Composition and method for treating benign prostatic hyperplasia, and method of preparing a diterpenoid composition for the same

Abstract

A method of preparing a diterpenoid composition is provided. The method includes performing an extraction of Rubus chingii Hu material to obtain an extract composition; suspending the extract composition and performing liquid-liquid extraction of the suspension to obtain a target extraction portion; separating the target extraction portion by macroporous resin adsorption and performing elution to obtain a target fraction; eluting the target fraction to obtain a target eluate; and drying the target eluate to obtain a powder product. A diterpenoid composition obtained by the method, an oral composition including the diterpenoid composition, a composition including at least one diterpenoid extract of Rubus chingii Hu, a use of the compositions in preparing a medicine for treatment of benign prostatic hyperplasia (BPH) in a subject, and a method of treating BPH in a subject are also provided.

Description

COMPOSITION AND METHOD FOR TREATING BENIGN PROSTATIC HYPERPLASIA, AND METHOD OF PREPARING A DITERPENOID COMPOSITION FOR THE SAMECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of Chinese Application No. 202410740954.X, filed on 7 June 2024, the disclosure of which is incorporated herein by reference in its entirety.FIELD OF THE INVENTION

[0002] The present invention relates generally to compositions and methods for treating benign prostatic hyperplasia (BPH) and, more specifically, to a method of preparing a botanical extract-containing composition for treating BPH in a subject. The botanical extract-containing composition of the present invention comprises, consists essentially of, or consists of, isolated diterpenoid(s).BACKGROUND OF THE INVENTION

[0003] Benign prostatic hyperplasia (abbreviated as “BPH”) is a medical condition suffered by men, and particularly prevalent amongst elderly men, characterized by abnormal proliferation of prostatic epithelial and stromal tissues, i.e. an enlarged but non-cancerous prostate gland. One of the primary symptoms of an enlarged prostate is urinary retention resulting in frequent urination. The urinary retention is caused by the inability to completely empty the bladder due to the enlarged prostate pressing against and thereby restriction flow from the bladder to the urethra and subsequently leading to a weakening of the bladder. Complications associated with benign prostatic hyperplasia include, in addition to acute or chronic urinary retention, urinary tract infections (UTIs), blood in the urine, bladder damage, and more significantly, kidney damage.

[0004] In view of the foregoing, there remains an opportunity to provide new and useful compositions and methods of treating benign prostatic hyperplasia (BPH). Such compositions and methods can offer potential preventative and therapeutic approaches for preventing and / or treating BPH. As such, there also remains an opportunity to provide new and useful methods of preparing compositions for prevention and / or treatment of BPH.BRIEF SUMMARY OF THE INVENTION

[0005] A method of preparing a diterpenoid composition is disclosed. The diterpenoid composition may also be referred to as a composition comprising, optionally consisting essentially of, or optionally consisting of, one or more diterpenoids obtained or isolated from Rubus chingii Hu.

[0006] The method includes performing an extraction of Rubus chingii Hu material with a first solvent composition to obtain an extract composition, suspending the extract composition in water to obtain a suspension and performing liquid-liquid extraction of the suspension with a plurality of extraction solvents to obtain a plurality of extraction portions including a target extraction portion, separating the target extraction portion by macroporous resin adsorption in a bed of a macroporous adsorption resin, followed by eluting the bed of macroporous adsorption resin including the separated target extraction portion with a first eluent composition and subsequently with a second eluent composition to obtain a target fraction, eluting the target fraction with a third eluent composition and subsequently with a fourth eluent composition to obtain a target eluate, and drying the target eluate, optionally under reduced pressure, to obtain a powder product.

[0007] A diterpenoid composition is also disclosed. The diterpenoid composition may comprise, may consist essentially of, or may be one or more isolated diterpenoids obtained by the method.

[0008] A composition for administration to a subject is also disclosed. The composition includes at least one botanical active component comprising at least one diterpenoid extract that is an extract of Rubus chingii Hu. The composition fiirther includes at least one a pharmaceutically acceptable additive. Optionally, the pharmaceutically acceptable additive is not naturally occurring. The botanical active component is present in the composition in an amount effective to treat benign prostatic hyperplasia (BPH) in the subject.

[0009] An oral composition for administration to a subject and including the diterpenoid composition, a use of the diterpenoid composition in preparing a medicine for treatment of BPH in a subject, and a method of treating BPH in a subject are also provided. The method includes administering an effective amount of the composition to the subject, the composition being the diterpenoid composition or the oral composition. In various embodiments, the composition is administered orally to the subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 is a flow chart showing a method of preparing a Rubus chingii Hu diterpenoid composition in accordance with embodiments of the disclosure;

[0011] Figure 2 is a plot showing a high-performance liquid chromatogram (HPLC) of the Rubus chingii Hu diterpenoid composition;

[0012] Figure 3 is a plot showing the average weight of laboratory rats as a fimction of time during a efficacy study of the Rubus chingii Hu diterpenoid composition;

[0013] Figure 4 is a plot showing the average wet weight of the prostates of the laboratory rats in various test groups;

[0014] Figure 5 is a plot showing the prostate index of the various test groups; and

[0015] Figure 6 is a plot showing the inhibition of cell proliferation (in proliferation%) of RWPE-1 cells treated with DHT.DETAILED DESCRIPTION OF THE CURRENT EMBODIMENTS

[0016] A method of preparing a diterpenoid composition is disclosed (which may be referred to herein simply as “the composition”). The composition comprises at least one botanical active component. The at least one botanical active may be one or more isolated diterpenoids obtained from an extract of Rubus chingii Hu. The composition and its method of preparation are described below, followed by description of associated uses and methods.

[0017] As will be understood in view of this disclosure, the composition is not particularly limited aside from the botanical active component and, in particular, the extract(s) thereof (e.g., the isolated diterpenoid(s)), as well as the related methods of obtaining the botanical active component. As such, the composition may be formulated, for example, as or in an oral composition or as or in a nutraceutical, pharmaceutical, or supplement, and may be utilized as a unique and stand-alone therapeutic or in combination with other therapeutics compatible therewith.

