Formulation of a mixture of free-B-ring flavonoids and flavans as a therapeutic agent

Abstract

The present invention provides a novel composition of matter comprised of a mixture of two specific classes of compoundsFree-B-ring flavonoids and flavansfor use in the prevention and treatment of diseases and conditions mediated by the COX-2 and 5-LO pathways. The present invention further provides a novel method for simultaneously inhibiting the cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LO) enzymes, and reducing cox-2 mRNA production. Finally, the present invention includes a method for weight loss and blood glucose control. The methods of this invention are comprised of administering to a host in need thereof an effective amount of the composition of this invention together with a pharmaceutically acceptable carrier. This invention relates generally to the prevention and treatment of diseases and conditions mediated by the cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LO) pathways, including but not limited to the relief joint discomfort and pain associated with conditions such as osteoarthritis, rheumatoid arthritis, and other injuries that result from overuse.

Claims

1. A composition comprised of a mixture of an extract derived from Scutellaria enriched for Free-B-ring flavonoids containing baicalin and an extract derived from Acacia enriched for flavans containing catechin or epicatechin, wherein the ratio of Free-B-ring flavonoid:flavan in the composition is about 85:15.

2. The composition of claim 1, wherein at least 80% of the total weight of the composition comprises Free-B-ring flavonoids and flavans.

3. The composition of claim 1, wherein the major active components of the mixture are the Free-B-ring flavonoids and the flavans.

4. The composition of claim 1, wherein the major active ingredients of the mixture are baicalin, catechin, and epicatechin.

5. The composition of claim 1, wherein the Free-B-ring flavonoids and the flavans are enriched from a plant part selected from the group consisting of stems, stem barks, trunks, trunk barks, twigs, tubers, roots, root barks, young shoots, seeds, rhizomes, flowers and other reproductive organs, leaves and other aerial parts.

6. The composition of claim 1, wherein the Free-B-ring flavonoids are enriched from Scutellaria baicalensis or Scutellaria orthocalyx.

7. The composition claim 1, wherein said flavans are enriched from a plant species selected from the group consisting of the Acacia catechu, Acacia concinna, Acacia farnesiana, Acacia Senegal, Acacia speciosa, Acacia arabica, A. caesia, A. pennata, A. sinuata. A. mearnsii, A. picnantha, A. dealbata, A. auriculiformis, A. holoserecia and A. mangium.

8. The composition of claim 1, wherein the Free-B-ring flavonoids are enriched from Scutellaria baicalensis and the flavans are enriched from Acacia catechu.

9. The composition of claim 1, further comprising an adjuvant, excipient or carrier.

10. The composition of claim 9, wherein the adjuvant, excipient or carrier is selected from one or more of calcium-based salts, silica, boron, histidine, glucosamine sulfate, chondroitin sulfate, copper gluconate, cellulose, dextrose, saline, water, oil, vitamin D, and shark and bovine cartilage.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 depicts graphically the inhibition of COX-1 and COX-2 by HTP fractions from Acacia catechu. The extracts were prepared and fractionated as described in Examples 1 and 3. The extracts were examined for their inhibition of the peroxidase activity of recombinant ovine COX-1 (.square-solid.) or ovine COX-2 (.diamond-solid.) as described in Example 2. The data is presented as percent of untreated control.

(2) FIG. 2 depicts graphically the inhibition of COX-1 and COX-2 by HTP fractions from Scutellaria baicalensis. The extracts were prepared and fractionated as described in Examples 1 and 3. The extracts were examined for their inhibition of the peroxidase activity of recombinant ovine COX-1 (.square-solid.) or ovine COX-2 (.diamond-solid.) as described in Example 2. The data is presented as percent of untreated control.

(3) FIG. 3 depicts a HPLC chromatogram of a standardized extract isolated from the roots of Scutellaria baicalensis (lot # RM052302-01) having a Free-B-ring flavonoid content of 82.2%. Ten structures were elucidated using HPLC/PDA/MS as baicalin, wogonin-7-glucuronide, oroxylin A 7-glucuronide, baicalein, wogonin, chrysin-7-glucuronide, norwogonin-7-glucuronide, scutellarin, chrysin and oroxylin A.

(4) FIG. 4 depicts graphically a profile of the inhibition of COX-1 and COX-2 by the baicalein, which was isolated and purified from Scutellaria baicalensis. The compound was examined for its inhibition of the peroxidase activity of recombinant ovine COX-1 (.diamond-solid.) and ovine COX-2 (.square-solid.). The data is presented as percent inhibition of assays without inhibitor vs. inhibitor concentration (g/mL). The IC.sub.50 for COX-1 was calculated as 0.18 g/mL/unit of enzyme while the IC.sub.50 for COX-2 was calculated as 0.28 g/mL/unit.

(5) FIG. 5 depicts graphically a profile of the inhibition of COX-1 and COX-2 by the baicalin, which was isolated and purified from Scutellaria baicalensis. The compound was examined for its inhibition of the peroxidase activity of recombinant ovine COX-1 (.diamond-solid.) and ovine COX-2 (.square-solid.). The data is presented as percent inhibition of assays without inhibitor vs. inhibitor concentration (g/mL). The IC.sub.50 for COX-1 was determined to be 0.44 g/mL/unit of enzyme while that of COX-2 was determined to be 0.28 g/mL/unit.

(6) FIG. 6 depicts graphically a profile of the inhibition of COX-1 and COX-2 by a standardized Free-B-ring flavonoid extract (83% baicalin based on HPLC) isolated from Scutellaria baicalensis. The extract was examined for its inhibition of the peroxidase activity of recombinant ovine COX-1 (.diamond-solid.) and ovine COX-2 (.square-solid.). The data is presented as percent inhibition of assays without inhibitor vs. inhibitor concentration (g/mL). The IC.sub.50 for COX-1 was calculated as 0.24 g/mL/unit of enzyme while the IC.sub.50 for COX-2 was calculated as 0.48 g/mL/unit.

(7) FIG. 7 depicts graphically a profile of the inhibition of COX-1 and COX-2 by catechin, which was isolated and purified from Acacia catechu. The compound was examined for its inhibition of the peroxidase activity of recombinant ovine COX-1 (.diamond-solid.) and ovine COX-2 (.square-solid.). The data is presented as percent inhibition of assays without inhibitor vs. inhibitor concentration (g/mL). The IC.sub.50 for COX-1 was determined to be 0.11 g/mL/unit of enzyme while the IC.sub.50 for COX-2 was determined as 0.42 g/mL/unit.

(8) FIG. 8 depicts graphically a profile of the inhibition of COX-1 and COX-2 by a standardized flavan extract containing 50% total catechins isolated from Acacia catechu. The extract was examined for its inhibition of the peroxidase activity of recombinant ovine COX-1 (.diamond-solid.) and ovine COX-2 (.square-solid.). The data is presented as percent inhibition of assays without inhibitor vs. inhibitor concentration (g/mL). The IC.sub.50 for COX-1 was calculated as 0.17 g/mL/unit of enzyme while the IC.sub.50 for COX-2 was determined to be 0.41 g/mL/unit.

(9) FIG. 9 depicts the HPLC chromatogram of the flavans extracted from Acacia catechu with 80% MeOH in water.

(10) FIG. 10 depicts graphically a profile of the inhibition of 5-LO by the purified flavan catechin from Acacia catechu. The compound was examined for its inhibition of recombinant potato 5-lipoxygenase activity (+). The data is presented as percent inhibition of assays without inhibitor vs. inhibitor concentration (g/mL). The IC.sub.50 for 5-LO was 1.38 g/mL/unit of enzyme.

(11) FIG. 11 depicts graphically a profile of the inhibition of COX-1 and COX-2 by the Univestin composition produced through combination of the extracts of Free-B-ring flavonoids and flavans in a ratio of 85:15 as described in Example 14. Univestin was examined for its inhibition of the peroxidase activity of recombinant ovine COX-1 (.diamond-solid.) and ovine COX-2 (.square-solid.). The data is presented as percent inhibition of assays without inhibitor vs. inhibitor concentration (g/mL). The IC.sub.50 for COX-1 was 0.76 g/mL/unit of enzyme while the IC.sub.50 for COX-2 was 0.80 g/mL/unit.

(12) FIG. 12 depicts graphically a profile of the inhibition of COX-1 and COX-2 by the Univestin composition produced through combination of Free-B-ring flavonoids and flavans extracts in a ratio of 50:50 as described in Example 14. Univestin was examined for its inhibition of the peroxidase activity of recombinant ovine COX-1 (.diamond-solid.) and ovine COX-2 (.square-solid.). The data is presented as percent inhibition vs. inhibitor concentration (g/mL). The IC.sub.50 for COX-1 was 0.38 g/mL/unit of enzyme while the IC.sub.50 for COX-2 was 0.84 g/mL/unit.

(13) FIG. 13 depicts graphically a profile of the inhibition of COX-1 and COX-2 by the Univestin composition produced through combination extracts of Free-B-ring flavonoids and flavans in a ratio of 20:80 as described in Example 14. Univestin was examined for its inhibition of the peroxidase activity of recombinant ovine COX-1 (.diamond-solid.) and ovine COX-2 (.square-solid.). The data is presented as percent inhibition of assays without inhibitor vs. inhibitor concentration (g/mL). The IC.sub.50 for COX-1 was 0.18 g/mL/unit of enzyme while the IC.sub.50 for COX-2 was 0.41 g/mL/unit.

(14) FIG. 14 depicts the effect of increasing concentrations of Univestin on the amount of LPS-induced newly synthesized LTB.sub.4 (.diamond-solid.) as determined by ELISA in THP-1 or HT-29 cells (ATCC). The activity of the combination extract is expressed as % inhibition of induced LTB.sub.4 synthesis.

(15) FIG. 15 compares the LTB.sub.4 levels as determined by ELISA that remain in HT-29 cells after treatment with 3 g/mL Univestin in non-induced cells to treatment with 3 g/mL ibuprofen as described in Example 16.

(16) FIG. 16 compares the effect of various concentrations of Univestin on cox-1 and cox-2 gene expression. The expression levels are standardized to 18S rRNA expression levels (internal control) and then normalized to the no-treatment, no-LPS condition. This Figure demonstrates a decrease in cox-2, but not cox-1 gene expression following LPS-stimulation and exposure to Univestin.

(17) FIG. 17 compares the effect of 3 g/mL Univestin on cox-1 and cox-2 gene expression with the equivalent concentration of other NSAIDs. The expression levels are standardized to 18S rRNA expression levels (internal control) and then normalized to the no-treatment, no-LPS condition.

(18) FIG. 18 illustrates graphically ear-swelling data as a measure of inhibition of inflammation. Univestin produced through the combination of standardized extracts of Free-B-ring flavonoids and flavans in a ratio of 80:20 was compared to untreated mice and mice given indomethacin (50 mg/kg) via oral gavage. The data is presented as the difference in micron measurement of the untreated vs. the treated ear lobe for each mouse.

(19) FIG. 19 shows the effect of 100 mg/kg of Univestin (80:20) ratio of standardized extracts of Free-B-ring flavonoids to flavans) on the AA injected ankles of mice (Univestin+arachidonic acid) compared to non-treated mice (no treatment+arachidonic acid), mice without AA injections (negative control) or mice that were injected with the liquid carrier (vehicle control).

(20) FIG. 20 illustrates graphically the 95% confidence interval for the pain index WOMAC score at baseline, 30, 60 and 90 days of treatment with Univestin at a dosage of 250 mg/day.

(21) FIG. 21 illustrates graphically the 95% confidence interval for the pain index WOMAC score at baseline, 30, 60 and 90 days of treatment with Univestin at a dosage of 500 mg/day.

(22) FIG. 22 illustrates graphically the 95% confidence interval for the pain index WOMAC score at baseline, 30, 60 and 90 days of treatment with celecoxib at a dosage of 200 mg/day.

(23) FIG. 23 illustrates graphically the 95% confidence interval for the pain index WOMAC score at baseline, 30, 60 and 90 days of treatment with the placebo.

(24) FIG. 24 illustrates graphically the 95% confidence interval for the stiffness index WOMAC score at baseline, 30, 60 and 90 days of treatment with Univestin at a dosage of 250 mg/day.

