The computerized method, software, and product of this invention implements a process for computationally designing proteins and/or associated molecular targets with desired quantitative stability/flexibility relationships (QSFR). QSFR is an innovative, high dimensional metric calculated using an improved Distance Constraint Model (DCM). This invention includes three distinct aspects: (i.) utilizing QSFR in computational molecular design of protein sequences, or variations of molecular structures, to have specific stability and flexibility relationships deemed important for biochemical function for specified thermodynamic and solvent conditions; (ii.) utilizing QSFR to optimize thermodynamic and/or solvent conditions necessary for a specified molecular system to exhibit protein function characteristics, such as improved catalytic efficiency; and (iii.) utilizing QSFR to computationally screen or design small molecules and/or design its putative receptor so as to exhibit a desired affect on protein function.

Computational protein design is an extremely compute intensive (NP-hard) search problem [1]. Current methods search for a global minimum in energy, not free energy, which are generally pairwise decomposable and (relative to free energies) quick to compute. Myriad conformational search algorithms (i.e. Dead-End Elimination, A*, genetic algorithms, simulated annealing, Monte Carlo sampling, etc.) are used to find the conformation with lowest energy [2]. In the past decade, the science of protein design has made tremendous strides, and several experimental investigations have confirmed the usefulness of the computational predictions [3-6]. For a complete history of the field, see the reviews by Tuchscherer et al. [7], Street and Mayo [8], Bornscheuer and Pohl [9], Pokala and Handel [10], Park et al. [11], and Cozzetto et al. [12]. Recently, a new protein design paradigm, implemented in the software RosettaDesign, has been introduced [13]. Unlike the previous conformational search algorithms, RosettaDesign is based on combined threading/refinement process that also identifies experimentally validated protein structures [6, 13, 14]. In addition to individual protein structures, it has been demonstrated that the above protein design strategies can be extended to engineer specific protein-protein interactions [13, 15, 16].
In all of the above current approaches, the search algorithms attempt to identify the best sequence/conformation pair that minimizes the energy function for a given problem. While these current approaches have enjoyed many successes, they are still not efficient or accurate enough for the majority of real-world applications. Due to computational cost, no method has ever attempted to design proteins with specific targeted free energy. Calculating free energies is prohibitive because current methods estimate conformational entropy from lengthy simulations that attempt to cover accessible phase space.
In this invention, the conformational entropy contributions are calculated without simulation for a given template structure. As such, free energy landscapes are calculated in computationally tractable timescales based on specified order parameters, as FIGS. 1 and 2 illustrate for a beta-hairpin turn. Here the free energy of a small molecular system is expressed as a function of the number of hydrogen bonds and native torsions present in the structure. Free energy landscapes can be calculated based on user-defined selection of order parameters. Order parameters characterize the general properties of a system. Locating where the lowest free energy regions are in the landscape provides information about stable or metastable thermodynamic states. As thermodynamic and solvent conditions change, the shape of the free energy landscape also changes. Multiple minimums will generally appear, as will saddles that are important for describing transitions between different thermodynamic states. The free energy landscape is perhaps the most important element to this invention because all physical observables that are thermodynamic quantities require knowing the free energy landscape. In addition, kinetic properties can be described based on saddle points from barrier heights between minima within the free energy landscape.
Related to conformational entropy is flexibility. Flexibility characteristics are critical to proper enzyme function [17]; when an enzyme becomes too rigid, key catalytic normal modes are restricted, resulting in reduced functional efficiency, or possibly, complete loss of function [18, 19]. This is the main reason that traditional protein design strategies are unlikely to robustly design functioning enzymes. For example, energetic minima are frequently associated with more rigid conformations. Meaning, enzymes with increased stability are frequently too stable (rigid) to catalyze their associated reaction. Conversely, a more flexible enzyme might catalyze a given reaction faster, but would rarely be in the appropriate thermodynamic state, making the rate increase moot.
Flexibility is marginally considered in currently employed state-of-the-art protein design methods [11]. Current approaches allow protein conformations to adjust during refinement steps using routine energy minimization algorithms, such as steepest descent, conjugate gradient or Monte Carlo sampling; for example, see [6, 20, 21]. However, dynamics that occur on much longer (functional) timescales have not been implemented into any of the currently used design algorithms, although they have been envisioned for sometime [22]. In fact, in a recent protein design review [23], Butterfoss and Kuhlman state: "A second major step is the design of dynamic properties into proteins." They go on to conclude, "The unique challenges faced in the advanced design of dynamic behavior remain to be described." Despite consensus on the importance of stability and flexibility for protein function, no computational method currently exists to design a protein-molecule-solvent system based on a priori targeted stability/flexibility profiles for a given solvent and/or thermodynamic condition. Moreover, no computational method exists to design optimal solvent/thermodynamic conditions to obtain desired stability/flexibility profiles for a given protein-molecule system. A protein-molecule system is used here to mean either a single protein, or a protein interacting with one or more molecules, for which the molecule may be a protein (i.e. protein-protein interactions are included here).
Employing a Distance Constraint Model (DCM) [22, 24-30] based on network rigidity, free energy decomposition, and ensemble averaging, this invention uses Quantitative Stability/Flexibility Relationships (QSFR), which provides key insight about enzyme function [31, 32], to guide rational protein design efforts. This invention leverages the DCM's unique ability to calculate QSFR metrics fast, and focuses on the high dimensional QSFR metric that encapsulates relevant information about protein function. This invention will allow design of protein-molecule-solvent systems to respond and function in a desired way by optimizing to target QSFR metrics. In the optimization process, a variety of search methods will be employed to find optimal protein-molecule-solvent systems with targeted QSFR metrics.
QSFR represents a high dimensional body of descriptive data that includes both mechanical and thermodynamic metrics. Common QSFR metrics include: (i.) both global and local thermodynamic quantities, (ii.) descriptions of backbone flexibility, (iii.) mechanical susceptibility, and (iv.) correlation functions that describe intramolecular couplings. We have demonstrated previously that several of the QSFR metrics (especially backbone flexibility) are conserved across a protein family [31, 33, 34], thus leading to conservation of function. For example, we have demonstrated that several QSFR metrics are conserved across an E. coli and T. thermophilus RNase H ortholog pair, which are explained by the stability/flexibility requirements mandated by the enzymes' conserved function. A variety of QSFR quantities that can be calculated are described in FIG. 3. All of these quantities can be calculated for specified thermodynamic states, which are associated with particular portions of the free energy landscape. A common thermodynamic state of interest is the native state of a protein, which is described by a minimum within the free energy landscape that represents many native-like conformations.
Exemplar plots of common QSFR metrics are provided in FIG. 42, which include DCM outputs for global, local, and pairwise outputs for the protein thioredoxin. In panel (a), the free energy landscape is provided for three different temperatures. At T=359K, the folded and unfolded minima are equally probable, indicating that this is the melting temperature. At T=349K, the native state basin is more favourable than the unfolded, whereas at T=369K the opposite is true. In addition, the rigid cluster susceptibility is also plotted (at T=359K). In panel (b), the probability to rotate of thioredoxin is plotted for the same three temperatures as in panel (a). Notice that the protein becomes more flexible at higher temperatures. In panel (c), the RS-RS cooperativity correlation plot is provided. The plot highlights residue pairs that have a high likelihood of being correlated in their susceptibility. For example, the dark region centered at {87, 12} indicates that these regions are very likely to be simultaneously susceptible, whereas the white region at {100, 75} indicates that there is no correlation. The solid white bands at residues 34, 40, 64, 68, and 76, which are all Proline residues, occurs because Proline is locked into a native torsion angle state due to its cyclic sidechain.
In addition to being essential to successful design of proteins with targeted stability/flexibility/function characteristics, long-timescale flexibility is also critical in the computational screening of protein-ligand interactions [35-38]. This is because very few protein receptors are consistent with the classical "lock-and-key" model [38]. As a consequence, myriad different strategies for dealing with flexibility have been introduced in the primary literature, including one based on the same graph theoretic methods employed by the DCM [39]. However, few methodologies deal with the thermodynamic consequences of protein-ligand association, despite that pronounced affects are well documented in the primary literature [40-43]. Similar to other commonly employed protein design strategies, this invention will utilize the myriad virtual ligand screening methodologies currently available. The main reason current methods neglect rigorous thermodynamic descriptions is because methods have not been available to accurately calculate free energies in tractable computing timescales. The main barrier to achieving this goal is non-additivity within free energies [44, 45], which the DCM elegantly resolves. As a consequence of the DCM strategy, it is uniquely able to quantitatively reproduce experimental heat capacity curves [25, 30-32].
The DCM also efficiently models environmental affects such as pH, ionic strength, co-solute concentration, and pressure, which are coupled to the various QSFR metrics. For example, proteins become unstable and will unfold at extreme pH, which, compared to proteins at intermediate pH values, results in drastically different QSFR properties. Specifically, the protein is likely to unfold at non-ideal thermodynamic/solvent conditions, resulting in a less compact and more flexible structure with fewer mechanical couplings between sites. Conversely, solvent conditions that stabilize the protein will generally have the opposite effects.
As described above, there are many protein design strategies that focus on finding minimum energy structures to increase stability. However, stability is a property of free energy, which includes both energetic and entropic contributions. Stability is important for reproducibility in molecular recognition and ensuring that desired molecular conformations are populated sufficiently; however, when considering function, stability is only one aspect of consideration. Protein flexibility is also critical for mediating function. Increasing stability by better contacts (the focus of current protein design) frequently will reduce flexibility, which can result in reduction or complete loss of catalytic efficiency. Despite overwhelming consensus in the biophysics/biochemistry communities regarding the importance of flexibility to function, it is ignored in current protein design efforts (by pragmatic necessity). This invention defines an innovative process for incorporating stability, flexibility and their relationships, which lays the foundation for a new protein design paradigm.
The high dimensional QSFR metric characterizes thermodynamic, mechanical and kinetic properties and their relationships in proteins and other macromolecules. There is no limit to the number of quantities that can be calculated and included in the QSFR metric. For example, quantitative characterization of key motions critical to catalysis (related to catalytic normal modes) can be identified in a computationally tractable way. A QSFR metric translates rigidity and flexibility correlation functions into an elastic network model, from which vibrational normal modes can be calculated. In contrast to all currently employed elastic network models, the normal modes calculated in this invention are dependent upon thermodynamic and solvent conditions, which themselves are dependent, as are all other quantities, on the shape of the free energy landscape. As the thermodynamic state of the protein changes, so does its characteristic intramolecular interactions that appear in the structure. Therefore, mechanical response and thermodynamic response are coupled.
QSFR metrics encapsulate relevant information regarding protein stability and molecular cooperativity important to function. Consequently, QSFR measures can be used to distinguish molecular mechanisms that control stability and function. Through judicious selection of QSFR targets, catalytic and functional specificities can be computationally selected in rational design strategies. Some fundamental QSFR measures that can be currently calculated by a prototype DCM, are listed in FIGS. 3 and 4.
It is instructive to focus on one such QSFR descriptor. Consider heat capacity (Cp). Molecular dynamics (MD) and Monte Carlo (MC) all-atom simulations are unable to accurately predict Cp curves. There is of course the issue that current force field parameterization is simply inaccurate. However, even if using current MD/MC all-atom simulations with the most accurate force-field model available could lead to predictions within 30% error from experimental measurements (so far unrealizable), this success would be a moot point due to extreme computational cost. Conservative estimate suggest it would take far more than 100 continuous years running the simulation in parallel using 100,000 CPUs for a moderate size protein of only 200 residues! However, MD/MC methods are commonly employed on reduced models that are simplified variants of all-atom models, such as Go-like models, which do produce a peak in predicted Cp curves that correspond to the protein folding transition. Using reduced models that treat solvent implicitly is essential to make calculations much faster. For example, Go-like models typically take a few months of CPU time using 60 CPUs to produce a Cp curve for small proteins of about 80 residues. Unfortunately, this speed is still far too slow to be of any practical value for protein design applications. Worse still, a more serious issue is the corresponding loss of accuracy. Predicted Cp curves using Go-like models do not resemble experimental measurements. We are unaware of any results yielding quantitative agreement to experimental data. Fast methods are also a moot point if they are not accurate.
Typically, MD/MC predicted Cp curves using reduced models contain errors that are an order of magnitude or more off when considering key features such as the peak location that defines the melting temperature, the peak height and the width of the Cp curve. In contrast, a DCM prototype reproduces experimental Cp curves typically within a few percent error in a matter of 30 days on a single CPU for a moderate size (200 residue) protein. This prototype DCM has major drawbacks because it has been oversimplified, and we usually refer to it as a minimal DCM. The minimal DCM has three phenomenological fitting parameters in the theory. Fortunately, this limitation is not enough to make the overall approach impractical. Practical protein design can be performed even when the employed model requires a few adjustable parameters. Interestingly, most of the 30 days of CPU time is a result of the fitting procedure to reproduce the Cp curve. If model parameters were transferable, fitting would be eliminated, and the total calculation time becomes less than a few hours on a single CPU to produce a high dimensional QSFR metric using the methods in the minimal DCM. Moreover, nontrivial technical improvements (see U.S. patent application Ser. No. 12/344,192, Jacobs 2008) to the minimal DCM will make statistical sampling methods obsolete, thereby speeding calculations up by an estimated factor of 100 or more, while gaining accuracy. It is envisioned that with the help of a more sophisticated modelling scheme that is calculated faster, in a more accurate fashion, a transferable set of parameters will be successfully constructed. Under these conditions, a host of QSFR properties (Cp is just one of them) should be generated in matter of a few minutes on a single CPU for a moderate size protein (200 residues) on today's computers.
FIG. 5 shows the essential components that are utilized to calculate the DCM for sophisticated models that are both more accurate and can be calculated much faster than the minimal DCM. This invention is based on the ability to provide a Flexibility And Stability Test (FAST) in very fast compute times. FAST provides both thermodynamic and mechanical information, and the relationships that are important to characterize the mechanisms that facilitate function. Given a variety of input structures, QSFR metrics are rapidly generated. New structures are explored in a design setting, ranked based on free energies for stability, and other criteria that characterize flexibility, molecular cooperativity, allosteric mechanisms, where the predicted QSFR response is subject to thermodynamic and solvent conditions.