[0018] The composition is useful for treating, preventing, and / or ameliorating various conditions, such as those associated with benign prostatic hyperplasia (BPH). Specifically, as will be appreciated in view of the description and examples below, the composition of the present embodiments is believed to be capable of preventing, inhibiting and / or treating BPH in a subject.

[0019] As such, the composition may be utilized to treat (i.e., slow, prevent, etc.) conditions generally associated with BPH, such as but not limited to acute or chronic urinary retention, frequent urination, urinary tract infections (UTIs), weakening of the bladder, and kidney disease.

[0020] As introduced above, the composition comprises a botanical active component. More specifically, the botanical active component comprises, optionally consists essentially of, or optionally consists of, at least one extract of Rubus chingii Hu. In various embodiments, the botanical active component may be one or more isolated diterpenoid extract(s) of the plant Rubus chingii Hu, particularly the fruit of Rubus chingii Hu.Rubus chingii Hu

[0021] In some embodiments, the botanical active component, and thus the composition, comprises the extract of Rubus chingii Hu fruit, i.e., an extract comprising, optionally consisting essentially of, or optionally consisting of, material from the fruit-bearing plant species Rubus chingii Hu. In various embodiments, the Rubus chingii Hu fruit extract is notparticularly limited, and may comprise or be any fruit extract or combination of fruit extracts from Rubus chingii Hu fruit obtained by the methods disclosed herein and suitable for use in the embodiments herein. The fruit may be, but is not limited to the unripe fruit of the plant Rubus chingii Hu. More specifically, exemplary Rubus chingii Hu fruit extracts include those capable of treating or preventing BPH, or eliciting / exhibiting any other such activities described herein as part of the botanical active component.

[0022] Rubus chingii Hu is a member of the Rosaceae family and has been reported to contain various bioactive components, such as terpenoids, flavonoids, coumarins, steroids, alkaloids, phenylpropanoids, phenolics, and organic acids. Rubus chingii Hu may simply be referred to as R. chingii, or via various other names such as Rubus chingii var. chingii, Fructus Rubi, Chinese Raspberry, Palmleaf Raspberry Fruit, “Fu Pen Zi”, or

[0023] In certain embodiments, the extract of Rubus chingii Hu is obtained by extracting fruit or fruit-based plant material of Rubus chingii Hu. The fruit may be fresh or dried, typically dried to prevent decay. The fruit may be unripe, just turning from unripe to ripe, or may be ripe. For example, the fruit may be harvested when it turns from green to yellow. The fruit may also be slightly scalded in hot water or slightly steamed prior to drying. The dried fruit may then be fiirther processed such as crushed and / or ground into a powder prior to extraction, the crushed or powdered fruit is then fiirther processed to form the extract as like described below.

[0024] In various embodiments, the botanical active component consists of the extract of Rubus chingii Hu fruit. In fiirther or other embodiments, the composition is substantially to completely free of components obtained from non-fruit-based plant material of Rubus chingii Hu. In these embodiments, the non-fruit-based plant material of Rubus chingii Hu can be, for example, the root, stem, bark, rhizome, flower, leaf, or stem of a Rubus chingii Hu plant. Without being bound by any particular theory, it is believed that the fruit of Rubus chingii Hu is most usefiil for treatment or prevention of BPH; whereas other parts of Rubus chingii Hu are less usefiil or not usefiil.

[0025] Suitable fruit extracts may be processed (e.g., defatted, partially defatted, ground, dried, precipitated, washed, filtered, mesh-sorted, extracted, distilled, concentrated, etc.) to obtain the Rubus chingii Hu fruit extract. Likewise, Rubus chingii Hu fruit may be extracted in raw form, or processed prior to extraction of the Rubus chingii Hu fruit extract (e.g., used in raw form, suspended form, dehydrated form, concentrated form, etc.).

[0026] The amount of the Rubus chingii Hu extract utilized in the botanical active component may vary, and will be selected based on the number and types of components being utilized in the botanical active component. In certain embodiments, the botanical active component comprises from 1 to 2000 mg of the Rubus chingii Hu extract, such as from 1 to 1000,optionally of from 2 to 800, optionally of from 20 to 750, or optionally of from 50 to 500, mg. However, amounts outside these ranges may also be utilized. For example, in certain embodiments, the botanical active component includes the Rubus chingii Hu extract in an amount of at least 1, optionally of at least about 20, optionally of at least about 50, optionally of at least 100, optionally of at least 250, optionally of at least 500, optionally of at least 1000, or optionally of at least 1500, mg. In these or other embodiments, an upper boundary may be selected such that the botanical active component comprises the Rubus chingii Hu extract in an amount of <100, <250, <500, <750, <1000, <2000, <5000 mg. In various embodiments, the botanical active component can include an amount of Rubus chingii Hu extract optionally in an amount of greater than 1, optionally greater than 5, optionally greater than 10, optionally greater than 25, optionally greater than 50, optionally greater than 75, optionally greater than 80, or optionally greater than 95, wt.%, based on the total weight of the botanical active component. In such embodiments, an upper boundary may be selected to be generally <10, <20, <30, <40, <50, <60, <70, <80, <90, and <99 wt.%, respectively, based on the total weight of the botanical active component.

[0027] In certain embodiments, the botanical active component comprises more than one Rubus chingii Hu extract, such as 2, 3, 4, or more Rubus chingii Hu extracts. In such embodiments, each Rubus chingii Hu extract is independently selected, may be the same as or different from any other Rubus chingii Hu extract, and each utilized in an amount as described above. In various embodiments, each of the Rubus chingii Hu extracts is a specific diterpenoid extract obtained from the fruit of Rubus chingii Hu.