(25) FIG. 25 illustrates graphically the 95% confidence interval for the stiffness index WOMAC score at baseline, 30, 60 and 90 days of treatment with Univestin at a dosage of 500 mg/day.

(26) FIG. 26 illustrates graphically the 95% confidence interval for the stiffness index WOMAC score at baseline, 30, 60 and 90 days of treatment with celecoxib at a dosage of 200 mg/day.

(27) FIG. 27 illustrates graphically the 95% confidence interval for the stiffness index WOMAC score at baseline, 30, 60 and 90 days of treatment with the placebo.

(28) FIG. 28 illustrates graphically the 95% confidence interval for the functional impairment index WOMAC score at baseline, 30, 60 and 90 days of treatment with Univestin at a dosage of 250 mg/day.

(29) FIG. 29 illustrates graphically the 95% confidence interval for the functional impairment index WOMAC score at baseline, 30, 60 and 90 days of treatment with Univestin at a dosage of 500 mg/day.

(30) FIG. 30 illustrates graphically the 95% confidence interval for the functional impairment index WOMAC score at baseline, 30, 60 and 90 days of treatment with celecoxib at a dosage of 200 mg/day.

(31) FIG. 31 illustrates graphically the 95% confidence interval for the functional impairment index WOMAC score at baseline, 30, 60 and 90 days of treatment with the placebo.

(32) FIG. 32 shows the effect of Univestin at doses of 250 and 500 mg/day on decreasing BMI compared to celecoxib at 200 mg/day and the placebo.

(33) FIG. 33 shows the effect of Univestin at doses of 250 and 500 mg/day on decreasing weight compared to celecoxib at 200 mg/day and the placebo.

(34) FIG. 34 shows the effect of Univestin at doses of 250 and 500 mg/day on lowering blood glucose compared to placebo.

DETAILED DESCRIPTION OF THE INVENTION

(35) Various terms are used herein to refer to aspects of the present invention. To aid in the clarification of the description of the components of this invention, the following definitions are provided.

(36) It is to be noted that the term a or an entity refers to one or more of that entity; for example, a flavonoid refers to one or more flavonoids. As such, the terms a or an, one or more and at least one are used interchangeably herein.

(37) Free-B-ring Flavonoids as used herein are a specific class of flavonoids, which have no substitute groups on the aromatic B ring, as illustrated by the following general structure:

(38) ##STR00006##

(39) wherein

(40) R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are independently selected from the group consisting of H, OH, SH, OR, SR, NH.sub.2, NHR, NR.sub.2, NR.sub.3.sup.+X.sup., a carbon, oxygen, nitrogen or sulfur, glycoside of a single or a combination of multiple sugars including, but not limited to aldopentoses, methyl-aldopentose, aldohexoses, ketohexose and their chemical derivatives thereof;

(41) wherein

(42) R is an alkyl group having between 1-10 carbon atoms; and

(43) X is selected from the group of pharmaceutically acceptable counter anions including, but not limited to hydroxyl, chloride, iodide, sulfate, phosphate, acetate, fluoride, carbonate, etc.

(44) Flavans are a specific class of flavonoids, which can be generally represented by the following general structure:

(45) ##STR00007##

(46) wherein

(47) R.sup.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are independently selected from the group consisting of H, OH, SH, OCH.sub.3, SCH.sub.3, OR, SR, NH.sub.2, NRH, NR.sub.2, NR.sub.3.sup.+X.sup., esters of substitution groups, including, but not limited to, gallate, acetate, cinnamoyl and hydroxyl-cinnamoyl esters, trihydroxybenzoyl esters and caffeoyl esters and their chemical derivatives thereof; carbon, oxygen, nitrogen or sulfur glycoside of a single or a combination of multiple sugars including, but not limited to, aldopentoses, methyl aldopentose, aldohexoses, ketohexose and their chemical derivatives thereof; dimer, trimer and other polymerized flavans;

(48) wherein

(49) R is an alkyl group having between 1-10 carbon atoms; and

(50) X is selected from the group of pharmaceutically acceptable counter anions including, but not limited to hydroxyl, chloride, iodide, sulfate, phosphate, acetate, fluoride, carbonate, etc.

(51) Gene expression refers to the transcription of a gene to mRNA.

(52) Protein expression refers to the translation of mRNA to a protein.

(53) RT-qPCR is a method for reverse transcribing (RT) an mRNA molecule into a cDNA molecule and then quantitatively evaluating the level of gene expression using a polymerase chain reaction (PCR) coupled with a fluorescent reporter.

(54) Therapeutic as used herein, includes treatment and/or prophylaxis. When used, therapeutic refers to humans as well as other animals.

(55) Pharmaceutically or therapeutically effective dose or amount refers to a dosage level sufficient to induce a desired biological result. That result may be the alleviation of the signs, symptoms or causes of a disease or any other alteration of a biological system that is desired.

(56) Placebo refers to the substitution of the pharmaceutically or therapeutically effective dose or amount sufficient to induce a desired biological that may alleviate the signs, symptoms or causes of a disease with a non-active substance.

(57) A host or patient is a living subject, human or animal, into which the compositions described herein are administered.

(58) Note that throughout this application various citations are provided. Each citation is specifically incorporated herein in its entirety by reference.

(59) The present invention includes a novel composition of matter comprised of a mixture of Free-B-ring flavonoids and flavans. This novel composition of matter is referred to herein as Univestin. The ratio of Free-B-ring flavonoids to flavans in the composition of matter can be adjusted based on the indications and the specific requirements with respect to prevention and treatment of a specific disease or condition. Generally, the ratio of Free-B-ring flavonoids to flavans can be in the range of 99:1 Free-B-ring flavonoids:flavans to 1:99 of Free-B-ring flavonoids:flavans. In specific embodiments of the present invention, the ratio of Free-B-ring flavonoids to flavans is selected from the group consisting of approximately 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80 and 10:90. In a preferred embodiment of the invention, the ratio of Free-B-ring flavonoids:flavans in the composition of matter is approximately 85:15. In a preferred embodiment the Free-B-ring flavonoids are isolated from a plant or plants in the Scutellaria genus of plants and flavans are isolated from a plant or plants in the Acacia genus of plants.

(60) In one embodiment of the present invention, the standardized Free-B-ring flavonoid extract is comprised of the active compounds with a purity of between 1-99% (by weight) of total Free-B-ring flavonoids as defined in examples 5, 7 and 13; Tables 5, 7, 8 and 9 and FIG. 3. Baicalin is the major active component in the extract, which accounts for approximately 50-90% (by weight) of the total Free-B-ring flavonoids. In a preferred embodiment, the standardized extract contains >70% total Free-B-ring flavonoids in which >75% of the Free-B-ring flavonoids is baicalin.

(61) In one embodiment, the standardized flavan extract is comprised of the active compounds with a purity of between 1-99% (by weight) total flavans as defined in Example 8, 9 and 12; Tables 4, 6 and 9 and FIG. 9. Catechin is the major active component in the extract and accounts for 50-90% (by weight) of the total flavans. In a preferred embodiment, the standardized flavan extract contains >50% total flavans in which >70% of flavans is catechin.

(62) In one embodiment Univestin is be produced by mixing the above two extracts or synthetic compounds in a ratio from 99:1 to 1:99. The preferred ratio of Free-B-ring flavonoids to flavans is 85:15 Free-B-ring flavonoids:flavans as defined in Example 14.

(63) The concentration of Free-B-ring flavonoids in Univestin can be from about 1% to 99% and the concentration of flavans in Univestin can be from 99% to 1%. In a preferred embodiment of the invention, the concentration of total Free-B-ring flavonoids in Univestin is approximately 75% with a baicalin content of approximately 60% of total weight of the Univestin; and the concentration of total flavans in Univestin is approximately 10% with a catechin content of approximately 9%. In this embodiment, the total active components (Free-B-ring flavonoids plus flavans) in Univestin are >80% of the total weight.

(64) The present invention also includes methods that are effective in simultaneously inhibiting both the COX-2 and 5-LO enzymes. The method for the simultaneous dual inhibition of the COX-2 and 5-LO pathways is comprised of administering a composition comprising a mixture of Free-B-ring flavonoids and flavans synthesized and/or isolated from a single plant or multiple plants to a host in need thereof. The ratio of Free-B-ring flavonoids to flavans in the composition can be in the range of 99:1 Free-B-ring flavonoids:flavans to 1:99 of Free-B-ring flavonoids:flavans. In specific embodiments of the present invention, the ratio of Free-B-ring flavonoids to flavans is selected from the group consisting of approximately 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80 and 10:90. In a preferred embodiment of the invention, the ratio of Free-B-ring flavonoids:flavans in the composition of matter is approximately 85:15. In a preferred embodiment, the Free-B-ring flavonoids are isolated from a plant or plants in the Scutellaria genus of plants and flavans are isolated from a plant or plants in the Acacia genus of plants.

(65) The present further includes methods for the prevention and treatment of COX-2 and 5-LO mediated diseases and conditions. The method for preventing and treating COX-2 and 5-LO mediated diseases and conditions is comprised of administering to a host in need thereof an effective amount of a composition comprising a mixture of Free-B-ring flavonoids and flavans synthesized and/or isolated from a single plant or multiple plants together with a pharmaceutically acceptable carrier. The ratio of Free-B-ring flavonoids to flavans can be in the range of 99:1 Free-B-ring flavonoids:flavans to 1:99 of Free-B-ring flavonoids:flavans. In specific embodiments of the present invention, the ratio of Free-B-ring flavonoids to flavans is selected from the group consisting of approximately 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80 and 10:90. In a preferred embodiment of the invention, the ratio of Free-B-ring flavonoids:flavans in the composition of matter is approximately 85:15. In a preferred embodiment, the Free-B-ring flavonoids are isolated from a plant or plants in the Scutellaria genus of plants and flavans are isolated from a plant or plants in the Acacia genus of plants.

(66) In yet a further embodiment, the present includes a method for treating general joint pain and stiffness, improving mobility and physical function and preventing and treating pathological conditions of osteoarthritis and rheumatoid arthritis. The method for preventing and treating joint pain and stiffness, improving mobility and physical function and preventing and treating pathological conditions of osteoarthritis, and rheumatoid arthritis is comprised of by administering to a host in need thereof an effective amount of a composition comprising a mixture of Free-B-ring flavonoids and flavans synthesized and/or isolated from a single plant or multiple plants together with a pharmaceutically acceptable carrier. The ratio of Free-B-ring flavonoids to flavans can be in the range of 99:1 to 1:99 Free-B-ring flavonoids:flavans. In specific embodiments of the present invention, the ratio of Free-B-ring flavonoids to flavans is selected from the group consisting of approximately 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80 and 10:90. In a preferred embodiment of the invention, the ratio of Free-B-ring flavonoids:flavans in the composition of matter is approximately 85:15. In a preferred embodiment, the Free-B-ring flavonoids are isolated from a plant or plants in the Scutellaria genus of plants and flavans are isolated from a plant or plants in the Acacia genus of plants.

(67) The present invention also includes a method for modulating the production of mRNA implicated in pain pathways said method comprising administering to a host in need thereof an effective amount of a composition comprising a mixture of Free-B-ring flavonoids and flavans synthesized and/or isolated from a single plant or multiple plants and optionally a pharmaceutically acceptable carrier. The ratio of Free-B-ring flavonoids to flavans can be in the range of 99:1 to 1:99 Free-B-ring flavonoids:flavans. In specific embodiments of the present invention, the ratio of Free-B-ring flavonoids to flavans is selected from the group consisting of approximately 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80 and 10:90. In a preferred embodiment of the invention, the ratio of Free-B-ring flavonoids:flavans in the composition of matter is approximately 85:15. In a preferred embodiment the Free-B-ring flavonoids are isolated from a plant or plants in the Scutellaria genus of plants and flavans are isolated from a plant or plants in the Acacia genus of plants.

