Methods of isolating amyloid-inhibiting compounds and use of compounds isolated fromand related plants
원문보기
IPC분류정보
국가/구분
United States(US) Patent
등록
국제특허분류(IPC7판)
A61K-036/00
A61K-031/35
출원번호
US-0624435
(2003-07-21)
등록번호
US-7314642
(2008-01-01)
발명자
/ 주소
Castillo,Gerardo
Choi,Paula Y.
Nguyen,Beth
Snow,Alan D.
출원인 / 주소
Castillo,Gerardo
Choi,Paula Y.
Nguyen,Beth
Snow,Alan D.
인용정보
피인용 횟수 :
1인용 특허 :
6
초록▼
Assay-guided affinity fractionation and reverse phase high pressure liquid chromatography (HPLC) methodology to isolate, test and characterize the most active water-soluble ingredients within Cat's Claw, or Uncaria tomentos. These components appear to account for the majority of the amyloid or A^
Assay-guided affinity fractionation and reverse phase high pressure liquid chromatography (HPLC) methodology to isolate, test and characterize the most active water-soluble ingredients within Cat's Claw, or Uncaria tomentos. These components appear to account for the majority of the amyloid or Aβ fibrillogenesis inhibitory activity. Individual fractions and/or compounds as isolated by HPLC are tested in relevant in vitro and/or animal models, and found to consistently demonstrate inhibition of amyloid or Aβ fibrillogenesis. Related extraction methods are disclosed.
대표청구항▼
We claim: 1. A method for isolating compounds that possess amyloid inhibitory activity from plant matter of the genus Uncaria, the method comprising the steps: a) preparing a polar solvent extract of Uncaria plant matter, where the polar solvent extraction is selected from one of the extraction met
We claim: 1. A method for isolating compounds that possess amyloid inhibitory activity from plant matter of the genus Uncaria, the method comprising the steps: a) preparing a polar solvent extract of Uncaria plant matter, where the polar solvent extraction is selected from one of the extraction methods from the group of extraction methods consisting of extraction with water, extraction with a water solution of a polar alcohol, extraction with a water solution of acetonitrile and extraction with a water solution of another polar organic solvent and running the extract through a first column that comprises hydroxy group containing resin, resin having hydrophobic characteristics but without any hydroxy groups, or a mixture of both; b) eluting the first column with distilled water, followed by eluting with not more than 2-4 column bed volume washings with a dilute polar alcohol/water solution having an alcohol/water ratio not greater than 50/50, and discarding any eluate; c) eluting the first column with one or more column bed volume washings of a polar alcohol/water solution having an alcohol/water ratio between 50/50 and substantially pure alcohol, and collecting and drying the eluted volumes to a dried material. 2. The method of claim 1 wherein the column that comprises hydroxy containing resin, resin having hydrophobic characteristics but without any hydroxy groups, or a mixture of both is a column selected from the group of columns consisting of carbon-containing columns, Tris-acrylate column, LH-20 column, and Affi-prep 10 gel column. 3. The method of claim 1 wherein the polar alcohol/water solution has an alcohol/water ratio of 75/25 or higher. 4. The method of claim 1 wherein the washing in step (c) is effected with substantially pure methanol. 5. The method of claim 1 wherein the plant matter of the genus Uncaria is taken from one or more of the plants of the various Uncaria species plant group consisting of tomentosa, attenuata, elliptica, guianensis, pteropoda, bernaysli, ferra DC, kawakamii, rhyncophylla, calophylla, gambir, and orientalis. 6. The method of claim 1 wherein the plant matter of the genus Uncaria is taken from Uncaria tomentosa. 7. The method of claim 6 wherein the Uncaria tomentosa plant matter is taken from one or more of the group of plant parts consisting of inner bark and root. 8. The method of claim 1 further comprising the steps: d) applying an aqueous solution of the dried material from step (c) to a second column comprising a hydrophobic resin, the second column having been preparatorily equilibrated in a solvent comprising about 95% water/5% acetonitrile, referred to herein as solvent A, and then eluting the second column with more solvent A and discarding the eluate; e) eluting the second column with a mixture of solvent A containing 10-15% of a solvent comprising about 95% acetonitrile/5% water, referred to herein as solvent B, and collecting and drying the eluted volumes to a dried material. 