WO2005003145A1 - Shanzhuyu extract and uses thereof - Google Patents

Abstract

Provided are compositions and formulations containing an SZY extract or its active ingredients, loganin, morroniside, and cornuside. Also disclosed is a method of treating pain, autoimmune diseases, and inflammation using such compositions and formulations.

Description

SHANZHUYU EXTRACT AND USES THEREOF

CROSS REFERENCE OF RELATED APPLICATION The present application claims the benefit of Chinese patent application Serial No.

03145774.6 filed on July 3, 2003, entitled the same, which is explicitly incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0001] This invention relates to a ShanZhuYu extract and its use in treating pain, autoimmune disorders, or inflammation.

BACKGROUND OF THE INVENTION

[0002] ShanZhuYu (Cornus officinalis Sieb et Zu cc; hereinafter referred to as "SZY") is a traditional Chinese medicine used for toning up the liver and kidneys, constricting semen, and checking perspiration. Although it has been extensively studied, reports on the pharmacological activities of its active ingredients vary due to differences in isolated ingredients.

SUMMARY OF THE INVENTION

[0003] This invention relates to an extract of SZY, its active ingredients (loganin, morroniside, and cornuside), and their use for treating pain, autoimmune disorders, or inflammation.

[0004] In one aspect, the invention features an extract of SZY. The extract contains (or consists essentially of) 5-20% by weight loganin, 5-25% by weight morroniside, and 0.5-5% by weight cornuside. In particular, the extract may include (or consist essentially of) 9.7% by weight loganin, 17.5% by weight morroniside, and 2.6% by weight cornuside. The extract may contain (or consist essentially of) at least 50% by weight polyphenols.

[0005] An SZY extract of the invention can be produced, e.g., by boiling a mixture of SZY and water, filtering the mixture, passing the mixture through a macroporous resin column, and eluting the mixture with water, 10% ethanol, and 80-95% ethanol successively. The method of producing the SZY extract is within the invention.

[0006] In another aspect, the invention provides a composition consisting essentially of at least two members of loganin, morroniside, or cornuside. Also within the invention is a composition containing (or consisting essentially of) at least two members of loganin, morroniside, or cornuside, wherein at least a portion of one of the members in the composition is isolated. Such compositions may be produced, for example, by combining at least two members of non-isolated loganin, morroniside, or cornuside; or isolated loganin, morroniside, or cornuside.

[0007] A pharmaceutically acceptable carrier can be added to an SZY extract of the invention or its active ingredients to produce a pharmaceutical composition. Examples of such compositions include: a composition containing isolated morroniside and a pharmaceutically acceptable carrier; a composition containing isolated cornuside and a pharmaceutically acceptable carrier; a composition consisting essentially of a pharmaceutically acceptable carrier and at least two members of loganin, morroniside, or cornuside; and a composition containing (or consisting essentially of) a pharmaceutically acceptable carrier and at least two members of loganin, morroniside, or cornuside, wherein at least a portion of one of the members in the composition is isolated. [0008] The invention also features a method of treating pain, an autoimmune disorder, or inflammation by administering to a subject in need thereof an effective amount of the SZY extract, its active ingredients (alone or in combination), and the compositions delineated herein. The subject (e.g., a mammal such as human) may be one who is identified as being in need of treatment for pain, an autoimmune disorder, or inflammation. [0009] The SZY extract and its active ingredients may be administered alone or as a part of a composition, e.g., a composition delineated herein, through a parenteral or non-parenteral route. For example, they can be administered orally, sublingually, topically, subcutaneously, intradermally, mucosally, via an acupuncture point, nasally, intratracheally, intramuscularly, intraurethrally, vaginally, rectally, or intravenously. [0010] Examples of the pain to be treated include, but are not limited to, primary hyperalgesia, secondary hyperalgesia, myofascial pain, intractable myofascial pain, osteoarthritis pain, headache, back pain, neck pain, homotopic pain, inflammatory pain, ulcerative pain, neuropathic pain, nociceptive pain, cancerous pain, visceral spasm pain, and muscular pain.

[0011] Examples of the autoimmune disorders to be treated include, but are not limited to, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes, myasthenia gravis, chronic ulcerative colitis, pernicious anemia and chronic atrophic gastritis, Good Pasture's Syndrome, pemphigus, pemphigoid, primary biliary cirrhosis, multiple sclerosis, Guillain-Barre Syndrome, systemic lupus erythematosis, Sjogren's Syndrome, scleroderma, necrotizing arteritis, Wegener's granulomatosis, rheumatoid arthritis, nephritis, nephritic syndrome, organ transplantation, bone marrow transplantation, dermatitis medicamentosa, measles, phytophoto dermatitis, anaphylactic shock, or asthma.

[0012] Examples of the inflammation to be treated include, but are not limited to, cirrhotic inflammation, serous inflammation, fibrosing inflammation, suppurative inflammation, hemorrhagic inflammation, catarrhal inflammation, regenerative inflammation, or chronic granulomatous inflammation. [0013] Furthermore, the invention features a packaged product. The product includes a container, an extract of SZY disposed in the container, and a legend (e.g., a label or product insert) associated with the container and indicating administration of the extract for pain relief.

[0014] Also within the invention is a method of treating pain. The method involves administering to a subject in need thereof (e.g., a subject identified as being in need of treatment for pain) an effective amount of an extract of SZY.

[0015] The details of one or more embodiments of the invention are set forth in the accompanying drawings and description below. Other features, objects, and advantages of the invention will be apparent from the drawings and the description, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS [0016] Figure 1. HPLC chromatogram of SZY extract;

[0017] Figure 2. Inhibition of SZY extract and cornuside on TNF-α expression;

[0018] Figure 3. Inhibition of SZY extract and cornuside on IL 1-β expression;

[0019] Figure 4. Effects of TGCO (SZY extract) on injected footpad swelling in AA rats; and

[0020] Figure 5. Effects of TGCO (SZY extract) on non-injected footpad swelling in AA rats. DETAILED DESCRIPTION

[0021] This invention is based in part on an unexpected discovery that loganin, morroniside, and cornuside are present in an SZY extract which has pain-relieving, immunosuppressive, and anti-inflammatory activities. Thus, the SZY extract and its active ingredients (alone or in combination) are useful for treating pain, autoimmune disorders, or inflammation. [0022] An SZY extract of the invention contains 5-20% (e.g., 9.7%) by weight loganin, 5-25% (e.g., 17.5%) by weight morroniside, and 0.5-5% (e.g., 2.6%) by weight cornuside. The percentage of polyphenols in the extract may be at least 50% (i.e., any integer% between 50% and 100%, inclusive) by weight, as determined using the hide powder method, which has been the official method for polyphenol analysis (American Leather Chemist Association). The amount of polyphenols is determined by preparation of polyphenols solutions, absorption of the polyphenols on chromated hide powder, and subsequent determination of residual materials by gravimetric analysis.

[0023] The extract can be prepared according to the method described in Example 1 below, or by any other equivalent method. Briefly, SZY is boiled in water. The mixture is then filtered and passed through a purification column. The extract is eluted, e.g., with water, 10% ethanol, and 80-95% ethanol successively. The extract can then be dried, if desired.