(68) The Free-B-ring flavonoids that can be used in accordance with the method of this invention include compounds illustrated by the general structure set forth above. The Free-B-ring flavonoids of this invention may be obtained by synthetic methods or may be isolated from the family of plants including, but not limited to Annonaceae, Asteraceae, Bignoniaceae, Combretaceae, Compositae, Euphorbiaceae, Labiatae, Lauranceae, Leguminosae, Moraceae, Pinaceae, Pteridaceae, Sinopteridaceae, Ulmaceae, and Zingiberaceae. The Free-B-ring flavonoids can also be extracted, concentrated, and purified from the genera of high plants, including but not limited to Desmos, Achyrocline, Oroxylum, Buchenavia, Anaphalis, Cotula, Gnaphalium, Helichrysum, Centaurea, Eupatorium, Baccharis, Sapium, Scutellaria, Molsa, Colebrookea, Stachys, Origanum, Ziziphora, Lindera, Actinodaphne, Acacia, Derris, Glycyrrhiza, Millettia, Pongamia, Tephrosia, Artocarpus, Ficus, Pityrogramma, Notholaena, Pinus, Ulmus, and Alpinia.

(69) The Free-B-ring flavonoids can be found in different parts of plants, including but not limited to stems, stem barks, twigs, tubers, roots, root barks, young shoots, seeds, rhizomes, flowers and other reproductive organs, leaves and other aerial parts. Methods for the isolation and purification of Free-B-ring flavonoids are described in U.S. application Ser. No. 10/091,362, filed Mar. 1, 2002, entitled Identification of Free-B-ring Flavonoids as Potent COX-2 Inhibitors, which is incorporated herein by reference in its entirety.

(70) The flavans that can be used in accordance with the method of this invention include compounds illustrated by the general structure set forth above. The flavans of this invention may be obtained by synthetic methods or may be isolated from a plant or plants selected from the Acacia genus of plants. In a preferred embodiment, the plant is selected from the group consisting of Acacia catechu, A. concinna, A. farnesiana, A. Senegal, A. speciosa, A. arabica, A. caesia, A. pennata, A. sinuata. A. mearnsii, A. picnantha, A. dealbata, A. auriculiformis, A. holoserecia and A. mangium.

(71) The flavans can be found in different parts of plants, including but not limited to stems, stem barks, trunks, trunk barks, twigs, tubers, roots, root barks, young shoots, seeds, rhizomes, flowers and other reproductive organs, leaves and other aerial parts. Methods for the isolation and purification of flavans are described in U.S. application Ser. No. 10/104,477, filed Mar. 22, 2002, entitled Isolation of a Dual COX-2 and 5-Lipoxygenase Inhibitor from Acacia, which is incorporated herein by reference in its entirety.

(72) The present invention implements a strategy that combines a series of in vivo studies as well as in vitro biochemical, cellular, and gene expression screens to identify active plant extracts and components that specifically inhibit COX-2 and 5-LO enzymatic activity, and impact cox-2, but not cox-1 mRNA production. The methods used herein to identify active plant extracts and components that specifically inhibit COX-2 and 5-LO pathways are described in Examples 1 to 13 (FIGS. 1-10). These methods are described in greater detail in U.S. application Ser. No. 10/091,362, filed Mar. 1, 2002, entitled Identification of Free-B-ring Flavonoids as Potent COX-2 Inhibitors and U.S. application Ser. No. 10/104,477, filed Mar. 22, 2002, entitled Isolation of a Dual COX-2 and 5-Lipoxygenase Inhibitor from Acacia, each of which is specifically incorporated herein by reference in its entirety.

(73) These studies resulted in the discovery of a novel composition of matter referred to herein as Univestin, which is comprised of a proprietary blending of two standardized extracts, which contain Free-B-ring flavonoids and flavans, respectively. A general example for preparing such a composition is provided in Example 14 using two standardized extracts isolated from Acacia and Scutellaria, respectively, together with one or more excipients. The Acacia extract used in Example 14 contained >60% total flavans, as catechin and epicatechin, and the Scutellaria extract contained >70% Free-B-ring flavonoids, which was primarily baicalin. The Scutellaria extract contained other minor amounts of Free-B-ring flavonoids as set forth in Table 11. One or more excipients are optionally added to the composition of matter. The amount of excipient added can be adjusted based on the actual active content of each ingredient desired. A blending table for each individual batch of product must be generated based on the product specification and QC results for individual batch of ingredients. Additional amounts of active ingredients in the range of 2-5% are recommended to meet the product specification. Example 14 illustrates a blending table that was generated for one batch of Univestin (Lot#G1702-COX-2). Different blending ratios of the formulated Univestin product were tested for their ability to inhibit COX-2 and 5-LO enzyme activities, and to reduce cox mRNA production as described in Examples 15-17.

(74) The COX-2 inhibition assay relied on the activity of the enzyme peroxidase in the presence of heme and arachidonic acid. In order to screen for compounds that inhibited COX-1 and COX-2 activity, a high throughput, in vitro assay was developed that utilized inhibition of the peroxidase activity of both enzymes as illustrated in Examples 2 and 6. After isolating plant fractions that inhibited COX-2 activity in the screening process, the two individual standardized extracts, one composed primarily of Free-B-ring flavonoids (isolated from Scutellaria) and the other of flavans (isolated from Acacia), were compared, as well as, purified components from each extract and different ratios of the combined extracts by titrating against a fixed amount of the COX-1 and COX-2 enzymes. This study revealed that the purified Free-B-ring flavonoids, baicalin and baicalein isolated from Scutellaria baicalensis and the purified flavan, catechin isolated from Acacia catechu, inhibited COX-2 and 5-LO activity. Additionally, each of the individual standardized extracts, which contained concentrations of Free-B-ring flavonoids in the range of 10-90% (based on HPLC) and flavans in the range of 10-90% (based on HPLC), also inhibited COX-2 and 5-LO activity. Finally, the study revealed that compositions containing mixtures of each of the individual standardized extracts having ratios of Free-B-ring flavonoids to flavans of approximately 80:20, 50:50, and 20:80, were also all highly effective at inhibiting COX-2 enzymatic activity in vitro. The results are set forth in FIGS. 11-13).

(75) Example 16 describes cell assays performed that targeted inhibition of compounds in the breakdown of arachidonic acid in the 5-LO pathway, namely LTB.sub.4. The results are set forth in FIGS. 14 and 15.

(76) Example 17 describes an experiment performed to determine differential inhibition of the cox-2 gene by Univestin. Gene expression data was obtained for the inhibition of cox-1 and cox-2 mRNA production in a semi-quantitative RT-qPCR assay. The results are set forth in FIGS. 16 and 17. With reference to FIG. 16 it can be seen that Univestin inhibited cox-2 mRNA production without effecting cox-1 gene expression. In addition, when compared with other COX-2 inhibitor drugs, Univestin was able to decrease LPS-stimulated increases in cox-1 and cox-2 gene expression. Importantly, celecoxib and ibuprofen both increased cox-2 gene expression (FIG. 17).

(77) In vivo efficacy was demonstrated by the application of skin irritating substances, such as AA, to the ears of mice and measuring the reduction of swelling in mice treated with Univestin as described in Example 18. The results are set forth in FIG. 18. Additionally, efficacy at the site of inflammation and pain, was determined by the injection of an irritant into the ankle joints of mice and measuring the reduction of swelling in mice treated with Univestin, as described in Example 19. The results are set forth in FIG. 19.

(78) Individual standardized extracts containing concentrations of Free-B-ring flavonoids in the range of 10-99% (based on HPLC) and flavans in the range of 10-99% (based on HPLC) as well as the product Univestin were tested for toxicity in mice with chronic and acute administration (data not shown). In the chronic administration protocol, mice were fed the test articles by oral gavage with daily doses of 90 mg/kg (equivalent to the human daily dose of 500 mg), 450 mg/kg (five times the daily-dose equivalent) and 900 mg/kg (ten times the daily-dose equivalent). Mice showed no adverse effects in terms of weight gain, physical appearance or behavior. Gross necropsy results showed no organ abnormalities and histology of the stomach, kidney, and liver showed no differences compared to untreated control mice. Full blood work measuring electrolytes, blood proteins, blood enzymes, and liver enzymes showed no abnormalities compared to the untreated control mice. In the acute protocol, individual standardized extracts containing concentrations of Free-B-ring flavonoids in the range of 10-99% (based on HPLC) and flavans in the range of 10-99% (based on HPLC) as well as the product Univestin given at 2 grams/kg (20 times the daily-dose equivalent) showed no abnormalities in weight gain, appearance, behavior, gross necropsy appearance of organs, histology of stomach, kidney, and liver or blood work.

(79) Example 20 describes a clinical study performed to evaluate the efficacy of Univestin on the relief of pain caused by rheumatoid arthritis or osteoarthritis of the knee and/or hip. The study was a single-center, randomized, double-blind, placebo-controlled study. Sixty subjects (n=60) with rheumatoid arthritis or osteoarthritis of the knee and/or hip were randomly placed into four groups and treated for 90 days with a placebo, Univestin (250 mg/day or 500 mg/day) or Celebrex (also known as celecoxib) (200 mg/day). The Univestin, as illustrated in Example 14, Table 11, consisted of a proprietary blend of standardized extract of Scutellaria baicalensis Georgi with a baicalin content of 82.2% (w/w) and total Free-B-ring Flavonoids >90% (w/w) and a standardized extract of Acacia catechu with a total flavan content of 77.2% (w/w) in a ratio of 85:15. Celebrex is a trade name for a prescription drug that is a COX-2 selective inhibitor. Table 12 sets forth the WOMAC index scores for pain, stiffness and function before treatment (baseline scores) and at 30, 60 and 90 days. Table 13 sets forth the absolute changes in WOMAC index scores for pain, stiffness and function after treatment for 30, 60 and 90 days. FIGS. 20-31 illustrate the results of this study graphically plotting the 95% confidence intervals for all data.

(80) As shown in the FIGS. 20 to 31, the WOMAC composite scores and individual subscores, related to pain, stiffness and physical function exhibited significant improvements during administration of Univestin compared to the placebo group. Further, Univestin exhibited a similar effectiveness on pain relieve, better effectiveness at decreasing stiffness, and marked improvement of physical function compared to the prescription drug Celebrex. The greatest significance can be seen in comparing each dose of Univestin to the placebo and celecoxib in relieving pain, stiffness and functional impairment associated with osteoarthritis or rheumatoid arthritis.

(81) Multiple post-hoc comparisons for each treatment group pairs within the Analysis of Variance models showed that Univestin at 500 mg/day was significantly more effective than celecoxib at 200 mg/day for the reduction of pain caused osteoarthritis during the 30 days (p=0.020) of treatment. In addition, the administration of a dose of 500 mg/day of Univestin was also significantly more effective than the placebo for the reduction of pain within 30 days (p=0.044), 60 days (p=0.032) and 90 days (p=0.001) of treatment. Celecoxib at 200 mg/day showed significance for the reduction of pain vs. the placebo at 60 days (p=0.009) of treatment. At 90 days, the 500 mg/day Univestin dose showed significantly higher effectiveness compared to the 250 mg/day dose within 90 days (p=0.038) of treatment.

(82) Univestin at 250 mg/day was significantly more effective than the placebo for the reduction of stiffness caused by osteoarthritis, within 30 days (p=0.00), 60 days (p=0.027) and 90 days (p=0.015) of treatment. In addition, Univestin at a dose of 500 mg/day was significantly more effective than placebo for reduction of stiffness caused by osteoarthritis, within 30 days (p=0.001) and 90 days (p=0.005) of treatment. Celecoxib at 200 mg/day showed significantly more effectiveness than the placebo for the reduction of stiffness caused by osteoarthritis only at 30 days (p=0.023) of treatment.

(83) For reduction of functional impairment caused by osteoarthritis, Univestin was significantly more effective than celecoxib at 200 mg/day within 30 days (p=0.010) of treatment. In addition, the 250 mg/day dose of Univestin was also significantly more effective than placebo for the reduction of functional impairment caused by osteoarthritis within 30 days (p=0.010), 60 days (p=0.043) and 90 days (p=0.039) of treatment. The 500 mg/day dose of Univestin was more effective than celecoxib at 200 mg/day within 30 days (p=0.015), 60 days (p=0.043) and 90 days (0.039) of treatment. Finally, the 500 mg/day dose of Univestin was also significantly more effective than placebo for the reduction of functional impairment caused by osteoarthritis within 30 days (p=0.015), 60 days (p=0.016) and 90 days (p=0.003) of treatment.

