US 201302.95140A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/029514.0 A1 Shifrine (43) Pub. Date: Nov. 7, 2013

(54) OLFACTION (52) U.S. Cl. CPC ...... A61K 36/062 (2013.01); A61K 39/36 (71) Applicant: Moshe Shifrine, Santa Fe, NM (US) (2013.01) USPC ...... 424/275.1; 424/195.15 (72) Inventor: Moshe Shifrine, Santa Fe, NM (US) (21) Appl. No.: 13/864,520 (57) ABSTRACT A composition of an olfactory stimulus mechanism for stimu (22) Filed: Apr. 17, 2013 lating the production of testosterone. A method of increasing O O the level of testosterone in an individual by smelling the Related U.S. Application Data extract, causing in the truffle extract to (60) Provisional application No. 61/641,358, filed on May attach to the olfactory bulb in the individual, and inducing 2, 2012, provisional application No. 61/734,551, filed testosterone production. A method of preventing the onset of on Dec. 7, 2012. Alzheimer's disease in an individual by the individual smell ing the truffle extract, and inducing testosterone production in Publication Classification the individual. A method of treating symptoms of menopause in a woman by the woman Smelling the truffle extract, and (51) Int. Cl. inducing testosterone production in the woman. A method of A6 IK36/062 (2006.01) treating andropause in a man by a man Smelling the truffle A6 IK39/36 (2006.01) extract, and inducing testosterone production in the man. US 2013/0295 140 A1 Nov. 7, 2013

TESTOSTERONE OLFACTION 0007 Treatments for low testosterone currently include hormone replacement therapy (HRT or TRT testosterone BACKGROUND OF THE INVENTION replacement therapy). Testosterone can be given to men as an intramuscular injection, patch or gel placed on the skin, or a 0001 1. Technical Field putty applied to the gums of the mouth. Some experts Suggest 0002 The present invention relates to hormone treat that testosterone replacement therapy might reduce the need ments. More specifically, the present invention relates to tes for the medications entirely. Unfortunately, tOSterOne treatmentS. there is no current treatment available for women. Some 0003 2. Background Art women resort to using patches available for men and cutting 0004 Testosterone is a compound needed and produced them to a size more appropriate of a dose for women, but this by the body in both men and women for several different has questionable accuracy and safety. functions. It is needed for the development and maintenance 0008 ANDROGEL(R) (Abbott Laboratories) is one prod of sex organs and characteristics, as well as for muscle uct on the market for TRT for men that contains synthetic growth, bone development, and bone maintenance. The pro testosterone, and when applied to the skin, it increases the duction of testosterone is regulated by the brain through the concentration of testosterone. The dose used is 100 times as release of hormones. Testosterone is produced in the testes, what is present in the blood. The use of ANDROGEL(R) can 95% and 5% in the adrenals. To stimulate its production the produce unwanted side effects, such as causing irritation to hypothalamus releases to the pituitary gland a gonadotropin the skin due to the carrier used which includes many chemi releasing hormone (GnRH), which, in turn, causes the pitu cals to facilitate penetration through the skin. It is also an itary to produce a follicle-stimulating hormone (FSH) and a expensive drug. (LH) collectively known as gonadotro 0009 Olfaction, the sense of smell, is critical for the sur pins. LH is released into the blood stream and travels to the vival of almost all creatures. Humans are able to distinguish testes to trigger the production of testosterone from choles over 10,000 different odor molecules. With every inhalation, terol (). When the testosterone level gets too high the pitu currents of air swirl through the nostrils over the bony turbi itary slows the release of LH to slow it down. The actual nates in the nose that contain receptors in the olfactory epi testosterone production occurs in the Leydig cells in the testes thelium. The cilia projecting from the olfactory knob contain and adrenals in men and also in women production occurs in receptors for odorants. The cilia project from the knob the and adrenals. Healthy young men produce about 6 directly into the atmosphere. The interaction of the right milligrams of testosterone per day and healthy women pro molecule with the right receptor causes a structural transfor duce 0.3 mg of testosterone per day (/20" of what men pro mation of the receptor, which gives rise to an electrical signal duce). to the olfactory bulb and thence to the areas in the limbic brain 0005 While age can naturally decrease testosterone lev that perceive it as the original Smell. els, many individuals suffer from lower than normal levels of 0010. In 2000 Shinohara, et al. published a study on the testosterone. Low levels of testosterone can be caused by effect of on the pulsating secretion of the lutein hypogonadism (a problem with the organs that produce test izing hormone (LH) in human females. The Subjects, female