9. The method of claim 8 wherein the second column comprising a hydrophobic resin is a column selected from the group of columns consisting of C18 SPE, a flash chromatography column, other HPLC columns, and other carbon-containing columns. 10. The method of claim 1 or 8 further comprising the steps: f) making one or more injections of a solution of the dried material of step (c) or the dried material of step (e) in a solvent selected from the group of solvents consisting of water, water/dilute alcohol and a solvent comprising about 95% water/5% acetonitrile, referred to herein as solvent A, and no more than 10% of a solvent, comprising about 95% acetonitrile/5% water, referred to herein as solvent B, into an HPLC instrument having a diode array uv/vis detector with a graphic display, the HPLC instrument further comprising a reverse-phase column; g) eluting the material through the HPLC column using a solvent gradient profile as follows: 10% solvent B for about the first 20 minutes from start of elution, 10 to 100% solvent B gradient for about minutes 20 to 30 from start of elution, and 100 to 10% solvent B gradient for about minutes 30 to 32 from start of elution, while observing the uv/vis detector graphic display during the elution gradient overtime, and separating fractions of the eluate at elution times corresponding to times associated with the graphic display peaks. 11. The method of claim 10, wherein the reverse-phase column has dimensions of about 2.2 cm��25 cm and contains about 95 ml of C18 reverse phase resin, wherein the solution of the dried material is a solution of about 50 mg of the dried material of step (c) in about 1-2 ml of solvent A, wherein the step of injecting the solution of dried material into the HPLC may be repeated, wherein a HPLC column solution gradient flow rate is set to about 5 mls per minute, and further wherein the solvent gradient profile is 10% solvent B for 0 to 20 minutes, followed by 10 to 100% solvent B gradient for minutes 20 to 30, and 100% to 10% solvent B gradient from minutes 30 to 31; such that fractions G though N of the eluate are collected at the following times: fraction G (13-14 minutes), fraction H (17-20 minutes), fraction I (21 minutes), fraction K1 (24 minutes), fraction K2 (25 minutes), fraction L (26-27 minutes), fraction M (27-28 minutes), and fraction N (28-29 minutes). 12. The method of claim 10, wherein the reverse-phase column with dimensions of 1.0 cm��25.0 cm containing 20 ml of C18 reverse phase resin, wherein the solution of the dried material of step (c) is a solution of 50 μg of the dried material in 50-100 μl of solvent A, wherein the step of injecting the solution into the HPLC is repeated multiple times, wherein a HPLC column solution gradient flow rate is set to about 1.5 mls per minute, and further wherein the solvent gradient profile is 10% solvent B for 0 to 20 minutes, followed by 10 to 100% solvent B gradient for minutes 20 to 30, and 100% to 10% solvent B gradient from minutes 30 to 31; such that fractions G though O of the eluate are collected at the following times: fraction G (12-13 minutes), fraction H (15 minutes), fraction I (16 minutes), fraction K1 (20 minutes), fraction K2 (21 minutes), fraction L (21-23 minutes), fraction M (23 minutes), fraction N (24 minutes), and fraction O (26-27 minutes). 13. The method of claim 10 wherein steps (f) and (g) are as follows: f) injecting a solution of 1 gram of the dried material of step (c) in 5-10 ml of solvent A into an HPLC instrument having a Varian model 320 uv/vis detector set at 230 nm with a graphic display, the HPLC further comprising a 4.14 cm��25 cm Varian Dynamax column further comprising 380 ml of C-18 reverse phase resin, the column fitted to a Varian Prostar 215 solvent delivery system; g) eluting the HPLC column at a solution gradient flow rate of about 50 ml/minute, and further wherein the solvent gradient profile is with a solvent C/solvent D gradient as follows: 0-4 minutes, 25% D; 4-11 minutes, 25-30% D gradient; 11-14 minutes, 30-90% D gradient; 14-17 minutes, 90% D; and 17-19 minutes, 90-25% D gradient, where C is water and D is methanol, such that fractions G through O of the eluate are separated at elution times corresponding to times associated with the graphic display peaks. 14. The method of claim 8 wherein the aqueous solution of a dried material from step (c) is further prepared by the following steps: 1) dissolving the dried material in water at 80 mg/ml and applying 5 ml at a time to a disposable C18 SPE column (10 gram) equilibrated in a first solvent comprising about 95% water/5% acetonitrile/0.1% TFA; 2) washing with 3 column bed volumes of the first solvent and discarding the eluate; 3) eluting with 3 column bed volumes of the first solvent further comprising about 12.5% of a second solvent comprising about 95% acetonitrile/5% water/0.1% TFA; and 4) lyophilizing the corresponding fractions using a freeze-dryer. 