(84) These results suggest that Univestin, particularly at a dosage of 500 mg/day, is much more effective than the placebo and celecoxib at relieving pain, stiffness and improving functional impairment caused by osteoarthritis. Additionally, Univestin administered at a dosage of 250 mg/day is also very effective at relieving stiffness and improving functional impairment caused by osteoarthritis compared to the placebo and celecoxib. Celecoxib also showed only marginal improvement overall in relieving pain, stiffness and functional impairment caused by osteoarthritis.

(85) In addition to the effects of Univestin on pain, stiffness and functional impairment caused by osteoarthritis, Example 21 shows a measurable effect by Univesti on body mass index (BMI) and weight loss. While not limited by theory, this effect may be due to an increase in mobility as a result of the administration of an anti-inflammatory or may also be due to a specific mechanism that increases metabolism or reduces the utilization of fats and carbohydrates in the body. Table 14 shows the effect of Univestin administered at a dose of 250 and 500 mg/day as well as celecoxib and placebo on weight and BMI after 30 and 90 days of treatment. The results are illustrated graphically in FIGS. 32 and 33. With reference to FIGS. 32 and 33, it can be seen that Univestin administered at a dosage of both 250 and 500 mg/day resulted in a significant drop in weight and BMI after thirty days, with weight loss almost doubling after 90 days. Celecoxib had a smaller effect on weight and BMI as compared to Univestin.

(86) Multiple post-hoc comparisons for each treatment group pairs with the Analysis of Variance models were also performed for weight loss and BMI as described in Example 21. These analyses showed that Univestin at 250 mg/day and 500 mg/day doses caused statistically significant weight loss (p=0.011 vs. p=0.118) against the placebo after 30 days of treatment. Celecoxib did not cause significant weight loss against placebo at 30 days. The weight loss continued throughout 90 days of treatment with Univestin at 250 and 500 mg/day with statistical significance versus placebo (p=0.001 and 0.01 receptively). Celecoxib still did not show significance relative to the placebo. The decrease of BMI followed similar trends for the 250 mg/day dose of Univestin which was significant relative to the placebo after 30 days (p=0.008) as well as 90 days (p=0.001). The 500 mg/day dose of Univestin showed decreasing of BMI without statistical significance at 30 days of treatment. However, the decrease of BMI reached statistical significance 90 days of treatment (p=0.011). Again, after 90 days of treatment, the celecoxib treatment group showed no statistically significant changes in BMI versus placebo.

(87) Example 22 suggests that administration of Univestin may affect blood glucose levels as well as its effect on weight loss and BMI. Measurable differences in blood glucose levels are detected with 30 days of initiating treatment with Univestin. At 90 days, both the 250 and 500 mg/day Univestin treated groups showed significant drops in blood glucose levels. The effect of celecoxib on blood glucose was less dramatic. The results are set forth in Table 15 and illustrated graphically in FIG. 34.

(88) Once again multiple post-hoc comparisons for each treatment group pairs with the Analysis of Variance models were also performed for blood glucose as described in Example 22. Only the 500 mg/day dose of Univestin showed statistically relevant significance versus the placebo group (after 30 days, p=0.028; after 90 days, p=0.022). The 250 mg/day dose of Univestin, however, showed clinically significant changes in blood glucose levels versus the placebo.

(89) The applicant believes that U.S. application Ser. No. 10/104,477, filed Mar. 22, 2002, entitled Isolation of a Dual COX-2 and 5-Lipoxygenase Inhibitor from Acacia, is the first report of a composition of matter isolated from the Acacia genus of plants that demonstrates dual specificity for COX-2 and 5-LO and that U.S. application Ser. No. 10/091,362, filed Mar. 1, 2002, entitled Identification of Free-B-ring Flavonoids as Potent COX-2 Inhibitors, is the first report of a correlation between Free-B-ring flavonoid structure and COX-2 inhibitory activity. These discoveries led to a novel blending of two classes of specific compoundsFree-B-ring flavonoids and flavansto produce a composition of matter, referred to herein as Univestin, which can be used for alleviating joint pain and stiffness, improving mobility and physical function and preventing and treating the pathological conditions of osteoarthritis, and rheumatoid arthritis.

(90) While not limited by theory, the identified mechanism of action of this formulation is believed to be the direct inhibition of both the peroxidase activity of the COX-2 enzyme and the 5-LO enzyme activity, together with a decrease in the mRNA production of each of these enzymes. Univestin can also be utilized to prevent and treat COX-2 and 5-LO mediated diseases and conditions, including, but are not limited to osteoarthritis, rheumatoid arthritis, menstrual cramps, arteriosclerosis, heart attack, obesity, diabetes, syndrome X, Alzheimer's disease, respiratory allergic reaction, chronic venous insufficiency, hemorrhoids, Systemic Lupus Erythromatosis, psoriasis, chronic tension headache, migraine headaches, inflammatory bowl disease; topical infections caused by virus, bacteria, fungus, sunburn, thermal burns, contact dermatitis, melanoma and carcinoma. Finally, Univestin has been found in human clinical study that it can cause weight loss and reduce blood glucose level due to improvement of flexibility, mobility and increase physical activity.

(91) The present invention is also directed toward therapeutic compositions comprising the therapeutic agents of the present invention. The therapeutic agents of the instant invention can be administered by any suitable means, including, for example, parenteral, topical, oral or local administration, such as intradermally, by injection, or by aerosol. The particular mode of administration will depend on the condition to be treated. It is contemplated that administration of the agents of the present invention may be via any bodily fluid, or any target or any tissue accessible through a body fluid. In the preferred embodiment of the invention, the agent is administered by injection. Such injection can be locally administered to any affected area. A therapeutic composition can be administered in a variety of unit dosage forms depending upon the method of administration. For example, unit dosage forms suitable for oral administration of an animal include powder, tablets, pills and capsules. Preferred delivery methods for a therapeutic composition of the present invention include intravenous administration and local administration by, for example, injection or topical administration. A therapeutic reagent of the present invention can be administered to any animal, preferably to mammals, and more preferably to humans.

(92) For particular modes of delivery, a therapeutic composition of the present invention can be formulated so as to include other components such as a pharmaceutically acceptable excipient, an adjuvant, and/or a carrier. For example, compositions of the present invention can be formulated in an excipient that the animal to be treated can tolerate. Examples of such excipients, include but are not limited to cellulose, silicon dioxide, dextrates, sucrose, sodium starch glycolate, calcium phosphate, calcium sulfate, water, saline, Ringer's solution, dextrose solution, mannitol, Hank's solution, and other aqueous physiologically balanced salt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used. Other useful formulations include suspensions containing viscosity-enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Examples of buffers include phosphate buffer, bicarbonate buffer, Tris buffer, histidine, citrate, and glycine, or mixtures thereof, while examples of preservatives include thimerosal, m- or o-cresol, formalin and benzyl alcohol. Standard formulations can either be liquid injectables or solids, which can be taken up in a suitable liquid as a suspension or solution for injection. Thus, in a non-liquid formulation, the excipient can comprise dextrose, human serum albumin, preservatives, etc., to which sterile water or saline can be added prior to administration.

(93) In one embodiment of the present invention, the composition can also include an adjuvant or a carrier. Adjuvants are typically substances that generally enhance the function of the formula in preventing and treating indications related to COX & LO pathways. Suitable adjuvants include, but are not limited to, Freund's adjuvant; other bacterial cell wall components; aluminum-based salts; calcium-based salts; silica; boron, histidine, glucosamine sulfates, Chondroitin sulfate, copper gluconate, polynucleotides; vitamin D, vitamin K, toxoids; shark and bovine cartilage; serum proteins; viral coat proteins; other bacterial-derived preparations; gamma interferon; block copolymer adjuvants, such as Hunter's Titermax adjuvant (Vaxcel, Inc. Norcross, Ga.); Ribi adjuvants (available from Ribi ImmunoChem Research, Inc., Hamilton, Mont.); and saponins and their derivatives, such as Quil A (available from Superfos Biosector A/S, Denmark). Carriers are typically compounds that increase the half-life of a therapeutic composition in the treated animal. Suitable carriers include, but are not limited to, polymeric controlled release formulations, biodegradable implants, liposomes, bacteria, viruses, oils, esters, and glycols.

(94) One embodiment of the present invention is a controlled release formulation that is capable of slowly releasing a composition of the present invention into an animal. As used herein, a controlled release formulation comprises a composition of the present invention in a controlled release vehicle. Suitable controlled release vehicles include, but are not limited to, biocompatible polymers, other polymeric matrices, capsules, microcapsules, microparticles, bolus preparations, osmotic pumps, diffusion devices, liposomes, lipospheres, and transdermal delivery systems. Other controlled release formulations of the present invention include liquids that, upon administration to an animal, form a solid or a gel in situ. Preferred controlled release formulations are biodegradable (i.e., bioerodible).

(95) Once the therapeutic composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or dehydrated or lyophilized powder; or directly capsulated and/or tableted with other inert carriers for oral administration. Such formulations may be stored either in a ready to use form or requiring reconstitution immediately prior to administration. The manner of administering formulations containing the compositions for systemic delivery may be via oral, subcutaneous, intramuscular, intravenous, intranasal or vaginal or rectal suppository.

(96) The amount of the composition that will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder of condition, which can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness or advancement of the disease or condition, and should be decided according to the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curved derived from in vitro or animal model test systems. For example, an effective amount of the composition can readily be determined by administering graded doses of the composition and observing the desired effect.

(97) The method of treatment according to this invention comprises administering internally or topically to a patient in need thereof a therapeutically effective amount of the composition comprised of a mixture of Free-B-ring flavonoids and flavans. The purity of the mixture includes, but is not limited to 0.01% to 100%, depending on the methodology used to obtain the compound(s). In a preferred embodiment, doses of the mixture of Free-B-ring flavonoids and flavans and pharmaceutical compositions containing the same are an efficacious, nontoxic quantity generally selected from the range of 0.01 to 200 mg/kg of body weight. Persons skilled in the art using routine clinical testing are able to determine optimum doses for the particular ailment being treated.

(98) The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.

EXAMPLES

Example 1

Preparation of Organic and Aqueous Extracts from Acacia and Scutellaria Plants

(99) Plant material from Acacia catechu (L) Willd. barks, Scutellaria orthocalyx roots, Scutellaria baicalensis roots or Scutellaria lateriflora whole plant was ground to a particle size of no larger than 2 mm. Dried ground plant material (60 g) was then transferred to an Erlenmeyer flask and methanol:dichloromethane (1:1) (600 mL) was added. The mixture was shaken for one hour, filtered and the biomass was extracted again with methanol:dichloromethane (1:1) (600 mL). The organic extracts were combined and evaporated under vacuum to provide the organic extract (see Table 1 below). After organic extraction, the biomass was air dried and extracted once with ultra pure water (600 mL). The aqueous solution was filtered and freeze-dried to provide the aqueous extract (see Table 1 below).

(100) TABLE-US-00001 TABLE 1 Yield of Organic and Aqueous Extracts of Acacia and Scutellaria Species Organic Aqueous Plant Source Amount Extract Extract Acacia catechu barks 60 g 27.2 g 10.8 g Scutellaria orthocalyx roots 60 g 4.04 g 8.95 g Scutellaria baicalensis roots 60 g 9.18 g 7.18 g Scutellaria lateriflora 60 g 6.54 g 4.08 g (whole plant)

Example 2

Inhibition of COX-2 and COX-1 Peroxidase Activity by Plant Extracts from Acacia Catechu, Various Scutellaria Species and Other Plants

(101) The bioassay directed screening process for the identification of specific COX-2 inhibitors was designed to assay the peroxidase activity of the enzyme as described below.

(102) Peroxidase Assay.

(103) The assay to detect inhibitors of COX-2 was modified for a high throughput platform (Raz). Briefly, recombinant ovine COX-2 (Cayman) in peroxidase buffer (100 mM TBS, 5 mM EDTA, 1 M Heme, 1 mg epinephrine, 0.094% 10 phenol) was incubated with extract (1:500 dilution) for 15 minutes. Quantablu (Pierce) substrate was added and allowed to develop for 45 minutes at 25 C. Luminescence was then read using a Wallac Victor 2 plate reader. The results are presented in Table 2.

(104) Table 2 sets forth the inhibition of enzyme by the organic and aqueous extracts obtained from five plant species, including the bark of Acacia catechu, roots of two Scutellaria species and extracts from three other plant species, which are comprised of structurally similar Free-B-ring flavonoids. Data is presented as the percent of peroxidase activity relative to the recombinant ovine COX-2 enzyme and substrate alone. The percent inhibition by the organic extract ranged from 30% to 90%.

