Patent Application
20080261896
The invention relates to a testosterone generating and metabolizing enhancer having novel components, and more particularly to a testosterone generating and metabolizing enhancer that may be applied in medicines, substances for external uses on skin, and foods for mammals including humans.
Traditionally, it has been known that fresh stems of herbs of the Cistanche family are effective for treating infertility, impotence, and constipation (please refer to Patent Citation 1). Moreover, preparations made from the fresh stems of the perennial herbs may be used to nutritionally supplement the bloodstream and the kidneys. These perennial herbs have been widely cultivated in the northwestern region of China, and are known as “ginseng from the deserts”. Among the cultivated herbs of the Cistanche family, the holoparasitic herb Cistanche tubulosa (Schrenk.) Wight is the most widely cultivated.
Previously, Japanese researchers have systematically studied the chemical compositions and pharmaceutical properties of the herbs of the Cistanche family, and found that phenylethanoid glycosides are the main active components of these perennial herbs (for example, refer to Non-patent Citation 1-3). The active components may serve as effective anti-oxidants, metabolizing enhancers, memory enhancers, and sexual drive enhancers. Many researchers have further studied the medicinal properties of phenylethanoid glycosides.
Basing on the aforesaid studies, the inventor of the invention had carried out relevant tests, in which rats were fed phenylethanoid glycosides and extracts from herbs of the Cistanche family comprising phenylethanoid glycosides, and the resulted showed that testosterone concentration in the seminal vesicles of the rats was increased. In addition, it was found that after feeding mice the aforesaid substances, testosterone concentration in the bloodstream of the mice was also increased, which indicated that phenylethanoid glycosides and extracts from herbs of the Cistanche family comprising phenylethanoid glycosides may promote the gene expression of turning cholesterol into the enzymes of testosterone, as well as promoting the gene expression of synthesizing 5α-reductase (which allows testosterone to be metabolized into dihydrotestosterone); the results formed the basis for the present invention. In other words, a major objective of the invention is to propose a testosterone generating enhancer, which may enhance the gene expression of testosterone generation, and consequently increase testosterone concentration in the seminal vesicle and the bloodstream. Additionally, another objective of the invention is to propose a testosterone metabolizing enhancer, which may enhance the gene expression of 5α-reductase (which allows testosterone to be metabolized into dihydrotestosterone), thereby promoting the metabolism of testosterone into dihydrotestosterone.
In order to solve the aforesaid problems, the invention proposes:
(1) A testosterone generating enhancer comprising phenylethanoid glycosides as effective components.
The invention will be further elucidated hereafter. The testosterone generating and metabolizing enhancer of the invention is characterized in that phenylethanoid glycosides are included as the effective components. In addition, “the testosterone generating and metabolizing enhancer” of the invention refers to both “the testosterone generating enhancer” and “the testosterone metabolizing enhancer”.
The aforesaid phenylethanoid glycosides refer to the following compound having the formula (1).
The phenylethanoid glycosides are not limited to the aforesaid scope, and may also include components like echinacoside, acteoside, 2′-acetylacteoside, campneoside I, campneoside II, cistantubuloside A, cistantubuloside B1, cistantubuloside B2, cistantubuloside C1, cistantubuloside C2, crenatoside, decaffeoylacteoside, isoacteoside, rhodioloside, syringalide A 3′-α-L-rhamnopyranoside, and tubuloside A. In addition, the invention may use only one of the aforesaid compounds, or use two or more of the aforesaid compounds.
The phenylethanoid glycosides of the testosterone generating and metabolizing enhancer preferably comprise at least one of echinacoside and acteoside, and more preferably comprise both echinacoside and acteoside, which further enhances the generation and metabolism of testosterone.
