Patent Application
20080008818
We believe that one skilled in the art can, based upon the description herein, use the present invention to its fullest extent. The following specific embodiments are to be construed as merely illustrative, and do not serve to limit the remainder of the disclosure in any way whatsoever.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Also, all publications, patent applications, patents, and other references mentioned herein are incorporated by reference. Unless otherwise indicated, a percentage refers to a percentage by weight (i.e., % (W/W)).
“Enhancing the elasticity or the structural integrity” means increasing or improving the synthesis of elastic fibers, preventing the loss, or retarding the loss of elasticity or structural integrity of the tissue, including but not limited to, treating sagging, lax and loose tissue, tightening skin or mucosal or viscero-elastic tissues.
The loss of elasticity or tissue structure integrity may result from a number of factors, including but not limited to: disease, aging, hormonal changes, mechanical trauma, environmental damage, or the result of an application of products, such as a cosmetics or pharmaceuticals, to the tissue.
“Reducing triglyceride or uric acid” means, respectively, reducing the level, or preventing the increase in the level, of triglycerides in serum or of uric acid in the serum and/or urine.
The increase in triglyceride or uric acid levels in the body may be a result of a number of factors, including but not limited to disease, aging, hormonal changes, environmental damage, genetic factors, nutritional imbalance, and the like.
“Mucosal tissues” are tissues that express elastin and are composed in part of cells of mesenchymal and epithelial origin. Examples of mucosal tissues include, but are not limited to, vaginal, oral, corneal, nasal, rectal, and viscero-elastic tissues. Examples of viscero-elastic tissues are those that line the respiratory track, blood vessel walls, the gastro-intestinal track, the urinal track, the bladder, and the reproductive track.
“Urogenital tissues” are tissues of the bladder, the urinal track and the reproductive track, including but not limited to the vagina, clitoris, external genitalia, uterus, bladder, and urethra.
“Vessel walls” are walls of vessels of the circulatory system that function to transport blood or lymph throughout the body. The most important types of blood vessels, arteries and veins, carry blood away from or towards the heart, respectively.
A “product” is a product in finished packaged form. In one embodiment of the products of this invention, the package is a container such as a paper, cardboard, plastic, metal or glass bottle or jar containing the composition or a “blister pack” for containing unit dosages of the composition. The product may further contain additional packaging such as a plastic or cardboard box for storing such container. In one embodiment, the product contains instructions directing the user to ingest the composition (e.g., for the purposes set forth herein). Such instructions may be printed on the container, label insert or on any additional packaging.
Various methods of using the compositions of this invention and statements that may be used to “promote” the sale of such compositions are listed below. What is meant by “promoting” is promoting, advertising, or marketing and such promoting relates to various methods of using the compositions of the invention in accordance with this invention. Examples of promoting include, but are not limited to, written, visual, or verbal statements made on the product or in stores, magazines, newspaper, radio, television, internet, and the like. Examples of such statements include, but are not limited to, “enhances skin elasticity or structural integrity,” “improving visible and tactilely perceptible manifestations of the skin,” “increases skin elasticity or structure,” “restores skin elasticity or structure,” “treats sagging of lax skin,” “enhances vaginal elasticity,” “enhances sexual satisfaction,” “increases vaginal elasticity,” “restores vaginal elasticity,” “strengthen vaginal wall,” “prevents or treats vaginal prolapse,” “reduces incontinence episodes,” “strengthen bladder wall,” “improves bladder compliance,” “enhances gum elasticity,” “increases gum elasticity,” “restores gum elasticity,” “enhances alveolar wall elasticity,” “increases alveolar wall elasticity,” “enhances the healthy look of the gums,” “restores alveolar wall elasticity,” “decreases the risk of hemorrhoids”, “Prevents or reduces pain and discomfort from hemorrhoids”, “reduces the severity of hemorrhoids,” “decreases the risk of cardiovascular diseases,” “decreases the risk of pancreatitis,” “reduces pancreatitis,” “decreases the risk of thrombosis,” “reduces thrombosis,” “lowers C-reactive protein (CRP),” “decreases systolic and diastolic blood pressure,” “lowers tissue edema,” “reduces gout,” “reduces tumor lysis syndrome,” “slows or reverses age-related metabolic syndromes,” and “slows or reverses disease-related metabolic syndromes,” “enhances vessel wall strength,” “improves fragile skin,” “decreases bruising,” and “decreases inflammatory cell extravasation.”