osterone, i.e. the ovaries or testes), secondary hypogonadism college students, had their upper lip exposed to androstenol (a problem with regulation of testosterone), or tertiary and their pulsating frequency of LH was measured. The fre hypogonadism (a problem with the hypothalamus). Each of quency of the pulse in the was decreased. This these problems can be caused by a variety of factors, includ is a direct proof that Sniffing androstenol affects the LH pulse. ing aging, injury, disease, cancer therapies such as chemo In a subsequent study (Shinohara, et al. 2001) it was deter therapy and radiation therapy, chromosomal abnormalities, mined that axillary (putatively androstenol) drug use, and obesity. In particular, secondary and tertiary modulated the LH of humans. hypogonadism are caused by diseases that affect the hypo thalamic-pituitary-gonadal axis (i.e. the system of the hypo 0011 Preti, et al. (2003) showed that male axillary extracts thalamus, pituitary gland, and gonads). The hypothalamus (underarm secretions) affect the pulsating secretion of LH produces gonadotropin-releasing hormone (GnRH), the ante and the mood of the women participants in this experiment. rior portion of the pituitary gland produces luteinizing hor 0012. From these studies, it is evident that presenting mone (LH) and follicle-stimulating hormone (FSH), and the human hormones through the olfactory system elicits a physi gonads produce testosterone. ologic response. 0006 Low levels oftestosterone, commensurate with high 0013 Another study (unpublished work by Shifrine) levels of , are also associated with stress, depression showed that pigs fed cultured truffle powder (i.e. French and other psychological disorders. This occurs through inter grown in hydroponics and on grain) in their feed connections linking the components of the body in stress exhibited improved feed efficiency, significantly lowered apparatus i.e. the nerves, hormonal glands (HPA axis) and the feed consumption, significantly lowered fat, and increased immune system. Other symptoms of low testosterone in men leanness of carcasses. The very low dose of 1 gram dried include decreased libido, infertility, erectile dysfunction, loss powdered cultured truffle per ton of feed suggested that the of hair, decreased muscle mass, osteoporosis, and, in women, effect of the truffle was due through inhalation and not inges include hot flashes, irritability, decreased libido, sleep distur tion. This suggests a “testosterone response'. However, at the bances, loss of muscle mass, osteoporosis, and loss of body time of this study, testosterone had not been reported in sci hair. To make matters worse for aging men, many conven entific literature to be present in truffles. Androstenol, shown tional antidepressant medications suppress testosterone pro to be contained intruffles, on the other hand was isolated from duction and thus libido. As women go through menopause, pigtestes by Prelog & Ruziaka (1944). It was later discovered they also have many of these problems because of lower in truffles and offered as an explanation for the ability of pigs levels of testosterone. to detect this hypogeous mushroom (Claus et al., 1981). US 2013/0295 140 A1 Nov. 7, 2013

0014. In 2006, Applicant discovered that, in addition to ca. 6x10" molecules. There are ample molecules to coverall androstenol, the truffle culture produces testosterone, which receptors in the nose. Since pigs are the best experi might explain the testosterone effect in pigs, and the reason mental animals for humans (Simon and Maibach, 2000) it is truffles are reputed to have aphrodisiac potential. At the con reasonable to extrapolate the data from the pig study centration of 1.7 ug of testosterone in agram of dry weight of described above to humans. Based on the finding of testoster truffles (truffle powder, from truffles grown on grain), which one, in the cultured truffles, the present invention is based on is equivalent to ca. 6x10" molecules, there are ample mol the finding that cultured truffle testosterone scent activates ecules to coverall androgen receptors in the nose. Since pigs androgenic receptors in the brain of men and women with are the best experimental animals for humans (Simon and Subsequent initiation of its anabolic pathway. Maibach, 2000) it is reasonable to extrapolate the data from 0024 Preferably, the composition is prepared by harvest the pig study to humans. ing truffles, drying, and then milling the truffles to a fine 0.015 Therefore, there remains a need for a safe and effec powder. The fine powder can then be combined with any tive method of increasing testosterone in both men and appropriate excipients as further described below, or be used women who suffer from low levels of testosterone. in nutraceutical formulations. A tincture extract of 0.5 g to 1 g or more of truffles can be administered; however, even SUMMARY OF THE INVENTION Smaller amounts such as picograms or less of the truffles can 0016. The present invention provides for a composition be used. that stimulates the production of testosterone through olfac 0025. Alternatively, any other composition that can pro tion. vide olfaction of testosterone can be used. For example, the 0017. The present invention provides for a method of composition can be pollen of the pine tree Pinus silvestris that increasing the level of testosterone in an individual by smell contains testosterone. While the truffle extract is further ing the truffle extract, causing androgens in the truffle extract described in the methods herein, it should be understood that to attach to the olfactory bulb in the individual, and inducing one skilled in the art can use any other composition that testosterone production. provides olfaction of testosterone in the same manner. 