[0024] Loganin is an iridoid glycoside found in Cornus officinalic Sieb.et Zucc (Li et al., Daozong Shipin Gongye Keji 23(10):45-47, 2002), Hydrangea hortensis (Khalifa et al., Journal of Pharmaceutical Sciences 28:221-229, 2001), and Strychnos cathayensis (Cheng et al., Journal of the Chinese Chemical Society (Taipei, Taiwan) 48(2):235-239, 2001). It has been shown to be an anti-inflammatory agent (Maria del Carmen Recio et al., Planta Med. 60:232-234, 1994). The structure of loganin is shown below:

Loganin (C₁₇H₂₆O₁₀), MW 390.39 Da, melting point 221-222°C

[0025] Morroniside is an iridoid glycoside found in Cornus officinalic Sieb.et Zucc (Endo et al., Yakugaku Zasshi 93(l):30-32, 1973) and Lonicera morrowii (D eshiro et al., Planta Medica 58(1):109, 1992). The structure of morroniside is as follows:

Morroniside (C₁₇H₂₆O_n), MW 406.39 Da

[0026] Cornuside is an iridoid glycoside found in Cornus officinalic Sieb.et Zucc (Hatano et al., Phytochemistry 29(9):2975-2978, 1990). Unexpectedly, cornuside has been found to inhibit expression of TNF-α and IL 1-β, two of the cellular factors involved in arthritis. TNF-α and IL 1-β protein levels can be determined using enzyme-linked immunosorbent assays (ELISA). For example, they can be measured by using TNF-α (Bender Medsytems, Vienna, Austria) and IL 1-β (Jingmei Biotech, Shanghai, China) ELISA kits, respectively, following the manufacture's instructions. TNF-α and IL 1-β mRNA levels can be determined by RT-PCR analysis of total cellular RNA. For example, total cellular RNA can be purified with RNAeasy^® Mini kit (Qigen, Valencia, CA), and the transcription of TNF-α and IL 1-β can be analyzed using Access RT-PCR System (Promega, Madison, WI). The structure of cornuside is shown below:

Cornuside (C₂₄H₃₀O₁₄), MW 542.49 Da [0027] As the biological functions of loganin, morroniside, and cornuside differ, the proportion of each ingredient in the SZY extract may be adjusted as needed, e.g., by further purification of the extract, or by adding to the extract an isolated (including chemically synthesized) ingredient. Alternatively, a composition may be formed by mixing two or three of the isolated ingredients in desired ratios. A mixture of at least two members of loganin, morroniside, cornuside (e.g., an SZY extract) may be particularly useful, for example, by exhibiting higher anti-inflammatory, immunosuppressive, or pain-relieving activities than each of the three individual ingredients due to synergistic interactions.

[0028] As used herein, the term "isolated" refers to a state in which a compound has a higher purity than in its natural state. More specifically, isolated loganin is at least 21% (i.e., any integer% between 21% and 100%, inclusive) pure by dry weight, isolated morroniside is at least 26% (i.e., any integer% between 26% and 100%, inclusive) pure by dry weight, and isolated cornuside is at least 6% (i.e., any integer% between 6% and 100%, inclusive) pure by dry weight. Purity o f a c ompound c an b e m easured b y any appropriate s tandard m ethod, e .g., b y column chromatography or HPLC analysis.

[0029] Typically, a pharmaceutically acceptable carrier is added to the SZY extract or its active ingredients to facilitate their administration. Preferably, the composition contains 0.1-99.9% by weight the extract or its active ingredients. A "pharmaceutically acceptable carrier" does not destroy t he p harmacological a ctivities o f t he e xtract o r i ts i ngredients, a nd i s n on-toxic w hen administered in doses sufficient to deliver an effective amount of the extract or its ingredients.

[0030] Pharmaceutically acceptable carriers that may be used include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-α-tocopherol polyethyleneglycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, and wool fat. Cyclodextrins such as α-, β-, and γ-cyclodextrin, or chemically modified derivatives such as hydroxyalkylcyclodextrins, including 2- and

3-hydroxypropyl-β-cyclodextrins, o r o ther s olubilized d erivatives m ay a lso b e a dvantageously used to enhance delivery of the extract or its active ingredients.

[0031] Other pharmaceutically acceptable additives such as fillers (e.g., anhydrous lactose, starch, lactose beads, and glucose), binders (e.g., microstalline cellulose), disintegrating agents (e.g., cross-linked sodium carboxymefhyl starch, low-substituted hydroxypropyl c ellulose, and cross-linked PVP), lubricating agents (e.g., magnesium stearate), absorption-promoting agents, flavoring agents, sweetening agents, diluting agents, excipients, wetting agents, solvents, solublizing agents, and coloring agents may also be included in the composition.

[0032] The SZY extract, its active ingredients, and the compositions delineated herein may be administered parenterally or non-parenterally. For oral administration, the SZY extract, its active ingredients, and the compositions may be in the form of pills, granules, capsules, suspensions, or solutions. For parenteral administration, the SZY extract, its active ingredients, and the compositions may be in the form of injectable suspensions, creams, ointments, patches, or sprays. The term "parenteral," as used herein, includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. Other administration routes include oral, topical, rectal, nasal, buccal, vaginal, sublingual, intradermal, mucosal, intratracheal, or intraurethral routes. The SZY extract, its active ingredients, and the compositions may also be administered via inhalation spray or an implanted reservoir, or through an acupuncture point.

[0033] For oral administration, the SZY extract, its active ingredients, and the compositions may be in any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions, solutions, microcapsules, pills, lozenges, granules, and powders. In the case of tablets, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions and/or emulsions are administered orally, the SZY extract or its active ingredients may be suspended or dissolved in an oily phase and combined with emulsifying and/or suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added. [0034] Due to its acidic pH (i.e., pH 2-3), the SZY extract may cause irritation to the stomach and intestines (e.g., stomach upset, pain, nausea, or vomiting). To reduce the gastrointestinal irritation, an alkaline substance can be co-administered with the extract or its active ingredients. By "co-administration" is meant that the alkaline substance may be administered before or after administration of the extract or its ingredients, or administered concurrently with the extract or its ingredients; and the extract or its ingredients and the alkaline substance may be administered either individually or as a mixture.

[0035] The alkaline substance should be chosen such that it does not cause damage to the extract or its active ingredients, thereby reducing their pharmacological activities. Exemplary alkaline substances include bicarbonate salts (e.g., sodium bicarbonate or potassium bicarbonate, and preferably sodium bicarbonate), carbonate salts (e.g., sodium carbonate or calcium carbonate, and p referably s odium c arbonate), metal hydroxides (e.g., sodium hydroxide), orbasic amino acids (e.g., arginine, lysine or histidine, and preferably lysine). The amount of the alkaline substance should be sufficient for reducing the gastrointestinal irritation caused by the SZY extract or its ingredients, e.g., judged by lessened or cured stomach upset, pain, nausea, or vomiting. When the alkaline substance and the extract or its ingredients are administered individually, the amount of the alkaline substance relative to the amount of the extract or its ingredients may be adjusted according to the age and weight of the subject to be treated and the level of the gastrointestinal irritation caused by the extract or its ingredients. When the alkaline substance and the extract or its ingredients are administered as a mixture, the pH of the mixture should be in the range of 4-10, preferably 6-8, and most preferably 7. To prepare a mixed composition, a pharmaceutically acceptable carrier and the SZY extract or its ingredients may be dissolved in water, and the pH of the solution is adjusted to 4-10 by adding an alkaline substance. The solution can then be dried, if needed. Alternatively, once the relative amounts of the extract or its ingredients, the pharmaceutically acceptable carrier, and the alkaline substance have been determined, e.g., according to the just-described method, the composition may be prepared by mixing the three components in the pre-determined ratio.