The preparation of phenylethanoid glycosides is not limited in any way, and may be obtained by synthesis or extracted from plants. Moreover, if phenylethanoid glycosides are extracted from plants, the plants used as the raw materials for such extraction are not limited in any way, either. For example, the plants used for extraction may include Rehmannia glutinosa (Gaertn.)Libosch. Ex Fisch. Et Mey, plants of genus Stachys (family Lamiaceae), fruits of Piper Linn. (family Piperaceae), and herbs of the Cistanche family. Furthermore, the phenylethanoid glycosides may be extracted from only one of the aforesaid plants, or from two or more of the aforesaid plants. Among the aforesaid plants, the herbs of the Cistanche family are most preferably used for the extraction; this is because the phenylethanoid glycosides from the herbs of the Cistanche family not only include echinacoside and acteoside, but also include 2′-acetylacteoside, campneoside I, campneoside II, cistantubuloside A, cistantubuloside B1, cistantubuloside B2, cistantubuloside C1, cistantubuloside C2, decaffeoylacteoside, isoacteoside, rhodioloside, syringalide A 3′-α-L-rhamnopyranoside, and tubuloside A, and may further enhance the generation and metabolism of testosterone. The plants of the Cistanche family used as the raw materials for extraction are not limited in any way. For example, the plants used for extraction may include Cistanche tubulosa, Cistanche salsa, Aeginetia indica, and Boschniakia rossica, but are not limited thereto. The invention may use only one of the aforesaid plants, or use two or more of the aforesaid plants. However, the holoparasitic plant Cistanche tubulosa is the most preferably used.
The relationship between a composition of the aforesaid phenylethanoid glycosides and the above-mentioned formula (1) is shown below, while the chemical structure of crenatoside is as indicated in the formula (2). Said preparations basically comprise very small or minute amount of components other than the echinacoside and acteoside described above.
The compounds shown in the above-mentioned Table 1 may be identified by using high performance liquid chromatography (HPLC). The conditions for using HPLC are shown below. The stationary phase of HPLC was based on stearyl-silance bonded silica gel, while the mobile phase was based on acetonitrile-0.05M phosphoric acid solution (gradient elution was 4:96 15:85), and the flow rate was 1 ml/min; the detection wavelength was set at 330 nm.
The other testosterone generating and metabolizing enhancers of the invention are characterized in that extracts from the plants of the Cistanche family are included as the effective components.
The plants of the Cistanche family used as the raw materials were not limited to any particular plants. For example, the plants may include Cistanche tubulosa, Cistanche salsa, Aeginetia indica, and Boschniakia rossica, but are not limited thereto. The invention may use only one of the aforesaid plants, or use two or more of the aforesaid plants. However, the holoparasitic plant Cistanche tubulosa is the most preferably used.
Furthermore, said “extracts” are preferably comprised of phenylethanoid glycosides. It should be noted that said phenylethanoid glycosides are not limited to any particular compounds, and may include echinacoside, acteoside, 2′-acetylacteoside, campneoside I, campneoside II, cistantubuloside A, cistantubuloside B1, cistantubuloside B2, cistantubuloside C1, cistantubuloside C2, crenatoside, decaffeoylacteoside, isoacteoside, rhodioloside, syringalide A 3′-α-L-rhamnopyranoside, and tubuloside A. In addition, the aforesaid components may be comprised of only one of the aforesaid compounds, or of two or more of the aforesaid compounds. The components preferably comprise at least one of echinacoside and acteoside, and more preferably comprise both echinacoside and acteoside.
The concentration of echinacoside is not specifically limited; assuming that a total weight of the extracts of a plant of the Cistanche family (such as Cistanche tubulosa) was 100 wt %, echinacoside may make up 10-70 wt %, and preferably makes up 15-50 wt %, or more preferably makes up 20-40 wt %, with 23-38 wt % as the most preferable percentage. Moreover, the concentration of acteoside is not specifically limited; assuming that a total weight of the extracts of a plant of the Cistanche family was 100 wt %, acteoside may make up 1-40 wt %, and preferably makes up 5-30 wt %, or more preferably makes up 7-20 wt %, with 8-10 wt % as the most preferable percentage.
The extraction from the plants of the Cistanche family is not limited to any particular parts of the plants, and may be from leaves, roots, and stems; as long as a desired effect is achieved. The extraction is preferably obtained from stems of the plants of the Cistanche family, and especially from fresh stems.
Methods for extracting from the plants of the Cistanche family are not specifically limited, and may be carried out by using solvent extraction and supercritical fluid extraction.
When using the method of solvent extraction, the solvents used are not limited to any particular solvents, but polar solvents are preferably used. The polar solvents are not particularly limited to any solvents, and may be water, methanol, ethanol, isopropanol, acetone, 1,3-butanediol, ethylene glycol, propylene glycol, glycerin, acetic acid, ethyl acetate, and ether. The extraction may use only one of the aforesaid solvents, or use two or more of the aforesaid solvents. Preferably, the extraction is followed by dilution, concentration, drying, and purification; this is because these treatments facilitate the production of phenylethanoid glycosides extracts at high concentration. The purification step may be carried out by using active carbon processing, resin adsorption processing, ion-exchange resins, and liquidi-liquid reversed flow partitioning.