[0028] The Rubus chingii Hu extract may be utilized in any form, such as neat (i.e., absent solvents, carrier vehicles, diluents, etc.), or disposed in a carrier vehicle, such as a solvent or dispersant. The carrier vehicle, if present, may comprise an aqueous solvent (e.g., water), an organic solvent, fluid, or oil, or the like, or combinations thereof. When utilized, the carrier vehicle will be selected based on the particular components of the botanical active component and / or the composition, such as the particular Rubus chingii Hu extract(s) utilized. It will be appreciated that the Rubus chingii Hu extract may be combined with the carrier vehicle, if utilized, prior to, during, or after being combined with any other components of the botanical active component and / or composition.Method of Preparing the Rubus chingii Hu Composition

[0029] With reference to Figure 1, in the present method an extraction of Rubus chingii Hu plant material is first performed with a first solvent composition to obtain an extract composition. In specific embodiments, the first solvent composition includes an alcohol, preferably ethanol (EtOH). In certain embodiments, the first solvent composition is preferably80% ethanol. Optionally, the first solvent composition is between 0% and 90% ethanol, between 10% and 90% ethanol, between 20% and 90% ethanol, between 30% and 90% ethanol, between 40% and 90% ethanol, between 50% and 90% ethanol, between 60% and 90% ethanol, between 70% and 90% ethanol, between 80% and 90% ethanol, or between 75% and 85% ethanol. The ratio of Rubus chingii Hu material to liquid may be set at approximately 1:8 (grams of plant material per liter of solvent; “g:L”), optionally between 1 :3 and 1 :20, optionally between 1:4 and 1:15, optionally between 1:5 and 1:12, optionally between 1:6 and 1:10, optionally between 1:7 and 1:9, or optionally between 1:7.5 and 1:8.5, g:L.

[0030] In various embodiments, the extraction is performed by hot reflux, and the extraction may be performed twice sequentially, with each extraction being performed for at least 1 hour, optionally at least 1.5 hours, at least 2 hours, at least 2.5 hours, or at least 3 hours. Optionally, the extraction is performed for a time period of from 1.5 to 2.5 hours, optionally of from 1.6 and 2.4 hours, of from 1.7 and 2.3 hours, of from 1.8 and 2.2 hours, or of from 1.9 and 2.1 hours. Preferably, the extraction is performed for at least, or approximately, 2 hours.

[0031] The extract composition obtained from the extraction step above is subsequently subjected to one or more enrichment steps to isolate and purify the desired components from the extract composition. The first such enrichment step is liquid-liquid extraction. Particularly, the extract composition obtained above is suspended in water to obtain a suspension, and a plurality of extraction solvents are used successively to obtain a plurality of extraction portions including a target extraction portion. In various embodiments, the extraction solvents include petroleum ether (PE), dichloromethane (DCM), n-butanol (NB), and water. PE and DCM remove small polar component part such as triterpenoids and phenols, and water removes large polar component parts such as polysaccharides, proteins, and pigments. NB extracts the diterpenoids from the suspension, and as such, the NB portion so obtained is the target extraction portion, whereas the PE, DCM, and water portions may be discarded.

[0032] The target NB extraction portion is then preliminarily separated by macroporous resin adsorption in a bed of a macroporous adsorption resin. In various embodiments, the macroporous adsorption resin is DI 01 macroporous adsorption resin. Next, the macroporous resin bed is eluted with a first eluent composition. In specific embodiments, the first eluent composition includes an alcohol, preferably ethanol (EtOH). In certain embodiments, the first eluent composition is preferably 30% ethanol. Optionally, the first eluent composition is between 20% and 40% ethanol, between 22.5% and 27.5% ethanol, between 25% and 35% ethanol, between 27.5% and 32.5% ethanol, or between 29% and 31% ethanol. The first eluent composition may be used to elute approximately 9 times the bed volume (BV), optionally 8 to 10 times the bed volume, or optionally 7 to 11 times the bed volume. The first eluentcomposition removes flavonoids, phenols, and saponins. Subsequently, the macroporous resin bed is eluted with a second eluent composition. In specific embodiments, the second eluent composition includes an alcohol, preferably ethanol (EtOH).

[0033] In certain embodiments, the second eluent composition is preferably 60% ethanol. Optionally, the second eluent composition is between 50% and 70% ethanol, between 52.5% and 67.5% ethanol, between 55% and 65% ethanol, between 57.5% and 62.5% ethanol, or between 59% and 61% ethanol. The second eluent composition may be used to elute approximately 2 times the bed volume (BV), optionally 1.5 to 2.5 times the bed volume, or optionally 1 to 3 times the bed volume. The product (eluate) of the second elution with the second eluent composition is the target fraction; the product (eluate) of the first elution with the first eluent composition may be discarded.

[0034] The target fraction is then subjected to polyamide column chromatography using a third eluent composition and subsequently a fourth eluent composition to obtain a target eluate. Particularly, the target fraction is loaded in a polyamide column and first eluted with the third eluent composition. In specific embodiments, the third eluent composition is water. The third eluent composition may be used to elute 1 times the bed volume (B V) of the polyamide column, optionally 1 to 2 times the bed volume, or optionally 1 to 3 times the bed volume. The product (eluate) of the elution with the third eluent composition is not the desired product and may be discarded.

[0035] Next, the polyamide column is eluted with the fourth eluent composition. In specific embodiments, the fourth eluent composition includes an alcohol, preferably ethanol (EtOH). In certain embodiments, the fourth eluent composition is preferably 30% ethanol. Optionally, the fourth eluent composition is between 10% and 40% ethanol, between 20% and 40% ethanol, between 22.5% and 37.5%, between 25% and 35% ethanol, between 27.5% and 32.5% ethanol, or between 29% and 31% ethanol. The fourth eluent composition may be used to elute 3 times the bed volume (BV) of the polyamide column, optionally 2.5 to 3.5 times the bed volume, or optionally 2 to 4 times the bed volume. The product obtained by elution with the fourth eluent composition is the target eluate that includes the target, isolated diterpenoid(s) of Rubus chingii Hu.

[0036] The target composition including the target diterpenoid(s) in the target eluate is then purified by drying to obtain a target powder. Optionally, drying is conducted under reduced pressure to facilitate purification.Definitions

[0037] In order to provide a clear and consistent understanding of the specification and claims, the following definitions are provided.