[0036] Additionally, the SZY extract or its active ingredients (with or without an alkaline substance) maybe enclosed with an enteric layer, which does not dissolve at the acidic pH of the stomach but leaches out in the small intestines. The enteric layer therefore prevents the enclosed active components from being damaged in the stomach. Examples of enteric layers include enteric hard gelatin capsules, enteric coatings, enteric microcapsules, or enteric composite gelatin capsules. The enteric layers can be produced using, e.g., hydroxyalkyl celluloses phthalate with plasticizing, surface activating, and coloring agents; hydrox propylcellulose, polyethyleneglycol, cellulose acetate, phthalate with plasticizing and anti-oxidizing agents; cellulose acetate phthalate; or acetone.

[0037] The SZY extract, its active ingredients, and the compositions of the invention may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and or suspensions. Other commonly used surfactants such as Tweens or Spans and/or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation. [0038] The SZY extract, its active ingredients, and the compositions of the invention may also be administered in the form of suppositories for rectal administration. These compositions can be p epared b y m ixing the S ZY extract o r i ts a ctive i ngredients w ith a su itable n on-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax, and polyethylene glycols. [0039] Topical administration of the SZY extract, its active ingredients, and the compositions of the invention is useful when the desired treatment involves areas or organs readily accessible by topical application. For application topically to the skin, the SZY extract or its active ingredients should be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier. Carriers for topical administration of the SZY extract or its ingredients include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene or polyoxypropylene compound, emulsifying wax, and water. Alternatively, the composition can be formulated with a suitable lotion or cream containing the active components suspended or dissolved in a carrier with suitable emulsifying agents. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water. The extract or its active ingredients may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topically transdermal patches are also included in this invention.

[0040] The SZY extract, its ingredients, and the compositions of the invention may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.

[0041] The SZY extract, its active ingredients, and the compositions of the invention can be administered using an implantable device. Implantable devices and related technology are known in the art and are useful as delivery systems where a continuous, or timed-release delivery of the extract, its ingredients, or the compositions is desired. Additionally, the implantable device delivery system is useful for targeting specific points of delivery (e.g., localized sites and organs). See, e.g., Negrin et al., Biomaterials 22(6):563, 2001. Timed-release technology involving alternate delivery methods can also be used in this invention. For example, timed-release formulations based on polymer technologies, sustained-release techniques and encapsulation techniques (e.g., polymeric and liposomal) can also be used for delivery o f the extract, its ingredients, or the compositions delineated herein. [0042] Also within the invention is a patch to deliver the SZY extract, its ingredients, or the compositions delineated herein. A patch includes a material layer (e.g., polymeric, cloth, gauze, and bandage) and the compositions of the invention. One side of the material layer can have a protective layer adhered to it to resist passage of the extract, its ingredients, or the compositions. The patch can additionally include an adhesive to hold the patch in place on a subject. An adhesive is a composition, including those of either natural or synthetic origin, that when contacted with the skin of a subject, temporarily adheres to the skin. It can be water resistant. The adhesive can be placed on the patch to hold it in contact with the skin of the subject for an extended period of time. The adhesive can be made of a tackiness, or adhesive strength, such that it holds the device in place subject to incidental contact, however, upon an affirmative act (e.g., ripping, peeling, or other intentional removal) the adhesive gives way to the external pressure placed on the d evice or the adhesive itself, and allows for breaking of the adhesion contact. The adhesive can be pressure-sensitive, i.e., it can allow for positioning of the adhesive (and the device to be adhered to the skin) against the skin by the application of pressure (e.g., pushing and rubbing) on the adhesive or device.

[0043] When the SZY extract, its active ingredients, the compositions, and the formulations of the invention are combined with one or more additional therapeutic or prophylactic agents, both the additional agent and the SZY extract, its ingredients, and the composition should be present at dosage levels of between about 1 to 100%, and more preferably between about 5 to 95% of the dosage normally administered in a monotherapy regimen. The additional agents may be administered separately, as part of a multiple dose regimen, from the extract, its ingredients, or the compositions of this invention. Alternatively, those agents may be part of a single dosage form, mixed together with the extract, its ingredients, or the composition of this invention in a single formulation. [0044] Packaged products can be manufactured by disposing the SZY extract, its active ingredients, the compositions and the formulations delineated herein in a container (e.g., bottle, canister, t ube, t in, e tc), and p rinting d osing i nformation o n a 1 egend ( e.g., a 1 abel o r p roduct insert) associated with the container. The container is made of any material suitable for holding the extract, its active ingredients, the compositions and the formulations delineated herein, including glass, plastic, metal or polymer. [0045] The invention further provides a method of treating pain, autoimmune disorders, and inflammation, including administering to a subject in need thereof an effective amount of the SZY extract, its ingredients, and the compositions of the invention. For treatment of pain, other SZY extract preparations may also be used. A subject to be treated may be identified as being in need of treatment for pain, an autoimmune disorder, or inflammation. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional, and can be subjective (e.g., opinion) or objective (e.g., measurable by a test or diagnostic method). The term "treating" is defined as administration of a substance to a subject with the purpose to cure, alleviate, relieve, remedy, prevent, or ameliorate a disorder, symptoms of the disorder, a disease state secondary to the disorder, or predisposition toward the disorder. An "effective amount" is an amount of the substance that is capable of producing a medically desirable result as delineated herein in a treated subject. The medically desirable result may be objective (i.e., measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect).

[0046] The pain to be treated includes, but is not limited to, primary hyperalgesia, secondary hyperalgesia, myofascial pain, intractable myofascial pain, osteoarthritis pain, headache, back pain, neck pain, homotopic pain, inflammatory pain, ulcerative pain, neuropathic pain, nociceptive pain, cancerous pain, visceral spasm pain, or muscular pain.

[0047] The autoimmune disorders to be treated include, but are not limited to, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes, myasthenia gravis, chronic ulcerative c olitis, p ernicious anemia and chronic a trophic gastritis, Good P asture's S yndrome, pemphigus, pemphigoid, primary biliary cirrhosis, multiple sclerosis, Guillain-Barre Syndrome, systemic lupus eryfhematosis, Sjogren's Syndrome, scleroderma, necrotizing arteritis, Wegener's granulomatosis, rheumatoid arthritis, nephritis, nephritic syndrome, organ transplantation, bone marrow transplantation, dermatitis medicamentosa, measles, phytophoto dermatitis, anaphylactic shock, or asthma.

[0048] The inflammation to be treated includes, but is not limited to, cirrhotic inflammation, serous inflammation, fibrosing inflammation, suppurative inflammation, hemorrhagic inflammation, catarrhal inflammation, regenerative inflammation, or chronic granulomatous inflammation. [0049] The effective amount of the SZY extract or its ingredients is between 1 and 300 mg/kg body weight per day. The effective amount can be any specific amount within the aforementioned range, wherein the lower boundary is any number of mg/kg body weight between 1 and 299, inclusive, and the upper boundary is any number of mg/kg body weight between 2 and 300, inclusive. The effective amount is useful in a monotherapy or in combination therapy for the treatment of pain, autoimmune disorders, or inflammation. As the skilled artisan will appreciate, lower or higher doses than those recited above may be required. Effective amounts and treatment r egimens for any p articular s ubject ( e.g., a m animal s uch a s human) will depend upon a variety of factors, including the activity of the specific extract or its ingredients employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, condition or symptoms, the subject's disposition to the disease, condition or symptoms, and the judgment of the treating physician or veterinarian.

[0050] In order that the invention described herein may be more readily understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting this invention in any manner. All references cited herein are expressly incorporated by reference in their entirety.