As used herein, “ingestible composition” means a composition that is intended to be ingested. Examples of forms of ingestible compositions include, but are not limited to, tablets, pills, capsules, powders, granules, solutions or suspensions, and drops. Such compositions may be swallowed whole or may be in chewable form. An “ingestible composition,” may also be in the form of a confectionary or a food product such as a cookie, candy, food bar, chewing gum, yogurt additive, sprinkles, tea, juice or other drink, liquid shake or the like. Ingestible compositions do not include compositions intended to be topically administered to the skin or oral/vaginal cavity.
As used herein, “pharmaceutically-acceptable” means that the ingredients which the term describes are suitable for ingesting without undue toxicity, incompatibility, instability, irritation, allergic response, and the like.
As used herein, “safe and effective amount” means an amount of the extract or of the composition sufficient to induce the desired effect, but low enough to avoid serious side effects. The safe and effective amount of the compound, extract, or composition will vary with e.g. the age, health and environmental exposure of the end user, the duration and nature of the treatment, the specific extract, ingredient, or composition employed, the particular pharmaceutically-acceptable carrier utilized, and like factors.
In one embodiment, partially-denatured whole soybean extract of the compositions of this invention is a substance derived from the soybean, containing ingredients naturally found in soybeans, at the relative concentrations as found in the beans, with the exception of water, in which said extract contains active BBI but reduced or eliminated trypsin inhibitory activity. The partially denatured soy extract of this invention may be in the form of a fluid (e.g., soymilk) or a solid (e.g., a soybean powder or soymilk powder). When in the form of a fluid, the term “soy” refers to the solid constituents of the fluid that are derived from the soybean. The soy may be in the form of soybean powder. Soybean powder may be made by grinding dry soybeans. The soybean powder may be lyophilized. Soymilk and soymilk powder are also useful soy products. Soymilk is a combination of solids derived from soybeans and water, the mixture of which has some or all of the insoluble, constituents filtered off.
Soymilk powder is evaporated soymilk, which in one embodiment, is in a lyophilized, or freeze-dried or spray-dried form. Procedures for manufacturing soymilk include, but are not limited to, the following three: (a) soymilk may be made by placing soybeans into water to allow them to absorb the water. The swelled beans are then ground and additional water is then added. The mixture may then be filtered to remove any insoluble residue. A method according to this invention, utilizing this procedure, is described more fully below. Soymilk may also be prepared from soybean powder. Soybean powder is thoroughly mixed with water (e.g., depending upon the efficiency of the mixing process, for at least one hour), and then followed by filtering the resulting mixture to remove insoluble residues. (c) soymilk may also be reconstituted from soymilk powder by adding water. The soymilk useful in the compositions of this invention may comprise from about 1% to about 50%, by weight (more preferably, from about 5% to about 20%, by weight) of solids from the soybean.
Another example is the use of soymilk powder, made from lyophilized, spray dried or freeze-dried soymilk, with the addition of water and following with or without additional filtration or homogenization. Other methods of soybean extraction could also be used to create the active ingredients in the formulations described below. For example, the active ingredients could be extracted from ground soybeans using ethanol/water mixtures, followed by the removal of the ethanol from the extract, in such ways that the BBI activity of the soybean will be retained, but the STI activity will be removed or substantially reduced.
The soymilk useful in the compositions of this invention may comprise from about 1% to about 50%, by weight (more preferably, from about 5% to about 20%, by weight) of solids from the soybean. The abovementioned processes yield a substrate which may be processed, in accordance with the methods of this invention, to yield the novel compositions of this invention having reduced trypsin inhibition while retaining BBI activity.
Other known active ingredients of soy include, but are not limited to, isoflavones, phytoestrogens, genistein, daidzein, glycitein, saponins, and phytosterols. The soy products useful in the compositions of this invention may be produced from all soybean species, regardless of their geographic origin, genetic origin, sun exposure, harvest time and the like. However, specific strains, geographic origins or growth conditions might be preferred. For example, preferred soybean strains are those particularly rich in their BBI content, or particularly low in their STI content. Growth conditions resulting in BBI enrichment or in STI reduction in the bean are also preferred.