[0038] The terms “composition” or “formulation” refer to a product that treats, improves, promotes, increases, manages, controls, maintains, optimizes, modifies, reduces, inhibits, or prevents a particular condition associated with a natural state, biological process or disease or disorder. For example, a composition or a formulation prevents, inhibits, and / or improves at least one condition of benign prostatic hyperplasia (BPH). The terms composition and formulation include, but are not limited to, pharmaceutical (i.e., drug), over-the-counter (OTC), cosmetic, food, food ingredient or dietary supplement compositions that include an effective amount of an extract, at least one component thereof, or a mixture thereof. Exemplary compositions and / or formulations include pills, tablets, capsules, powder, bar, beverage, or other liquid. Preferred compositions are formulated for oral administration / ingestion.

[0039] As used herein, the term “effective amount” or “therapeutically effective amount” of a pure compound, composition, extract, extract mixture, component of the extract, and / or active agent or ingredient, or a combination thereof refers to an amount effective at dosages and for periods of time sufficient to achieve a desired result. For example, the “effective amount” or “therapeutically effective amount” refers to that amount of a pure compound, composition, extract, botanical extract, extract mixture, botanical extract mixture, component of the extract, and / or active agent or ingredient, or a combination thereof of this invention which, when administered to a subject (e.g., mammal, such as a human), is sufficient to effect treatment, such as preventing, inhibiting, or improving BPH. The amount of a composition, extract, botanical extract, extract mixture, botanical extract mixture, component of the extract, and / or active agent or ingredient of this disclosure that constitutes an “effective amount” or “therapeutically effective treatment” will vary depending on the active agent or the compound, the condition being treated and its severity, the manner of administration, the duration of treatment, or the age of the subject to be treated, but can be determined routinely by one of ordinary skill in the art having regard to his own knowledge and to this disclosure.

[0040] The term “pharmaceutically acceptable” means those drugs, medicaments, extracts or inert ingredients, which are suitable for use in humans and lower animals without undue toxicity, incompatibility, instability, irritation, and the like, commensurate with a reasonable benefit / risk ratio.

[0041] The terms “applying” and “administering” are defined as providing a composition to a subject via a route known in the art, including but not limited to intravenous, intra-arterial, oral, parenteral, buccal, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, or intraperitoneal routes of administration. In preferred embodiments, oral routes of administering the described composition are suitable.

[0042] The terms “minimize,” “reduce,” “suppress,” “decrease” and / or “inhibit” refer to a decrease or reduction in BPH and its conditions or downstream effects, in the presence of a botanical (or plant) ingredient or botanical extract as described herein, when compared to BPH in the absence of a botanical ingredient or botanical extract as described herein, such as in a control sample. The degree of decrease or inhibition of BPH or its conditions, will vary with the nature and quantity of a botanical ingredient or botanical extract present, but will be evident, e.g., as a detectable decrease in BPH; desirably a degree of decrease greater than about 5%, about 10%, about 15%, about 20%, about 25%, about 50%, about 75%, about 90%, about 95% or about 99% (or any degree of decrease in the range of from about 5% to about 99%) as compared to BPH in the absence of the botanical ingredient or botanical extract. For example, a composition comprising a plant ingredient or plant extract of Rubus chingii Hu can minimize or reduce BPH in a subject.

[0043] As used herein, the term “subject” or “individual” includes mammals to which a composition may be administered. Non-limiting examples of mammals include humans, nonhuman primates, rodents (including transgenic and non-transgenic mice) or the like. In some embodiments, the subject is a mammal, and in some embodiments, the subject is human. Compositions (or Formulations)

[0044] The oral composition may include any amount of the botanical active component (e.g., one or more diterpenoid extracts of Rubus chingii Hu), which will be selected based on the number and types of components being utilized in the oral composition as a whole. In general, the botanical active component is present in the oral composition in an amount effective to inhibit, prevent, or reduce BPH in a subject.

[0045] In certain embodiments, the oral composition comprises the botanical active component in an amount of from 1 to 5000, optionally of from 2 to 2000, optionally of from 5 to 1750, optionally of from 10 to 1500, optionally of from 15 to 1250, optionally of from 20 to 1000, optionally of from 25 to 750, optionally of from 30 to 500, optionally of from 35 to 500, optionally of from 40 to 500, optionally of from 45 to 450, optionally of from 50 to 450, or optionally of from 50 to 400, mg. However, amounts outside and / or overlapping with these ranges may also be utilized. For example, it is to be appreciated that the ranges described above with respect to the amount of each botanical extract in the botanical active component may equally apply to the amount of each botanical extract in the oral composition as a whole, such as when the botanical active component consists of but one of the botanical extracts.

[0046] In general, the oral composition is not limited in terms of formulation, peripheral ingredients, form, number of fimctions, etc., aside from comprising the botanical activecomponent and the botanical extract(s) thereof. Rather, the composition may be varied, and may be formulated in any fashion consistent with this disclosure.

[0047] Typically, the oral composition is formulated or otherwise adapted for administration to a mammalian subject (e.g., a human). For example, in various embodiments, the oral composition is adapted to be consumed and / or orally administered to a human subject. Thus, the oral composition is formulated for oral administration to the subject. In such embodiments, the oral composition may also be referred to as an ingestible oral composition, and typically comprises at least one pharmaceutically acceptable additive in addition to the botanical active component. In specific embodiments, the pharmaceutically acceptable additive is not naturally occurring, such as those described by example below. In other words, the pharmaceutically acceptable additive is not a product of nature in these specific embodiments. In other embodiments, the pharmaceutically acceptable additive is selected from conventional additives understood in the art, and can be used in conventional amounts.

[0048] As such, it should be appreciated that the particular additives, carriers, adjuvants, fillers, etc. present in or combined with the oral composition may vary. Moreover, the physical form of the oral composition is not limited, and will be selected based on the particular components of the oral composition, a desired use of the oral composition, etc. As such, as will be understood in view of the description herein, the oral composition may be formulated as a liquid, dry powder, suspension, emulsion, gel, paste, etc., and combinations thereof. In certain embodiments, the oral composition is formulated as a sterile, non-pyrogenic liquid solution or suspension, a coated capsule, a suppository, a lyophilized powder, a transdermal patch, a softgel, or other forms are known. Other examples of suitable forms include solids, gels, liquids, powders, or other such preparations where the botanical active component is disposed in an appropriate carrier vehicle, such as any of those described herein.