Example 1 Preparation of SZY extract and determination of the amount of polyphenols thereof

[0051] 500 g SZY was placed in 5000 mL water. The mixture was lightly boiled 3 times for 2 hours each. The mixture was filtered, combined, and allowed to stand still until use.

[0052] A column was packed with D101 macroporous resin. The weight ratio of the resin to the raw SZY material was 1 :2. The mixture described above was run through the column at 20 ml/min, and was eluted with water, 10% ethanol, and 95% ethanol successively. 48 g SZY exact was obtained by collecting and drying the eluted material.

[0053] Above SZY extract 2.0076 g(W) was dissolved in 500 mL water entirely. 25mL was taken out, concentrated and dried in 105°C for 3 hours to provide T!=0.0966g. At the same time, 6g hide powder (slightly chromated) was added to 100 mL above solution, shook for 15 minutes. The solid was filtrated off and 25 mL of the filtrate was concentrated and dried at 105°C for 3 hours to provide T₂=0.0561g. On the other hand, 6g hide powder was added to lOOmL distilled water and shook for 15 minutes. The solid was filtrated off and 25 mL of the filtrate was concentrated and dried at 105°C for 3 hours to provide T₀=0.0155g. W: the total weight of sample T\ the weight of tannin and non-tannin determined T : the weight of non-tannin determined and the hide powder dissolved T₀: the weight of the hide powder dissolved Tannin quantity%=(Tι -T₂+T₀)^χ 10/Wx 100% =55.3%

Example 2 Isolation and identification of three ingredients in SZY extract

1. Isolation and identification of loganin

[0054] (1) 100 g Lonicera morrowii was extracted 3 times in 500 ml of 95% ethanol. The extracts were combined and then concentrated down to 6 g. 34 mg loganin was obtained by repeated silica gel chromatography purification of the concentrated extract.

[0055] (2) lOOg Cornus officinalis was extracted 3 times in 500 ml of 95% ethanol. The extracts were combined and then concentrated down to 10 g. 875 mg loganin was obtained by repeated silica gel chromatography purification of the concentrated extract.

[0056] The following spectral data was obtained for the isolated loganin:

[α]_D ²⁵ = -81° (c 0.1, H₂O), UV (λ_max, nm) 238(4.0); IR: (ran ¹, KBr): 3350, 3300, 1715, 1650, 1440, 1300, 1080; 1H NMR (D₂O, 300 MHz) δ ppm: 7.42(s, H-3), 5.39(d, J=3.6 Hz, H-l), 4.14(m, H-7),

3J3(s, OCH3), 3.05(m, H-5), 2.16(m, H-6a), 2.10(m, H-9), 1.88(m, H-8), 1.73(m, H-6b), 1.06(d, J=6.9 Hz, H-10), 4.78(d, J=7.8 Hz, H-l'), 3.90(d, J=10.8, H-6a'), 3.50(m, H-6b'), 3.36-3.47(m, H-3-H5'), 3.28(t, J=8Hz, H-2').

¹³C NMR (D₂O, 75MHz) δ ppm: 170.3(C-11), 151.2(C-3), 113.3(C-4), 96.9(C-1), 74.6(C-7), 52.0(C-12), 45.2(C-9), 40.6(C-6), 40.4(C-8), 30.1(C-5), 12.3(C-10), 98.9(C-1'), 76.6(C-5'), 76.0(C-3'), 73.0(C-2'), 69.9(C-4'), 61.0(C-6').

2. Isolation and identification of morroniside

[0057] (1) 100 g Lonicera morrowii was extracted 3 times in 500 ml of 95% ethanol. The extracts were combined and then concentrated down to 7 g. 55 mg morroniside was obtained by repeated silica gel chromatography purification of the concentrated extract.

[0058] (2) 1 00 g C ornus o fficinalis w as e xtracted 3 t imes i n 500 m 1 o f 95% e thanol. The extracts were combined and then concentrated down to 7 g. 643 mg morroniside was obtained by repeated silica gel chromatography purification of the concentrated extract.

[0059] The following spectral data was obtained for the isolated morroniside:

[α]_D ²⁵ = -70° (c 1, EtOH), UV (λ_max, nm) 238(4.0); IR: (cm^"1, KBr): 3350, 3300, 1710, 1650, 1080;

1H NMR (D₂O, 300 MHz) δ ppm: 7.57(s, H-3), 5.90(D, J=9.3 Hz, H-l), 4.99(d, J=1.0 Hz, H-7), 4.91(d, J=7.8 Hz, H-l'), 4.36(m, H-8), 3.93(dd, J-2.0, 12.4 Hz, H-6'a), 3.3-3.8(m, H-2', 3', 4', 5', 6'b), 3.0(dt, J=12.8, 4.6 Hz, H-5), 2.05(m, H-6b), 1.90(m, H-6b), 1.85*m, H-9), 1.38(d, J=3.8 Hz, CH3), 3.74(s, OCH3).

3. Isolation and identification of cornuside

[0060] 100 g Cornus officinalis was extracted 3 times in 500 ml of 95% ethanol. The extracts were combined and then concentrated down to 10 g. 23 mg morroniside was obtained by repeated silica gel chromatography purification of the concentrated extract.

[0061] The following spectral data was obtained for the isolated cornuside:

[α]^D = -90°(c 1.0, CH₃OH); UV (λ_max, nm) 275(4.0); IR (cm^"1, KBr): 3350, 2980, 1720, 1640, 1600, 1400, 1020;

1H NMR (acetone-d₆ + D₂O, 300 MHz) δ ppm: 7.43(s, H-3), 7.04(d, J=2.1Hz, H-2', H-6'), 5.73(ddd, J=9.0, 1 1.5, 1 7.7 Hz, H-8), 5.40(d, J=6.6Hz, H-l), 5 .25(dd, J=11.4, 1 7.4Hz, H-10),

4J2(d, J=7.8 Hz, H-l"), 4.12(m, H-7), 3.82(dd, j=1.8, 12.3 Hz, H-6"a), 3.61(dd, J=5.4, 12.0,

H-6"b), 3.50(s, OCH₃), 3.20-3.42(m, H-2", 3", 4", 5"), 2.86(m, H-5), 2.634(m, H-9), 1.90(m,

H-6).

¹³C NMR (acetone-d₆+ D₂O, 75 MHz) δ ppm: 168.6(C-11), 167.5(C-7'), 153.1(C-8), 145.7(C-3), 134.8(C-2', 6'), 121.0(C-3', 4', 5'), 119.7(C-1'), 110.7(C-4), 109.7(C-10), 99.5(C-1"), 97.2(C-1), 77.3(C-5"), 76.8(C-3"), 73.6(C-2"), 70.5(C-4"), 63.7(C-7), 61.8(C-6"), 51.7(OCH3), 44.3(C-9), 30.7(C-6), 30.4(C-5). Example 3 HPLC analysis of SZY extract

[0062] The SZY extract prepared according to Example 1 was subjected to HPLC analysis using Agilent 1100 HPLC system with DAD detector under the following conditions: wavelength - 240 nm, column - Zorbax SCI 8 4.6* 150mm, flow rate - lml/min, mobile phases - CH₃CN and aqueous 0.1% H₃PO , CH₃CN gradient - 0 to 25% within 50 min, 25% to 100% within 5 min, 100% for 10 min, and 100% to 0% within 5 min. The reference compounds of loganin, morroniside and cornuside were each estimated to be more than 96% pure by HPLC analysis. The SZY extract was found to contain 9.7% loganin (retention time = 20.4 min), 17.5% morroniside (retention time = 26.5 min), and 2.6% cornuside (retention time = 41.9 min). See Figure 1.