An embodiment for preparing the aforesaid extracts is described in details below, but the preparation of the extracts is not limited to the following embodiment.
The preparation of the extracts comprised of an extraction step and a purification step. In the first step of the preparation, stems (more preferably to be fresh stems) from a plant of the Cistanche family (such as the holoparasitic plant Cistanche tubulosa) were sliced into thin slices, or pulverized into minute particles or powder. The resulted slices, minute particles or powder were immersed in solvents like water, and low-grade fatty alcohols like methanol or ethanol, or a mixed solution thereof. The extraction step was carried out under room temperature. Subsequently, the mixed solution was filtered, and extracts were obtained by concentrating the filtered solution under reduced pressure or in vacuum. In the second step of the preparation, the extracts were heated in water, and then purified by allowing the extracts to pass through adsorption columns filled with Type D-101 or Type AB-8 large-hole adsorption resins. This was followed by eluting the adsorption columns with water, methanol, ethanol, mixed solutions of water and methanol or water and ethanol as the eluting solvents. The eluting process might be done by using the aforesaid eluting solvents of a predetermined concentration, or the aforesaid eluting solvents might be used according to a concentration gradient. The eluting liquids were collected and concentrated, and then dried by using common drying methods. The aforesaid extracts were obtained after the eluting liquids were dried. The resulted extracts were comprised of phenylethanoid glycosides.
The testosterone generating and metabolizing enhancer of the invention may be used as a raw material of a variety of foods and beverages. For example, the foods and beverages may include edible oils (salad oils), snacks (chewing gums, candies, caramel, chocolates, biscuits, bite-sized cookies, jellies, chewing sweets, and desserts), noodles (soba noodles, oolong noodles, and ramen noodles), dairy foods (milk, ice cream, and yoghurt), seasoning foods (miso sauces and bean pastes), soups, drinks (juices, coffee, red tea, tea, carbonated drinks, and sport drinks), which are the general foods, and may further include health foods (tablets and capsules) and nutritional supplements (nutritional drinks). The aforesaid foods and beverages may be adequately supplemented with the testosterone generating and metabolizing enhancer of the invention.
The foods and beverages may have various ingredients added thereto. For instance, the ingredients may include edible food additives like glucose, fructose, sucrose, maltose, sorbitol, stevioside, corn syrup, lactose, citric acid, tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbic acid, dl-α-vitamin E, sodium erythorbate, glycerin, propylene glycol, glycerin fatty acid esters, polyglycerol ester of fatty acids, sucrose fatty acid esters, sorbitol fatty acid esters, propylene glycol fatty acid esters, Arabia gum, carrageenan, casein protein, gelatin, pectin, agar, vitamin B group, nicotinamide, calcium pantothenate, amino acids, calcium salts, colorants, aroma chemicals, and preservatives. In addition, the testosterone generating and metabolizing enhancer of the invention may have other anti-oxidative substances or health foods added thereto to serve as health supplements. The health supplements may include anti-oxidative substances, ascorbic acid of reducing property (vitamin C), vitamin E, glutathione of reducing property, tocotrienol, derivatives of vitamin A, lycopene, β-zeaxanthin, astaxanthin, zeaxanthin, fucoxanthin, uric acid, ubiquinone, coenzyme Q10, folic acid, extracts of garlic, allicin, sesamolin, cellulose, catechin, isoflavone, chalcone, tannic acid, flavone, coumarin, isocoumarin, bilberry extracts, arbutin, tannins, anthocyanin, apple polyphenols, grape seed extracts, ellagic acid, kojic