[0049] The oral composition can be prepared using various methods. For example, actives of the oral composition (such as the botanical extract(s)), and optionally one or more inactives (such as one or more conventional components, additives, excipients, etc.), can be mixed or blended and compressed or compounded utilizing various techniques understood in the art. The oral composition of this disclosure is not limited to a particular order of manufacturing steps or method of manufacture.

[0050] Optionally, the composition may include one or more additional components such as additives or other components. Excipients can be further classified as other components. Specifically, excipients used in oral solid dosage forms have been classified based on their functionality into groups such as diluents, disintegrants, binders, compression aids, granulating agents, glidants, lubricants, release-controlling polymers, stabilizers (such as antioxidants,chelators, and pH-modifiers), film-coating polymers, coating agents, vehicles, plasticizers, surfactants, colorants, sweeteners, and flavors.

[0051] In various embodiments, the oral composition comprises at least one component selected from the group consisting of binders, lubricants, glidants, and combinations thereof. In certain embodiments, the oral composition includes one or more compounds including, but not limited to, methylcellulose, hydroxypropyl methylcellulose, ethyl cellulose, cellulose acetate phthalate, acacia, gums, wax, glycerol monostearate, acrylic acid polymers and copolymers, methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate, ethyl methacrylate, lactose, calcium sulfate, calcium phosphate dibasic, sugar, microcrystalline cellulose (MCC), starch, sodium starch glycolate, polyvinylpyrrolidone, polyethylene glycol, and magnesium stearate. Combinations of such components can be utilized, and such components and other components used in conventional tablets are understood in the art.

[0052] As used herein, “diluents” may be inert substances added to increase the bulk of the oral composition to make a tablet a practical size for compression. As such, they may also be referred to as bulking agents. Commonly used diluents include, but are not limited to, microcrystalline cellulose (MCC), wood cellulose, com starch, modified com starch, (tri)calcium phosphate, calcium sulfate, lactose, kaolin, mannitol, sodium chloride, dry starch, (powdered) sugar, dextrose, mannitol, sorbitol, and the like. The diluent / bulking agent may be used alone or in various mixtures, and utilized in any amount known in the art for oral compositions.

[0053] As used herein, “flavoring agents” are compounds designed to give the oral composition a more palatable taste. Flavoring agents vary considerably in their chemical structure, ranging from simple esters, alcohols, and aldehydes to carbohydrates and complex volatile oils. Synthetic flavors of almost any desired type are now available and are well known in the art. If hard taste, acid taste or bitter taste derived from starting materials may be suppressed by seasoning or flavoring, the acidulant (e.g., citric acid, tartaric acid, malic acid, ascorbic acid, etc.), the sweetener (e.g., sodium saccharin, dipotassium glycyrrhizinate, aspartame, stevia, thaumatin, etc.), or the perfume (e.g., various fruit perfumes containing lemon oil, orange oil or strawberry, and yoghurt, mint, menthol, etc.) may be included in the oral composition. The flavoring agent may be used alone or in various mixtures, and utilized in any amount known in the art for oral compositions.

[0054] As used herein, “lubricants” are materials that perform a number of fimctions relating to oral compositions. In certain embodiments, like tablet manufacture, the lubricants perform one or more functions such as improving the rate of flow of the tablet granulation, preventing adhesion of the tablet material to the surface of dies and punches, reducing interparticle friction,and facilitating the ejection of the tablets from a die cavity. Examples of suitable lubricants include, but are not limited to, zinc stearate, gum arabic powder, cacao butter, carnauba wax, carmellosecalcium, carmellosesodium, caropeptide, aqueous silicon dioxide, dried aluminum hydroxide gel, glycerin, magnesium silicate, light anhydrous silicic acid, light liquid paraffin, crystalline cellulose, hardened oil, synthetic aluminum silicate, sesame oil, flour starch, white beeswax, magnesium oxide, dimethyl polysiloxane, potassium sodium tartrate, sucrose fatty acid ester, glycerin fatty acid ester, silicon resin, aluminum hydroxide gel, stearyl alcohol, stearic acid, aluminum stearate, calcium stearate, polyoxyl stearate, magnesium stearate, cetanol, gelatin, talc, magnesium carbonate, precipitated calcium carbonate, cornstarch, lactose, hard fat, saccharose, potato starch, hydroxypropylcellulose, fiimaric acid, sodium stearyl fumarate, polyethylene glycol, polyoxyethylene polyoxypropylene glycol, polysorbate, beeswax, magnesium aluminometasilicate, methylcellulose, Japan wax, glycerin monostearate, sodium lauryl sulfate, calcium sulfate, magnesium sulfate, liquid paraffin, phosphoric acid, palmitic acid, and hydrogenated vegetable oils and fats. The lubricant may be used alone or in various mixtures, and utilized in any amount known in the art for oral compositions.

[0055] As used herein, “binders” are agents used to impart cohesive qualities to powdered materials. Binders, or “granulators” as they are sometimes known, impart a cohesiveness to the tablet formulation, which ensures the tablet remaining intact after compression, as well as improving the free-flowing qualities by the formulation of granules of desired hardness and size. Materials commonly used as binders include starch, such as com starch and pregelatinized starch; gelatin; sugars, such as sucrose, glucose, dextrose, molasses, and lactose; natural and synthetic gums, such as gum acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone (PVP), Veegum, microcrystalline cellulose, microcrystalline dextrose, amylose, larch arabogalactan, ethyl cellulose, cellulose acetate, and the like. The binder may be used alone or in various mixtures, and utilized in any amount known in the art for oral compositions.

[0056] As used herein, “colorants” are agents that give the oral composition a more pleasing appearance, and in addition help the manufacturer to control the product during its preparation and help the user to identify the product. Any of the approved certified water-soluble FD&C dyes, mixtures thereof, or their corresponding lakes may be used to color tablets. A color lake is the combination by adsorption of a water-soluble dye to a hydrous oxide of a heavy metal, resulting in an insoluble form of the dye. The colorant may be used alone or in various mixtures, and utilized in any amount known in the art for oral compositions.