Example 4 Pharmacolofiical tests of SZY extract

1. Twisting test

Materials:

[0063] Animals: NTH mice, female, 18-22 g (body weight), provided by Department of Health, Beijing Biological Products Research Institute.

[0064] Test composition: SZY extract, prepared according to Example 1, dissolved in distilled water, pH adjusted to 7.2 with NaHCO₃.

[0065] Positive control composition: ibuprofen, 0.1 g/pill, daily dose 1.2 g, provided by Shenyang Dongling Pharmaceuticals, Ltd., registration No. 9808020.

[0066] Reagents: 0.6% HOAc, saline-HOAc solution (0.3 ml 100% HOAc in 50 ml saline, pH 7-7.2), and saline.

Methods and results:

[0067] 55 mice were divided into 5 groups (11 mice/group). Each group was respectively administered with the following compositions for 5 days: saline, ibuprofen (0.2 g/kg), high dose of SZY extract (0.32 g/kg), medium dose of SZY extract (0.16 g/kg), and low dose of SZY extract (0.08 g/kg). One hour after administration of the compositions on the 5 day, 0.6% HOAc (0.2 ml/mouse) was injected intraperitoneally. The number of twisting mice and the number of twists were observed for 20 minutes and recorded (Table 1). Pain relief percentage was calculated as: pain relief percentage = [(number of twists in control group - number of twists in test group) / number of twists in control group] x 100% Table 1 Effects of SZY extract on pain caused by intraperitoneal injection of HOAc in mice ~ No. of -_^ i s No. of twists No. of twisting Pain relief Group _ mi •ce Dose (g^o/kg °)' ( _X.+, s .) mice ° percentage ( ,_a%_/.) Saline 11 —• 24±16.78 11 0 Ibuprofen 11 0.2 1.818±3.46** 4 64 SZY extract (low dose) 11 0.08 0.364±0.771** 3 73 SZY extract (medium dose) 11 0.16 0.818±1.403** 3 73 SYZ extract (high dose) 11 0.32 1.091±2.58** 2 82

** P<0.01 when compared with the saline group.

2. Hot plate test

Materials:

[0068] Animals: Kunming mice, female, 18-20 g (body weight), purchased from the Chinese Academy of Preventive Medicine, Institute of Epidemiology and Microbiology.

[0069] Test composition: SZY extract, prepared according to Example 1, dissolved in distilled water, pH adjusted to 7.2 with NaHCO₃.

[0070] Positive control composition: ibuprofen, 0.1 g/pill, daily dose 1.2 g, provided by Shenyang Dongling Pharmaceuticals, Ltd., registration No. 9808020.

[0071] Water bath: manufactured by Beijing Changfeng Scientific Instruments Company.

Methods and results: [0072] Water was added into the water bath such that it was in contact with a plate. The water temperature was adjusted to 55±0.5°C, and the plate was pre-heated for 10 minutes.

[0073] Each of 80 mice was placed on the hot plate. The threshold pain value was defined as the time period (in seconds) from when a mouse was placed on the hot plate to when the mouse started to lick its feet. Mice with threshold pain values of less than 5 seconds or more than 30 seconds and mice that jumped were excluded from the test.

[0074] 70 qualified mice were randomly divided into 5 groups (14 mice/group): saline group, ibuprofen (0.2 g/kg) group, high dose SZY extract (0.32 g/kg) group, medium dose SZY extract (0.16 g/kg) group, and low dose SZY extract (0.08 g/kg) group. For each mouse, the threshold pain value was measured twice, and the average value was used as the threshold value prior to administration of a composition.

[0075] Each group of mice was respectively administered with saline, ibuprofen, and different doses of SZY extract for 4 days. On the 4^th day, 30, 60, and 90 minutes after administration of the compositions, the threshold pain values were determined for each group of mice and subjected to the t-test (Table 2). The results indicate that the SZY extract is a potent pain-reliever. Table 2 Pain-relieving effects of SZY extract in mice (hot plate method) .... ,. ._ Threshold pain „ No. of Dose . , Group . ₍ ι_r value before Threshold pain values after administration ° administration Time 30 min 60 min 90 min Saline 11 —— 18.04±2.797 18.61±2.34 18.36±3.39 18.14±3.18 Ibuprofen 11 0.2 18.39±2.795 32.43±7.197** 28.25±7.51** 26.07±8.56** SZY extract 11 0.08 18.86±2.22 29.14±7.199** (low dose) 26.43±6.33** 24.29±6.696** SZY extract 11 0.16

(medium dose) 18.72-fc2.39 31.07±7.82** 27.5±6.297** 25.29±6.91** SZY extract 11 0.32 18.14±2.57 33.68±6.41** 28.04±6.81** 25.36±7.53** (high dose) ** PO.01 when compared with the saline group.

Example 5 Comparison of anti-inflammatory activities of SZY extract and cornuside

Materials:

[0076] Cells: PBMC.

[0077] Test compositions: SZY extract and cornuside.

[0078] Positive control composition: Dexamethason.

[0079] Reagents: Ficoll-Paque Plus (Amersham Bioscience), LPS and dexamethason (CalBiochem.), TNF-α ELISA Kit and ILl-β ELISA Kit (Jingmei), DMSO (Sigma).

Method:

[0080] PBMC cells were isolated from fresh blood using Ficoll-Paque Plus reagent according to the manufacturer's protocol. Cells were suspended in 10% FBS containing RPMI 1640 medium. 100 μl of 1x10^s cells/ml was seeded in each well of a 96-well plate. Three wells were used for each reaction, and the total number of cells in each well was lxlO⁴.

[0081] 10 μl of the SZY extract or cornuside was added into cells at a final concentration of 10, 100, or 300 μg/ml. Dexamethason was used as a positive control at a final concentration of lOμM. 10 μl of medium was added into cells as a negative control. The plate was incubated in a 37°C, 5% CO₂ incubator for 15 minutes. 10 μl of 100 μg/ml LPS was added to each well except for the negative controls. Cells were then incubated overnight.

[0082] The p late was spun at 1 000 rpm for 1 5 m inutes. Supernatant as transferred from each w ell t o t he w ells i n a n ew p late. Concentrations o f T NF-α and IL 1 -β w ere m easured using the assay kits.

Results:

[0083] It was found that the SZY extract has higher anti-inflammatory activities than the three individual ingredients, suggesting that synergistic interactions occur among the three ingredients in the SZY extract (see, e.g., Tables 3 and 4, and Figures 2 and 3).

Example 6 Preparation of enteric capsules containing SZY extract

[0084] SZY extract powder (prepared according to Example 1) is packed in enteric hard gelatin capsules. These capsules do not disintegrate in the acidic gastric environment.

Example 7 Preparation of enteric tablets containing SZY extract

[0085] SZY extract powder (80% by weight, prepared according to Example 1) is mixed with soluble cellulose powder (20% by weight). The mixture is made into tablets with different shapes and sizes and coated with enteric layers. These enteric tablets do not disintegrate in the acidic gastric environment.

Example 8 Preparation of multilayer enteric capsules containing SZY extract

[0086] SZY extract powder (85% by weight SZY, prepared according to Example 1) is mixed with soluble cellulose powder (15% by weight). The mixture is first enclosed in enteric microcapsules, and then in enteric hard gelatin capsules. The enteric composite gelatin capsules do not disintegrate in the acidic gastric environment.