acid, Seabuckthorn extracts, vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, vitamin D, vitamin E, vitamin P, choline, nicotinamide, pantothenic acid, calcium folinate, EPA, oligosaccharides, food fibers, squalene, soy lecithin, taurine, Dunaliella, proteins, octacosanol, DHA, egg yolk lecithin, linoleic acid, lactoferrin, magnesium, zinc, chromium, selenium, potassium, ferrihemoglobin, extracts of oyster flesh, deacetylated chitin, chito-oligosacchrides, collagen, chondoritin, tulips, licorice, Fructus lycii, cinnamon, crab apples, fresh ginger, Ganoderma lucidum, extracts of shellfish, Trionychidae species, licorice, Fructus lycii, cinnamon, crab apples, fresh ginger, Ganoderma lucidum, Plantago asiatica, Chrysanthemum, Camomiles, dandelion, Hibiscus, honey, spinach, royal jelly, limes, lavender, fruits of wild rose, Rosemary, Sage, Bifidobacterium, faecalis, spore-bearing lactic acid bacteria, wheat germ oil, castor oil, perilla oil, soy oil, middle-chain fatty acids, Agaricus, extracts of Ginkgo leaves, tulips, chondoritin, extracts of germ, Ganoderma lucidum, onions, DHA, EPA, DPA, sweet tea, Cordycrps sinensis (Berk.) Sacc., garlic, bee larvae, papaya, Pu'er tea, propolis, Acer nikoense Maxim, Hericium erinaceum, royal jelly, extracts of Saw palms, hyaluronic acid, collagen, GABA, seal oil, shark cartilage, glucosamine, lecithin, phosphatidylserine, Panax notoginseng (Burk.), mulberry leaves, extracts of soy beans, plants of the Echinacea genus, Siberian ginseng, extracts of barley, olive leaves, olive fruits, Gymnema, Lagerstroemia speciosa (L.)Pers., Salacia, Garcinia, deacetylated chitin, Hypericum perforatum, Fructus Jujubae, ginseng, Passiflora edulis, cauliflower, placenta, Coix lacryma-jobi, grape seeds, peanut peels, bilberry, Black Cohosh, Milk Thistle, bay tree, Sage, Rosemary, Apocynum venetum L., black vinegar, Bitter melon, Maca, safflower, flax, oolong tea, Oxytropis grandiflora, caffeine, capsaicin, xylooligosaccharide, glucosamine, soba, plants of the Citrus genus, food fibers, proteins, dried plum, spirulina, fresh leaves of barley, nucleic acids, yeasts, mushrooms, plum flesh, amino acids, extracts of sharks, noni, oyster flesh, Trionychidae species, Agaricus campestris, Plantago asiatica, cherries, pineapples, bananas, peaches, apricots, melons, strawberries, berries, oranges, fucoidan, Phellinus linteus, cranberries, chondroitin sulfate, zinc, iron, ceramide, silk peptide, glycine, nicotinic acid, Chaste Tree, ceramide, L-cysteine, L-camitine, leaves of red grape, maize, Equisetum debile Roxb, biotin, Gotu Kola, Lonicera caerulea L., pycnogenol, Petasites japonicus, Rheum palmatum L., plants of the Syring a species, Rosemary, catechins, Pu'er tea, citric acid, beer yeast, Melilot, black soybean, fresh ginger, Rhizoma Curcumae Aeruginosae, Bacillus natto, red yeast rice, tocotrienol, lactoferrin, cinnamon, F. tataricum, cocoa powder, Citrus grandis seed extracts, perilla seed extracts, Litchi seed extracts, evening primrose extracts, brown rice extracts, α-lipoic acid, GABA, extracts of raw coffee beans, extracts of Petasites japonicus, kiwi seed extracts, Unshu orange extracts, fresh ginger extracts, and astaxanthin.
For the preparation process, the testosterone generating and metabolizing enhancer (if it is the extract, the extract should be process with cyclodextrin) of the invention may be directly made into powder, granules, tablets, or solutions by using spray drying or freeze drying, so that the products may be readily included into foods (such as instant foods). Moreover, the testosterone generating and metabolizing enhancer or extracts of the invention may also be mixed with adhesive agents like Arabia gum, so as to make the products into powder or granules, and add into solid foods. For the extracts, it may be directly added into beverages, or dissolved in solutions like water, ethanol, glycerin, or a mixture thereof, before adding the extracts into beverages.