Computer implemented system for protein and drug target design utilizing quantified stability and flexibility relationships to control function

The University of North Carolina at Charlotte


A four-stroke engine comprising an engine frame including a block and a head, the block forming at least one cylinder, and a crankcase. A piston is mounted for reciprocation in the cylinder, and a crank and connecting rod are mounted in the crankcase and connected to the piston. The head supports an intake valve, an exhaust valve, and a valve actuating mechanism, and a valve cover forms an enclosure with the head that encloses the valves and mechanism. The crankcase includes a fuel inlet port and an outlet port, and a duct connects the outlet port to the enclosure in the valve cover. The inlet port is connected to a supply of a combustible mixture comprising fuel, lubricating oil and air. During engine operation, the mixture flows through the crankcase from the fuel inlet port to the outlet port, the piston functioning as a pump to move the mixture. The oil in the mixture lubricates the engine parts in the crankcase. From the outlet port, the mixture flows through the duct to the valve cover and to the intake valve. The enclosure formed by the valve cover contains an oil mist around the valves and the valve actuating mechanism. Valves may be provided at the fuel inlet and outlet ports of the crankcase to achieve crankcase compression of the mixture, and the duct may form a plenum or surge tank containing the mixture under pressure. The duct is preferably separate from the block portion.

This invention relates to an internal combustion (IC) engine, and more particularly to an IC engine particularly suited for use in hand-held (portable) tools.
Relatively small size IC engines are well known and are commonly used to power tools such as chain saws, blowers, line trimmers, etc. Since such tools are normally carried and used by a single person, the engine must be light weight and capable of operation in different orientations (sideways or straight up, for example).
At the present time, most or all engines for this purpose are two-stroke air-cooled engines because they have a good power vs. weight and size ratios, do not have a complex construction, and they are all position or orientation engines. The latter feature is made possible because such engines utilize a diaphragm-type carburetor and engine lubrication is accomplished by adding lubrication oil to the fuel (typically a 40:1 fuel-to-oil mixture).
While two-stroke engines of this type work well, they have certain drawbacks. The fuel consumption rate is relatively high and the operating noise level is also high. A very important disadvantage is that the emissions levels of such engines are quite high because the exhaust includes a sizable amount of fresh fuel. The State of California regulations effective in 1995 limit the amounts of hydrocarbons and carbon monoxide that may be produced, and most or all two-stroke engines presently in use will not be able to meet the California standards, and it is expected that those standards will soon be adopted by other states and countries.
Four-stroke IC engines are, of course, also well known and they generally have lower hydrocarbon and carbon monoxide emissions than two-stroke engines. This is true because four-stroke engines exchange the exhaust and fresh fuel/air mixture in a more positive manner with the use of valves. Four-stroke engines also in general have lower noise levels.
Relatively small four-stroke engines are available and have been used in, for example, model or hobby aircraft. While such engines are sufficiently small to be used in portable tools, they would not be satisfactory because they have a relatively complex and light duty construction. Four-stroke engines normally have an oil sump in a crankcase at the bottom of the engine and an oil pump for moving the oil to the moving parts such as the overhead valves and the valve actuating mechanisms. This type of lubricating system is not satisfactory for all-position use.
The Y. Imagawa et al. U.S. Pat. No. 5,176,116, dated Jan. 5, 1993, described a lubrication system for a portable four-stroke engine, wherein some of the engine parts are lubricated by oil in a crankcase and other parts by grease which is packed around moving parts. It is questionable whether grease will provide satisfactory lubrication for engine parts that become very hot during use. In any event it is doubtful that grease is satisfactory for long-term use in an engine in field and garden use because the grease should be periodically cleaned out and repacked. This is not practical in engines used, for example, in home gardening tools.
It is therefore a general object of the present invention to provide an improved four-stroke engine that avoids the foregoing problems.


Provided is a low yield ratio, high strength and high toughness steel plate having excellent strain ageing resistance equivalent to API 5L X70 Grade or lower and a method for manufacturing the same. The steel plate has a metallographic microstructure that is a three-phase microstructure including bainite, M-A constituent, and quasi-polygonal ferrite, the area fraction of the bainite being 5% to 70%, the area fraction of the M-A constituent being 3% to 20%, the remainder being the quasi-polygonal ferrite, the equivalent circle diameter of the M-A constituent being 3.0 μm or less. The steel plate has a yield ratio of 85% or less and a Charpy impact test absorbed energy of 200 J or more at −30° C. before or after being subjected to strain ageing treatment at a temperature of 250° C. or lower for 30 minutes or less.