Example 9 Disintegration test of enteric formulations of SZY extract

[0087] Enteric capsules or tablets prepared according to Example 6, 7, or 8 are subjected to a disintegration test following the protocol described in Chinese Pharmacopoeia, edition 1990. These samples do not disintegrate in artificial gastric juice for 2 hrs. They disintegrate in artificial intestinal juice after 15 minutes and pass through a sieve completely. Therefore, the enteric layer will not dissolve in the acidic gastric environment to cause irritation to the stomach, but will allow release of the SZY extract in the small intestines.

Example 10 Effects of alkaline substances on SZY extract activities and gastrointestinal irritation [0088] When the SZY extract prepared according to Example 1 was administered orally to mice, it caused fierce twisting and vomiting such that the feeding could hardly be completed. To prepare a composition containing both the SZY extract and an alkaline substance, the SZY extract was dissolved in 10% DMSO/PBS buffer to a final concentration of 10, 30, 100, or 300ug/ml. The pH values of these solutions were adjusted to 4, 6, 7, or 10 with sodium bicarbonate. When these solutions were administered orally to mice, no resistance to the feeding appeared. These results demonstrate that irritation caused by an SZY extract can be reduced by co-administration of sodium bicarbonate.

[0089] Effects of sodium bicarbonate on the activities of the SZY extract were determined by measuring the amount of TNF-α protein according to the method described above after administration of the extract with or without sodium bicarbonate (Table 3). The results indicate that the extract activities are not affected by addition of sodium bicarbonate.

Example 11 Preparation of pills containing SZY extract SZY extract 30 g Starch 3 g Starch broth (10%) as needed Citric acid 0.15 g Talcum powder 1.5 g

[0090] SZY extract (prepared according to Example 1) is mixed with starch. 10% starch broth is added to the mixture. Granules are prepared using a 14-mesh nylon sieve, dried at 70-80°C, passed through a 10-12-mesh wire sieve, and mixed with talcum powder. Pills are casted using a 12 mm die.

Example 12 Preparation of injection powders containing SZY extract SZY extract 20 g NaOH 40 g

[0091] SZY extract (prepared according to Example 1) is dissolved in distilled water. The pH of the solution is adjusted to 7.4. The solution is filtered through a 0.22 um filter under sterile conditions, filled in 10 ml vials, pre-cooled at a temperature 10-20°C below its melting point, and placed in a freeze-dryer of less than -45°C. After crystals appeared, the solution is lyophilized, and the vials are capped.

Example 13 Preparation of sprays containing SZY extract SZY extract 21.5 g Vc l g Ethanol 296.5 g ^" Fι₂ as needed [0092] SZY extract (prepared according to Example 1) is dissolved in ethanol. The spraying agent F₁₂ is added to 50% of the total volume (which could then be adjusted according to the spraying power). Antioxidant Vc is finally added to make the spray.

Example 14 Effects of total glycoside from Cornus Officinalis (SZY extract) on adjuvant arthritis

Materials

1. Animals

[0093] Male Wistar rats weighing 140-170g were obtained from Chinese Academy of Sciencer animal center (Shanghai, China), and maintained with free access to pellet food and water in plastic cages at 21±2°C, and kept on a 12h light/dark cycle. This study complied with current ethical regulation on animal research of this institute, and all rats used in the experiment received humane care.

2. Drugs and reagents

[0094] Total glycosides of Cornus Officinalis (TGCO, SZY extract), pilotscale mixed by eight batches of sample (Hutchison MediPhama Limited Chemical department), neutralized to pH7.2 by 1% NaHCO before administrated; Dexamethasone hydrochloric acid (Dex, Tianjin Pharmaceutical Company, Tianjin, China, Lot20021020 0.75mg/tablet). Tabellae Glucosidoram Tripterygii Totorum (TGT, Shanghai Fudan Fuhua limited, Shanghai, China, 10 mg/tablet, Lot 030103); Reagents used in this study were as follows: Bacilllus Calmette Guerin (BCQ Shanghai Institute of Biological Products, Shanghai, China, 200301001); Paraffin oil (China National Medicine Group Shanghai Chemical Reagent Company)

3. Equipments Plethysmometer (Ugo Basile. Italy)

Balance (Metier, AB104-N, 0.0 lg, 0-2100g)

Method and results

1. Adjuvant arthritis in rats induced by Freunds's complete adjuvant (FCA)

[0095] BCG was heat-killed at 80 °C for 1 h and then dissolved in sterilized liquid paraffin to make FCA with a concentration of 10 mg/ml. Rats were injected intradermally with lOOμl of FCA in the left hind paw.

2. In vivo drug delivery

[0096] Rats were grouped randomly into seven groups, such as: Normal, Model, Dexamethasone (DEX, 0.125 mg/kg), Tabellae Glucosidorum Tripterygii Totorum (TGT, 10 mg/kg) and Total glycosides of Cornus Officinalis (TGCO, 60, 120, 240 mg/kg ). TGCO were prepared fresh daily and administered to the rats from day 0 to day 24 (prevention administration) or from day 10 to day 28 (treatment administration). Control naϊve and untreated arthritic rats received p.o. normal saline only. A total of 8~12 rats were evaluated in each treatment group.

3. Effects of TGCO on Limb swelling of adjuvant arthritis rats

[0097] The injection day was regarded as day 0. The thickness in adjuvant-injected and non-injected hind paw was pre-measured before the injection and then determined at 3-4 days intervals with a Plefhysmometer (Ugo Basile. Comerio. Italy). The inflammatory reaction was evaluated by the footpad swelling of hind paw volume after FCA injection. Footpad swelling (%)=(volume after FCA injection - volume before FCA injection)/ volume before FCA injection. Injected or non-injected footpads swelling were shown in table 6 and figure 4 or table 7 and figure 5.

[0098] As shown in table 6 and figure 4, DEX, TGT and TGCO (60, 120, 240 mg/kg) significantly inhibited injected footpad swelling administered on the day of sensitization, whatever, only DEX, TGT and TGCO (240 mg/kg) significantly inhibited footpad swelling administered on the tenth day after sensitization(P<0.05 or PO.01).

Table 6 Effects of TGCO on injected footpad swelling in AA rats (mean ±s.e.m) Days after Footpad swelling (%) sensitization

Prevention Normal (10) Model (12) DEX (12) TGT (12) TGCO-60(12) TGCO-20(12) TGCO-40(12)

Day l 1.8±0.7** 69.8±2.6 63.4±2.2 70.5±4.2 68.3±2.0 61.0±2.6* 60.6±4.2

Day 4 10.5±1.4** 54.8±7.2 33.3±2.9* 37.9±3.3* 43.5±2.6 34.1±2.5* 31.8- .0**

Day 7 14.7±1.9** 48.5±2.7 25.8±2.2** 38.4±3.4** 47.0±2.0 33.8±1.4** 34.0±2.0**

Day 11 I .3±2.1** 119.2±7.8 38.0±5.9** 85.3±4.2** 99.6±4.1* 83.9±4.1** 81.0±6.6**

Day 14 23.3±2.3** 173.0±12.3 30.7±3.7** 127.0±8.8** 146.7±3.5 134.7±8.5* 129.3±7.6**

Day 18 28.9±2.0** 194.3±10.1 39.7±5.5** 157.7±6.9** 162.9±3.9** 157.8±6.9** 151.6±6.5**

Day 21 19.1±4.1** 189.4±13.0 27.2±2.9** 153.6±7.1* 140.2±5.0** 138.5±7.9** 129.3±6.4**

Day 24 23.0±2.1** 187.2±16.5 34.7±4.9** 144.9±7.7* 146.2±5.5* 144.3±9.4 125.7±8.5**