The testosterone generating and metabolizing enhancer of the invention may also be used as raw materials for medicines (which includes oral medicines and medicines for external applications). The aforesaid medicines can be manufactured by appropriately adding the testosterone generating and metabolizing enhancer of the invention into raw materials for making the medicines. The aforesaid medicines may be applied to humans, and also other mammals other than the humans. The raw materials that may be used in combination with the testosterone generating and metabolizing enhancer of the invention for making the medicines include: emulsifying agents (glucose, lactose, sugar, sodium chloride, starch, calcium carbonate, kaolin, crystal cellulose, cocoa butter, plant hardening oil, kaolin, and talc powder), adhesive agents (distilled water, normal saline solution, ethanol solution, sugar solution, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, potassium phosphate, and polyvinylpyrrolidone), disintegrating agents (sodium alginate, agar, sodium bicarbonate, calcium carbonate, dodecyl sodium sulfate, glycerol stearate, starch, lactose, Arabia gum powder, gelatin, and ethanol), anti-disintegrating agents (sugar, stearate, cocoa butter, and hydrogenated oils), absorption promoters (quaternary ammonium hydroxide and dodecyl sodium sulfate), adsorption agents (glycerin, starch, lactose, kaolin, clay, and silicate), and lubricants (fine talc powder, stearate, and polyethylene glycol).
The testosterone generating and metabolizing enhancer of the invention may be orally administered as slices, tablets, soft capsules, hard capsules, granules, powder, and particles. In addition, the testosterone generating and metabolizing enhancer may also be administered as solution-based doses, and the solution-based doses may be administered orally or non-orally. For the non-oral administration, the testosterone generating and metabolizing enhancer may be dissolved in appropriate solvents like ethanol or water, and used as cataplasms, washing solutions, pastes, tinctures, and ointments. The aqueous solutions of the testosterone generating and metabolizing enhancer may be used directly, or have dissipating agents, suspending agents, or stabilizing agents added thereto, before being administered as cataplasms, washing solutions, pastes, tinctures, and ointments.
The administered dosage may vary according to administrative methods, patients' conditions, and patients' age. But an adult is generally administered a daily effective dosage of 5-400 mg, and a child is administered a daily effective dosage of 0.5-200 mg.
When the testosterone generating and metabolizing enhancer is applied for medicinal uses, a ratio thereof may be altered according to the types of administration. Generally, when the orally administered dosage is absorbed through membranes, a ratio for adding the testosterone generating and metabolizing enhancer may be approximately 0.01-10 wt %; but when it is not administered orally, a ratio for adding the testosterone generating and metabolizing enhancer may be approximately 0.01-20 wt %. Because the administrative dosage varies according to different conditions; in some cases, the administrative dosage may be sufficient even if it is smaller than the aforesaid ranges of administrative dosage, or the administrative dosage may have to be more than the aforesaid ranges of administrative dosage. In addition to the testosterone generating and metabolizing enhancer, the medical composition may also comprise other known compounds commonly used for medical purposes, as well as compounds that are commonly used in the orally administered drugs. The compounds may include lactose, starch, hydroxypropyl cellulose, kaolin, talc powder, and calcium carbonate.
The testosterone generating and metabolizing enhancer may also be included into animal feeds for mammals. The testosterone generating and metabolizing enhancer may be included in said animal feeds the same way it is included in the aforesaid foods and beverages. The animal feeds are not limited to uses for any particular animals. For instance, the animal feeds may be formulated to feed farm animals like cattle and pigs, and companion animals like dogs, cats, and hamsters. The animal feeds may include flour and meat as ingredients. Said flour may be comprised of wheat powder, rice powder, rye powder, oat powder, barley powder, grain powder, corn powder, and soy powder; said flour may also be comprised of two or more of the aforesaid powder. The use of said flour may provide the necessary carbohydrates for the companion animals. Among the aforesaid powder, the wheat powder is preferably used. The wheat powder may be used alone or used with high-grade flour, middle-grade flour, and low-grade flour; the wheat powder may also be used with any other types of flour. The elasticity of the heat-processed animal feeds may be adjusted by combining the wheat powder with wheat gran and soy proteins. After the heat treatment, the lattice-like structures in wheat bran will become enlarged, which helps improve its taste.
The meat used in the invention is not specifically limited, and may use chicken, pork, beef, mutton, goat meat, rabbit meat, turkey, and horse meat; chicken is preferably used. The meat may be obtained by using the conventional methods for slaughtering and cutting commonly available farm animals. The deterioration of quality in animal feeds having middle level or low level of water is due to the oxidation of fat, and the solution is to use lean meat that has less or no amount of fat. The composition of meat in the animal feeds not only provides animal proteins of decent quality for the animals being fed, but also improves the behavior of the companion animals.