In recent years, steels for welded structures have been required to have low yield strength and high uniform elongation in addition to high strength and high toughness from an earthquake-proof point of view. In general, it is known that steel is enabled to have low yield strength and high uniform elongation in such a manner that the metallographic microstructure of the steel is transformed into a microstructure in which a hard phase such as bainite or martensite is adequately dispersed in ferrite, which is a soft phase. The term "uniform elongation" as used herein is also called even elongation and refers to the limit of the permanent elongation of a parallel portion of a specimen uniformly deformed in a tensile test. The uniform elongation is usually determined in the form of the permanent elongation corresponding to the maximum tensile load.
As for manufacturing methods capable of obtaining a microstructure in which a hard phase is adequately dispersed in a soft phase as described above, Patent Literature 1 discloses a heat treatment method in which quenching (Q′) from the two-phase, (γ+α) temperature range of ferrite and austenite is performed between quenching (Q) and tempering (T).
As for methods in which the number of manufacturing steps is not increased, Patent Literature 2 discloses a method in which after rolling is finished at the Ar3 transformation temperature or higher, the start of accelerated cooling is delayed until the temperature of a steel material decreases to or below the Ar3 transformation temperature, at which ferrite is produced.
As for techniques for achieving low yield ratio without performing such heat treatment as disclosed in Patent Literature 1 or 2, Patent Literature 3 discloses a method in which low yield ratio is achieved in such a manner that after the rolling of a steel material is finished at the Ar3 transformation temperature or higher, the rate of accelerated cooling and the finishing cooling temperature are controlled such that a two-phase microstructure consisting of acicular ferrite and martensite is produced.
Furthermore, as for techniques for achieving low yield ratio and excellent welded heat affected zone (HAZ) toughness, Patent Literature 4 discloses a method in which a three-phase microstructure consisting of ferrite, bainite, and Martensite-Austenite constituent (island martensite, Martensitic Islands or M-A constituent, hereinafter called M-A constituent) is produced in such a manner that Ti/N and/or the Ca—O—S balance is controlled.
Patent Literature 5 discloses a technique in which low yield ratio and high uniform elongation are achieved by the addition of an alloying element such as Cu, Ni, or Mo.
On the other hand, welded steel pipes such as UOE steel pipes used for line pipes and electric welded tubes are manufactured in such a manner that steel plates are cold-formed into pipes, abutting surfaces thereof are welded, and the outer surfaces of the tubes are usually subjected to coating such as polyethylene coating or powder epoxy coating in view of corrosion resistance. Therefore, there is a problem in that the steel pipes have a yield ratio greater than the yield ratio of the steel plates because strain ageing is caused by the strain during pipe making and the heat during coating and the yield stress is increased. In order to cope with such a problem, Patent Literatures 6 and 7 each disclose a steel pipe which has excellent strain ageing resistance, low yield ratio, high strength, and high toughness and which makes use of fine precipitates of composite carbides containing Ti and Mo or fine precipitates of composite carbides containing two or more of Ti, Nb, and V and also disclose a method for manufacturing the steelpipe.

Low yield ratio, high strength and high toughness steel plate and method for manufacturing the same


Methods for preparing an estrogenic preparation and isolating estrogenic compounds from a plant, such as an Epimedium plant, are provided. Also provided are estrogenic compounds from Epimedium plant that have been isolated and characterized, and methods for their use in modulating estrogen receptors and in treating conditions mediated by estrogen receptors, such as menopause and estrogen-dependent cancers. Also provided are preparations from Epimedium that are enriched for estrogenic compounds, and methods for their use in modulating estrogen receptors and preventing and treating conditions that are mediated by estrogen receptors.