“Denaturation” is defined as the change in the physical and the physiological properties of a protein, that is brought about by heat, X-rays, organic solvents or other chemicals. These changes include loss of activity (e.g. for enzymes) and loss (or alteration) of antigenicity (in the case of antigens).
A “partially denatured whole soybean extract” is a composition extracted or derived from the whole soybean in which the processing for the derivation of such soy extract (e.g., the temperature, duration at certain temperature, extraction media) selectively and substantially inactivates the trypsin inhibitory activity of soybean trypsin inhibitor (STI) protein, while preserving the activity of the Bowman-Birk inhibitor (BBI).
Preferably, the soy products utilized in the compositions and methods of this invention should have a microbial content of less than about 1,000 cfu per gram (such as less than about 100 cfu per gram) of the legume product. In order to achieve such low microbial content, the soy products of this invention may be exposed to gamma irradiation in accordance with the procedures set forth in U.S. Pat. Nos. 6,555,143 (Issued Apr. 29, 2003) and U.S. patent application Ser. No. 09/796,054 filed Feb. 28, 2001, which are both incorporated herein by reference,. The soy product may be exposed to between about 2 to about 30 kGy of gamma irradiation, such as between about 5 and about 10 kGy of gamma irradiation. Surprisingly, such treatment reduces the microbial content of the legume product of this invention, while maintaining its biological activity (e.g. retaining BBI activity). The treatment of legume products with gamma irradiation maintains the cosmetic elegance of the legume product, such as maintained natural colors and does not induce significant malodors.
Other anti-microbial processes that also maintain the protease inhibitory activity of the legume product that can be practiced alone or in combination with gamma irradiation, include, but are not limited to, exposure to x-rays, high energy electron or proton beams, ultraviolet radiation, hydrostatic pressure, and addition of chemical agents possessing antimicrobial activity that do not affect the biological activity profile of the compositions of this invention, and combinations thereof.
The partially denatured whole soybean extract of this invention may be combined with continues coggygria extract and/or with malva sylvestris extract in order to achieve additional efficacy in promoting elasticity in the tissues of the body. Compositions containing coggygria extract and malva sylvestris extract are described in copending patent applications U.S. Ser. No. 10/973,313 filed Oct. 26, 2004, Ser. No. 11/313/079 filed Dec. 20, 2005, Ser. No. 11/248,465 filed Oct. 12, 2005 and Ser. No. 11/387,892 filed Mar. 23, 2006, which are hereby incorporated herein by reference.
Preferably, partially denatured whole soybean extract is present in the composition in an amount from about 0.001% to about 20% by weight, in particular in an amount from about 0.01% to about 10% by weight. Unless stated otherwise, the weight of the extract refers to the dry weight of the extract.
The ingestible compositions useful in the present invention involve formulations suitable for ingesting by the mammal, such as a human, in need to such treatment. In one embodiment, the compositions contain a safe and effective amount of (i) partially denatured whole soybean extract and (ii) a pharmaceutically-acceptable carrier.
In one embodiment, the extracts of this invention are combined with foods, confectionary or food supplements; said foods, confectionary or food supplements were not processed to temperature higher than 90’ C. after the addition of extracts of this invention, and said food supplements having a nutritional or physiological effect whose purpose is to supplement the normal diet. Said nutritional supplements are in the form of botanical extracts, synthetic molecules and mixtures thereof. Said nutritional supplements include, but are not limited to proteins, vitamins (such as vitamin A or its derivatives or different forms of tocopherols), Minerals (such as Zinc, Iron or Selenium), antioxidants (such as ascorbate, lycopenes or carotenes), Fatty acids (such as omega-3 fatty acids). Other examples of nutritional supplements that can we combined with extracts of this invention include, but are not limited to Soy extracts, pomegranate juice, green tea and phenolic compounds and synthetic derivatives of resveratrol and the like. Said foods and confectionary include, but are not limited to candy, chocolate, chewing gum, bars, snack foods, dairy products such as yogurt, pastry and baked goods and the like. However, these products should not be heated to a temperature higher than 90° C. after the addition of extracts of this invention.