(105) TABLE-US-00002 TABLE 2 Inhibition of COX-2 Peroxidase activity by various species Inhibition of Inhibition of COX-2 by COX-2 by Plant Source organic extract aqueous extract Acacia catechu (bark) 75% 30% Scutellaria orthocalyx (root) 55% 77% Scutellaria baicalensis (root) 75% 0% Desmodium sambuense (whole plant) 55% 39% Eucaluptus globulus (leaf) 30% 10% Murica nana (leaf) 90% 0%

(106) Comparison of the relative inhibition of the COX-1 and COX-2 isoforms requires the generation of IC.sub.50 values for each of these enzymes. The IC.sub.50 is defined as the concentration at which 50% inhibition of enzyme activity in relation to the control is achieved by a particular inhibitor. In these experiments, IC.sub.50 values were found to range from 6 to 50 g/mL and 7 to 80 g/mL for the COX-2 and COX-1 enzymes, respectively, as set forth in Table 3. Comparison of the IC.sub.50 values of COX-2 and COX-1 demonstrates the specificity of the organic extracts from various plants for each of these enzymes. The organic extract of Scutellaria lateriflora for example, shows preferential inhibition of COX-2 over COX-1 with IC.sub.50 values of 30 and 80 g/mL, respectively. While some extracts demonstrate preferential inhibition of COX-2, others do not. Examination of the HTP fractions and purified compounds from these fractions is necessary to determine the true specificity of inhibition for these extracts and compounds.

(107) TABLE-US-00003 TABLE 3 IC.sub.50 Values of Organic Extracts for Human and Ovine COX-2 and COX-1 IC.sub.50 IC.sub.50 IC.sub.50 Human Ovine Ovine COX-2 COX-2 COX-1 Plant Source (g/mL) (g/mL) (g/mL) Acacia catechu (bark) 3 6.25 2.5 Scutellaria orthocalyx (root) Not done 10 10 Scutellaria baicalensis (root) 30 20 20 Scutellaria lateriflora (whole plant) 20 30 80 Eucaluptus globulus (leaf) Not done 50 50 Murica nana (leaf) 5 6 7

Example 3

HTP Fractionation of Active Extracts

(108) Organic extract (400 mg) from active plant was loaded onto a prepacked flash column. (2 cm ID8.2 cm, 10 g silica gel). The column was eluted using a Hitachi high throughput purification (HTP) system with a gradient mobile phase of (A) 50:50 EtOAc:hexane and (B) methanol from 100% A to 100% B in 30 minutes at a flow rate of 5 mL/min. The separation was monitored using a broadband wavelength UV detector and the fractions were collected in a 96-deep-well plate at 1.9 mL/well using a Gilson fraction collector. The sample plate was dried under low vacuum and centrifugation. DMSO (1.5 mL) was used to dissolve the samples in each cell and a portion (100 L was taken for the COX inhibition assay.

(109) Aqueous extract (750 mg) from active plant was dissolved in water (5 mL), filtered through a 1 m syringe filter and transferred to a 4 mL High Pressure Liquid Chromatography (HPLC) vial. The solution was then injected by an autosampler onto a prepacked reverse phase column (C-18, 15 m particle size, 2.5 cm ID10 cm with precolumn insert). The column was eluted using a Hitachi high throughput purification (HTP) system with a gradient mobile phase of (A) water and (B) methanol from 100% A to 100% B in 20 minutes, followed by 100% methanol for 5 minutes at a flow rate of 10 mL/min. The separation was monitored using a broadband wavelength UV detector and the fractions were collected in a 96-deep-well plate at 1.9 mL/well using a Gilson fraction collector. The sample plate was freeze-dried. Ultra pure water (1.5 mL) was used to dissolve samples in each cell and a portion (100 L) was taken for the COX inhibition assay.

Example 4

Inhibition of COX Peroxidase Activity by HTP Fractions from Acacia and Scutellaria Species

(110) Individual bioactive organic extracts were further characterized by examining each of the HTP fractions for the ability to inhibit the peroxidase activity of both COX-1 and COX-2 recombinant enzymes. The results are presented in FIGS. 1 and 2, which depict the inhibition of COX-2 and COX-1 activity by HTP fractions from organic extracts of the bark of Acacia catechu and the roots of Scutellaria baicalensis isolated as described in Examples 1 and 3 and assayed as described in Example 2. The profiles depicted in FIGS. 1 and 2 show multiple peaks of inhibition that indicate multiple active components in each extract. Several active peaks are very selective for COX-2. Other Scutellaria sp. including Scutellaria orthocalyx and Scutellaria lateriflora demonstrate a similar peak of inhibition (data not shown). However, both the COX-1 and COX-2 enzymes demonstrate multiple peaks of inhibition suggesting that there is more than one molecule contributing to the initial inhibition profiles.

Example 5

Isolation and Purification of the Active Free-B-Ring Flavonoids from the Organic Extract of Scutellaria

(111) The organic extract (5 g) from the roots of Scutellaria orthocalyx, isolated as described in Example 1, was loaded onto prepacked flash column (120 g silica, 40 m particle size 32-60 m, 25 cm4 cm) and eluted with a gradient mobile phase of (A) 50:50 EtOAc:hexane and (B) methanol from 100% A to 100% B in 60 minutes at a flow rate of 15 mL/min. The fractions were collected in test tubes at 10 mL/fraction. The solvent was evaporated under vacuum and the sample in each fraction was dissolved in 1 mL of DMSO and an aliquot of 20 L was transferred to a 96 well shallow dish plate and tested for COX inhibitory activity. Based on the COX assay results, active fractions #31 to #39 were combined and evaporated. Analysis by HPLC/PDA and LC/MS showed a major compound with a retention times of 8.9 minutes and a MS peak at 272 m/e. The product was further purified on a C18 semi-preparation column (25 cm1 cm), with a gradient mobile phase of (A) water and (B) methanol, over a period of 45 minutes at a flow rate of 5 mL/minute. Eighty-eight fractions were collected to yield 5.6 mg of light yellow solid. Purity was determined by HPLC/PDA and LC/MS, and comparison with standards and NMR data. .sup.1H NMR: ppm. (DMSO-d6) 8.088 (2H, m, H-3,5), 7.577 (3H, m, H-2,4,6), 6.932 (1H, s, H-8), 6.613 (1H, s, H-3). MS: [M+1]+=271 m/e. The compound was identified as baicalein. The IC.sub.50 of baicalein against the COX-2 enzyme was determined to be 10 g/mL.

(112) Using preparative C-18 column chromatography, other Free-B-ring flavonoids were isolated and identified using a standardized extract isolated from the roots of Scutellaria baicalensis (lot # RM052302-01), having a Free-B-ring flavonoid content of 82.2%. Eleven structures were elucidated using HPLC/PDA/MS as illustrated in FIG. 3. With reference to FIG. 3, the eleven compounds identified were baicalin, wogonin-7-glucuronide, oroxylin A 7-glucuronide, baicalein, wogonin, chrysin-7-glucuronide, 5-methyl-wogonin-7-glucuronide, scutellarin, norwogonin, chrysin and oroxylin A.

Example 6

COX Inhibition of Purified Free-B-Ring Flavonoids

(113) Several Free-B-ring flavonoids have been obtained and tested at a concentration of 20 g/mL for COX-2 inhibitory activity using the methods described in Example 2. The results are summarized in Table 4.

(114) Measurement of the IC.sub.50 of baicalein, baicalin and a standardized Free-B-ring flavonoid extract isolated from the roots of Scutellaria baicalensis was performed using the following method. A cleavable, peroxide chromophore was included in the assay to visualize the peroxidase activity of each enzyme in the presence of arachidonic acid as a cofactor. Typically, the assays were performed in a 96-well format. Each inhibitor, taken from a 10 mg/mL stock in 100% DMSO, was tested in triplicate at room temperature using the following range of concentrations: 0, 0.1, 1, 5, 10, 20, 50, 100, and 500 g/mL. To each well, 150 L of 100 mM Tris-HCl, pH 7.5 was added along with 10 L of 22 M Hematin diluted in tris buffer, 10 L of inhibitor diluted in DMSO, and 25 units of either COX-1 or COX-2 enzyme. The components were mixed for 10 seconds on a rotating platform, after which 20 L of 2 mM N,N,NN-Tetramethyl-p-phenylenediamine dihydrochloride (TMPD) and 20 L of 1.1 mM AA was added to initiate the reaction. The plate was shaken for 10 seconds and then incubated for 5 minutes before reading the absorbance at 570 nm. The inhibitor concentration vs. percentage inhibition was plotted and the IC.sub.50 determined by taking the half-maximal point along the isotherm and intersecting the concentration on the x-axis. The IC.sub.50 was then normalized to the number of enzyme units in the assay. The dose response and IC.sub.50 results for baicalein, baicalin and a standardized Free-B-ring flavonoid extract isolated from the roots of Scutellaria baicalensis are provided in FIGS. 4, 5 and 6, respectively.

(115) TABLE-US-00004 TABLE 4 Inhibition of COX Enzymatic Activity by Purified Free-B-ring Flavonoids Inhibition Inhibition Free-B-ring Flavonoids of COX-1 of COX-2 Baicalein 107% 109% 5,6-Dihydroxy-7-methoxyflavone 75% 59% 7,8-Dihydroxyflavone 74% 63% Baicalin 95% 97% Wogonin 16% 12%

Example 7

HPLC Quantification of Free-B-Ring Flavonoids in Active Extracts Isolated from Scutellaria Orthocalyx (Roots), Scutellaria Baicalensis (Roots) and Oroxylum Indicum (Seeds)

(116) The presence and quantity of Free-B-ring flavonoids in five active extracts isolated from three different plant species have been confirmed and are set forth in the Table 5. The Free-B-ring flavonoids were quantitatively analyzed by HPLC using a Luna C-18 column (2504.5 mm, 5 m) a using 1% phosphoric acid and acetonitrile gradient from 80% to 20% in 22 minutes. The Free-B-ring flavonoids were detected using a UV detector at 254 nm and identified based on retention time by comparison with Free-B-ring flavonoid standards.

(117) TABLE-US-00005 TABLE 5 Free-B-ring Flavonoid Content in Active Plant Extracts % Total Weight Extractible amount of % Free-B-ring of from Free-B-ring Flavonoids in Active Extracts Extract BioMass Flavonoids Extract S. orthocalyx 8.95 g 14.9% 0.2 mg 0.6% (aqueous extract) S. orthocalyx 3.43 g 5.7% 1.95 mg 6.4% (organic extract) S. baicalensis 7.18 g 12.0% 0.03 mg 0.07% (aqueous extract) S. baicalensis 9.18 g 15.3% 20.3 mg 35.5% (organic extract) Oroxylum indicum 6.58 g 11.0% 0.4 mg 2.2% (organic extract)

Example 8

Isolation and Purification of Active Compounds from the Organic Extract of Acacia Catechu

(118) The organic extract (5 g) from the roots of A. catechu, isolated as described in Example 1, was loaded onto prepacked flash column (120 g silica, 40 m particle size 32-60 m, 25 cm4 cm) and eluted with a gradient mobile phase of (A) 50:50 EtOAc:hexane and (B) methanol from 100% A to 100% B in 60 minutes at a flow rate of 15 mL/min. The fractions were collected in test tubes at 10 mL/fraction. The solvent was evaporated under vacuum and the sample in each fraction was dissolved in DMSO (1 mL) and an aliquot of 20 L was transferred to a 96 well shallow dish plate and tested for COX inhibitory activity. Based upon the COX assay results, active fractions #32 to #41 were combined and evaporated to yield 2.6 g of solid. Analysis by HPLC/PDA and LC/MS showed two major compounds with retention times of 15.8 and 16.1 minutes, respectively. The product was further purified on a C18 semi-preparatory column (25 cm1 cm), loaded with 212.4 mg of product and eluted with a gradient mobile phase of (A) water and (B) acetonitrile (ACN), over a period of 60 minutes at a flow rate of 5 mL/minute. Eighty-eight fractions were collected and two active compounds were isolated. Compound 1 (11.5 mg) and Compound 2 (16.6 mg). Purity was determined by HPLC/PDA and LC/MS data by comparison with standards (catechin and epicatechin) and NMR data.