The current invention concerns methods of preparing an estrogenic preparation and isolating an estrogenic compound from a plant, such as an Epimedium plant, and uses thereof for selectively modulating the function of estrogen receptors.
There is an ever-increasing interest in herbal or natural-source remedies or medications. Many individuals would rather use such products than conventional pharmaceutical preparations. Additionally, medicinal substances derived from natural products can provide commercial or industrial opportunities for local populations in areas where medicinal plants grow or are cultivated. Moreover, compounds identified as the active ingredients in natural products form an important basis for pharmaceutical research.
Estrogens are steroid hormones that regulate physiological processes such as the growth, differentiation, and functioning of many target tissues by regulating the expression of genes under control of estrogen-responsive elements. Tissues that express estrogen receptors include tissues of the female and male reproductive systems such as the mammary gland, uterus, vagina, ovary, testes, epididymis, and prostate (For reviews see: Pettersson, et al., 2001; Conneely, 2001). Estrogens have been increasingly shown to have important regulatory roles in the central nervous system and cardiovascular system and in physiological processes such as the maintenance of bone, lipid and fat metabolism, and atherosclerosis formation. Of the several physiological estrogens in women, 17-beta-estradiol (hereinafter referred to as xe2x80x9cestradiolxe2x80x9d) is the most potent.
xe2x80x9cPhytoestrogensxe2x80x9d are a broad group of plant-derived compounds of non-steroidal structure that can behave as estrogen mimics (For reviews: Kurzer, et al., 1997; Zava, et al., 1998; Setchell, 1998; Tham, et al., 1998). A typical feature of the chemical structure of phytoestrogens is the presence of a phenolic ring that, with few exceptions, is a prerequisite for binding to the estrogen receptor. Phytoestrogenic substances have been demonstrated to have estrogenic or anti-estrogenic activity and epidemiological data suggests that these substances may be useful for treatment of a variety of health problems that are correlated with estrogenic deficiency, including: premature ovarian failure; menopausal syndromes; osteoporosis; menstrual irregularities; pre-menstrual syndrome; cardiovascular disease; atherosclerosis, coronary artery disease and strokes; and cancer. These potential health benefits are consistent with epidemiological evidence that rates of heart disease, hormone-dependent cancers, osteoporotic fractures and menopausal symptoms are more favorable among populations that consume plant-based diets, especially among cultures with diets rich in soy products.
The biological effects of estrogens are mediated via estrogen receptors (ER), of which there are at least two types, i.e. ER-alpha and ER-beta (Pettersson, et al., 2001; Conneely, 2001). Estrogens are retained in target cells by the estrogen receptors (ERs). The ERs belong to the steroid-receptor superfamily of nuclear transcription factors, whose members include receptors for progesterone (PR), glucocorticoids (GR) and androgens (AR). The ERs, in common with other members of the steroid-receptor superfamily, are organized into domains that are responsible for specific functions. The N-terminal transactivation domain has a ligand-independent activation function. The DNA-binding domain (DBD) enables the receptor to bind to its cognate target site consisting of an inverted repeat of two half-sites with the consensus motif AGGTCA (or closely related sequences) spaced by 3 basepairs and referred to as an estrogen response element (ERE). The DBDs of ER-alpha and ER-beta share approximately 97% sequence homology but significant differences in amino-acid sequences are found in the N-terminal, and ligand-binding domains. Both ER-alpha and ER-beta bind to EREs in promoter regions of target cells. In addition ERs can regulate AP-1 enhancer elements, by acting on the transcription factors Fos and. Jun. The ligand-binding domain (LBD) also harbors a nuclear localization signal as well as sequences necessary for dimerization and transcriptional activation. Upon estrogen binding, ER undergoes a conformational change allowing the receptor to interact with chromatin and to modulate transcription of target genes. Modulation of gene expression leads to changes in the level of expression of corresponding proteins, which in turn bring about the myriad physiological activities associated with estrogens.
Although both ER-alpha and ER-beta bind specifically to the same estrogen response element (ERE), there are important differences in their actions in cellular systems and in their response to agonists and antagonists (Enmark, et al., 1998, Barkhem, et al., 1998). ER-beta requires approximately five- to ten-fold higher concentrations of estradiol than ER-alpha for maximum transactivation activity to occur and ER-beta is only approximately 30% as efficient as ER-alpha in a variety of reporter gene systems. The anti-estrogen tamoxifen is a mixed agonist/antagonist for ER-alpha but is a pure antagonist for ER-beta. ER-alpha and ER-beta can form functional DNA-binding heterodimeric complexes, and in these complexes ER-beta appears to be the dominant partner, given that activity is repressed at low concentrations of estradiol and in the presence of tamoxifen. Notably, ER-alpha and ER-beta have opposite effects at AP-1 and SP-1 sites (Paech, et al., 1997), in the presence of both agonists and antagonists. ER-alpha and ER-beta also bind to the same ligands with different affinity. In addition, after binding to estrogens and anti-estrogens, ER-alpha and ER-beta use different enhancer elements such as estrogen responsive element and AP1 sites in promoter regions of genes. Taken together, these studies suggest that ER-alpha and ER-beta may activate different downstream effects.
There are substantial differences in the distribution of ER-alpha and ER-beta in estrogen responsive tissues. ER-beta is expressed in many human tissues including the central nervous system, the cardiovascular system, the immune system, the urogenital tract, the gastrointestinal tract, the kidneys and the lungs (Omoto, et al., 2001). In contrast ER-alpha seems to predominate in reproductive tissues such as the uterus and breast, although smaller amounts of ER-beta are also present in these tissues. It is possible that ER-beta is the more widely expressed estrogen receptor in the body.
In tissues where both ER-alpha and ER-beta are co-expressed, it has been found in many cases that ER-beta opposes the actions of ER-alpha.
Phytochemicals and Phytoestrogens
Phytoestrogens may have partial estrogenic and anti-estrogenic activity due to their competition with endogenous estradiol for estrogen receptors (For reviews: Kurzer, et al., 1997; Zava, et al., 1998; Setchell, 1998; Tham, et al., 1998). The majority of phytoestrogens found in plants (including herbs and legumes) can be categorized into two primary classes: flavonoids and lignans. These substances have a structural similarity to the estrogens, estradiol and diethylstilbesterol, especially in relation to their xe2x80x94OH groups.
Flavonoids represent a family of phytochemicals with disparate functions, including: deterring herbivores, acting as antibacterial/antifungal agents and stimulating the formation of symbiotic relationships with nitrogen-fixing bacteria. The family of flavonoids is often subclassified on the basis of structural features into: flavones, isoflavones or coumestans. Isoflavones make up the most common form of phytoestrogens. Typically, phytoestrogens exhibit weak estrogenic activity, i.e. on the order of 100- to 1000-fold weaker activity compared to estradiol, but since these compounds may be present in the body in concentrations of 100-fold higher than endogenous estrogens, they may be present in the body at levels sufficient to compete with endogenous estrogen or compensate for estrogen deficiency. Two isoflavonoids, genistein and daidzein, found in soybean tissue have been described as being 100-1000 times less active than estradiol, and are therefore effective as agonists of ER-alpha at 100-1000 times greater concentrations.
Flavones with estrogenic activity are uncommon and known examples include 8-prenylnaringenin and 6-prenylnarigenin from hops, 6-hydroxyflavone and luteolin. In particular the prenylated flavone, 8-prenylnaringenin, isolated from hops has been described to be a potent phytoestrogen with an activity which is greater than other established plant estrogens (Milligan, et al., 1999).
Phytoestrogens may have favourable effects on the risk of cardiovascular disease and are thought to be hypocholesterolemic, anticarcinogenic, antiproliferative, antiosteoporotic, and hormone altering. These health benefits may be attributable to a variety of mechanisms, including ER action.
Epimedium Herb
Epimedium herb (Yin Yang Huo) is a traditional Chinese herb consisting of the dried aerial part of E. sagitatum (Sieb. et Zucc.), E. koreanum Nakai (Fam.Berberidaceae), E. pubescens Maxm., E. wushanense T. S. Ying, or E. brevicorum Maxim or mixtures thereof. The herb is listed in Pharmacopoeia of the People""s Republic of China (1997) as having action to xe2x80x9creinforce the kidney yang, strengthen tendons and bones, and relieve rheumatic conditionsxe2x80x9d and indicated xe2x80x9cfor impotence, seminal emission, weakness of the limbs, rheumatoid arthralgia with numbness and muscle contracture, and climacteric hypertensionxe2x80x9d.
Several compounds including flavones and isoflavones have been isolated from Epimedium herb. These compounds have been found to have disparate physiological activities. Compounds isolated from the aerial parts of Epimedium herb include icariin, phenooxychromones, flavonoids, chrysoeriol, quercetin, apigenin, apigenin 7,4xe2x80x2-dimethyl ether, kaempferol, luteolin, and brevicornin (Liang, et al., 1997a,).
Luteolin, a flavone, has been described as having estrogenic activity with a relative potency of 58% compared to genistein (Rosenberg Zand, et al., 2000). Apigenin and quercetin have also been described as being estrogenic with relative potencies of 16% and 10% respectively compared to genistein.
Other flavonoids and flavanol glycosides described in Epimedium herb include epimedokoreanoside I, epimedin B and C, beta-anhydroicaritin, tricin, and Baohuoside III, V, and VI, some of which flavonol glycosides were reported to have immuno-modulatory activity (Liang, et al., 1997b).
Chen et al. (1996) described the isolation of 5 new prenyflavones, Yin Yang Huo A, B, C, D, and E, some of which (i.e. Yin Yang Huo A and B) were described as having significant anti-platelet activity.
A flavone analogue, Baihuoside I, isolated from Epimedium herb was described as being an anti-rejection agent, effective at prolonging mouse heart allograft survival (U.S. Pat. No. 6,071,883: Chen et al., 2000). Assessment of the mice for appearance, behaviour, biochemistry, hematology and histology revealed no side effects even at the intra-peritoneal dose of 32 mg/kg/day for 14 days.
Icariin-containing preparations have been isolated from aerial parts of plants of the genus Epimedium (U.S. Pat. No. 6,123,944: Chen et al., 2000). These icariin-containing preparations were stated to be effective in treating osteoporosis. Bone phosphorus and calcium contents, bone mineral density and the femur strength of treated ovariectomized rats were increased significantly compared to control groups.
Icariin compounds have been described as displaying anti-hepatotoxic activity in cultured rat hepatocytes (Lee et al., 1995).
Methanol extract of E. sagitatum was stated to show neurite outgrowth activity on cultured PC12H cells. Bioassay guided fractionation yielded six prenylated flavonol glycosides, ikarisiside A, icarisid II, epimedoside A, icariin, epimedin B and epimedokoreanosid-1 as the active ingredients (Kuroda, 2000).
Complex mixtures of herbs comprising Epimedium grandiforum have been described as a method to treat hot flushes (U.S. Pat. No. 5,847,084, Yng Wong, 1999; U.S. Pat. No. 6,238,707, Chun, 2001).
Many women perceive prescription estrogens as being unnatural and there is an increasing interest in the use of plant-derived estrogens for treatment of post-menopausal problems and other conditions associated with estrogen receptors or estrogen-deficiency (Glazier, et al., 2001). Thus, there is a need to identify naturally occurring compounds that interact with and activate ER, especially ER-beta. Natural estrogen receptor activating compounds are needed for the development of therapeutic compositions and/or neutraceutical applications. Pure chemical entities, isolated and purified from herbs can also be developed as pharmaceutical agents.
The present invention provides methods of preparing an estrogenic preparation and isolating an estrogenic compound from a plant, such as an Epimedium plant, and the use of the estrogenic preparation or the isolated estrogenic compound in modulating an estrogen receptor.
In one aspect, the invention provides a method for modulating an estrogen receptor comprising contacting said estrogen receptor with a compound having the formula I:
In a further aspect, the invention provides a compound having the formula II:
It will be seen that formula II has two chiral carbon atoms and that the invention extends to all stereoisomers, diastereomers, enantiomers and racemates of formula II.
In a further aspect, the invention provides a compound having the formula III:
It will be seen that formula III has two chiral carbon atoms and that the invention extends to all stereoisomers, diastereomers, enantiomers and racemates of formula III.
In a further aspect, the invention provides a method for modulating an estrogen receptor comprising contacting an estrogen receptor with a compound having the formula III.
In a further aspect, the invention provides a pharmaceutical composition, nutritional supplement, food product or beverage, comprising a compound having the formula III.
In a further aspect, the invention provides a commerical package comprising a pharmaceutical composition, nutritional supplement, food product or beverage, comprising a compound having the formula III together with instructions for use in treating a condition mediated by an estrogen receptor.
In a further aspect, the invention provides a method for preparing a preparation having estrogenic activity from a plant, said method comprising:
(a) extracting the plant or a part thereof with a solvent to obtain an extract;
(b) fractionating said extract to obtain a plurality of fractions;
(c) assaying estrogen receptor agonist activity in the fractions from (b); and
(d) collecting a fraction from (c) that is enriched for estrogen receptor agonist activity by about 2-fold or more relative to said extract.
In a further aspect, the invention provides a preparation prepared according to the method described herein from a plant selected from the group consisting of Dioscorea opposita; Anemarrhena asphodeloides; Cuscuta chinensis; and Psoralea corylifoloa.
In a further aspect, the invention provides a preparation having estrogenic activity obtained from an Epimedium plant, wherein said preparation comprises between about 0.002% to 99.9% by weight of a compound having the formula III. The invention further provides a pharmaceutical composition, nutritional supplement, food product or beverage comprising such a preparation. The invention further provides a commercial package comprising such a preparation together with instructions for use for modulating an estrogen receptor.
In a further aspect, the invention provides a method for preventing or treating a condition mediated by an estrogen receptor in a subject comprising administering to a subject in need thereof a therapeutically effective amount of:
(i) a compound of the formula:
(ii) a compound of the formula:
(iii) a mixture of said compound in (i) and said compound in (ii); or
(iv) a preparation having estrogenic activity obtained from an Epimedium plant, wherein said preparation comprises between about 0.002% to 99.9% of a compound of the formula:
Methods for preparing estrogenic preparations and isolating estrogenic compounds from a plant, such as an Epimedium plant, are herein described.
By xe2x80x9cestrogenicxe2x80x9d we mean having the property of being able to stimulate or activate estrogen receptors, including homodimers of ER-alpha and ER-beta subunits and heterodimers comprised of ER-alpha and ER-beta subunits. Thus, xe2x80x9cestrogenic extractxe2x80x9d, xe2x80x9cestrogenic preparationxe2x80x9d and xe2x80x9cestrogenic compoundxe2x80x9d refer to extracts, preparations or compounds, respectively, that are capable of stimulating estrogen receptors. Estrogenic preparations and compounds may be described herein as having xe2x80x9cestrogen receptor agonist activityxe2x80x9d or xe2x80x9cestrogenic activityxe2x80x9d, particularly in the context of describing results of assays and in characterizing fractions obtained by the methods herein described. An estrogenic compound may also be described herein as being an xe2x80x9cestrogen receptor agonistxe2x80x9d.
In the context of the present invention, the expression xe2x80x9cEpimedium plantxe2x80x9d means a plant (or part(s) thereof) of the genus Epimedium and includes Epimedium herb (i.e. the traditional Chinese herb, Epimedium herb or xe2x80x9cYinyanghuoxe2x80x9d) and mixtures of plants of different Epimedium species.
xe2x80x9cCrude extractxe2x80x9d of a plant refers to the extract obtained by extracting the plant or part(s) thereof with a solvent, without fractionating the extract to enrich for estrogenic compounds. In the present context, the expression xe2x80x9cES extractxe2x80x9d means an estrogenic extract of Epimedium plant, and xe2x80x9cES crude extractxe2x80x9d means estrogenic crude extract of Epimedium plant.
A compound that shows stronger agonist activity with a receptor type over others may be described as being xe2x80x9cselectivexe2x80x9d for that receptor. For example, a compound that shows stronger agonist activity with one of the ER subtypes, ER-alpha and ER-beta, over the other may be described as being xe2x80x9cselectivexe2x80x9d for that subtype.