0018. The present invention provides for a method of pre 0026. The composition can be in the form of a pharmaceu venting the onset of Alzheimer's disease in an individual by tical composition or a nutraceutical composition. The phar the individual Smelling the truffle extract, and inducing test maceutical or nutraceutical composition is preferably a topi osterone production in the individual. cal composition for application to the skin. The composition 0019. The present invention provides for a method of can be applied to any part of the skin, such as, but not limited treating symptoms of menopause in a woman by the woman to, the wrist or the upper lip, much like a perfume. The truffle Smelling the truffle extract, and inducing testosterone produc extract can be added to a pharmaceutically/nutraceutically tion in the woman. acceptable carrier of or oil that still allows for the scent of the truffle to be detected. Preferably, the carrier is 0020. The present invention also provides for a method of alcohol because it evaporates easily from the skin; however, treating andropause in a man by a man Smelling the truffle any other appropriate carrier (such as a wax or a cream) can be extract, and inducing testosterone production in the man. used. Furthermore, while not preferred in combination with the alcohol carrier, any other pharmaceutical or nutraceutical DETAILED DESCRIPTION OF THE INVENTION excipients can also be used where appropriate. The amount of 0021. The present invention provides generally for a com truffle extract in a pharmaceutically or nutraceutically effec position that stimulates the production of testosterone tive amount can be determined by one skilled in the art but through transmucosal administration (administration via any generally is in an amount to increase the levels of testosterone mucosal surface of the body, and preferably by olfaction) and in an individual and return the testosterone levels to a normal a method of treating low testosterone levels in both men and level. women with the olfaction. This is a unique method that is used 0027. The composition is administered to an individual, as a testosterone replacement therapy. preferably a mammal, such as, but not limited to, humans and 0022 Preferably, the composition of the present invention pigs. One preferred time of administration is in the evening or is a truffle extract that provides an olfactory stimulus. Truffles night before the individual goes to sleep. Testosterone has a are mushrooms, and more specifically, the fruiting bodies of circadian rhythm so that it is highest in the morning and Subterranean mushrooms. Truffles are generally found near lowest at night. Therefore, administration of the composition tree roots of beech, poplar, oak, birch, and pine trees. Any type during the lowest natural levels of testosterone can have a of truffle can be used to obtain the truffle extract, such as, but greater effect on the individual. The individual using the not limited to, white truffle (Tuber magnatum, Tuber magna composition can be any age, but it is preferable to use the tum pico, or Tuber borchii), black truffle (Tuber melano compound at an age when testosterone levels are lower than sporum), black Summer or burgundy truffle (Tuber aestivum/ normal. For example, at ages 50 to 60, testosterone levels can uncinatum), Tuber macrosporum, Tuber Oregonense, Tuber be approximately half of the levels at age 13 (the age at which gibbosum, and Tuber lyoni. The truffles are preferably cul testosterone levels are generally highest). Use at the ages of tured truffles, but wild truffles can also be used. Cultured 50 to 60 can be beneficial for the prevention of libido as well truffles (in particular Tuber melanosporum) have been found as Alzheimer's disease. Alzheimer's disease generally has a to be identical to wild truffles by DNA analysis. The extract 15-year period between the onset and the development of can also be derived from truffle mycelium grown in vitro. symptoms, and therefore, early administration of the compo 0023 Truffles naturally contain testosterone, and the sition before any symptoms can help in the prevention of present invention provides for an extract from a truffle that Alzheimer's disease (also further described below). can be smelled by an individual. One gram of dry weight of 0028. The composition of the present invention is admin truffles contains 1.7 ug of testosterone, which is equivalent to istered and dosed in accordance with good medical practice, US 2013/0295 140 A1 Nov. 7, 2013