Treatment Normal (10) Model (12) DEX (12) TGT (12) TGCO-60(12) TGCO-20(12) TGCO-40(12)

Day 10 14.7±1.3** 87.4±7.2 92.4±9.6 90.1±10.5 90.7±6.6 92.7±8.4 88.0±8.8

Day 13 22.5±2.4** 148.21±13.9 77.0±7.4** 133.3±20.1 138.8±13.7 131.4±8.6 114.9±15.6

Day 17 16.1±1.7** 198.5±12.0 71.4±8.5** 197.7±16.9 191.0±8.9 196.7±8.7 143.7±16.4*

Day 20 18.9±1.7** 177.3±11.3 48.6±5.0** 151.3±14.9 175.0±14.7 159.6±11.3 126.8±13.0**

Day 24 18.9±1.8** 176.4±16.6 44.7±5.7** 141.5±15.3 170.4±11.1 168.4±15.0 133.2±15.6** Vs. Model, *P<0.05, ** PO.01

[0099] As shown in table 7 and figure 5, DEX, TGT and TGCO (120, 240 mg/kg) significantly inhibited non-injected footpad swelling administered whatever from the day of sensitization or from the tenth day after sensitization(P<0.05 or P<0.01). Inhibitive rate of footpad swelling of DEX (0.125 mg/kg, p.o.), TGT (10 mg/kg, p.o.) and TGCO (240 mg/kg, p.o.) was respectively about 90-100%, 40-50%, 40-60%. Table 7 Effects of TGCO on non-injected footpad swelling in AA rats (mean ±s.e.m) Days after sensitization Footpad swelling

Prevention Normal Model DEX TGT TGCO-60 TGCO- 120 TGCO-240

Day 11 21.1±1.2* 32.9±4.4 2.7±24** 13.1±3.1** 13.6±2.1** 7.2±3.7** 6.6±2.8**

Day 14 25.2±1.5** 47.3±5.3 1.6±1.9** 24.3±3.4** 33.3±4.7 21.7±3.9** 23.0±2.9**

Day 18 29.0±1.6** 95.0±6.1 0.1±2.2** 58.0±7.5** 58.1±8.2** 47.6±7.7** 39.1±6.8**

Day 21 24.2±2.5** 87.6±11.7 5.6±24** 58.7±7.5* 58.6±12.0 49.3±6.6* 444±5.5**

Day 24 23.2±1.6** 90.8±11.0 2.0±1.6** 51.1±6.1 ** 62.4±9.1 44.4±8.3* 394±7.1 **

Treatment Normal Model DEX TGT TGCO-60 TGCO-120 TGCO-240

Day 10 16.3±1.6 14.5±2.5 17.8±5.3 124±3.7 13.3±3.6 11.6±2.7 14.2±4.3

Day 13 20.6±1.8* 374±6.8 12.6±3.2** 17.6±5.8 33.6±6.4 29.6±6.1 24.2±6.5

Day 17 23.9±1.7** 106.0±11.8 12.6±2.7** 35.0±13.7** 48.3±11.5** 43.3±9.9** 43.0±10.9**

Day 20 20.0±2.1** 99.4±10.1 7.8±3.2** 41.1±15.8** 49.4±10.6** 46.4±10.3** 44.0±13.2**

Day 24 28.0±1.6** 146.2±19.7 10.6±3.0** 36.9±13.5** 70.6±16.7** 66.6±16.5** 64.0±18.8** Vs. Model, *P<0.05, ** P<0.01

4. Effects of TGCO on severity of adjuvant arthritis rats [0100] The severity of pathological changes of ear, nose, tail and four paws in adjuvant arthritis rats was scored at 3-4 days intervals from the eleven day after sensitization, as follows: ear: 0, normal, 1 small nodule or red, 2 severe nodule; nose: 0, normal, 1, connective tissue swelling; tail: 0, normal, 1 rheumatoid nodules, 2 severe nodule; footpad: 0, normal, 1, erythema and mild swelling confined to the ankle joint or toes; 2, erythema and mild swelling extending from the ankle to the midfoot; 3, erythema and severe selling extending from the ankle to the metatarsal joints; and 4, ankylosing deformation with joint swelling. The disease score for each rat was calculated as the sum of the scores for ear, nose, tail and four paws.

[0101] As shown in table 8 : the severity of pathological changes of ear, nose, tail and four paws in adjuvant arthritis rats in the every footpad was scored on the 24th day after sensitization, TGCO (60, 120, and 240 mg/kg), DEX, TGT dissolved in water were given p.o. for 24 (prevention) or 14 (treatment) days from the day 0 or day 10 of the second immunization by FCA (day 0). The result was that 60, 120 and 240 mg/kg of astilbin, 0.125 mg/kg of DEX, and 10 mg/kg of TGT all significantly inhibited clinical scores (table 2). TGCO (240 mg/kg, p.o.) inhibited clinic score to the same degree as TGT (10 mg/kg, p.o.).

Table 8 Effects of TGCO on severity of adjuvant arthritis rats (Mean±s.e.m) Group n Dose /kg) Clinic score (day 24) Prevention /Treatment Prevention Treatment Normal 10/10 - 0 0 Model 12/11 - 13.9*0.5 13.8±0J DEX 12/10 0.125 1.4±0.3** 1.9*0.2** TGT 12/8 10 9J±0J** 7.3*0.9** TGCO-60 12/11 60 11.3*0.5** 9.8*0.7** TGCO-120 12/10 120 10.6*0.4** 9.7.1:1.3* TGCO-240 12/11 240 9.6*0.6** 7.9*0.9** Vs. Model, *P<0.05, ** P<0.01

5. Effects of TGCO on body weight of adjuvant arthritis rats

[0102] The injection day was regarded as day 0. The body weight of rats was determined at 3-4 days intervals. As shown in table 9, compared with normal group, weight gain of model group significantly reduced (PO.01). Compared with the arthritic control group, DEX, TGT and TGCO (60 mg/kg) therapeutically administered significantly increased weight, but those groups preventively administered had no significant effect on weight gain and only showed a little increase.

Table 9 Effects of TGCO on body weight of adjuvant arthritis rats (Mean±s.cm) Days after Body weight increase (g) sensitization

Prevention Normal Model Dex TGT TGCO-60 TGCO-120 TGCO-240

Day 4 39.0*2.4** 21.7*2.6 22.5*1.6 22.5*2.2 20.4*1.1 22.5*2.0 20.0*2.2 Day 8 61.0*1.8** 35.0±2.5 29.6*2.3 40.4*2.5 29.2*1.6 29.6*2.5 28.3*2.2 Day 14 93.0*4.0** 20.4*3.0 20.8*5.4 26.3*3.3 16.3*2.1 16.8*1.7 16.3*3.2 Day 21 105.0*5.2** 14.6*2.7 18.2*4.9 23.3*3.7 13.8*3.1 15.0*2.3 20.4*2.7 Day 24 110.0*5.2** 12.1*3.0 20.6*2.7 23.3*3.7 13.3*2.8 16.0*2.2 20.4*2.7

Treatment Normal Model Dex TGT TGCO-60 TGCO-120 TGCO-240

Day 10 65.5*2.4** 31.8*2.8 36.0*4.3 39.4*2.6 33.2*1.8 34.0*3.0 32.3*3.0 Day 13 80.0*3.9** 23.2*3.7 23.0*5.1 31.3*3.8 18.2*1.5 20.5*3.0 21.8*4.3 Day 17 92.5±4.7** 14.1*2.1 30.0*5.3* 26.3*3.9** 18.2*1.9 17.0*2.8 20.5*4.8 Day 20 106.5*5.2** 14.5*2.4 31.0*5.5* 25.0*4.3* 25.5*2.1* 19.5*2.3 20.5*4.9 Day 24 115.5*5.7** 16.4*2.3 37.5*5.4** 31.3*5.8* 27.7*2.9* 20.5*3.1 25.5*4.9 Vs. Model, *P<0.05_: , ** PO.01