In one embodiment, the ingestible compositions herein contain, per dosage unit, e.g., tablet, capsule, powder, injection, teaspoonful and the like, or a food or a confectionary dose unit, an amount of the extract(s) necessary to deliver an effective dose as described above. Preferably, the ingestible compositions herein contains, per dosage unit, e.g., tablet, chewable tablet, capsule, powder, teaspoonful and the like, of from about 5 to about 2500 mg, such as from about 1 to about 2000 mg, and may be given at a dosage of from about 10 mg/kg/day to about 5 g/kg/day, such as from about 0.1 gr/kg/day to about 1 gr/kg/day. More preferably, a dosage of about 500 to about 1000 mg/kg/day should be used. In another embodiment the ingestible compositions herein may be incorporated into foods and confectionary products, from about 5 to about 5000 mg per food or confectionary product unit.
The preferred dosages, however, may be varied depending upon the requirement of the individuals, the severity of the condition being treated, and the extract(s) being employed. The use of either daily administration or post-periodic dosing may be employed. In one embodiment, the compositions of this invention may be provided in the form of tablets, such as those containing 50, 100, 250, 500, 1000, 2000 and/or 5000 milligrams of the extract(s) for the symptomatic adjustment of the dosage to the individual to be treated. The extract(s) may be administered on a regimen of 1 to 4 times per day. Advantageously, the compositions may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily.
Optimal dosages to be administered may be readily determined by those skilled in the art, and will vary with the particular extract(s) used, the mode of administration, the strength of the preparation, and the advancement of the disease/condition being treated. In addition, factors associated with the particular individual being treated, including individual's age, weight, diet and time of administration, will result in the need to adjust dosages.
Ingestible compositions containing one or more of the extracts of the invention described herein can be prepared by intimately mixing the extract(s) with a pharmaceutically-acceptable carrier according to conventional pharmaceutical compounding techniques, providing that the extracts of this invention are not exposed to a temperature higher than 90° C. The carrier may take a wide variety of forms depending upon the type of formulation. Thus for liquid preparations such as suspensions, elixirs and solutions, suitable carriers and additives include water, glycols, oils alcohols, flavoring agents, preservatives, stabilizers, coloring agents and the like; and for solid preparations, such as powders, capsules and tablets, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like. Solid oral preparations may also be coated with substances such as sugars or be enteric-coated so as to modulate major site of absorption.
For preparing solid compositions such as tablets, the principal extract(s) is mixed with a pharmaceutically-acceptable carrier, e.g. conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutically-acceptable diluents, e.g. water, to form a solid preformulation composition containing a homogeneous mixture of the extract(s). When referring to these preformulation compositions as homogeneous, it is meant that the extract(s) is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective dosage forms such as tablets, pills and capsules. This solid preformulation composition may then subdivided into unit dosage forms of the type described above. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of material can be used for such enteric layers or coatings, such materials including a number of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
The liquid forms in which the novel compositions of the present invention include aqueous and/or organic (e.g., ethanol) solutions, suitably flavored syrups, aqueous or suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil or peanut oil, as well as elixirs and similar pharmaceutically-acceptable vehicles, when such vehicle have no effect on BBI activity. Suitable dispersing or suspending agents for aqueous suspensions, include synthetic and natural gums such as tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose, methylcellulose, polyvinyl-pyrolidone or gelatin.
One skilled in the art will recognize that, both in vivo and in vitro trials using suitable, known and generally accepted cell and/or animal models are predictive of the ability of an extract to treat or prevent a given condition. One skilled in the art will further recognize that human clinical trails including first-in-human, dose ranging and efficacy trials, in healthy individuals and/or those suffering from a given condition or disorder, may be completed according to methods well known in the clinical and medical arts.
The following is a description of the preparation of various extracts of the present invention. As used in the subsequent Examples, the weight percentage of extract refers to the weight of the soybean powder.
Extract 1A: Devansoy “high sucrose” non-denatured soybean powder was purchased from Dupont, Wilmington Del. 2% non-denatured soybean extract suspension was made in PBS, with brief sonication of 3 repeats of 45 seconds on ice, and heat-denatured for 1 hour at 90° C. (Extract 1A). Extract 1B: Soy preparation was also made by suspending non-denatured soybean powder (conventional non-GMO soybeans, Natural Products, Inc., Grinnell, IA) in 1× PBS at a concentration of 2% (w/v). The suspension was sonicated 3-4 times for 10 seconds at 70% power (using Sonics CV33, Newtown, Conn.) to make a homogeneous suspension and heated at different temperatures and durations (Extract 1B). The homogeneous suspension of extracts 1B was then diluted to 0.2% soy (w/v) as a working solution. STI and BBI (Sigma, St Louis, Mo.) were dissolved in 1× PBS at a concentration of 0.05% (w/v), respectively. Preparations of Soy (0.2%, w/v), STI (0.05%, w/v), and BBI (0.05%, w/v) were then incubated at different temperature (0-100° C.) for 1 hour. The heat-treated samples were later tested for STI and BBI activity using trypsin and chymotrypsin inhibition assays, respectively.