Though the animal feeds for the companion animals may be fabricated by using a variety of methods, the preferable methods include the following: The animal feeds may be fabricated with a mixture (referred to as “raw dough” hereafter) of raw materials that include flour (with wheat powder as the preferable choice) and meat, and heated after being shaped. The raw dough has flour and meat in a composition thereof, and a ratio between the flour and meat is not specifically limited. Generally, the composition has approximately 5-60% of flour and 5-80% of meat, while the preferably percentage of flour is 10-50%, and the preferable percentage of meat is 20-50%; the raw dough also has water of a desired amount in the composition. When an Aw adjusting agent is used, the Aw adjusting agent takes up 5-30% in the composition, and is preferably between 10-20%. The percentage of water may be adjusted according to the levels that the raw dough is mixed, stirred, and shaped, and also according to the percentage of flour, meat, and the Aw adjusting agent. The methods for fabricating the raw dough is not specifically limited, and the meat is preferably made into fillings by using cutting machines or grinders. In order to sufficiently fill the meat used for making the fillings with bubbles, the meat is preferably sliced into thin slices. The meat used for making fillings are added with flour, water, and the Aw adjusting agent according to actual needs, and then sufficiently stirred to fill in bubbles, so as to make raw dough that contains bubbles. Foaming agents may also be added when fabricating the raw dough, and is preferably used when the aforesaid grain powder except the wheat powder is used. The addition of the foaming agents allows tiny bubbles to be evenly filled into the raw dough. The foaming agents may be all types of foaming agents, but the foaming agents based on soy proteins and/or soy proteins that have been digested with enzymes are preferably used, which is for stabilizing the bubbles.
After shaping and heating the raw dough described above, the animal feeds for the aforesaid companion animals are obtained consequently. When shaping the raw dough, the aforesaid animal feeds may be shaped into appropriate shapes according to factors like facilitating the eating process of the companion animals, and facilitating the feeding process of the owners of such animals. For example, the animal feeds may be shaped into slices, rods, round slices, rings, and heart-shape. Moreover, the raw dough that have been fabricated by using identical methods may be colored with colorants of different tones of color; vegetable and fruits may also be added thereto in order to fabricate raw dough that possess a variety of appearances, and the various raw dough can be combined to form multiple layers or concentric circles as the final shapes. Methods for heating the shaped raw dough are not specifically limited, and may be carried out by using ovens or microwave ovens. The heating methods are commonly known and can be done by the commonly known procedures. After heating, the animal feeds usually comprise approximately 20-40% of water therein. The raw dough also becomes blown up because the heating treatment has caused water therein to evaporate and the bubbles to expand; additionally, because water was evaporated quickly, the Aw of the raw dough was lowered and thus making it more preservable. Furthermore, if wheat powder was used as the flour, the heat treatment would fix the lattice-like structures in wheat bran of the wheat powder, which helps improve its taste. By using an oven for heating the raw dough, a resulted animal feed will appear in a unique color tone (light brown) or be imparted with a unique smell. When a microwave oven is used for heating, because the raw dough is heated from within, the raw dough will become evenly blown, and a resulted animal feed will have evenly distributed bubbles therein. When carrying out the heating treatment, the Aw of the resulted animal feed is preferably between 0.6-0.9. By keeping the Aw within this range, the animal feed may be made more preservable.
The animal feeds fabricated by using the aforesaid methods are made bread-like, and because the animal feeds have soft taste, soft texture, and adequate elasticity, the animal feeds are suitable for serving as the food for the companion animals that have weaker teeth; including puppies, older dogs, and cats. For packing, adequate amounts of the aforesaid animal feeds should be packed into packages, followed by sealing in order to make the animal feeds into final products. Said packages are preferably airtight packing materials that do not allow oxygen to pass through. Methods for packing include vacuum packing and packing in packages filled with active carbon, but the preferable method is to use packages simultaneously filled with deoxidants (For example, Ageless™) and active carbon. This packing method or similar ones protects the animal feeds from spoiling resulted from oxidation and microbial growth within an expiry date.
The testosterone generating and metabolizing enhancer of the invention may also be used on skin externally (such as in cosmetics, medicines, and external medical applications), and the substances for external uses on skin may inhibit the actions of hyaluronidase. The substances for external uses on skin may be applied to humans, and may also be applied to mammals other than the humans. The testosterone generating and metabolizing enhancer may be applied in substances for external uses on skin, which includes lotions, bars of soap, facial cleansers, body cleansers, skin creams, lotions, toners, toning lotions, shaving creams, shaving toners, makeup oils, sun-blocking toners, foundation powder, foundation, perfumes, masks, nail polishes, enamels, nail polish removers, brow dyes, blushes, eye creams, eye shadows, mascaras, eyeliners, lipsticks, lip glosses, shampoos, hair conditioners, hair dyes, dissipating solutions, and washing solutions. In addition, the testosterone generating and metabolizing enhancer may be applied as medicines or for external medical uses in forms of ointments, creams, and external-use liquids.