(119) Compound 1.

(120) .sup.13C NMR: ppm (DMSO-d6) 27.84 (C4), 66.27 (C3), 80.96 (C2), 93.78 (C9), 95.05 (C7), 99.00 (C5), 114.48 (C12), 115.01 (C15), 118.36 (C16), 130.55 (C11), 144.79 (C14), 155.31 (C6), 156.12 (C10), 156.41 (C8). .sup.1H NMR: ppm. (DMSO-d6) 9.150 (1H, s, OH), 8.911 (1H, s, OH), 8.835 (1H, s, OH), 8.788 (1H, s, OH), 6.706 (1H, d, J=2 Hz, H2), 6.670 (1H, d, J=8.0 Hz, H-6), 6.578 (1H, dd, J=2, 8 Hz, H-5), 5.873 (1H, d, J=2 Hz, H8), 5.670 (1H, d, J=2 Hz, H6), 4.839 (1H, d, J=4 Hz, OH), 4.461 (1H, d, J=7.3 Hz, H2), 3.798 (1H, m, H3), 2.625 (1H, m, H4b), 2.490 (1H, m, H4a). MS: [M+1].sup.+=291 m/e. This compound was identified as catechin.

(121) Compound 2.

(122) .sup.13C NMR: ppm. (DMSO-d6) 28.17 (C4), 64.87 (C3), 78.02 (C2), 94.03 (C9), 95.02 (C7), 98.44 (C5), 114.70 (C12), 114.85 (C15), 117.90 (C16), 130.56 (C11), 144.39 (C14), 155.72 (C6), 156.19 (C10), 156.48 (C8). .sup.1H NMR: ppm. (DMSO-d6) 9.083 (1H, s, OH), 8.873 (1H, s, OH), 8.777 (1H, s, OH), 8.694 (1H, s, OH), 6.876 (1H, d, J=2 Hz, H2), 6.646 (2H, s, H-5, 6), 5.876 (1H, d, J=2 Hz, H8), 5.700 (1H, d, J=2 Hz, H6), 4.718 (1H, s, OH), 4.640 (1H, d, J=4.5 Hz, H2), 3.987 (1H, d, J=4.5 Hz, H3), 2.663 (1H, dd, J=4.6, 6.3 Hz, H4b), 2.463 (1H, dd, J=4.6, 6.3 Hz, H4a). MS: [M+1].sup.+=291 m/e. This compound was identified as epicatechin.

(123) The dose response and IC.sub.50 results for catechin and a standardized flavan extract isolated from the bark of A. catechu are illustrated in FIGS. 7 and 8, using the method described in Example 6. The IC.sub.50 values of epicatechin against the COX-1 and COX-2 enzymes are 7 g/mL and 20 g/mL, respectively.

Example 9

HPLC Quantification of Active Extracts from Acacia Catechu

(124) The flavan content in the organic and aqueous extracts isolated from Acacia catechu were quantified by HPLC using a PhotoDiode Array detector (HPLC/PDA) and a Luna C18 column (250 mm4.6 mm). The flavans were eluted from the column using an acetonitrile gradient from 10% to 30% ACN over a period of 20 minutes, followed by 60% ACN for five minutes. The results are set forth in Table 6. A profile of the HPLC purification is shown in FIG. 9. The flavans were quantified based on retention time and PDA data using catechin and epicatechin as standards. The retention times for the two major flavans were 12.73 minutes and 15.76 minutes, respectively.

(125) TABLE-US-00006 TABLE 6 Free-B-ring Flavonoid Content in Active Plant Extracts Active Extracts from Weight of % Extractible % Flavans bark of A. catechu Extract from BioMass in Extract Aqueous Extract 10.8 g 18.0% 0.998% Organic Extract 27.2 g 45.3% 30.37%

Example 10

In Vitro Study of COX Inhibitory Activity of Organic Extracts from Acacia Catechu and Scutellaria Species

(126) In vitro efficacy and COX-2 specificity of organic extracts isolated from Acacia catechu and various Scutellaria species were tested in cell-based systems for their ability to inhibit the generation of AA metabolites. Cell lines HOSC, which constitutively express COX-2 and THP-1, which express COX-1 were tested for their ability to generate PGE.sub.2 in the presence of AA.

(127) COX-2 Cell Based Assay.

(128) HOSC (ATCC#8304-CRL) cells were cultured to 80-90% confluence. The cells were trypsinized, washed and resuspended in 10 mL at 110.sup.6 cells/mL in tissue culture media (MEM). The cell suspension (200 L) was plated out in 96-well tissue culture plates and incubated for 2 hours at 37 C. and 5% CO.sub.2. The media was then replaced with new HOSC media containing 1 ng/mL IL-1b and incubated overnight. The media was removed again and replaced with 190 mL HOSC media. Test compounds were then added in 10 L of HOSC media and were incubated for 15 minutes at 37 C. Arachidonic acid in HOSC media (20 mL, 100 M) was added and the mixture was incubated for 10 minutes on a shaker at room temperature. Supernatant (20 L) was transferred to new plates containing 190 L/well of 100 M indomethacin in ELISA buffer. The supernatants were analyzed as described below by ELISA.

(129) COX-1 Cell Based Assay.

(130) THP-1 cells were suspended to a volume of 30 mL (510.sup.5 cells/mL). TPA was added to a final concentration of 10 nM TPA and cultured for 48 hours to differentiate cells to macrophage (adherent). The cells were resuspended in HBSS (25 mL) and added to 96-well plates in 200 mL volumes at 510.sup.5 cells/well. The test compounds in RPMI 1640 (10 L) were then added and incubated for 15 minutes at 37 C. Arachidonic acid in RPMI (20 L) was then added and the mixture was incubated for 10 minutes on a shaker at room temperature. Supernatant (20 L) was added to ELISA buffer (190 L) containing indomethacin (100 M). The supernatants were then analyzed by ELISA, as described below.

(131) COX-2 Whole Blood Assay.

(132) Peripheral blood from normal healthy donors was collected by venipuncture. Whole blood (500 L) was incubated with test compounds and extracts for 15 minutes at 37 C. Lipopolysaccharide (LPS, from E. coli serotype 0111:B4) was added to a final concentration of 100 g/mL and cultured overnight at 37 C. The blood was centrifuged (12,000g) and the plasma was collected. Plasma (100 L) was added to methanol (400 L) to precipitate proteins. Supernatants were measured for PGE.sub.2 production by ELISA. This procedure is a modification of the methods described by Brideau et al. (1996) Inflamm. Res. 45:68-74.

(133) COX-1 Whole Blood Assay.

(134) Fresh blood was collected in tubes not containing anti-coagulants and immediately aliquoted into 500 L aliquots in siliconized microcentrifuge tubes. Test samples were added, vortexed and allowed to clot for 1 hour at 37 C. The samples were then centrifuged (12,000g) and the plasma was collected. The plasma (100 L) was added to methanol (400 L) to precipitate proteins. Supernatants were measured for TXB.sub.2 production by ELISA. This procedure is a modification of the methods described by Brideau et al. (1996) Inflamm. Res. 45:68-74.

(135) ELISA Assays.

(136) Immunolon-4 ELISA plates were coated with capture antibody 0.5-4 g/mL in carbonate buffer (pH 9.2) overnight at 4 C. The plates were washed and incubated for 2 hours with blocking buffer (PBS+1% BSA) at room temperature. The plates were washed again and test sample (100 L) was added and incubated for 1 hour at room temperature while shaking. Peroxidase conjugated secondary antibody was added in a 50 L volume containing 0.5-4 mg/mL and incubated for 1 hour at room temperature while shaking. The plates were then washed three times and TMB substrate (100 L) was added. The plates were allowed to develop for 30 minutes, after which the reaction was stopped by the addition of 1 M phosphoric acid (100 L). The plates were then read at 450 nm using a Wallac Victor 2 plate reader.

(137) Cytotoxicity.

(138) Cellular cytotoxicity was assessed using a colorimetric kit (Oxford Biochemical Research) that measures the release of lactate dehydrogenase in damaged cells. Assays were completed according to manufacturer's directions. Both purified flavans and standardized extract from Acacia catechu were tested. No cytotoxicity was observed for any of the tested compounds.

(139) The results of the assays are set forth in Table 7. The data are presented as IC.sub.50 values for direct comparison. With reference to Table 5, IC.sub.50 values are generally lower for COX-1 than COX-2. Additionally, whole blood was also measured for the differential inhibition of PGE.sub.2 generation (a measure of COX-2 in this system) or thromboxane B2 (TXB.sub.2) (a measure of COX-1 activation). Referring to Table 7, these studies clearly demonstrate specificity for COX-2 inhibition within the assays based on whole blood cells. However, studies using the THP-1 and HOSC-based model system actually showed greater selectivity for COX-1. Possible reasons for this discrepancy are the fundamental differences between immortalized cell lines that constitutively express each of the enzymes and primary cells derived from whole blood that are induced to express COX enzymes. Primary cells are a more relevant model to study inflammation in vivo. Additionally, the compounds used to identify COX-1 vs. COX-2 activity vary in each of these systems and consequently are not directly comparable.

(140) TABLE-US-00007 TABLE 7 Inhibition of COX Activity in Cell Systems by Organic Extracts Cell Line Based Assay Whole Blood Assay Plant Source of the IC.sub.50 IC.sub.50 IC.sub.50 organic extracts IC.sub.50 COX-2 COX-1 COX-2 COX-1 A. catechu (bark) 78 g/mL 22 g/mL 40 g/mL >50 g/mL S. orthocalyx (root) 50 g/mL 18 g/mL 10 g/mL >50 g/mL S. baicalensis (root) 82 g/mL 40 g/mL 20 g/mL 8 g/mL S. lateriflora 60 g/mL 30 g/mL 8 g/mL 20 g/mL (whole plant)

Example 11

Inhibition of 5-Lipoxygenase by the Catechin from Acacia Catechu

(141) As noted above, one of the most important pathways involved in the inflammatory response is produced by non-heme, iron-containing lipoxygenases (5-LO, 12-LO, and 15-LO), which catalyze the addition of molecular oxygen onto fatty acids such as AA (AA) to produce the hydroperoxides 5-, 12- and 15-HPETE, which are then converted to leukotrienes. There were early indications that the flavan extract from A. catechu may provide some degree of 5-LO inhibition, thereby preventing the formation of 5-HPETE. A Lipoxygenase Inhibitor Screening Assay Kit (Cayman Chemical, Inc., Cat#760700) was used to assess whether the purified flavan catechin from Acacia catechu directly inhibited 5-LO in vitro. The 15-LO from soybeans normally used in the kit was replaced with potato 5-LO, after a buffer change from phosphate to a tris-based buffer using microfiltration was performed. This assay detects the formation of hydroperoxides through an oxygen sensing chromagen. Briefly, the assay was performed in triplicate by adding 90 L of 0.17 units/L potato 5-LO, 20 L of 1.1 mM AA, 100 L of oxygen-sensing chromagen, and 10 L of purified flavan inhibitor to final concentrations ranging from 0 to 500 g/mL. The IC.sub.50 for 5-LO inhibition from catechin was determined to be 1.38 g/mL/unit of enzyme.

Example 12

Preparation of a Standardized Extract from Acacia Catechu

(142) Acacia catechu (500 mg of ground bark) was extracted with the following solvent systems. (1) 100% water, (2) 80:20 water:methanol, (3) 60:40 water:methanol, (4) 40:60 water:methanol, (5) 20:80 water:methanol, (6) 100% methanol, (7) 80:20 methanol:THF, (8) 60:40 methanol:THF. The extracts were concentrated and dried under low vacuum. The identification of the chemical components in each extract was achieved by HPLC using a PhotoDiode Array detector (HPLC/PDA) and a 250 mm4.6 mm C18 column. The chemical components were quantified based on retention time and PDA data using catechin and epicatechin as standards. The results are set forth in Table 8 and FIG. 9. As shown in Table 6, the flavan extract generated from solvent extraction with 80% methanol/water provided the best concentration of flavan components.