The inhibition of trypsin (representing STI and BBI activity) or chymotrypsin (representing BBI activity)-induced cleavage of a fluorescent casein peptide was measured using the EnzChek™ protease assay kit, following manufacturer's instructions (EnzChek™ Protease Assay Kits Product Information, Revised Mar. 15, 1999; Molecular Probes, Eugene Oreg.). Briefly, soy preparations (Extract 1B, 0.2%, w/v), or STI, or BBI solutions (0.05%) were incubated with 100 units of trypsin or 0.08 units of chymotrypsin (Sigma, St. Louis, Mo.) dissolved in digestion buffer provided in the assay kit. Then, 1.0 mg/ml stock solution of BODIPY FL casein was prepared by adding 0.2 mL of deionized water to the vials supplied with this substrate (provided in kit), then made to a final working concentration of 10 microgram/ml in digestion buffer. Following incubation of the trypsin or chymotrypsin, with or without the test material, with the BODIPY fluorescent casein substrate, at room temperature for one hour, fluorescence was measured (excitation 485 nm/emission 530 nm) using a SpectraMax Gemini microtiter plate reader (Molecular Devices Corporation, Sunnyvale, Calif.) and Softmax Pro 3.0 software (Molecular Devices Corporation). Each experiment was performed in three replicates. The results of the experiment are shown in Table I. These results demonstrate that it is possible to inhibit STI (trypsin inhibitory activity) without inhibiting BBI. These data suggest that heating non-denatured soy extracts to e.g. 90° C. for 1 hr, would reduce or eliminate trypsin inhibitory activity of STI but preserve the BBI activity of the soy preparation.
C57BL/6 female mice of age 5 weeks were purchased from Taconic Farms (Germantown, N.Y.). Mice were housed in appropriately sized cages in an environmentally controlled room with a 12-hour light-12-hour dark photoperiod and supplied with food and water ad libitum. Animal care was based on the “Guide for the Care and Use of Laboratory Animals”, NIH Publication No. 85-23. Animals were acclimated for 3 weeks before study starts, using a special Casein Based Diet (5K96 with low isoflavone content, purchased from Test Diet) and housed together to achieve synchronized estrous cycling. After acclimation, 200 μl of test materials (Extract 1A) were given orally, 5 days a week Monday through Friday. Skin and bladder samples following 12 and 22 weeks of treatment were obtained for histological analysis. Oral treatment continued for 22 weeks. During this period the animals appeared healthy, did not lose body weight, and did not show any signs of digestive distress.
The effect of selected partially denatured whole soybean extract on mouse bladder and skin was assessed on C57B1/6 mice. 12 weeks after the start of the oral treatments, mice were sacrificed, and bladder and skin samples were analyzed histologically for elastin fibers. Surprisingly, an increased amount of elastic fibers was observed in mouse bladders and skins from all mice treated orally with the partially denatured soybean extract, as compared to controls.
The ingestible treatments were continued for additional two months, and bladder and skin samples at week 22 after the start of the treatments were analyzed histologically for elastin fibers. Surprisingly, an even greater increase in elastic fibers was observed in mouse bladders and skins of mice treated with the partially denatured soybean extract, as compared to control and to results from week 12.
Elastin fiber density around bladder vessels was also evaluated. As shown in Table III, there was a considerable increase in the elastin fiber density around blood vessels in bladders of treated mice, as compared to controls.
This example demonstrates that the use of partially denatured whole soybean extracts is well tolerated, causes no digestive distress, and provides elastin and structural enhancement to body tissues.
Mice were treated as in Example 2, and the effect of selected partially denatured whole soybean extract on mouse plasma triglyceride and uric acid levels was assessed. Three months after the start of the oral treatments, mice were sedated by isoflurane gas inhalation, and blood was collected by heart puncture. Surprisingly, a major decrease in the levels of triglycerides and uric acid was observed in plasma from mice treated orally with the selected natural extracts, as compared to controls.