taking into account the clinical condition of the individual of Alzheimer's disease can be prevented or delayed by using patient, the site and method of administration, Scheduling of the truffle extract of the present invention and increasing the administration, patient age, sex, body weight and other fac levels of testosterone in the individual. The testosterone can tors known to medical practitioners. The pharmaceutically then regulate B-amyloid and prevent accumulation in the “effective amount” for purposes herein is thus determined by brain. Such considerations as are known in the art. The amount must 0033. The present invention provides a method of treating be effective to achieve improvement including but not limited symptoms of menopause in women by a woman Smelling the to improved Survival rate or more rapid recovery, or improve composition (i.e. truffle extract) and inducing the production ment or elimination of symptoms and other indicators as are of testosterone and thereby increasing levels of testosterone selected as appropriate measures by those skilled in the art. in a woman. Many women Suffer from Sweats, hot flushes, 0029. The doses can be single doses or multiple doses over inability to sleep, lack of sex drive, and lack of vitality during a period of several days. The treatment generally has a length menopause, and the truffle extract can prevent or lessen any or proportional to the length of the disease process and drug combinations of these symptoms. The olfaction of truffle can effectiveness and the patient species being treated. be used in addition to / treatment. It is 0030 The present invention provides for a method of ini generally known by doctors that testosterone is essentially in tiating the normal production of testosterone in an individual addition to estrogen/progesterone treatment. by Smelling the composition (i.e. truffle extract). Essentially, 0034. The present invention also provides a method of the levels of testosterone are increased in the individual. treating andropause in men by a man Smelling the composi Smelling the truffle induces testosterone production in the tion (i.e. truffle extract) and inducing the production of test individual when androgenic receptors in the brain are acti osterone and thereby increasing levels of testosterone in a vated. Androgens contained in the truffle extract travel to the man. Andropause is considered similar to menopause but nose upon Smelling and attach to androgen receptors in the occurring in men due to the loss of testosterone production, olfactory bulb. Preferably, the application is performed by and causes the symptoms described above when lower levels applying to the skin before an individual goes to sleep of testosterone are produced. because testosterone levels are at their lowest at this time. 0035. There are several advantages to this method. The 0031. By smelling the truffle extract, several things hap scent is a natural product, whereas current testosterone treat pen in an individual. Due to the increased testosterone levels, ments use only synthetic testosterone. The scent of the truffle vitality, well-being, mood, mortality, sexuality (libido drive initiates an immediate response of increasing testosterone and performance), muscle (lean body mass and muscle), levels through the stimulation of testosterone production, not immunity, bone mass and repair, platelet aggregation, cogni by adding testosterone to the body unlike other treatments. tion, blood pressure, HDL levels, and sleep qual The composition is easy to apply because it is applied to the ity are all improved. With regards to homeostasis, by increas skin much like the application of perfume and can be done ing testosterone levels, abdominal fat is reduced (fat without a physician. Furthermore, the composition cannot be Suppresses LH action and therefore testosterone production) overdosed. and incidents of diabetes and cardiovascular disease are 0036. The invention is further described in detail by refer reduced. ence to the following experimental examples. These 0032. The present invention also provides a method of examples are provided for the purpose of illustration only, and preventing the onset of Alzheimer's disease in an individual are not intended to be limiting unless otherwise specified. by the individual smelling the composition (i.e. truffle Thus, the invention should in no way be construed as being extract) and inducing testosterone production in the indi limited to the following examples, but rather, should be con vidual and thereby increasing levels of testosterone in an Strued to encompass any and all variations which become individual. Alzheimer's disease is commonly found in the evident as a result of the teaching provided herein. form of dementia (loss of memory and other intellectual functions), which is serious enough to interfere in daily life EXAMPLE 1. and presently does not have a cure. Available treatments focus on helping people maintain mental function, managing 0037. In order to prepare a nutritional supplement grade behavioral symptoms and slowing its development. 