In addition to the testosterone generating and metabolizing enhancer, the aforesaid substances for external uses on skin may also have the following substances, which do not impede the generation and metabolism of testosterone and may also be added into cosmetics and medicines for external uses on skin; including: oil, higher alcohol, fatty acid, ultraviolet absorber, powder, colorant, surfactant, polyhydric alcohol, sugar, polymer, biologically active ingredient, solvent, anti-oxidant, aroma chemical (perfume material), and preservatives. Examples of the aforesaid substances are listed below, though the invention is not limited to these examples.
Although particular examples of the invention will be described in details for purposes of illustration, the invention is not limited to the following examples.
Preparation of the testosterone generating and metabolizing enhancer of the present invention.
Preparation of extracts from the holoparasitic plant Cistanche tubulosa of the Cistanche family (abbreviated as C. tubulosa hereafter).
Methods for Extraction
10 kg of thinly sliced fresh stems of C. tubulosa were immersed in water that was eight times the weight of the thin slices for one hour, and then boiled for two hours; the boiled mixture was subsequently filtered to obtain a first filtered solution. Afterwards, leftovers from the first filtration were mixed with 40% ethanol that was four times the weight of the leftovers and boiled for four hours; the boiled mixture was filtered again to obtain a second filtered solution. Subsequently, leftovers from the second filtration were subjected to the aforesaid procedures for another two times in order to obtain a third filtered solution and a fourth filtered solution. The aforesaid four filtered solutions were then combined and concentrated in vacuum until a resulted concentrated solution had reached a weight of 1.05 (50° C.), thereby resulting in a final extract. Said final extract obtained by using the aforesaid method had a weight of 6.2 kg.
Methods for Purification
6 kg of the aforesaid final extract was dissolved in water that was half the weight of the final extract by heating, and then a resulted solution having the final extract was allowed to pass through the aforesaid adsorption columns filled with Type D-101 large-hole adsorption resins. Afterwards, water was firstly used to elute the adsorption columns, and a water eluting liquid that was twice the weight of said fresh stems was obtained; 20% ethanol was subsequently used to elute the adsorption columns and obtain a first eluting liquid of 20% ethanol that was also twice the weight of said fresh stems. Subsequently, said water eluting liquid underwent one more round of adsorption-elution steps, and a second eluting liquid of 20% ethanol was obtained. The two 20% ethanol eluting liquids were consequently combined, concentrated, and dried to obtain an extract having phenylethanoid glycosides (the first embodiment). The extract had a weight of 0.865 kg.
The concentrations of echinacoside and acteoside were determined by using HPLC, and the HPLC was set as follows: the stationary phase was based on stearyl-silance bonded silica gel; the mobile phase was based on methanol-0.15% acetic acid solution (30:70), and the flow rate was 1 ml/min; the detection wavelength was set at 330 nm.
After drying in vacuum for 24 hours at 60° C., the echinacoside and acteoside were dissolved in 50% methanol in order to make a control solution (in which 1 ml of solution has 0.1 mg of dissolved substances).
Using supersonic treatment, 50 mg of the extract having phenylethanoid glycosides was dissolved in 50% methanol of adequate amount in a 25 ml container, and then more 50% methanol was added into said solution until said solution reached a line indicating 25 ml. Precisely obtained 1 ml of said solution to place into a 10 ml container, and filled 50% methanol into the container until a line indicating 10 ml was reached. Said solution was filtered by using a 0.45 μm membrane to obtain a sample solution.
5 μl was separately obtained from the control solution and the sample solution and injected into a HPLC instrument, so as to respectively determine peak areas of echinacoside and acteoside, so that concentrations of echinacoside and acteoside could be calculated from the peak areas thereof. The outcome was that echinacoside occupied 29.3 wt % in the extract (the first embodiment), and acteoside took up 10.0 wt % in the extract (the first embodiment).