15. The method of claim 8 wherein the aqueous solution of a dried material from step (c) is further prepared by the following steps: 1) dissolving the lyophilized fractions at 5 grams in 20 ml water and applying 20 ml at a time to a flash chromatography column; 2) washing with 3 column bed volumes of a first solvent comprising about 95% water/5% acetonitrile/0.1% TFA and discarding the eluate; 3) eluting with 3 column bed volumes of the first solvent further comprising about 12.5% of a second solvent comprising about 95% acetonitrile/5% water/0.1% TFA; and 4) collecting and drying the next 3 column bed volumes of eluate. 16. The method of claim 1 wherein the preparation in step (a) of the extract of Uncaria is as follows: 1) adding 4000 ml of methanol to 1 kg of Uncaria tomentosa and mixing 2) centrifuging the mixture at ��2,500 g using a centrifuge for 30 minutes and collecting the supernatant; 3) extracting the insoluble material about 3 more times as steps (a) and (b) above; 4) combining the supernatants and evaporating to a dried extract, or to at least about 500 ml volume, using a rotary evaporator at 50�� C.; 5) washing the dried extract, or the 500 ml volume, 4 times with 300 ml of petroleum ether, and discarding the ether layer; 6) further evaporating any remaining methanol to dryness using a rotary evaporator at 50�� C.; 7) extracting the dried extract 5 times with 150 ml of distilled water, followed by centrifugation at 2,500��g for 30 minutes each time, and 8) combining the supernatants and then lyophilizing using a freeze-dryer. 17. The method of claim 16 wherein the further preparation of the extract of Uncaria from the resulting lyophilized extract includes the following additional steps: 9) dissolving the resulting lyophilized extract into about 500 ml of distilled water, and applying 50-100 ml portions to a 400 ml LH-20 column equilibrated with distilled water; 10) eluting the LH-20 column with 1,100 ml of distilled water (˜3 column volumes) and discarding the amber/yellow, non-active fractions; 11) eluting the LH-20 column with 1,100 ml of 100% methanol (˜3 column volumes) and collecting a set of active fractions and evaporating to dryness using a rotary evaporator at 50�� C. 18. The method of claim 1 further comprising the steps: d) applying an aqueous solution of the dried material from step (c) to a second column, eluting the material from the column with successive column volumes of water/methanol mixtures containing 0.1% TFA, beginning with 25% methanol and increasing to 100% menthol in 25% increments, and collecting and combining the fractions; e) separating, combining and drying a fraction to a dried material, referred to hereafter as compound H, by analytical HPLC, the fraction containing a peak occurring between 7-8 minutes from start of elution on a C18 column having dimensions of about 4.6 mm��25 cm, using an elution gradient of water for solvent A and methanol for solvent B, A and B each containing about 0.1% TFA, with detection at 280 nm, the gradient conditions being 0 to 9 min fro 25% to 36% B gradient, 3 to 10 min for 36 to 100% B gradient, 10 to 12 min for 100% B and 12 to 13 min for 100 to 25% B gradient, all at a flow rate of about 20 ml/min; f) making one or more injections of a solution of the dried material of step (e) above in a solvent comprising water/methanol 80/20 containing about 0.1% TFA and applied at about 150 mg/run to a preparative HPLC C-18 column with dimensions of about 21.4 mm��25 cm, using substantially the same elution gradient as used in step (e) above, with detection at 280 and 300 nm, the gradient conditions being 0 to 3 min for 20% to 25% B gradient, 3 to 9 min for 25 to 45% B gradient, 9 to 10 min for 45 to 100% B gradient, 10 to 12 min for 100% B and 12 to 13 min for 100 to 25% B gradient, all at a flow rate of about 20 ml/min, a compound H fraction eluting between 7-8 minutes from start of elution, and; g) repeating steps (e) and (f) above until the peak as seen on analytical HPLC in step (e) is relatively pure. 19. The method of claim 1 wherein the polar organic solvent of step (a) is selected from the group of polar organic solvents consisting of triethanolamine and acetone. 20. The method of claim 2 wherein the carbon-containing columns are selected from the group consisting of C2 column, C4 column and C18 column.
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