The terms xe2x80x9calpha estrogen receptorxe2x80x9d and xe2x80x9cER-alphaxe2x80x9d refer to homodimers comprised of ER-alpha subunits. The terms xe2x80x9cbeta estrogen receptorxe2x80x9d and xe2x80x9cER-betaxe2x80x9d refer to homodimers comprised of ER-beta subunits. It is expected that a compound that binds to ER-alpha and ER-beta will also bind to a heterodimeric estrogen receptor, ER-alpha/ER-beta, comprised of alpha and beta estrogen receptor subunits.
The estrogenic preparations and isolated estrogenic compounds from a plant, such as an Epimedium plant, are useful for modulating estrogen receptors. Therefore, the estrogenic preparations and isolated estrogenic compounds may be useful for treating conditions that are mediated by estrogen receptors. The expression xe2x80x9ccondition mediated by an estrogen receptorxe2x80x9d includes any condition that may benefit from administration of an endogenous estrogen receptor agonist to a subject. Such conditions include but are not limited to conditions that arise as a result of insufficient or deficient estrogen supply in a subject, for example conditions associated with menopause.
As used herein, the term xe2x80x9cmodulatexe2x80x9d means to alter the activity of a biologically active protein, such as the estrogen receptor protein. By xe2x80x9cmodulating an estrogen receptor proteinxe2x80x9d, we mean altering its biological activities, such as its binding to estrogen response elements (ERE) in the chromatin. Modulation of a biologically active protein can stimulate (i.e. activate) or inhibit activity of the protein.
Methods
Methods are described herein for preparing estrogenic preparations and isolated estrogenic compounds from a plant, such as an Epimedium plant. In one embodiment, these methods can be used to identify and isolate estrogenic compounds that are selective for a particular ER subtype, i.e. ER-alpha or ER-beta.
These methods have been used to isolate two estrogenic compounds from an Epimedium plant, Compound I (having a structure described by formula I, above) and Compound III (having a structure described by formula III, above).
The methods described herein may be used to identify and isolate additional estrogenic compounds from a plant, such as an Epimedium plant, based on their functional property as estrogen receptor agonists.
Estrogenic preparations and isolated estrogenic compounds may be prepared from a plant using any part thereof, including the whole plant, or various parts of the plant, including without limitation: plant cells, tissues, seeds, embryos, leaves, stems, roots, flowers, etc. The method described herein is not limited to the particular part of the plant used to prepare estrogenic preparations and isolated estrogenic compounds. Plants that may be used in the method described herein include without limitation Epimedium plant, Dioscorea opposita; Anemarrhena asphodeloides; Curculigo orchioides; Cuscuta chinensis; Polygonatum kingianum; Morinda officinalis; Psoralea corylifoloa; Angelica sinensis; Lycium chinense; and Radix astragali.
(i) Assaying Estrogenic Activity
The estrogenic properties of preparations and compounds isolated from a plant, such as an Epimedium plant, can be assayed with cell-based assays to measure the potency and purity and quality of the raw herbs and preparations and isolated compounds derived from it. Examples of cell-based assays for estrogenic activity include: estrogen-reporter gene assays in a cell line transfected with a reporter gene that is under regulatory control of an estrogen response element (ERE); and estrogen-stimulated growth assays, using cell lines whose growth are sensitive to estrogens such as MCF-7 cells. Estrogenic activity may also be assayed with non-cell-based assays, such as ligand-binding assays involving the use of purified receptors in affinity columns.
For example, in a suitable cell-based assay, tissue culture cells (such as Hela cells) are co-transfected (either transiently or stably) with a first plasmid comprising DNA encoding an estrogen receptor and a second plasmid comprising an estrogen-responsive promoter driving a reporter gene (such as luciferase, green fluorescent protein, and CAT (chloramphenical acetyltranferase)), whose transcription is regulated by an estrogen response element; the activity of the estrogenic preparation or a compound isolated therefrom can be assayed by monitoring the expression of reporter gene in test cells versus controls consisting of an estrogen receptor agonist (such as estradiol) and xe2x80x9cvehiclexe2x80x9d (the solvent in which the estrogenic preparation or isolated compound is dissolved). Other suitable cells for use in this assay include CV1, and COS-7 cells. Other suitable estrogen receptor agonists include estrone, estriol, and diethylstilbesterol.
(ii) Preparation of Estrogenic Preparations and Isolated Estrogenic Compounds from a Plant
For the initial extraction of estrogenic compounds from a plant, such as an Epimedium plant, the plant source (a whole plant or a part thereof, such as aerial parts) is extracted with a solvent such as water or alcohol (such as ethanol) or a mixture thereof. Other solvents may be used in the extraction process, including but not limited to acetone, butanol, and methanol. Polar organic solvents are generally preferred. The plant or part thereof may be crushed, ground, mashed, macerated, pulverized, triturated or otherwise broken up before extraction, and doing so may increase the efficiency of extraction. The plant source may be dried or fresh. Plant parts are typically extracted in solvent at a temperature ranging from, for example, room temperature to the boiling point of the solvent, for a period of time ranging from about one hour to about 3 days.
For example, Epimedium crude extract (ES crude extract) can be prepared by mixing crushed aerial parts of Epimedium plant with 100% ethanol and soaked for 5-7 days at 37xc2x0 C. Other methods such as boiling with water and extraction with varying concentrations of ethanol or methanol are also effective at extracting estrogenic compounds from a plant, such as an Epimedium plant.
Epimedium crude extract (ES crude extract) (100% ethanol) has several properties. It exhibits estrogenic activity but does not have progestogenic, glucocorticoid or androgenic activity at the concentrations tested. Thus ES crude extract can specifically activate the estrogen receptor at concentrations that do not significantly activate other closely-related members of the steroid receptor family. Moreover, ES crude extract can activate estrogen receptors when used at concentrations that do not antagonize estradiol action nor on the action of progestogens, glucocorticoids or androgens.
The ES crude extract thus obtained may be used directly to modulate estrogen receptors, for example as a control in assays for estrogenic activity. Typically, ES crude extract is fractionated to enrich for or isolate estrogenic compounds.
To enrich for estrogenic compounds, ES crude extract can be fractionated. Methods for fractionating estrogenic preparations and isolated estrogenic compounds from a plant, such as an Epimedium plant, may include the following steps or processes: solvent-solvent partitioning, solid-phase fractionations, thin-layer chromatography (TLC), flash chromatography, and high-performance or high pressure liquid chromatography (HPLC).
For example, ES crude extract can be fractionated by loading it on a chromatography column (for example, a bonded phase column such as a Diol solid phase column), eluting with a solvent gradient of increasing or decreasing polarity to obtain a plurality of fractions, some of which may be enriched for estrogen-receptor agonist activity. The fractions thus obtained can be assayed for the presence of estrogenic compounds by assaying for estrogen-receptor agonist activity, as described herein. To determine whether a fraction is selective for ER-alpha or ER-beta, the agonist activity of the fraction for ER-alpha and ER-beta is measured in cells expressing one or the other receptor subtype. Optionally, non-bioactive compounds (such as tannins, chlorophylls) can be removed from ES crude extract before fractionation by loading on a chromatography column packed with matrix, such as polyamide resin, and eluting with a solvent, such as methanol. The eluate thus obtained may be fractionated as described above.
Estrogenic fractions obtained from a first fractionation of ES crude extract may have estrogenic activity that is increased relative to crude extract by 0.5-fold to 10-fold, typically 2-fold to 6-fold (crude extract at 50 xcexcg/ml exhibits about 55% ER-alpha stimulating activity relative to estradiol at 1 nM). Estrogenic fractions thus obtained, i.e. that are enriched for estrogen receptor agonist activity, may be used directly as estrogenic preparations to modulate estrogen receptor activity. Alternatively, these fractions may be further fractionated to enrich for or isolate estrogenic compounds.
Compound I (MW: 338 Daltons) is a prenylated flavone having a structure described by formula I (above) that may be isolated from an Epimedium plant by: extracting the plant with solvent; fractionating the crude extract thus obtained to obtain a plurality of fractions; assaying the fractions thus obtained for estrogen receptor agonist activity; and collecting one or more fractions that is enriched for estrogen receptor agonist activity relative to said crude extract; further fractionating the estrogen receptor agonist enriched fraction(s), for example by high pressure liquid chromatography (HPLC); and collecting one or more fractions that are enriched for Compound I. The presence of Compound I in a fraction may be determined by negative mass spectrometry (i.e. high resolution electron spray ionization mass spectrum, or HRESIMS) or nuclear magnetic resonance (NMR). Compound I demonstrates estrogenic activity and can activate both ER-alpha and ER-beta. Thus Compound I, or estrogenic preparations enriched for Compound I, may be useful for modulating estrogen receptors.
Compound III (MW: 438 Daltons) is a prenylated flavone having a structure described by formula III (above) which can be isolated from an Epimedium plant and shows specificity for ER-beta. Compound III can be isolated, for example, from estrogenic fractions obtained by fractionating ES extract using solid phase chromatography (for example with Diol and C18 columns), and further fractionating the estrogenic fractions thus obtained to a second HPLC (such as a Luna Phenomenex C18) column using a gradient solvent system, such as an isocratic mobile phase of 43% ACN in H2O over 20 minutes. Fractions thus obtained may be assayed either to detect the presence of Compound III itself (by NMR or mass spectrophotometry) or by first assaying for ER-beta-selective agonist activity, then confirming the presence of Compound III by NMR or mass spectrophotometry.
Compound III may co-purify with Compound II. Compound II is a prenylated flavone having a structure corresponding to formula II (above). These compounds may be separated from each other by further fractionation, for example by Reverse Phase (RP) Amide HPLC column chromatography using a suitable solvent system (such as a gradient mobile phase from 50% ACN in H2O to 60% ACN in H2O over 10 minutes, followed by an isocratic mobile phase of 60% ACN in H2O for 10 minutes).
If the fractions are subjected to mass spectrometry or NMR analyses, patterns can be found which can be used for quality control purposes. For example, negative mass spectrometric (Mxe2x88x92H)xe2x88x92 (HRESIMS) examination may be used to detect whether Compound I (MW 338 Daltons), or Compound II and/or Compound III (both MW 438 Daltons) is present in a fraction. Similarly the gHSQC NMR chemical shifts and COSY gHMBC patterns of ES extracts may be used to standardize and classify the various types of ES extracts. Thus, once an estrogenic compound has been identified and characterized, it is then possible to isolate the compound by detecting its presence in fractions based on known physical properties (by methods such as NMR and Mass spectrophotometry), thereby avoiding the cell-based assay for estrogenic activity during the purification process. However, assays of estrogenic activity of the final product may be used to assure quality control.
Compound I, Compound II and Compound III, respectively, are preferably isolated from other components of Epimedium plant at a purity of between about 70% to 99.9% by weight, and preferably at a purity of at least about 75%, 80%, 85%, 90%, 95%, and 99.9% by weight.
An extract of Epimedium plant, or typically a fraction of such extract, that is enriched for Compound III can be used directly as a Compound III-containing preparation of Epimedium plant. Typically, the preparation will contain Compound III in an amount of at least 0.002% to 99.9% by weight, based on the Epimedium-derived portion of the preparation, and preferably at least 0.01%, even more preferably at least 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99.9% by weight Compound III, based on the Epimedium plant derived portion of the preparation. Crude extract of Epimedium contains about 0.001% of Compound III.
(iii) Epimedium Plants
Epimedium plants for use in preparing estrogenic preparations and isolated estrogenic compounds as described herein include any Epimedium plant or mixture of Epimedium plants of the genus Epimedium. Examples of species within the Epimedium genus that may be used for preparing preparations having estrogenic activity include, but are not limited to E. sagittatum, E. koreanum, E. brevicornum, E. pubescens, E. glandiflorum, E. wushanense, E. leptorrhizum, E. hunanense, E. acuminatum, E. davidii, E. fargesii, E. baicaliquizhounense, E. sutchuenense, E. caotum, E. glandolospilosum, E. zushanense, E. reticulatum, E. baojingenensis, E. simplicioflium, E. clongatum, E. ecalcaratum, E. truncatum, E. haiyangense and E. platypetalum. Epimedium plants may be obtained from herbariums, planters, wholesalers in China or their business associates overseas; mention is made of commercial retailer Eu Yan Sang, Singapore.
Preferred species of Epimedium include Epimedium brevicornum Maxis, Epimedium sagittatum (Sieb. Et Zucc.) Maxim, Epimedium pubescens Maxim, Epimedium wushanense T. S. Ying, and Epimedium koreanum Nakai (Fain. Berberidaceae).
Estrogenic preparations and isolated estrogenic compounds may be prepared from an Epimedium plant using any part thereof, including the whole plant, or various parts of the plant, including without limitation: plant cells, tissues, seeds, embryos, leaves, stems, roots, flowers, etc. Thus, the method of this invention is not limited to the particular part of the Epimedium plant used to prepare the estrogenic preparation or isolated estrogenic compound.
Estrogenic Compositions: Pharmaceutical Compositions, Nutritional Supplements, Food Products, and Beverages:
Estrogenic compositions (including pharmaceutical compositions, nutritional supplements, food products (i.e. supplemented or fortified food), or beverages) can be prepared that comprise as an active ingredient one or more of the following: a preparation having estrogenic activity obtained from an Epimedium plant, especially a Compound III-containing preparation as described above; isolated Compound I or an estrogenic extract of an Epimedium plant enriched for Compound I; or isolated Compound III or an estrogenic preparation of an Epimedium plant enriched for Compound III.
The compositions comprise, for example, approximately from 0.1% to 100%, preferably from approximately 1% to approximately 60%, of the active ingredient.
Pharmaceutical compositions are compositions for enteral (e.g. oral) administration, and also rectal or parenteral administration, also for topical administration to warm-blooded animals (particularly humans), the pharmacological active ingredient being present alone or together with customary pharmaceutical excipients, diluents or carriers.
Pharmaceutical compositions for enteral or parenteral administration are, for example, in unit dose forms, such as dragees, tablets, capsules or suppositories, and also ampoules. They are prepared in a manner known per se, for example by means of conventional mixing, granulating, confectioning, dissolving or lyophilising processes. For example, pharmaceutical compositions for oral administration can be obtained by combining the active ingredient with solid carriers, optionally granulating a resulting mixture and processing the mixture of granules, if desired or necessary after the addition of suitable excipients, into tablets or dragee cores.
Suitable carriers include, especially, fillers, such as sugars, for example lactose, saccharose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, also binders, such as starch pastes, using, for example, corn, wheat, rice or potato starch, gelatin, gum tragacanth, methyl-cellulose and/or polyvinylpyrrolidone, and, if desired, disintegrators, such as the above-mentioned starches, also carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar or alginic acid or a salt thereof, such as sodium alginate.
Excipients include, especially, flow conditioners and lubricants, for example silicic acid, talc, stearic acid or salts thereof, such as magnesium or calcium stearate, and/or polyethylene glycol. Dragee cores are provided with suitable coatings that may be resistant to gastric juices, there being used, inter alia, concentrated sugar solutions which may contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, coating solutions in suitable organic solvents or solvent mixtures, or, for the preparation of enteric coatings, solutions of suitable cellulose preparations, such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate. Colourings or pigments may be added to the tablets or dragee coatings, for example for identification purposes or to indicate different doses of active ingredient.
Further orally administrable pharmaceutical compositions include dry-filled capsules consisting of gelatin, and also soft sealed capsules consisting of gelatin and a plasticizer, such as glycerol or sorbitol. The dry-filled capsules may contain the active ingredient in the form of granules, for example in admixture with fillers, such as lactose, binders, such as starches, and/or glidants, such as talc or magnesium stearate, and optionally stabilisers. In soft capsules, the active ingredient is preferably dissolved or suspended in suitable liquids, such as fatty oils, paraffin oil or liquid polyethylene glycols, to which stabilisers may also be added.
