracts having estrogen like activity as measured by methods described herein may be further developed and used for therapeutics and nutriceutics in animals, including humans. It is to be appreciated that as described herein, a method to detect

120 estrogen activity in a plant and a method to measure estrogen activity in a plant can also refer to a plant extract, a herb, a herb extract, etc. Further, the detection and measurement methods as described herein can be used to detect and measure estrogen like bioactivity in other substances used to formulate remedies including minerals, organic compounds and other compounds. In an embodiment, features of this discussion and compositions

125 described herein can be incorporated into a formulation and administered in products in forms including a tablet, capsule, liquid drink, juice, oral powder, other ingestion methods, as well as absorbed by a suppository, topical applications including a lotion, skin cream, vaginal cream, spray, and taking forms including an emulsion and a suspension. In an embodiment, the formulation having dosing, varied per the recipient, is

130 utilized by a human, a plant, an animal and other organisms. By organism as described herein it is meant an individual form of life, including vegetable and animal, such as a plant, animal, bacterium, protist, and fungus. Some lower forms of life are so simple in structure as to be without organs, but are still conventionally referred to and intended to be defined herein as organisms, since they have different parts analogous in functions to 135 the organs of higher plants and animals.

The potency of herbal preparations can vary from batch to batch, which leads to difficulties in standardizing products. In an embodiment, the measurement methods of the present invention can be used to test herb batches before product compounding to

140 increase standardization. The methods of detection and measurement as described herein can lead to determining appropriate human and other animal or organism dosing (prescribed quantity) of herbs. In an embodiment, an effective amount of one of fo-ti, red clover and soy for ERT is determined by the detection and measurement methods as described herein. Further, as an example, the contents of a composition or a capsule

145 having one of fo-ti, red clover and soy can be analyzed for estradiol equivalents by the detection and measurement methods described herein. Also, the herbs named herein are known to be safe and generally accepted to be safe in generally accepted safe quantities. Ultimate dosing and distribution for human consumption at high levels of herb requires studies and evaluation by the Food and Drug Administration. Estrogen effects in women

150 using products developed by the methods described herein may further be detected by utilizing the recombinant cell bioassay described above. Further, monitoring a subject's response to the administration of a composition as described herein can be accomplished by examining a subjects menopausal symptoms, uterine lining thickness, assay of estradiol serum levels, and evaluating the subjects expressed feeling of well being.

155

A further understanding of the above description can be obtained by reference to the following specific examples and experiments, which are provided for illustrative purposes and are not intended to be limiting. The following experiments and procedures utilize, adapt and add procedures to the ultrasensitive recombinant cell bioassay, as described

160 above.

Chaste tree berry, black cohosh, red clover, dong quai, fo-ti, licorice, hops, soy, and soy aglycone (soy A) were analyzed for estrogen activity. Dried herb extracts were obtained from Natural Alternative International, San Marcos, CA. Sixty mg of each herb were 165 used for extraction, with the exception of soy and soy A, for which 3 mg were used. All herbs were extracted with 2 ml methanol (MeOH), except for licorice, which was extracted with 2 ml ether. Herb and solvent were vortexed for 2 min and centrifuged at 3000 rpm for 10 min. Supernatant was removed and filtered through 0.45 um glass syringe filter and stored at room temperature for use. It is to be appreciated that since

170 many herb varieties exist, extraction can require a variable range of herb to solvent. In an embodiment, a herb is combined with a solvent in a ratio ranging from 0.25 to 100 milligrams of herb with 1 to 20 milliliters of solvent. Further, in an alternative embodiment, when the herb is one of soy and soy having a glycone group removed (soy A), the herb may be combined with a solvent in a ratio ranging from 0.5 to 10

175 milligrams of herb with 1 to 20 milliliters of solvent. Additionally, other solvents can be used in the extraction method including ethanol, hexane, ethyl acetate, etc.

As used herein, estrogen activity is defined by estrogen receptor binding in a genetically engineered yeast system. A strain of Saccharomyces cerevisiae was transformed with

180 two plasmids. One plasmid contains the human estrogen receptor complementary DNA, and the other contains an estrogen response element upstream of the yeast iso-1- cytoclirome C promoter fused to the structural gene for beta-galactosidase (β- galactosidase). The transformed yeast was grown in selective media in the presence of estradiol extracted from plants. Beta-galactosidase activity was assayed and converted to

185 estradiol equivalent units by linear interpolation from a standard curve constructed by adding known amounts of estradiol to assay media. The sensitivity of the bioassay was 0.07 - 7 pmol/L (0.02 - 2 pg/mL).

Each herb extract was analyzed in triplicate and repeated over 3 consecutive assays.

190 Referring to the drawings wherein identical reference numerals denote the same elements throughout the various views, Fig. 1 shows estradiol equivalents that are calculated by interpolation from a standard curve constructed in assay media with known amounts of 17 beta-estradiol. Therefore, the results are expressed in estradiol equivalents in pmol/L per meg of dry herb extract. All data are expressed as the mean + SD. Comparisons between

195 groups were made using a MANOVA with post hoc correction (LSD). Differences were considered significant at P< 0.05.