5.3 mil tincture of organic mycelial biomass of Tuber melanosporum, lion people in the US have this disease. It is known that organic truffles were grown on organic rye medium in the incidence of Alzheimer's disease increases when testosterone dark, in a truffle/mushroom farm with hydroponics in the levels decrease. Pike Christian, et al., in Androgens, aging United States. Truffles were harvested and anatomically con and Alzheimer's disease' (2006) discussed new evidence that firmed to contain predominantly truffle mycelial biomass. one consequence of testosterone depletion in men is an Truffles thus grown were dried under sterile conditions and increased risk for the development of Alzheimer's disease ground into a fine powder. Five (5) g. of the truffle powder (AD). Furthermore, two candidate mechanisms were dis thus produced was placed in a 100 ml beaker with a magnetic cussed by which testosterone can affect AD pathogenesis. stirring bar on the bottom. One hundred (100) ml of 95% There are two possible mechanisms by which testosterone ethanol (USPAlcohol) was added to the truffle powder in the can affect AD pathogenesis. First, testosterone has been iden beaker. The beaker was then placed on a Corning stirrer/heat tified as an endogenous regulator off-amyloid, a protein that plate and the stirrer turned on, with stirring proceeding for abnormally accumulates in the AD brain and is implicated as thirty (30) minutes. The remaining powder in the beaker was a causal factor in the disease. Second, findings from several allowed to settle to the bottom of the beaker, and the alcohol different paradigms indicate that testosterone has both neu extract thus made was poured through a funnel lined with rotrophic and neuroprotective functions. These new findings filter paper into a storage bottle. The clear aromatic brownish Support the clinical evaluation of androgen-based therapies tincture thus produced was stored in a bottle at room tempera for the prevention and treatment of AD. Therefore, the onset ture, after having been tightly capped. US 2013/0295 140 A1 Nov. 7, 2013

0038 An aliquot (1-2 drops) of the tincture was adminis What is claimed is: tered to human Subjects as follows: ten human male Subjects, 1. A composition of an olfactory stimulus means for stimu ages 55-82, had saliva samples taken immediately upon lating the production of testosterone. awakening in the morning on the day prior to the start of the 2. The composition of claim 1, wherein said olfactory study, and the saliva samples were analyzed for testosterone stimulus means is further defined as a truffle extract. level utilizing fluorescent microscopy. Then on a daily basis 3. The composition of claim 1, wherein said truffle extract 1-2 drops of the tincture were administered by topical appli is derived from a truffle chosen from the group consisting of cation to the upper lip of the Subject, in the evening near the Tuber magnatum, Tuber magnatum pico, Tuber borchii, subject’s normal bedtime, after which the subject did not Tuber melanosporum, Tuber aestivum, Tuber macrosporum, wash or cover the skin thus topically treated. Administration Tuber Oregonense, Tuber gibbosum, and Tuber lyoni. once nightly continued for at least three days. Each subject 4. The composition of claim 1, wherein said composition had his morning saliva analyzed for testosterone 3-14 days includes 0.5 to 1 g of truffle extract. 5. The composition of claim 1, wherein said composition is after the onset of topical administration of the tincture; the a pharmaceutical composition and further includes a pharma increase in testosterone levels in picograms/ml is shown ceutically acceptable carrier. TABLE 1. All ten men 55+ in age had an increase in their 6. The composition of claim 5, wherein said pharmaceuti testosterone levels, on average by 100% (see TABLE 1). cal composition is a topical composition. 7. The composition of claim 5, wherein said pharmaceuti TABLE 1. cally acceptable carrier is chosen from the group consisting of Pre (pg/ml) Post (pg/ml) 3-14 alcohol, oil, wax, and cream. Name Age Baseline days 8. The composition of claim 1, wherein said composition is MS 82 64 87 a nutraceutical composition. AC 82 7 68 9. The composition of claim 1, wherein said olfactory Jim 74 64 100 stimulus means is further defined as pollen of Pinus silvestris. BM 73 61 69 JM 68 46 70 10. A method of increasing the level of testosterone in an JC 65 26 47 individual including the steps of JP 62 64 16S the individual smelling a truffle extract; TL 60 68 74 causing androgens in the truffle extract to attach to the WH 55 S4 293 TB 55 56 82 olfactory bulb in the individual; and TB (2nd exposure - 5 mths later) 55 52 275 inducing testosterone production in the individual. 11. The method of claim 10, wherein said smelling step is further defined as applying the truffle extract topically to the individual’s skin and smelling the truffle extract. EXAMPLE 2 12. The method of claim 11, wherein said applying step is performed before sleep. 