Rectally administrable pharmaceutical compositions, for example, suppositories that comprise a combination of the active ingredient and a suppository base are also provided. Suitable as suppository bases are, for example, natural or synthetic triglycerides, paraffin hydrocarbons, polyethylene glycols and higher alkanols. It is also possible to use gelatin rectal capsules that comprise a combination of the active ingredient and a base material. Suitable base materials are, for example, liquid triglycerides, polyethylene glycols and paraffin hydrocarbons.
Aqueous solutions of an active ingredient in water-soluble form, for example a water-soluble salt, are particularly suitable for parenteral administration. Also suitable for parenteral administration are suspensions of the active ingredient, such as corresponding oily injection suspensions, these being used suitable lipophilic solvents or vehicles, such as fatty oils, for example sesame oil, or synthetic fatty acid esters, for example ethyl oleate or triglycerides, or aqueous injection suspensions that contain viscosity-increasing substances, for example sodium carboxymethylcellulose, sorbitol and/or dextran and optionally also stabilisers.
The pharmaceutical compositions may be solid or a liquid preparation (such as a beverage, a sterile injectable solution, a powder, drops, a suspension, a syrup, a lotion, an ointment, a cream, a gel, or the like).
As in the case of the pharmaceutical compositions, the nutritional supplements, food products and beverages comprise, for example, approximately 0.1% to 100%, preferably from approximately 1% to 60%, of the active ingredient.
In one embodiment, the compounds, compositions and preparations of the invention comprise Compound I, Compound III or a mixture thereof in the absence of any other active ingredient, especially an estrogenic agent, e.g. from Epimedium.
The dose of the active ingredient may depend on various factors, such as the method of administration, species of animal, age and/or individual condition. The preferred route of administration is oral administration. The active ingredient may be administered in a range of about 0.3 xcexcg/kg to about 20 mg/kg body weight per dose, more preferably about 0.3 mg to about 3 mg/kg body weight per dose. Therefore, for a human subject with an average body weight of 60 kg, the active ingredient may be administered in a daily dose of between 18 xcexcg and 1.2 g, and preferred amounts to be administered to the human fall within 20 to 200 mg on a daily basis. Greater dosages can be administered for therapeutic reasons. Pharmaceutical compositions, nutritional supplements, food products and beverages of the present invention may be adapted for delivery of a daily dose of active ingredient of between about 18 xcexcg and about 1.2 g, more typically between about 20 mg to about 200 mg.
Modulating an Estrogen Receptor and Conditions Mediated by Estrogen Receptors
The estrogenic preparations and estrogenic compounds as described herein modulate (i.e. activate) estrogen receptors. Therefore, these preparations and compounds are useful for modulating an estrogen receptor and may be used to treat a condition mediated by an estrogen receptor, such conditions including but not being limited to: premature ovarian failure, menopause, osteoporosis, menstrual irregularities, hot flushes, genital tract atrophy, lack of libido, depression, abnormalities of lipid metabolism, cardiovascular diseases, cerebrovascular diseases, atherosclerosis, coronary artery disease, strokes, and cancer. In particular, estrogenic preparations and isolated estrogenic compounds from a plant, such as an Epimedium plant, isolated therefrom may be useful for treatment of menopausal and post-menopausal women.
Given the differences in tissue distribution and the fact that ER-beta and ER-alpha may have opposing actions when expressed in the same tissue, it is desirable to have compounds that express a preference, or selectivity, for one of the ER subtypes.
Differences in tissue distribution of ER-alpha and ER-beta may be important from a pharmacological point of view, for example with respect to hormone replacement therapy in post-menopausal women, which is a matter of public health that is of great concern, and treatment of prostatic hyperplasia and carcinoma in men. Estrogen replacement therapy in postmenopausal women is associated with increased risk of breast and uterine cancers. Development of ER-beta-selective agonists, i.e. agonists that bind ER-beta with higher affinity than ER-alpha, may be useful for regulating function of the central nervous system, the cardiovascular system, the urogenital tract and bone, while reducing or avoiding side-effects associated with ER-alpha activation. In particular, a compound such as Compound III that binds with higher affinity to ER-beta over ER-alpha may provide a useful alternative to conventional estrogen replacement therapy in post-menopausal women, especially in those women who have an increased risk of endometrial cancer and/or breast cancer. In men, ER-beta-selective agonists may be useful for the treatment of prostatic hyperplasia and carcinoma while avoiding or reducing side-effects associated with ER-alpha activation.
Compound I activates both the estrogen receptor (ER)-alpha and ER-beta. Isolated Compound I, or an preparation of a plant, such as an Epimedium plant, enriched for Compound I, can be used to treat a condition mediated by an estrogen receptor, such as: premature ovarian failure, menopause, osteoporosis, menstrual irregularities, hot flushes, genital tract atrophy, lack of libido, depression, abnormalities of lipid metabolism, cardiovascular diseases, cerebrovascular diseases, atherosclerosis, coronary artery disease, strokes, and cancer.
Compound II has no detectable estrogenic activity under the conditions described herein. Compound II is useful as a marker to identify fraction(s) of an Epimedium plant that contain Compound III, since Compound II is present in relatively large amounts in extracts of Epimedium plant, co-elutes with Compound III and is easily detected by MS and NMR. Compound II may have pharmacological activities observed in related prenylflavones, such as anti-platelet activity (Chen et al., 1996). Compound III activates ER-beta more strongly than it activates ER-alpha. Compound III, or preparations of an Epimedium plant enriched for Compound III, may be useful for treating conditions that are mediated by an estrogen receptor, and in particular, Compound III may be useful for treating a condition mediated by ER-beta while reducing or minimizing side-effects associated with activation of ER-alpha. Because Compound III activates ER-beta more strongly than ER-alpha, Compound III may be useful for regulating functions in tissues that express ER-beta as their predominant ER isoform, such as tissues of the central nervous system, the cardiovascular system, and the gastrointestinal and urogenital tracts, while reducing the incidence of side-effects resulting from stimulation of ER-alpha (such as breast cancer and endometrial cancer). Compound III can be further used to regulate anti-proliferative and anti-oxidant functions which are mediated at least in part through ER-beta. Thus Compound III may be used to treat hormone-dependent cancers like breast and endometrial cancers in women and prostate cancer in men, and other conditions associated with oxidative damage such as Alzheimer""s disease.
(i) Menopause
During the period of menopause and postmenopause, women often experience one or more symptoms due to declining estrogen levels, such as hot flushes, osteoporosis, depression, mood swings, sleeping disorders, atrophy of the breast and reproductive tissues, vaginal dryness and joint pain. An effective way of relieving menopausal symptoms involves estrogen replacement therapy. However, traditional hormone replacement therapy, using currently available forms of estrogens, brings about undesirable side effects, such as weight gain and breast tenderness. In addition, long-term estrogen therapy is also associated with a slight but significant increase in the risk of developing breast carcinoma and endometrial carcinoma. Epidemiological data show that a diet rich in phytoestrogen such as genistein found in soy, correlates with a reduction in the number of hot flushes (Tham, et al., 1998) and a lower incidence of breast cancer in Oriental women (Lee, et al., 1991). There is some evidence to suggest that phytoestrogens may have different pharmacological actions as compared to endogenous estradiol, and that phytoestrogens may selectively modulate the actions of estrogen receptors in cells (Diel, et al., 2001).
The differences in tissue distribution of ER-alpha and ER-beta may be important with respect to hormone replacement therapy in post-menopausal women, which is a great public health concern, and treatment of prostatic hyperplasia and carcinoma in men. Estrogen replacement therapy in postmenopausal women is associated with increased risk of breast and uterine cancers that may be mediated by ER-alpha. Development of alternative ER-agonists, in particular ER-beta-selective agonists (i.e. agonists that bind ER-beta with higher affinity than ER-alpha), may be useful for regulating function of the central nervous system, the cardiovascular system, the urogenital tract and bone, while reducing or avoiding side-effects, such as side-effects associated with ER-alpha activation. In particular, a compound that binds with higher affinity to ER-beta over ER-alpha may provide a useful alternative to conventional estrogen replacement therapy in post-menopausal women who have an increased risk of endometrial and breast cancers.
(ii) Breast and Mammary Cancer
Studies in the human and rodent breasts indicate that ER-beta is much more abundantly expressed than ER-alpha in these tissues. Studies in the breast of BERKO (ER-beta xe2x88x92/xe2x88x92 knockout) mice revealed abnormal epithelial growth and severe cystic breast disease as the mice age (Gustafsson, et al., 2000). In the human, ER-beta is expressed at high levels in stromal breast tissues but is significantly reduced in pre-invasive mammary tumors. This marked an early decrease in levels of ER-beta protein was associated with other criteria of cell proliferation in high grade ductal carcinoma-in-situ and suggest a protective effect of ER-beta against the mitogenic activity of estrogens in mammary premalignant tissue (Roger, et al., 2001). Ligands specific to ER-beta may therefore be of use for the prophylaxis or chemoprevention and treatment of breast carcinoma.
(iii) Cardiovascular System
Both ER-alpha and ER-beta are expressed in vascular tissue. However, in vascular tissue, experimental denudation of the endothelial layer leading to smooth muscle proliferation is accompanied by a huge increase (up to 80-fold) of ER-beta in smooth muscle cells, whereas expression of ER-alpha is unaffected. This result suggests that the protective effect of estrogens on vascular tissue inferred from epidemiological studies involves inhibition of smooth muscle proliferation mediated through ER-beta (Lindner, et al., 1998). Thus, drugs that regulate ER-beta action may be useful for treating diseases where vascular proliferation is important, such as strokes and coronary arterial disease.
(iv) Central Nervous System
In the central nervous system, estrogens are associated with a plethora of regulatory activities. For example, ER signaling regulates reproduction and sexual behavior.
In the human brain, the highest expression of ER-alpha has been found in restricted areas of the amygdala and hypothalamus. In contrast, ER-beta expression is low in these areas. ER-beta is most abundantly expressed in hippocampus, claustrum and cerebral cortex suggesting a role of ER-beta in the modulation of cognition, memory and motor function in the human (Osterlund, et al., 2000). The brains of BERKO mice, show several morphological abnormalities. There is a regional neuronal hypocellularity in the brain, with severe neuronal deficit in the somatosensory cortex, especially layers II, III, IV and V, and a remarkable proliferation of astroglial cells in the limbic system but not in the cortex. As BERKO mice age, the neuronal deficit becomes more pronounced and by 2 years there is degeneration of neuronal cell bodies throughout the brain, especially in the substantia nigra (Wang, et al., 2001). ER-beta is believed to play a role in neuronal survival, and consequently, compounds that activate ER-beta may be useful for the prevention and treatment of degenerative diseases of the central nervous system, such as Alzheimer""s disease and Parkinson""s disease, as well as disorders resulting from trauma and stroke in the brain. Thus administration of activators of ERs may affect important parameters of neural function, including cognition and memory.
(v) Urogenital Tract
Another tissue that expresses ER-beta is the urogenital tract. Both transitional epithelium in the bladder, and the epithelium of the urethra, the seminal vesicles, the prostate and kidney pelvis express significant quantities of ER-beta mRNA. The expression of ER-alpha in these tissues is less pronounced and the effect on urinary incontinence of estrogens might be mediated through ER-beta.
(vi) The Prostate Gland
Prostatic epithelial cells express ER-beta in normal rats and mice. In prostates from BERKO mice, androgen receptor levels are elevated and the tissue is hyperplastic, suggesting an important role of ER-beta in the male for regulating the growth of prostate tissue (Weihua, et al., 2001). Studies in human prostate cancer indicate that loss of ER-beta expression is associated with progression from normal prostate epithelium to prostate cancer (Horvath, et al., 2001). These data suggest that ligands that activate ER-beta may be useful in the prevention and/or clinical management of prostatic hyperplasia and prostate cancer. ER-beta-selective agonists may be useful for the treatment of prostatic hyperplasia and carcinoma while avoiding or reducing side-effects associated with ER-alpha activation, such as deep vein thrombosis, and strokes due to excessive blood clotting.
(vii) Reproduction
BERKO female mice have reduced fertility (Pettersson, et al., 2001). Poor reproductive capacity in BERKO mice is due to defects in both the ovary and uterus. Unlike ERKO (ER-alpha xe2x88x92/xe2x88x92 knockout) mice, where ovarian defects are the result of pituitary dysfunction, the defects of BERKO mice are in the ovary itself. There is a marked paucity of corpora lutea and an increase in the number of follicles with premature atresia. Uterine dysfunction also contributes to infertility in BERKO mice. ER-beta, but not hER-alpha, is expressed in the human testis. Some patients with azoospermia do not express ER-beta in the testis, suggesting that ER-beta agonists may be useful for treating certain kinds of male infertility. Thus discovery of ligands that can activate ER-beta specifically may be of use in treating both male and female infertility.
(viii) Cardiovascular Disease and Lipoprotein Metabolism
Epidemiological, clinical trial data, and basic science studies suggest a causal inverse relationship between phytoestrogens and cardiovascular disease. Populations with high intakes of dietary phytoestrogens have lower rates of cardiovascular disease (Tham, et al., 1998). Many epidemiological studies indicate that hormone replacement therapy is associated with reduction of the risks of coronary heart disease and has a possible cardioprotective role. Several human trials have reported cholesterol-lowering effects with phytoestrogens. Thus a significant reduction in total cholesterol was noted in premenopausal women when they consumed soy products with 45 mg conjugated isoflavones/day relative to levels during a control period when they were fed isoflavone-free soy products. In another study, hypercholesterolemic post-menopausal women were randomly assigned to 6 months of 40 g protein supplementation/day of casein or soy protein. Soy products lowered non high-density lipoprotein (non-HDL) cholesterol and increased HDL cholesterol significantly. Both soy groups experienced improved total cholesterol/low-density lipoprotein/cholesterol ratios.
(ix) Cancer
Both endogenous and exogenous sex-hormones have been associated with various cancers. High levels of biologically active androgen or estrogens are associated with increased risk of prostate cancer in men and with increased risk of ovarian and breast cancer in women. Despite the positive association of endogenous and exogenous estrogens with cancers, plant estrogens are inversely associated with cancer. It is well established that cancer risks differ strikingly in various populations of the world. Hormone-related cancers of the breast, ovary, endometrium and prostate have been reported to vary by as much as 5- to 20-fold between populations. The lowest rates were typically observed in Asian populations, which populations have plant-based diets with a high content of phytoestrogens. In a case-control study (Ingram D, Sanders K, Kolybaba M, Lopez D. Case-control study of phyto-oestrogens and breast cancer. Lancet. Oct. 4, 1997;350(9083):990-4), it was reported that a significant reduction in breast cancer risk among pre- and post menopausal women who consumed phytoestrogens. In a study of Asian-Americans, it was reported that tofu consumption was significantly and inversely associated with breast cancer (Tham 1998). A case-control study in Singapore showed that soy products were inversely and animal product positively associated with breast cancer in premenopausal women (Lee, et al., 1991). Soy and fiber consumption was also associated with a reduced risk of endometrial cancer. Thus discovery of new phyto-agonists which activate ERs, in particular ER-beta, may provide a valuable alternative treating many conditions in men and women. This is in relation to many important diseases that affect the cardiovascular health and cancer risk of aging populations.
The invention is further illustrated by the following non-limiting examples.