As shown in Table 1, measurable estrogenic activity, in vitro, was demonstrated in red clover, fo-ti, hops, soy, and licorice, whereas none was found in chaste tree berry, black

200 cohosh, or dong quai. As shown in the column denoted as Significance, herbs marked with different letters are statistically significantly different from each other, and those marked with the same letters are statistically significantly similar.

205

Table 1 - measured estrogenic activity in various herbs

210

Referring to Fig. 2, in an embodiment, among those herbs with detectable estrogen activity, a-glycosylated soy (Soy A) was found to contain the highest amount of estradiol

215 equivalents, 1453.7 + 389.1 pmol/lL per meg herb, compared to the others, including soy, 363.4 + 124.8 pmol/L per meg herb, (p < 0.001). The displayed bars represent mean + SE. Bars with different letters are significantly different from each other, p < 0.001 for Soy A, p < 0.05 for other herbs. Considerable estrogen bioactivity was identified in red clover and fo-ti. Licorice and hops showed significantly lower estrogen activity than fo-

220 ti. The detection of estrogen activity in fo-ti has not been previously reported. 225 In another embodiment and assay, results were obtained as shown in Table 2.

Table 2- measured estrogenic activity in various herbs, utilizing different solvents

230 These results are consistent with the relatively lower amounts of estrogenic activity in licorice and hops. The lack of measurable estrogen bioactivity in black cohosh may explain the failure of this herb to exhibit estrogen receptor binding or bioactivity in endometrial and breast cell culture systems.

235 Estrogen activity in fo-ti was equivalent to that observed for soy and red clover. Fo-ti was found to contain a large amount of estrogen activity, which has not previously been described. The preparations of fo-ti studied had estrogen activity of 409 + 55 pmol/L estradiol equivalents per meg of herb, which is approximately 1/300 of the activity of 17B-estradiol itself. The estrogen bioactivity of fo-ti was found to be approximately 240 1/300* that of estradiol itself. Fo-ti has not been previously studied by any known system. The estrogen bioactivity finding herein suggests that fo-ti is a candidate for alternative medicine therapy in menopausal women.

Producers of menopausal dietary supplementation products will find the detection and 245 measurement methods described herein useful. Further, the finding, through methods described herein, that a glycone group removed from soy results in a significant increase in the estrogen bioactivity of soy can be of use for dietary supplementation. Fig. 3 shows SoyA, which is constructed by removing the glucose moiety from the soy preparation. In an embodiment, SoyA, a phytoestrogen, also known as an isoflavone, is utilized in the 250 form of one of genistein and daidzein.

Difficulties in standardizing phytoestrogen content in herb preparations have contributed to the limitation of their use and scientific study. A wide variation of content can exist between herb preparations due to the source of herb and the processing used. Individual

255 batches of an herb for human compounding require estrogenic activity testing. For example, estrogen-like activity in fo-ti can vary based on the source and processing. The detection and measurement methods described herein can be utilized for such estrogenic like activity testing. In the present study, two separate lots of the same dried soy extract were compared and a two-fold difference was found, which did not reach statistical

260 significance, 165.2 + 44.1 pmol/L per meg herb, vs. 363.4 + 124.8 pmol L per meg herb.

Having disclosed exemplary embodiments, modifications and variations may be made to the disclosed embodiments while remaining within the spirit and scope of the invention as defined by the appended claims. For example, a yeast other than Saccharomyces 265 cerevisiae may be utilized, and a marker protein other than a gene for beta-galactosidase may be utilized in the methods described for detecting and measuring estrogen like bioactivity.

Claims

CLAIMS270 We claim:

1. An estrogen replacement therapy composition comprising: a plant extract including an effective amount of at least one of fo-ti (polygonum multiflorum), red clover (trifolium pretense L.) and soy.

275

2. The composition as in claim 1, wherein the soy has a glycone group removed.

3. The composition as in claim 2, wherein the soy having the glycone group removed is one of daidzein and genistein.

280

4. The composition as in claim 2, wherein the at least one of fo-ti (polygonum multiflorum), red clover (trifolium pretense L.) soy, and soy having a glycone group removed is incorporated into a formulation for one of a human, a plant and an animal organism that utilizes estrogen.

285

5. The composition as in claim 4, wherein the formulation is one of a liquid, juice, tablet, capsule, oral powder, suspension, emulsion, suppository, lotion, skin cream, vaginal cream and spray.

290 6. A method for estrogen replacement therapy comprising: administering to a subject in need of estrogen an effective amount of at least one of fo-ti (polygonum multiflorum), red clover (trifolium pretense L.) and soy.

7. The method as in claim 6, wherein the soy has a glycone group removed. 295

8. The method as in claim 6, wherein the soy having the glycone group removed is one of daidzein and genistein.

9. The method as in claim 6, wherein administering comprises at least one of 300 ingesting orally by tablet, capsule, liquid drink, juice, oral powder, absorbing by suppository, and topical applications including a lotion, skin cream, vaginal cream, spray, and taking forms including an emulsion and a suspension.

10. The method as in claim 6, further comprising monitoring the subjects response to 305 the administering by examining at least one of the subjects menopausal symptoms, uterine lining thickness, assay of estradiol serum levels, and evaluating the subjects expressed feeling of well being.

310