[0057] Other conventional ingredients that may optionally be present in the oral composition include preservatives, stabilizers, anti-adherents or silica flow conditioners or glidants, such as silicon dioxide. Such ingredients may be used alone or in various mixtures, and utilized in any amount known in the art for oral compositions.

[0058] It is to be appreciated that certain components or additives may be classified under different terms of art and just because a component or additive is classified under such a term does not mean that they are limited to that fimction. If utilized, the additive or additives may be present in the oral composition in various amounts. Additional ingredients for optional use in the oral composition, e.g., when adapted for topical or oral administration, are described in U.S. Patent Nos. 5,747,006; 5,980,904; 6,994,874; 7,060,304; 7,247,321; 7,348,034; 7,364,759; 7,700,110; 7,722,904; 8,202,556; 8,916,212; 9,445,975; 9,801,809; 10,307,366; 10,532,024; and 10,537,516; and in U.S. Publication Nos. 2006 / 0257509; 2007 / 0224154; 2008 / 0081082; 2008 / 0124409; 2013 / 0302265; 2017 / 0252293; 2017 / 0281666; 2018 / 0200285; 2019 / 0083566; 2019 / 0160117; 2020 / 0171117; 2020 / 0383898; 2021 / 0017240; and 2021 / 0212926; the disclosures of which are hereby incorporated by reference in their entirety. Method of Administration

[0059] The oral composition may be administered as needed, daily, several times per day or in any suitable regimen such that the desired outcome is achieved. In the method of this disclosure, the frequency of administration (e.g., oral administration) can depend on several factors, including the desired level of BPH treatment and / or prevention. Generally, a regimen includes administration of the oral composition once or twice daily to include an administration in the morning and / or an administration in the evening. The amount and / or frequency of administration of the oral composition may depend on several factors, including the level of desired results and the specific oral composition.

[0060] Treatment and / or prevention of BPH can be achieved by administering the formulations of the present invention internally. As an example, the formulation of the present invention can be internally administered with an acceptable carrier in the form of a pill, tablet, powder, bar, beverage, etc. Thus, the formulations described herein are useful in a wide variety of finished products, including pharmaceutical products, food products, and beverage compositions. Preferably, the products are usefiil for treatment and / or prevention of BPH, including reduction and / or prevention of symptoms associated with BPH.

[0061] When the formulations of the present invention are orally administered in the form of a liquid, the liquid may be water-based, milk-based, tea-based, fruit juice-based, or some combination thereof. Solid and liquid formulations for internal administration according to the present invention can further comprise thickeners, including xanthan gum, carboxymethyl-cellulose, carboxyethylcellulose, hydroxypropylcellulose, methylcellulose, microcrystalline cellulose, starches, dextrins, fermented whey, tofu, maltodextrins, polyols, including sugar alcohols (e.g., sorbitol and mannitol), carbohydrates (e.g., lactose), propylene glycol alginate, gellan gum, guar, pectin, tragacanth gum, gum acacia, locust bean gum, gum arabic, gelatin, as well as mixtures of these thickeners. These thickeners are typically included in the formulations of the present invention at levels up to about 0.1%, depending on the particular thickener involved and the viscosity effects desired.

[0062] The solid and liquid (food and beverage) formulations of the present invention can, and typically will, contain an effective amount of one or more sweeteners, including carbohydrate sweeteners and natural and / or artificial no / low calorie sweeteners. The amount of the sweetener used in the formulations of the present invention will vary, but typically depends on the type of sweetener used and the sweetness intensity desired.

[0063] Generally, the formulations of the present invention are orally administered at least on a daily basis for a period of time sufficient to bring about the desired level of improvement in BPH and its associated symptoms. Oral administration of the formulations of the invention may continue for any suitable period of time. More specifically, within a few hours to within a few days of the initial ingestion, a user may notice the BPH condition has improved. It should be appreciated that the frequency with which the formulations of the present invention should be ingested will vary depending on the desired level of BPH improvement or prevention. In particular, the degree of BPH improvement will vary directly with the total amount of the oral composition used.

[0064] Useful dosage forms can be prepared by methods and techniques that will be well understood by those of skill in the art and may include the use of additional ingredients in producing tablets, capsules, or liquid dosage forms.INDUSTRIAL APPLICABILITY

[0065] This disclosure provides new and usefill methods of preparing compositions including the botanical extracts described herein. The resulting diterpenoid compositions can offer potential preventive and therapeutic approaches for lowering the risks of and symptoms associated with BPH. As such, the compositions and methods described herein are usefiil for treating and preventing BPH.

[0066] General compositions and product lines provided by this disclosure relate to BPH and its treatment and prevention via the disclosed compositions, and specific examples include pills, tablets, and liquids utilizing such compositions.

[0067] The compositions and methods of this disclosure also provide potential preventive and therapeutic approaches for lowering the occurrence of frequent urination or other pathogenic complications caused by BPH.

[0068] The following examples, illustrating the compositions and methods of this disclosure, are intended to illustrate and not to limit the disclosure.EXAMPLES

[0069] The treatment efficacy and mechanism of the diterpenoid compositions obtained from Rubus chingii Hu via the disclosed preparation method was evaluated in male Sprague-Dawley rats. The results confirmed that the diterpenoid compositions obtained from Rubus chingii Hu had a positive effect in reducing prostate weight and prostate cell proliferation in rats.Preparation of Rubus chingii Hu Diterpenoid Composition

[0070] Rubus chingii Hu fruit was subjected to stepwise extraction and enrichment / isolation in accordance with the method described above and as shown in Figure 1. First, components of Rubus chingii Hu fruit were extracted two times by hot reflux, each for a period of two hours, using a solution of 80% ethanol in water and a ratio of material to liquid (grams to liter;“g:L”) of 1:8 The obtained Rubus chingii Hu fruit extract was then suspended in water for liquidliquid extraction and successively extracted with petroleum ether (PE), dichloromethane (DCM), water, and n-butanol (NB) to obtain their corresponding portions.