(143) TABLE-US-00008 TABLE 8 Solvents for Generating Standardized Flavan Extracts from Acacia catechu Total Extraction Weight of % Extractible amount of % Catechins Solvent Extract from BioMass Catechins in Extract 100% water 292.8 mg 58.56% 13 mg 12.02% water:methanol 282.9 mg 56.58% 13 mg 11.19% (80:20) water:methanol 287.6 mg 57.52% 15 mg 13.54% (60:40) water:methanol 264.8 mg 52.96% 19 mg 13.70% (40:60) water:methanol 222.8 mg 44.56% 15 mg 14.83% (20:80) 100% methanol 215.0 mg 43.00% 15 mg 12.73% methanol:THF 264.4 mg 52.88% 11 mg 8.81% (80:20) methanol:THF 259.9 mg 51.98% 15 mg 9.05% (60:40)

Example 13

Preparation of Standardized Free-B-Ring Flavonoid Extracts from Various Scutellaria Species

(144) Scutellaria orthocalyx (500 mg of ground root) was extracted twice with 25 mL of the following solvent systems. (1) 100% water, (2) 80:20 water:methanol, (3) 60:40 water:methanol, (4) 40:60 water:methanol, (5) 20:80 water:methanol, (6) 100% methanol, (7) 80:20 methanol:THF, (8) 60:40 methanol:THF. The extracts were combined, concentrated and dried under low vacuum. Identification of chemical components in each extract was performed by HPLC using a PhotoDiode Array detector (HPLC/PDA) and a 250 mm4.6 mm C18 column. The chemical components were quantified based on retention time and PDA data using baicalein, baicalin, scutellarein, and wogonin as standards. The results are set forth in Table 9.

(145) TABLE-US-00009 TABLE 9 Quantification of Free-B-ring Flavonoids Extracted from Scutellaria orthocalyx Weight Total % Extraction of % Extractible amount of Flavonoids Solvent Extract from BioMass Flavonoids in Extract 100% water 96 mg 19.2% 0.02 mg 0.20% Water:methanol 138.3 mg 27.7% 0.38 mg 0.38% (80:20) Water:methanol 169.5 mg 33.9% 0.78 mg 8.39% (60:40) Water:methanol 142.2 mg 28.4% 1.14 mg 11.26% (40:60) Water:methanol 104.5 mg 20.9% 0.94 mg 7.99% (20:80) 100% methanol 57.5 mg 11.5% 0.99 mg 10.42% methanol:THF 59.6 mg 11.9% 0.89 mg 8.76% (80:20) methanol:THF 58.8 mg 11.8% 1.10 mg 10.71% (60:40)

(146) Scutellaria baicalensis (1000 mg of ground root) was extracted twice using 50 mL of a mixture of methanol and water as follows: (1) 100% water, (2) 70:30 water:methanol, (3) 50:50 water:methanol, (4) 30:70 water:methanol, (5) 100% methanol. The extracts were combined, concentrated and dried under low vacuum. Identification of the chemical components was performed by HPLC using a PhotoDiode Array detector (HPLC/PDA), and a 250 mm4.6 mm C18 column. The chemical components in each extract were quantified based on retention time and PDA data using baicalein, baicalin, scutellarein, and wogonin standards. The results are set forth in Table 10.

(147) TABLE-US-00010 TABLE 10 Quantification of Free-B-ring Flavonoids Extracted from Scutellaria baicalensis % Weight Extractible Total % Extraction of from amount of Flavonoids Solvent Extract BioMass Flavonoids in Extract 100% water 277.5 mg 27.8% 1 mg 0.09% Water:methanol 338.6 mg 33.9% 1.19 mg 11.48% (70:30) Water:methanol 304.3 mg 30.4% 1.99 mg 18.93% (50:50) Water:methanol 293.9 mg 29.4% 2.29 mg 19.61% (30:70) 100% methanol 204.2 mg 20.4% 2.73 mg 24.51%

Example 14

Preparation of a Formulation with a Standardized Free-B-Ring Flavonoid Extract from the Roots of Scutellaria Baicalensis and a Standardized Flavan Extract from the Bark of Acacia Catechu

(148) A novel composition of matter, referred to herein as Univestin was formulated using two standardized extracts isolated from Acacia and Scutellaria, respectively, together with one or more excipients. A general example for preparing such a composition is set forth below. The Acacia extract used in this example contained >60% total flavans, as catechin and epicatechin, and the Scutellaria extract contained >70% Free-B-ring flavonoids, which was primarily baicalin. The Scutellaria extract contained other minor amounts of Free-B-ring flavonoids as set forth in Table 11. One or more exipients is added to the composition of matter. The ratio of flavan and Free-B-ring flavonoids can be adjusted based on the indications and the specific requirements with respect to inhibition of COX-2 vs. 5-LO and potency requirements of the product. The quantity of the excipients can be adjusted based on the actual active content of each ingredient. A blending table for each individual batch of product must be generated based on the product specification and QC results for individual batch of ingredients. Additional amounts of active ingredients in the range of 2-5% are recommended to meet the product specification. Table 11 illustrates a blending table that was generated for one batch of Univestin (Lot#G1702-COX-2).

(149) Scutellaria baicalensis root extract (38.5 kg) (lot # RM052302-01) having a Free-B-ring flavonoid content of 82.2% (baicalin); Acacia catechu bark extract (6.9 kg) (lot # RM052902-01) with total flavan content of 80.4%; and excipient (5.0 kg of Candex) were combined to provide a Univestin formulation (50.4 kg) having a blending ratio of 85:15. Table 9 provides the quantification of the active Free-B-ring flavonoids and flavans of this specific batch of Univestin (Lot#G1702-COX-2), determined using the methods provided in Examples 7 and 9.

(150) TABLE-US-00011 TABLE 11 Free-B-ring Flavonoid and Flavan Content of Univestin Formulation Active Components % Content 1. Flavonoids a. Baicalin 62.5% b. Minor Flavonoids i. Wogonin-7-glucuronide 6.7% ii. Oroxylin A 7-glucuronide 2.0% iii. Baicalein 1.5% iv. Wogonin 1.1% v. Chrysin-7-glucuronide 0.8% vi. 5-Methyl-wogonin-7-glucuronide 0.5% vii. Scutellarin 0.3% viii. Norwogonin 0.3% ix. Chrysin <0.2% x. Oroxylin A <0.2% c. Total Free-B-ring Flavonoids 75.7% 2. Flavans a. Catechin 9.9% b. Epicatechin 0.4% c. Subtotal Flavans 10.3% 3. Total Active Ingredients 86%

(151) With reference to Table 9, this specific batch of Univestin contains 86% total active ingredients, including 75.7% Free-B-ring flavonoids and 10.3% flavans. Two different dosage levels of final product in capsule form were produced from this batch of Univestin (50.0 kg): 125 mg per dose (60 capsules) and 250 mg per dose (60 capsules). The final product was evaluated in a human clinical trial as described in Example 15.

(152) Using the same approach, two other batches of Univestin were prepared using a combination of a standardized Free-B-ring flavonoid extract from Scutellaria baicalensis roots and a standardized flavan extract from Acacia catechu bark having a blending ratio of 50:50 and 20:80, respectively.

Example 15

Measurements of Dose Response and IC50 Values of COX Enzyme Inhibitions from Three Formulations of Univestin

(153) The three different formulations of Univestin are produced as provided in Example 14 were tested for COX-1 and COX-2 inhibitory activity as described in Example 6. All three formulation show significant dose response inhibition of COX enzyme activities as illustrated in FIGS. 11, 12 and 13).

Example 16

Measurements of Dose Response and IC50 Values of LO Enzyme Inhibition from a Formulation of Univestin

(154) A Univestin sample was produced as outlined in Example 14, using a combination of a standardized Free-B-ring flavonoid extract from Scutellaria baicalensis roots and a standardized flavan extract from Acacia catechu bark with a blending ratio of 80:20. The sample was titrated in tissue culture media containing THP-1 or HT-29 cells; monocyte cell lines that express COX-1, COX-2 and 5-LO. A competitive ELISA for LTB.sub.4 (LTB.sub.4; Neogen, Inc., Cat#406110) was used to assess the effect of Univestin on newly synthesized levels of LTB.sub.4 present in each cell line as a measure of Univestin's inhibitory effect on the 5-LO pathway. The assay was performed in duplicate by adding 160,000 to 180,000 cells per well in 6-well plates. Univestin was added to the THP-1 cultures at 3, 10, 30 and 100 g/mL and incubated overnight (12-15 hrs) at 37 C. with 5% CO.sub.2 in a humidified environment. The results are set forth in FIG. 14, which shows that the production of newly LPS-induced LTB.sub.4 was almost completely inhibited by the addition of Univestin to the THP-1 cultures between 3 and 10 g/mL.

(155) Univestin and ibuprofen, another known 5-LO inhibitor, were added to the HT-29 cells at 3 g/mL and incubated 48 hrs at 37 C. with 5% CO.sub.2 in a humidified environment. Each treated cell line was then harvested by centrifugation and disrupted by gentle dounce homogenization lysis in physiological buffers. As shown in FIG. 15, Univestin inhibited generation of 80% of the newly synthesized LTB.sub.4 in HT-29 cells. Ibuprofen only showed a 20% reduction in the amount of LTB.sub.4 over the same time period.

Example 17

Differential Inhibition of Cox-2 but not Cox-1 Gene Expression by Univestin Vs. Other NSAIDs

(156) To evaluate whether Univestin is operating on the genomic level, isolated human, peripheral blood monocytes (PBMCs) were stimulated with lipopolysaccharide (LPS), treated with Univestin as illustrated in Example 14, celecoxib, ibuprofen or acetaminophen, and the total RNA produced was then harvested and evaluated by semi-quantitative RT-qPCR. Specifically, the assay was constructed by adding 130,000 cells per well in 6-well plates. The cells were then stimulated with 10 ng/mL LPS and co-incubated with Univestin at 1, 3, 10, 30 and 100 g/mL and celecoxib, ibuprofen and acetaminophen at 3 g/mL for 18 hours at 37 C. with 5% CO.sub.2 in a humidified environment. Each cell-treatment condition was then harvested by centrifugation and total RNA produced was isolated using TRIzol reagent (Invitrogen Life Technologies, Cat#15596-026) and the recommended TRIzol reagent protocol. Total RNA was reverse transcribed using Moloney Murine Leukemia Virus reverse transcriptase (M-MLV RT; Promega Corp., Cat#M1701) using random hexamers (Promega Corp., Cat#C1181). qPCR experiments were performed on an ABI Prism7700 Sequence Detection System using pre-developed validated Assays-on-Demand products (AOD from Applied Biosystems, Inc., Cat#4331182) for 18S rRNA internal standard and gene specific assays. Gene specific expression values were standardized to their respective 18S rRNA gene expression values (internal control) and then the no-LPS no-drug treatment condition normalized to 100. Treatment conditions are relative to this null condition.

(157) Univestin decreased normalized gene expression of cox-2 by over 100-fold, while cox-1 normalized gene expression showed little variation. When PBMCs were treated with 3 g/mL of Univestin, celecoxib, ibuprofen or acetaminophen, only Univestin did not increase gene expression of cox-2. It is believed that this is the first report of changes in gene expression levels of eicosinoids, cytokines, chemokines and other genes implicated in pain and inflammation pathways following treatment with a mixture of Free-B-ring flavonoids and flavans using semi-quantitative RT-qPCR techniques. This work has been coupled work with ELISA-based assays to evaluate changes in protein levels as well as enzyme function assays to evaluate alterations in protein function. As a result of these studies, both genomic and proteomic coupled effects following treatment with Univestin have been demonstrated. Other studies cited in the literature have used protein specific methods to infer gene expression rather than show it directly. The results are set forth in FIGS. 16 and 17.

Example 18

Evaluation of the Efficacy of Univestin with In Vivo Mouse Ear Swelling Model

(158) In order to test whether Univestin could be used to treat inflammation in vivo, the composition, prepared as described in Example 14, was administered by oral gavage to 4-5 week old ICR mice (Harlan Labs) one day before treatment of their ears with AA. Test mice were fed dose equivalents of 50, 100 and 200 mg/kg of Univestin suspended in olive oil while control mice were fed only olive oil. The following day, 20 L of 330 mM AA in 95% alcohol was applied to one ear of each mouse, while alcohol was applied to the other ear as a control. Mice treated with Univestin showed a measurable dose response that tracked with increasing doses of Univestin, as demonstrated in FIG. 18. With reference to FIG. 18, the 200 mg/kg dose reduces swelling by over 50% as compared to the minus Univestin control. The 50 mg/kg dose of Univestin was as effective as the 50 mg/kg dose of another strong anti-inflammatory, indomethacin.