A pure product of echinacoside was used in Example 2 (manufactured by Sinphar Pharmaceutical Co., Ltd.), while a pure product of acteoside was used in Example 3 (manufactured by Sinphar Pharmaceutical Co., Ltd.).
Test 1: Testing testosterone generation of the mouse Leydig tumor cell line R2C.
Materials: The extract of C. tubulosa prepared in the aforesaid first embodiment, and said echinacoside and acteoside used in the second and the third embodiments. The mouse Leydig tumor cell line R2C was obtained from the Japan Health Sciences Foundation (Osaka), and a Testosterone EIA testing kit was used to test testosterone levels.
Method: The R2C cells were subcultured in a cell medium called Ham's F10, which comprised 15% bovine serum, 2.5% fetal bovine serum, 100 units/mL penicillin G, and 100 μg/mL streptomycin. The cells were inoculated on a 24-hole plate at a density of 5×105 cells/mL, and 500 μL of the cell medium was added into each hole. After culturing for 24 hours, 50 μL of solutions resulted from dissolving samples from the first to the third embodiments in the same cell medium was added into each hole. After culturing for another 4 fours, the cells were collected along with the cell medium and subjected to centrifugation, so as to collect supernatant that included the cell medium. The Testosterone EIA testing kit (manufactured by Cayman Chemical Corporate) was used to determine testosterone concentrations in the each tested sample, and the results were shown in
Results and Effects of Examples in Test 1: As shown in
Test 2: Testing testosterone levels in the bloodstream of mouse.
Method: 5-month-old male ddY mice were orally fed the C. tubulosa extract from the aforesaid first embodiment, and the regimen continued for two weeks. Afterwards, blood samples were collected from the mice in order to determine the testosterone levels therein, and blood samples were also collected from 6-week-old mice to serve as a control.
Results and Effects of Examples in Test 2: As revealed in Table 2, the feeding of the C. tubulosa extract (200 mg/kg and 400 mg/kg) increased levels of testosterone in the bloodstream of the mice.
Test 3: Effects on the expression of 5α-reductase in mice livers (microarray analysis)
Method: Firstly chose a 5-month-old mouse (from the control) from the aforesaid Test 2 and a mouse that was fed the C. tubulosa extract of the first embodiment only, and then removed the livers from the mice. Subsequently, thin slices of the livers (approximately 10 mg) were immersed in RNAlater (manufactured by Qiagen K. K.) for stabilization, and then Rneasy protect mini (manufactured by Qiagen K. K.) was used to extract and purify total RNA. This was followed by the use of genetic chips (Mouse 4302.0, manufactured by Affymetrix Inc.) to carry out microarray analysis for gene expression of the RNA; the result is shown below.
Results and Effects of Examples in Test 3: Referring to Table 3; in the liver of the mouse being fed the C. tubulosa extract (400 mg/kg) of the first embodiment, the gene expression of 5α-reductase was increased 2.24-fold as compared to the control. The result indicated that the C. tubulosa extract of the first embodiment was effective for enhancing the gene expression of 5α-reductase, which in turn enhanced the metabolism of testosterone into dihydrotestosterone.
Test 4: Testing effects of repeated feeding of the extract of the first embodiment on the gene expression of enzymes responsible for testosterone synthesis and metabolism (by using RT-PCR).
Method: 5-month-old male ddY mice were orally fed 400 mg/kg of the C. tubulosa extract (the extract of the first embodiment), and the regimen continued for two weeks before livers were removed from the mice; RNAlater was then used to stabilize the livers, and RNA was extracted from the livers by using a testing kit manufactured by Qiagen K. K. Using commonly available methods, c-DNA was fabricated via reverse transcription, and gene expression was determined via RT-PCR. The results are shown in
Results and Effects of the Embodiments in Test 4: The results shown in
Formulas for formulating the testosterone generating and metabolizing enhancer of the invention are listed below, though the invention is not limited to these formulas.
As described previously, a testosterone generating enhancer is proposed in the invention, which enhances the gene expression of enzymes related to testosterone synthesis, and increases testosterone levels in the seminal vesicle and the bloodstream. In addition, a testosterone metabolizing enhancer is also proposed in the invention, which enhances the gene expression of 5α-reductase (which metabolizes testosterone into dihydrotestosterone), such that the metabolism of testosterone into dihydrotestosterone is promoted subsequently.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2007-103561 | Apr 2007 | JP | national |
| 2007-127458 | May 2007 | JP | national |