[0071] PE and DCM were used to remove small polar parts such as triterpenoids and phenols, and water was used to remove large polar parts such as polysaccharides, proteins, and pigments. The NB portion was preliminarily separated using a DI 01 macroporous adsorption resin, and subsequently a solution of 30% ethanol in water was used to elute nine times of the bed volume (BV) to remove flavonoids, phenols, and saponins, followed by using a solution of 60% ethanol in water to elute two times of the B V.

[0072] Next, the resulting fraction was subjected to polyamide column chromatography. Pure water was first used to elute one times of the BV, and subsequently a solution of 30% ethanol in water was used to elute three times of the BV. The resulting fraction was purified / concentrated by drying under reduced pressure to obtain a dry powder including the target diterpenoids of Rubus chingii Hu.

[0073] The high-performance liquid chromatogram (HPLC) of the obtained Rubus chingii Hu extract composition as shown in Figure 2 indicates that the composition includes diterpenoids. Particularly, the obtained dry powder was dissolved in pure water at a concentration of 1 mg / mL, and the supernatant was filtered using a 0.22 pm nylon membrane and syringe filters before injection into a HPLC system. Elution was performed using acetonitrile (A) and an aqueous solution of 0.1% phosphoric acid (B) as the mobile phases withthe following gradient program: 0 - 10 min of 20% - 25% A; 10 - 15 min of 25% - 25% A; 15 - 45 min of 25% - 45% A. The column used was a Zorbax SB-C18 (4.6 mm x 250 mm, 5 pm), maintained at 30°C, the flow rate was 1.0 mL / min, the sample injection volume was 20 pL, and the detection A, was 210 nm.Evaluation of the Prepared Rubus chingii Hu Diterpenoid Composition

[0074] Male Sprague-Dawley rats were kept in the animal room for 7 days to adapt to the environment and undergo surgery. The rats were then anesthetized with Shutai combined with Serazine Hydrochloride (45 mg / kg and 10 mg / kg, IP), and surgery was initiated by injection of Meloxicam (1 mg / kg, SC) for pain relief and atropine (0.05 mg / kg, IM). The control group (“Ctrl”) were sutured after incision of the scrotum, while for the other groups, the testes and epididymis were removed, followed by hemostasis and suturing (suturing with the 3rd line and suturing with the 4th line). Penicillin sodium was injected to treat inflammation (200000 IU per animal, IM).

[0075] The rats were then weighed every two days, the average weight of the rats as a fimction of time being shown in Figure 3. According to their body weight, the control group (“Ctrl”) received daily injections of olive oil (0.1 mL / lOOg, SC), the other groups (“Model”; “PC”; “FDS”) received daily injections of testosterone propionate (10 mg / kg, 0.1 mL / lOOg, SC), the positive drug group (“PC”) received daily injections of finasteride (3 mg / kg, gavage), and the drug group (“FDS”) received daily injections of the Rubus chingii Hu diterpenoid composition (40 mg / kg, gavage).

[0076] After 28 consecutive days, blood was taken from the abdominal aorta to euthanize the rats, and the wet weight of the prostate was then measured. The average wet weight (PW) for each group (Ctrl, Model, FDS, PC) is shown in Figure 4. The prostate index (PI: PI=prostrate wet mass (PW)*1000 / rat body weight) for each group was calculated and shown in Figure 5.

[0077] In addition, cell proliferation was measured utilizing a CellTiter-Glo assay. RWPE-1 cells (human prostatic epithelial cells) were seeded into 96- well plates (9 x 103cells / well) and incubated for 24 hours. The following day, the cells were treated with 500 nM DHT (dihydrotestosterone), with or without various concentrations of the Rubus chingii Hu diterpenoid composition (FDS: 10, 25, 50, 75) present (0-75 pg / ml (cell survival rate > 80 %)), for 72 hours. After treatment, CTG solution was added to each well. The number of viable cells was determined by measuring the luminescence with a microplate reader. The results in terms of proliferation percentage are shown in Figure 6.

[0078] The terms “comprising” or “comprise” are used herein in their broadest sense to mean and encompass the notions of “including,” “include,” “consist(ing) essentially of,” and “consist(ing) of.” The use of “for example,” “e.g.,” “such as,” and “including” to list illustrativeexamples does not limit to only the listed examples. Thus, “for example” or “such as” means “for example, but not limited to” or “such as, but not limited to” and encompasses other similar or equivalent examples. The term “about” as used herein serves to reasonably encompass or describe minor variations in numerical values measured by instrumental analysis or as a result of sample handling. Such minor variations may be in the order of ±0-10, ±0-5, or ±0-2.5, % of the numerical values. Further, the term “about” applies to both numerical values when associated with a range of values. Moreover, the term “about” may apply to numerical values even when not explicitly stated.

[0079] Generally, as used herein a hyphen “-” or dash in a range of values is “to” or “through”; a “>” is “above” or “greater-than”; a “>” is “at least” or “greater-than or equal to”; a “<” is “below” or “less-than”; and a “<” is “at most” or “less-than or equal to.” On an individual basis, each of the aforementioned applications for patent, patents, and / or patent application publications, is expressly incorporated herein by reference in its entirety in one or more non-limiting embodiments.

[0080] It is to be understood that the appended claims are not limited to express and particular compounds, compositions, or methods described in the detailed description, which may vary between particular embodiments which fall within the scope of the appended claims. With respect to any Markush groups relied upon herein for describing particular features or aspects of various embodiments, it is to be appreciated that different, special, and / or unexpected results may be obtained from each member of the respective Markush group independent from all other Markush members. Each member of a Markush group may be relied upon individually and or in combination and provides adequate support for specific embodiments within the scope of the appended claims.