Example 19

Evaluation Efficacy of Univestin with In Vivo Mouse Ankle Joint Swelling Model

(159) Since Univestin is designed to target joint pain, a solution of 20 L of 100 mM AA in 95% ethanol was injected into the hind ankle joints of 4-5 week old ICR mice (Harlan Labs) to generate swelling. The test group was fed 100 mg/kg of Univestin suspended in olive oil 12 hours before while another group was not given Univestin. Control groups included mice that had not received arachidonic acid injections (negative control) and a group that had 95% ethanol without AA injected (vehicle control). These groups were also not given Univestin. The results are set forth in FIG. 19. With reference to FIG. 19, the mice given Univestin that were injected with AA showed background levels of swelling as compared to the controls and the untreated arachidonic injected group. These results demonstrate the effectiveness of Univestin for reducing swelling in joints, the site of action.

Example 20

Clinical Evaluation of the Efficacy of Free-B-Ring Flavonoids and Flavans on the Relief of Pain Caused by Rheumatoid Arthritis or Osteoarthritis of the Knee and/or Hip

(160) This clinical study was a single-center, randomized, double-blind, placebo-controlled study. Sixty subjects (n=60) with rheumatoid arthritis or osteoarthritis of the knee and/or hip were randomly placed into one of the following four groups:

(161) TABLE-US-00012 A.sub.0 Placebo n = 15 Placebo A.sub.1 Dose 1 n = 15 Univestin 250 mg/day (125 mg b.i.d.) A.sub.2 Dose 2 n = 15 Univestin 500 mg/day (250 mg b.i.d.) A.sub.3 Active Control n = 15 Celecoxib 200 mg/day (100 mg b.i.d.)

(162) The Univestin was prepared as described in Example 14. This specific batch of Univestin (lot#G1702-COX-2) contains 86% total active ingredients, including 75.7% Free-B-ring flavonoids and 10.3% flavans. Celecoxib, also known as Celebrex, is a trade name for a prescription drug that is a COX-2 selective inhibitor.

(163) Subjects were sex-matched and recruited from ages 40 to 75. Treatment consisted of oral administration for 90 days of the placebo or active compound (Univestin or celecoxib) according to the above dose schedule. Subjects taking NSAIDs engaged in a two-week washout period before beginning the study. Physical activity was not restricted, nor were the subjects given any advice as to diet. Subjects were free to withdraw from the trial at any time for any reason. The efficacy of the treatments was evaluated at 30, 60 and 90 days of oral administration by physicians, using the Western Ontario and McMaster Universities (WOMAC) Osteo-Arthritis Index (See Lingard et al. (2001) J. Bone & Joint Surg. 83:1856-1864; Soderman and Malchau (2000) Acta Orthop. Scand. 71(1):39-46). This protocol was reviewed and approved by an IRB board from University of Montreal.

(164) The WOMAC was administered to subjects preferably in the doctor's office. They were asked to read and respond to a questionnaire on their own or via proxy in the waiting room of the doctor's office or were interviewed by project personnel over the telephone and the data were transcribed in the computer database. This offered a stable environment among patients and reduced the possibility of bias due to different home environments among patients. Between groups differences for all measurements were evaluated with One-Way Analysis of Variance and Tukey's Least Significant Difference for multiple comparisons. All questions were assigned a weight from 0 to 4 depending on the severity of pain, stiffness or impaired function. These values were then converted to percentages normalized to 100 and reported as WOMAC scores. Higher values are indicative of greater impairment. Table 12 sets forth the mean WOMAC index scores for pain, stiffness and function for 250 mg and 500 mg per day Univestin compared to celecoxib at 200 mg per day and the placebo before treatment (baseline) and at 30, 60 and 90 days after treatment. The lower the score, the less pain and stiffness and better function a patient has.

(165) TABLE-US-00013 TABLE 12 WOMAC Index Scores at Baseline and at 30, 60 and 90 Days Univestin Univestin Celecoxib 250 500 200 Placebo WOMAC Std Std Std Std INDICE Mean Dev Mean Dev Mean Dev Mean Dev Pain-baseline 54.33 19.9 60.33 23.34 55 22.28 49.33 15.1 Pain-30 days 41.33 19.22 36 22.93 50 23.09 41.67 15.55 Pain-60 days 40.71 16.62 40.77 19.77 30 16.46 57.31 16.66 Pain-90 days 41.79 16.36 27.69 21.57 31.67 16.42 50 14.43 Stiffness- 63.33 26.92 61.67 23.84 47.5 21.75 46.67 21.37 baseline Stiffness-30 41.67 16.14 44.17 21.06 39.42 18.29 59.17 20.85 days Stiffness-60 37.5 18.99 39.42 19.66 37.5 29.76 46.15 24.68 days Stiffness-90 39.29 20.72 28.85 21.28 29.17 25.19 49.04 18.01 days Function- 58.41 22.74 62.92 17.68 49.38 10.33 52.82 8.29 baseline Function-30 42.09 14.51 47.59 17.18 48.43 9.29 51.88 14.8 days Function-60 41.47 7.75 41.59 7.34 41.23 9.12 49.64 7.16 days Function-90 42.44 17.08 38.12 13.21 44.41 11.06 50.95 12.73 days

(166) Table 13 sets forth the mean absolute change in WOMAC scores for pain, stiffness and function. They are expressed as the difference between the baseline and the scores given at 30, 60 and 90 days. The more negative the score, the greater the improvement.

(167) TABLE-US-00014 TABLE 13 Mean Absolute Change in WOMAC Scores at 30, 60 and 90 Days* Absolute Univestin 250 Univestin 500 Celecoxib 200 Placebo Change Mean Std Dev Mean Std Dev Mean Std Dev Mean Std Dev Pain-30 days 13 24.41 24.33 18.7 4.23 15.92 7.67 26.98 Pain-60 days 14.64 26.85 17.31 35.27 22.31 22.51 5 13.54 Pain-90 days 13.57 22.91 30.38 21.06 16.67 21.36 2.31 15.89 Stiffness-30 21.67 24.31 17.5 18.18 8.65 20.66 12.5 29.88 days Stiffness-60 28.57 27.05 21.15 33.61 9.62 29.82 1.92 30.55 days Stiffness-90 26.79 27.67 31.73 20.17 13.54 37.48 0.96 26.74 days Function-30 16.32 19.58 15.33 18.28 0.37 6.86 0.94 14.05 days Function-60 18.11 24.36 21.4 19.79 6.97 13.66 3.49 11.81 days Function-90 17.13 23.69 24.87 23.25 2.78 8.34 2.18 11.27 days *These data contain only subjects who completed the study.

(168) It is very difficult to ascribe a standard deviation to a group mean in a clinic trial due to the severe differences that appear in the data. Rather, confidence limits for the mean are preferred because they give a lower and upper limit for the mean and the narrower the interval, the more precise the estimate of the mean. Confidence limits are expressed in terms of a confidence coefficient. A 95% confidence interval is the most commonly used interval to describe a mean in this type of statistical analysis. This does not imply that there is a 95% probability that the interval contains the true mean. Instead, the level of confidence is associated with the method of calculating the interval. The confidence coefficient is simply the proportion of samples of a given size that may be expected to contain the true mean. That is, for a 95% confidence interval, if many samples are collected and the confidence interval computed, in the long run about 95% of these intervals would contain the true mean. With this in mind, the 95% confidence interval was computed for the WOMAC scores for pain, stiffness and function at 30, 60 and 90 days.

(169) Raw/non standardized scores for the WOMAC scores based on a five point Likert scale with a range between 1 and 5 were chosen to represent the final pain, stiffness and impaired function indices (FIGS. 20-31). Standardization to a scale between 0 and 100 was used in other sections for uniformity (see Tables 12 and 13) and to enhance the appreciation of the magnitudes of changes. However, given that all the figures are based on the same 1-5 point scales the raw data were plotted since they more accurately reflect the methods by which these scores were obtained from the patient questionnaires. In other words, since the patients were given a choice between 1 and 5 these representations better reflect the patient's response as opposed to the standardized or transformed score of 0-100 that does not reflect the patient's perception of possible range of answers.

(170) Clear trends exist showing that for the pain indice that Univestin at 250 and 500 mg/day reduced pain over the 90 day treatment period based on the patient responses. Celecoxib also reduces pain over this same period of time compared to the placebo, which does not. However, celecoxib does not seem to be as effective as Univestin at both dosages in reducing stiffness, since the confidence intervals heavily overlapped those of the placebo. Finally, Univestin at both doses clearly improved functional impairment, but celecoxib does not compared to placebo. The graphic representations contain all subjects even if they did not complete the study. Each confidence interval, however, is valid based on the number of subjects that were present at the time the WOMAC tests were taken so the trends still hold. These data are plotted in FIGS. 20 through 31.

Example 21

Clinical Evaluation of the Efficacy of Free-B-Ring Flavonoids and Flavans on BMI and Weight Loss Due to an Increase in Function

(171) Additional measurements taken during the clinical trial were height and weight. All subjects in all groups (see Example 20) were measured for height and weight at 30 and 90 days of treatment. The subjects were given no recommendations on diet or exercise in order not to bias the results toward reduction of BMI and weight loss. Table 14 illustrates the changes in weight and BMI that occurred after treatment for 30 and 90 days.

(172) TABLE-US-00015 TABLE 14 Change in Mean Weight (kg) and BMI (kg/m.sup.2) at 30 and 90 Days Group Univestin Univestin Celecoxib 250 500 200 Placebo Std Std Std Std Mean Dev Mean Dev Mean Dev Mean Dev Weight-30 days 3.60 3.76 2.40 3.31 2.00 3.08 .60 1.99 Weight-90 days 5.36 3.43 4.15 4.81 3.17 4.88 .08 1.50 BMI-30 days 1.28 1.33 .80 1.13 .68 1.06 .20 .64 BMI-90 days 1.84 1.14 1.39 1.64 1.07 1.67 .02 .54

(173) Based on these data, the 250 mg/day dose of Univestin gave the greatest amount of weight loss and change in BMI followed by the 500 mg/day dose of Univestin and then celecoxib. The placebo had no effect on weight or BMI.

(174) It is not believed that there are any other reports in the literature of anti-inflammatory compounds being used to effect weight loss or changes in BMI. Though the subjects were given no advice on exercise, the greater functional capabilities gained after treatment, especially with Univestin, may have allowed them to exercise more on their own accord. Alternatively, Univestin may be increasing thermogenesis, lipolysis, or causing an under utilization of carbohydrates or fat in the diet. FIGS. 32 and 33 illustrate the BMI and weight loss seen for Univestin after 30 and 90 days of treatment.

Example 22

Clinical Evaluation of the Efficacy of Free-B-Ring Flavonoids and Flavans on Lowering of Blood Glucose Due to an Increase in Function

(175) Blood glucose was also taken at 0 (baseline), 30 days and 90 days after treatment (see Example 20). These measurements were reported in mmole per liter. The data is also shown in mg/dL. Table 15 sets forth blood glucose levels after 30 and 90 days of treatment with Univestin at 250 and 500 mg/day.

(176) TABLE-US-00016 TABLE 15 Change in Blood Glucose after 30 and 90 days of Treatment Group Univestin 250 Univestin 500 Placebo Std Std Std mmol/L mg/dL Dev mmol/L mg/dL Dev mmol/L mg/dL Dev Glucose- 5.24 94.32 .74 5.09 91.62 .67 4.82 86.76 .80 Baseline Glucose- 5.10 91.80 .71 4.75 85.50 .55 5.08 91.44 .54 30 days Glucose- 4.88 87.84 .72 4034 78.12 .36 4.71 84.78 .56 90 days Percent 7.52 12.79 .94 Change By 90 days

(177) These data suggest that both the 250 and the 500 mg/day doses of Univestin are significantly lowering blood glucose levels over time. This impact may or may not be related to the loss of weight observed above or to the presumed increase in activity as functional impairment improved. It may also be possible that Univestin is acting directly to improve glucose metabolism by decreasing glucose tolerance or by utilizing carbohydrates more effectively.