Methods for preparing an estrogenic preparation and isolated estrogenic compounds from a plant and uses thereof


An information processing device which enables display of guide information made up of characters, images and map data related to destinations and facilities of interest, in association with user information made up of texts and images recorded by the user, is provided. User information in the form of text data as well as guide information is displayed in an explanation display part. A photograph or illustration of facilities is displayed in an image display part. A map showing routes to facilities of interest from principal public facilities is displayed in a map display part. Since the latitude and longitude is recorded in map data irrespective of whether its format is a vector format or image format, the position of the facilities can be displayed from positioning information acquired from other equipments. Moreover, the setting of destinations and navigation including route guide are possible.

1. Field of the Invention
This invention relates to an information processing device and an information processing method for electronically precessing various guide information such as character-based explanatory information about facilities and spots of interest like tourist facilities, places where one can enjoy events, and sight-seeing spots, information about routes and transportation, image information such as maps and photographs, and language information about languages used at and around the facilities and spots of interest. This invention also relates to an information processing program executed by hardware, which is the information processing device.
This application claims priority of Japanese Patent Application No. 2002-013309, filed on Jan. 22, 2002, the entirety of which is incorporated by reference herein.
2. Description of the Related Art
Conventionally, in traveling, the travelers must carry many bulky travel guidebooks, dictionaries and conversation guidebooks. While there are some portable terminals for displaying road maps thereon and thus performing navigation as tools for travel, only the place where the user is now is displayed on the map as in car navigation devices.
Although there are electronic travel guidebooks to read, there are no electronic travel guidebooks which display the place where the user is now or enable intake of information gathered by word of mouth or information gathered from friends, as POI (point of interest). Moreover, there are no electronic travel guidebooks interlocked with photographs taken by the user so that the user can enjoy both.