[0081] It is also to be understood that any ranges and subranges relied upon in describing various embodiments of the present invention independently and collectively fall within the scope of the appended claims, and are understood to describe and contemplate all ranges including whole and / or fractional values therein, even if such values are not expressly written herein. One of skill in the art readily recognizes that the enumerated ranges and subranges sufficiently describe and enable various embodiments of the present invention, and such ranges and subranges may be farther delineated into relevant halves, thirds, quarters, fifths, and so on. As just one example, a range “of from 0.1 to 0.9” may be birther delineated into a lower third, i.e., from 0.1 to 0.3, a middle third, i.e., from 0.4 to 0.6, and an upper third, i.e., from 0.7 to 0.9, which individually and collectively are within the scope of the appended claims, and may be relied upon individually and / or collectively and provide adequate support for specific embodiments within the scope of the appended claims. In addition, with respect to the languagewhich defines or modifies a range, such as “at least,” “greater than,” “less than,” “no more than,” and the like, it is to be understood that such language includes subranges and / or an upper or lower limit. As another example, a range of “at least 10” inherently includes a subrange of from at least 10 to 35, a subrange of from at least 10 to 25, a subrange of from 25 to 35, and so on, and each subrange may be relied upon individually and / or collectively and provides adequate support for specific embodiments within the scope of the appended claims. Finally, an individual number within a disclosed range may be relied upon and provides adequate support for specific embodiments within the scope of the appended claims. For example, a range “of from 1 to 9” includes various individual integers, such as 3, as well as individual numbers including a decimal point (or fraction), such as 4.1, which may be relied upon and provide adequate support for specific embodiments within the scope of the appended claims.

[0082] The present invention has been described herein in an illustrative manner, and it is to be understood that the terminology which has been used is intended to be in the nature of words of description rather than of limitation. Many modifications and variations of the present invention are possible in light of the above teachings. The present invention may be practiced otherwise than as specifically described within the scope of the appended claims. The subject matter of all combinations of independent and dependent claims, both single and multiple dependent, is herein expressly contemplated.

Claims

CLAIMSWhat is claimed is:

1. A method of preparing a diterpenoid composition, the method comprising: performing an extraction of Rubus chingii Hu material with a first solvent composition to obtain an extract composition; suspending the extract composition in water to obtain a suspension and performing liquid-liquid extraction of the suspension with a plurality of extraction solvents to obtain a plurality of extraction portions including a target extraction portion; separating the target extraction portion by macroporous resin adsorption in a bed of a macroporous adsorption resin; eluting the bed of macroporous adsorption resin including the separated target extraction portion with a first eluent composition and subsequently with a second eluent composition to obtain a target fraction; eluting the target fraction with a third eluent composition and subsequently with a fourth eluent composition to obtain a target eluate; and drying the target eluate, optionally under reduced pressure, to obtain a powder product.

2. The method according to claim 1, wherein the Rubus chingii Hu material is further defined as a fruit of Rubus chingii Hu.

3. The method according to claim 1 or 2, wherein: i) the first solvent composition comprises an alcohol; ii) the first eluent composition comprises an alcohol; iii) the second eluent composition comprises an alcohol; iv) the fourth eluent composition comprises an alcohol; or v) a combination of two or more of i) to iv).

4. The method according to claim 3, wherein the alcohol is ethanol.

5. The method according to claim 3 or 4, wherein: i) the first solvent composition is between 0 and 90% ethanol, or optionally is 80% ethanol; ii) the first eluent composition is 30% ethanol; iii) the second eluent composition is 60% ethanol;iv) the fourth eluent composition is between 10 and 30% ethanol, or optionally is 30% ethanol; or v) a combination of two or more of i) to iv).

6. The method according to any one of claims 1 to 5, wherein the third eluent composition is water.

7. The method according to any one of claims 1 to 6, wherein the extraction is performed by hot reflux.

8. The method according to any one of claims 1 to 7, wherein the extraction is performed two times in sequence, each for a time period of from 1.5 to 2.5 hours, or optionally for at least or approximately 2 hours.

9. The method according to any one of claims 1 to 8, wherein the plurality of extraction solvents for performing liquid-liquid extraction include petroleum ether (PE), dichloromethane (DCM), n-butanol (NB), and water.

10. The method according to claim 9, wherein the target extraction portion is obtained with n-butanol (NB).

11. The method according to any one of claims 1 to 10, wherein the macroporous adsorption resin is DI 01 macroporous adsorption resin.

12. The method according to any one of claims 1 to 11, wherein elution of the target fraction is performed via polyamide column chromatography.

13. A diterpenoid composition obtained by the method according to any one of claims 1 to 12, optionally wherein the diterpenoid composition comprises, consists essentially of, or is, one or more isolated diterpenoids.

14. An oral composition for oral administration to a subject, the oral composition comprising: the diterpenoid composition according to claim 13.

15. A composition for administration to a subject, the composition comprising: at least one botanical active component; and a pharmaceutically acceptable additive;wherein the botanical active component comprises at least one diterpenoid extract that is an extract of Rubus chingii Hu; and wherein the botanical active component is present in the composition in an amount effective to treat benign prostatic hyperplasia (BPH) in the subject.

16. The composition according to claim 15, wherein the botanical active component comprises the extract of Rubus chingii Hu fruit, or optionally wherein the botanical active component consists of the extract of Rubus chingii Hu fruit.

17. The composition according to claim 15 or 16, wherein the extract of Rubus chingii Hu fruit is obtained by alcohol extracting, optionally ethanol extracting, fruit or fruit-based plant material of Rubus chingii Hu.

18. The composition according to any one of claims 15 to 17, wherein the composition is substantially to completely free of components obtained from non-fruit-based plant material of Rubus chingii Hu.

19. The composition according to any one of claims 15 to 18, wherein the composition is farther defined as an oral composition that is formulated for oral administration to the subject.

20. The composition according to any one of claims 15 to 19, wherein the pharmaceutically acceptable additive is not naturally occurring.

21. Use of: i) the diterpenoid composition of claim 13; or ii) the composition according to any one of claims 15 to 20; in preparing a medicine for treatment of benign prostatic hyperplasia (BPH) in a subject.

22. A method of treating benign prostatic hyperplasia (BPH) in a subject, the method comprising administering an effective amount of a composition to the subject, wherein the composition is: i) the diterpenoid composition according to claim 13; ii) the oral composition according to claim 14; or iii) the composition according to any one of claims 15 to 20.

23. The method of claim 22, wherein the composition is administered orally to the subject.

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