0039 Twelve women who were in menopause/post 13. The method of claim 11, wherein said applying step is menopausal stage used the olfaction of truffle extract. The performed daily. above-described administration of the tincture to the upper lip 14. The method of claim 10, wherein said individual is at was followed for olfaction therapy for a sequence of days, and least 50 years old. it was reported that the administration removes common 15. The method of claim 10, wherein said inducing step symptoms including hot flashes, Sweating, inability to have a returns testosterone levels to a normal level. good nights sleep, anxiety, mood Swings, general lack of 16. The method of claim 10, further including the step of vitality, and reduced libido. Overall, the benefits of the improving vitality, well-being, mood, mortality, sexuality, present therapy are multiple, including without limitation muscle, immunity, bone mass and repair, platelet aggrega ease of use, virtual inability to overdose, and all the benefits tion, cognition, blood pressure, HDL cholesterol levels, and and reduced toxicities of (in the preferred embodiment) a sleep quality. natural, organically produced product. 17. The method of claim 10, further including the step of 0040. Throughout this application, various publications, reducing abdominal fat, reducing incidents of diabetes, and including United States patents, are referenced by author and reducing incidents of cardiovascular disease. year and patents by number. Full citations for the publications 18. The method of claim 10, wherein the truffle extract is are listed below. The disclosures of these publications and derived from a truffle chosen from the group consisting of patents in their entireties are hereby incorporated by refer Tuber magnatum, Tuber magnatum pico, Tuber borchii, ence into this application in order to more fully describe the Tuber melanosporum, Tuber aestivum, Tuber macrosporum, state of the art to which this invention pertains. Tuber Oregonense, Tuber gibbosum, and Tuber lyoni. 19. A method of preventing the onset of Alzheimer's dis 0041. The invention has been described in an illustrative ease in an individual including the steps of: manner, and it is to be understood that the terminology, which the individual smelling the truffle extract; has been used is intended to be in the nature of words of inducing testosterone production in the individual; and description rather than of limitation. preventing the onset of Alzheimer's disease. 0042. Obviously, many modifications and variations of the 20. The method of claim 19, wherein said smelling step is present invention are possible in light of the above teachings. further defined as applying the truffle extract topically to the It is, therefore, to be understood that within the scope of the individual’s skin and smelling the truffle extract. appended claims, the invention can be practiced otherwise 21. The method of claim 19, wherein said individual is at than as specifically described. least 50 years old. US 2013/0295 140 A1 Nov. 7, 2013

22. The method of claim 19, wherein said inducing step 28. The method of claim 25, wherein the truffle extract is returns testosterone levels to a normal level. derived from a truffle chosen from the group consisting of 23. The method of claim 19, further including the step of Tuber magnatum, Tuber magnatum pico, Tuber borchii, regulating B-amyloid and preventing accumulation in the Tuber melanosporum, Tuber aestivum, Tuber macrosporum, brain. Tuber Oregonense, Tuber gibbosum, and Tuber lyoni. 24. The method of claim 19, wherein the truffle extract is derived from a truffle chosen from the group consisting of 29. A method of treating andropause in a man including the Tuber magnatum, Tuber magnatum pico, Tuber borchii, steps of: Tuber melanosporum, Tuber aestivum, Tuber macrosporum, the man smelling the truffle extract; and Tuber Oregonense, Tuber gibbosum, and Tuber lyoni. 25. A method of treating symptoms of menopause in a inducing testosterone production in the man. woman including the steps of 30. The method of claim 29, further including the step of the woman smelling the truffle extract; treating symptoms chosen from the group consisting of inducing testosterone production in the woman; and sweats, hot flushes, inability to sleep, lack of sex drive, lack of treating symptoms of menopause. vitality, and combinations thereof. 26. The method of claim 25, wherein the symptoms treated are chosen from the group consisting of Sweats, hot flushes, 31. The method of claim 29, wherein the truffle extract is inability to sleep, lack of sex drive, lack of vitality, and com derived from a truffle chosen from the group consisting of binations thereof. Tuber magnatum, Tuber magnatum pico, Tuber borchii, 27. The method of claim 25, further including the step of Tuber melanosporum, Tuber aestivum, Tuber macrosporum, using a treatment chosen from the group consisting of estro Tuber Oregonense, Tuber gibbosum, and Tuber lyoni. gen, progesterone, and combinations thereof. k k k k k