Discussion:

[0103] Rheumatoid arthritis is an inflammatory disease of synovium, or lining of joint, results in pains, stiffness, swelling, joint damage, and loss of function of joints. It is reported about 0.32%-0.34% of Chinese population (about 4 million people) has rheumoid arthritis, and about 1% of the USA population (about 2.1% million people) has the disease. Adjuvant arthritis, AA, is an animal model resembling human rheumatoid arthritis and often used to study the disease. Shan Zhuyu (common macrocarpium fruit) is a famous Traditional Chinese Medicine, known for its tonic, analgesic, and diuretic activities. The first record of usage of Shan Zhuyu was in Shennong Bencao, as early as two thousand years ago before Han Dynasty. Recently, the extraction of Shan Zhuyu (SYZ) was found to be a good immunosuppressant agent. The present study examined the therapeutic effect of Total glycosides of Cornus Officinalis (TGCO) on adjuvant arthritis by its administration preventive or therapeutic. Result was shown that TGCO (120, 240 mg/kg) significantly inhibited injected or non-injected footpad swelling and reduce clinic score administered whatever preventive or therapeutic. Compared with model groups, body weight of all administrated group has a little increase. In conclusion, Total glycosides of Cornus Officinalis (TGCO) may become a beneficial agent in treating rheumatoid arthritis.

[0104] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

What is claimed is:

I . An extract of SZY, comprising 5-20% by weight loganin, 5-25% by weight morroniside, and 0.5-5% by weight cornuside. 2. The exfract of claim 1, comprising at least 50% by weight polyphenols.

3. The exfract of claim 1, consisting essentially of 5-20% by weight loganin, 5-25% by weight morroniside, and 0.5-5% by weight cornuside.

4. The exfract of claim 1, consisting essentially of at least 50% by weight polyphenols.

5. The exfract of claim 1, comprising 9.7% by weight loganin, 17.5% by weight morroniside, and 2.6% by weight cornuside.

6. The exfract of claim 5, comprising at least 50% by weight polyphenols.

7. The exfract of claim 5, consisting essentially of 9.7% by weight loganin, 17.5% by weight morroniside, and 2.6% by weight cornuside.

8. The extract of claim 5, consisting essentially of at least 50% by weight polyphenols. 9. An exfract of SZY, produced by a method comprising: boiling a mixture of SZY and water; filtering the mixture to obtain a filtrate; passing the filtrate through a macroporous resin column; and eluting the column with water, 10% ethanol, and 80-95% ethanol successively. 10. A composition, comprising isolated morroniside and a pharmaceutically acceptable carrier.

II. A composition, comprising isolated cornuside and a pharmaceutically acceptable carrier.

12. A composition, consisting essentially of at least two members of loganin, morroniside, or cornuside.

13. A composition, consisting e ssentially o f a pharmaceutically acceptable c arrier and at least two members of loganin, morroniside, or cornuside.

14. A composition, comprising at least two members of loganin, morroniside, or cornuside, wherem at least a portion of one of the members in the composition is isolated. 15. The composition of claim 14, further comprising a pharmaceutically acceptable carrier.

16. The composition of claim 14, consisting essentially of at least two members of loganin, morroniside, or cornuside, wherein at least a portion of one of the members in the composition is isolated.

17. A composition, c onsisting e ssentially o f a p harmaceutically a cceptable c airier and at least two members of loganin, morroniside, or cornuside, wherein at least a portion of one of the members in the composition is isolated.

18. A packaged product, comprising: a container; an extract of SZY disposed in the container; and a legend associated with the container and indicating administration of the extract for pain relief.

19. A method of treating pain, an autoimmune disorder, or inflammation, the method comprising administering to a subject in need thereof an effective amount of the extract of claim 1. 20. T he m ethod o f c laim 19, w herein t he e xtract i s a dministered a lone or as a p art o f a composition.

21. The method of claim 19, wherein the extract is administered parenterally or non-parenterally.

22. The method of claim 21, wherein the exfract is administered orally, sublingually, topically, subcutaneously, intradermally, mucosally, via an acupuncture point, nasally, intratracheally, intramuscularly, infraurethrally, vaginally, rectally, or intravenously.

23. The method of claim 19, wherein the subject is identified as being in need of treatment for pain, an autoimmune disorder, or inflammation.

24. The method of claim 19, wherein the pain is primary hyperalgesia, secondary hyperalgesia, myofascial pain, intractable myofascial pain, osteoarthritis pain, headache, back pain, neck pain, homotopic pain, inflammatory pain, ulcerative pain, neuropathic pain, nociceptive pain, cancerous pain, visceral spasm pain, or muscular pain.

25. The method of claim 19, wherein the autoimmune disorder is chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes, myasthenia gravis, chronic ulcerative colitis, pernicious anemia and chronic afrophic gastritis, Good Pasture's Syndrome, pemphigus, pemphigoid, primary biliary cirrhosis, multiple sclerosis, Guillain-Barre Syndrome, systemic lupus erythematosis, Sjogren's Syndrome, sclerodeπna, necrotizing arteritis, Wegener's granulomatosis, rheumatoid arthritis, nephritis, nephritic syndrome, organ transplantation, bone marrow transplantation, dermatitis medicamentosa, measles, phytophoto dermatitis, anaphylactic shock, or asthma. 26. The method o f claim 1 9, wherein the inflammation is cirrhotic inflammation, s erous inflammation, fibrosing inflammation, suppurative inflammation, hernorrhagic inflammation, catarrhal inflammation, regenerative inflammation, or chronic granulomatous inflammation.

27. A method of treating pain or an autoimmune disorder, the method comprising administering to a subject in need thereof an effective amount of loganin, morroniside, cornuside, or a combination thereof.

28. The method of claim 27, wherein the subject is identified as being in need of treatment for pain or an autoimmune disorder.

29. A method of treating inflammation, the method comprising administering to a subject in need thereof an effective amount of morroniside, cornuside, or a combination thereof. 30. The method of claim 29, wherein the subject is identified as being in need of treatment for inflammation.

31. A method of treating pain, an autoimmune disorder, or inflammation, the method comprising administering to a subject in need thereof an effective amount of the composition of claim 14.

32. The method of claim 31, wherein the subject is identified as being in need of treatment for pain, an autoimmune disorder, or inflammation.

33. A method of treating pain, the method comprising administering to a subject in need thereof an effective amount of an exfract of SZY of claiml.

34. The method of claim 33, wherein the subject is identified as being in need of treatment for pain. 35. Amethod of producing an exfract of SZY of claiml, the method comprising: boiling a mixture of SZY and water; filtering the mixture to obtain a filtrate; passing the filtrate through a macroporous resin column; and eluting the column with water, 10% ethanol, and 80-95% ethanol successively. 36. A method of producing a composition of claimlO, the method comprising combining isolated morroniside with a pharmaceutically acceptable carrier.

37. A method of producing a composition of claiml 1, the method comprising combining isolated cornuside with a pharmaceutically acceptable carrier.

38. A method of producing a composition of claiml4, the method comprising combining at least two members of loganin, morroniside, or cornuside, wherein at least a portion of one of the members in the composition is isolated.