Information processing device, information processing method and information processing program


An image pickup apparatus including: an image pickup element; an image pickup optical system for forming an image of an object by using a beam; a phase modulating element disposed on a pupil plane, for modulating a phase state of the beam; a modulating element controller for controlling the phase modulating element; and an image pickup element controller for dividing the image pickup element into multiple areas, and reading, for each of the multiple divided areas, information on the image in a time-sharing manner, in which, when the image pickup element controller reads the information on the image formed on the image pickup element by an area-by-area basis for the multiple divided areas in the time-sharing manner, the modulating element controller controls the phase modulating element so as to reduce a wavefront aberration in the divided area from which the information is to be read.

In order to correct aberrations in image pickup optical systems, there are used various methods. For example, in an image pickup optical system having a wide angle of field, an aspherical lens is used to perform aberration correction with respect to a beam having a large angle of field. As the image pickup angle of field becomes larger (as the angle of field becomes wider), there occur more frequently coma, field curvature/astigmatism and the like. Thus, in order to correct those aberrations, the number of lenses is increased, and aspherical lenses are used.
Optical elements (lenses) used in an image pickup optical system are unchangeable in shape. For this reason, even if the surface of a lens has a curvature or an aspherical shape optimal for a given angle of field (zoom position) or a focus position, this shape is not necessarily optimal for another angle of field (zoom position) or another focus position.
To address this, there is conventionally known an image pickup apparatus in which aberration correction is performed with an effect similar to the effect obtained when the curvature and the aspherical coefficient are dynamically changed, by interposing a reflective phase modulating element in the optical system and performing optimal wavefront modulation according to each zoom position, focus position or the like (PTL 1). Further, there is known a pickup apparatus for an optical disc in which aberration generated due to a thickness error, a tilt or the like of the optical disc is corrected by using a liquid crystal element (PTL 2).
In general, all aberrations in an optical system can be expressed as a pupil aberration at a pupil of the optical system. Accordingly, there is known an imaging optical system in which a phase modulating element is interposed at the pupil of the optical system to correct the spherical aberration, and conversely add the spherical aberration to obtain a soft-focus effect (PTL 3).
Further, as for the method in which the shape of the reflective phase modulating element is changed in synchronization with the zoom position or the focus position to correct aberration in an image pickup optical system, as the angle of field becomes wider, the aberration correction for the entire screen becomes more difficult with only a single aspherical shape. For this reason, this method tends to leave many residual aberrations.


A piezoresistive elevator button assembly comprises a polymer film piezoresistive element sandwiched between a button and a button pressure plate, and circuitry for sensing a change in the resistance of the piezoresistive element. The piezoresistive element is held in place by a high temperature potting compound to create a button less sensitive to fire or heat. A compensating spring is placed between the piezoresistive element and the buttom pressure plate to eliminate the effect on that element due to temperature cycling of the button assembly elements. A frustoconical housing of the button employing catch teeth allows for little movement; the use of the polymer film piezoresistive element allows a signal to be generated though buttom movement is nearly imperceptible.

It is desirable that an elevator button have a number of characteristics not required in other buttons. Such a button must be able to withstand a greater impact force because the average passenger often does not push an elevator button, but rather he/she strikes it. A vandal may strike it even harder. Second, elevator buttons must be resistant to heat so that they are not damaged in a fire nor register calls in a fire. They must be similarly unresponsive to flame. Finally, it is desirable, both for aesthetic reasons and functional reasons that the button not have perceptible motion to register a call for service. An elevator button with no moving mechanical parts will be less likely to be subject to the need for repair.
Three are two types of piezoelements commonly used for pushbuttons: piezoresistive and piezoelectric.
Buttons using a piezoelectric element are based on the piezoelectric effect, i.e., the material in the button generates a voltage potential when subjected to pressure. Such a button is disclosed in U.S. Pat. No. 4,805,739, "Elevator Control Switch and Position Indicator Assembly", by Lind et al, issued Feb. 21, 1989. Buttons incorporating piezoresistive elements use the fact that the resistivity of the piezoelement changes with pressure.
One problem with piezoelectric materials is that they are brittle, a characteristic wholly unsuited to elevator buttons because of the daily rigors to which they are subjected. Such buttons are defenseless against vandals. The brittleness of piezoelectric materials lends them to being easily shattered.
A second problem with piezoelectric buttons is that they produce an output signal that is difficult to process with digital circuitry. The output of the piezoelectric element, when subjected to a flat load curve such as a step function, is a signal similar to the output from a high past filter where the time constant is much smaller than the time period of an input step function. Third, piezoelectrics are particularly sensitive to damage in a fire due to their heat sensitivity.
Attempts have been made to produce an elevator button with the desirable qualities mentioned above. Such an attempt is found in "Pathless Piezobuttons For Industrial Applications", components report 14 (1975) No. 5, which displays a piezoceramic film utilizing the piezoelectric effect. In the reference, the travel involved by the finger pressure is less than 1 micrometer. Hence, the name "pathless". The piezoelectric material in this button does not have the inherent temperature resistant characteristics needed for an elevator button. It therefore requires a flame-protective material for the button housing in order to overcome this deficiency. The reference discloses a piezoelectric member located perpendicular to the button face and a button backing having support members which secure the element vertically.
Other buttons similarly are unable to meet the needs of elevator systems. U.S. Pat. No. 4,267,725 discloses a vertical piezoresistive element braced at its center by a set screw such that depression of the button face at the upper tip of the element, and transverse to it, causes the element to generate a signal proportional to bending of the element. The set screw serves to ensure that the bending is only in one direction. This button fails to fulfill the need for a button with no observable mechanical movement since the button face of the reference must descend in order for the piezoelement to bend and thus give a signal. The nature of thin film silicon is such that a large amount of deformation is required to change the resistance of the element significantly enough that it can be measured. Such deformation is achieved, with the least amount of force, by bowing the element; it could not be so easily achieved by applying pressure transverse to the element on a flat surface. The reference requires a large number of complexly interrelated moving parts because there is no allowance to choose not to bow. Accordingly, this design is undesirable.
In another button, disclosed in U.S. Pat. No. 4,644,315, a cantilevered piezoresistive element is used. In one embodiment the cantilevered element is vertical while in another the cantilevered element is horizontal. This reference discloses a donut-shaped magnet connected to a plunger on a button face such that the magnet descends with the button face to deflect an oppositely-poled magnet mounted on the tip of a cantilever having a piezoresistor hinge. This button, however, does not meet the need for an elevator button having no mechanical movement. Further, the use of the magnet/hinge apparatus and the need to position the magnet/hinge assembly with great precision make for a button that is costly. The same factors make it likely to be difficult to manufacture insofar as setting the magnet/hinge arrangement such that it interferes with the donut-shaped magnet so much as to sense its movement, but not so much as to be directly in the path of the donut-shaped magnet to as it descent. Also, this reference does not achieve a signal output readily susceptible of processing through digital circuitry. The second embodiment shows the cantilever mounted in horizontal fashion. This tends to produce more equal forces of attraction and repulsion leading to more symmetrical output pulse shapes. However, again, the fine tuning of the positioning of the hinge/magnet arrangement as well as the need for moving parts make this an undesirable button.
Finally, U.S. Pat. No. 2,632,062, "Semiconductor Transducer", is predicated upon the discovery that a PN junction, when subjected to pressure, changes resistance. The patent shows in several embodiments, various elements, shaped like pencil tips, pressing upon a piezoresistive PN junction. It also shows one or more axles, in other embodiments, passing through one or more PN junctions. Pressure upon the axles subjects the PN junction to pressure, yielding a change in resistance. This reference does not satisfy the needs of elevator button. The silicon material is not resilient. The pencil tip used to stress the PN junction would not lead to a button with a long life, while the axle embodiment is quite inappropriate for use in an elevator--it does not disclose a button.


A communication unit includes a plurality of antennas and controls, in transmitting signal using the plurality of antennas, the transmission directivity of the plurality of antennas based on a known signal from the communication terminal. A radio resource allocation unit allocates, to the communication terminal, an uplink frequency band for use in transmission of the known signal from a plurality of unit frequency bands lined in a frequency direction. In newly allocating the uplink frequency band to the communication terminal, the radio resource allocation unit newly determines the uplink frequency band based on elapsed time since the last use of the unit frequency band as the uplink frequency band.

In a communication system in which a TDMA/TDD (Time Division Multiple Access/Time Division Duplexing) scheme is adopted, such as PHS (Personal Handyphon System) and XGP (eXtended Global Platform) also called next-generation PHS, a base station may adopt an adaptive array antenna scheme of adaptively controlling the directivity of an array antenna composed of a plurality of antennas. Patent Documents 1 and 2 disclose the technique regarding the adaptive array antenna scheme. The base station adopting the adaptive array antenna scheme controls the reception directivity and transmission directivity of the array antenna based on a known signal from a communication terminal.

Base station and frequency band allocation method