Methods of isolating amyloid-inhibiting compounds and use of compounds isolated from Uncaria tomentosa and related plants
First Claim
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1. An Uncaria extract composition made according to a method comprising the steps of:
- 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 a 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.
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Abstract
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.
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25 Claims
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1. An Uncaria extract composition made according to a method comprising the steps of:
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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 a 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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 18, 19, 20, 21, 22, 23, 24, 25)
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11. An Uncaria extract composition made according to a method comprising the steps of:
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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 a 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; d) applying an aqueous solution of the dried material 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, combining and drying the fractions to a dried material; and e) making one or more injections of a solution of the dried material of step (d) 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 Dynamax 5 μ
C-18 column with dimensions of about 21.4 mm×
25 cm, with detection at 280 and 300 nm, the gradient conditions being 0 to 3 min for 20% to 25% B gradient, 3 to 9 mm for 25 to 45% B gradient, all at a flow rate of about 20 ml/min, and collecting a fraction eluting between 7-8 minutes from start of elution. - View Dependent Claims (12, 13)
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14. An Uncaria extract composition made according to a method comprising the steps of:
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a) adding 4000 ml of methanol to 1 kg of Uncaria tomentosa and mixing b) centrifuging the mixture at ×
2,500 g using a centrifuge for 30 minutes and collecting the supernatant;c) extracting the insoluble material about 3 more times as steps a and b above; d) combining the supernatants and evaporating to dryness (or until about 500 ml volume is reached) using a rotary evaporator at 50°
C.,e) taking the powdered extract (or about 500 ml volume), washing 4 times with 300 ml of petroleum ether, and discarding the ether layer, f) evaporating the methanol to dryness using a rotary evaporator at 50°
C.;g) extracting the solid material 5 times with 150 ml of distilled water, followed by centrifugation at 2,500×
g for 30 minutes each time;h) combining the supernatants and then lyophilizing using a freeze-dryer; i) 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. j) eluting the LH-20 column with 1,100 ml of distilled water (˜
3 column volumes) and discarding the amber/yellow, non-active fractions;k) 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.;l) dissolving the fractions of step k in water (80 mg/ml) and applying 5 ml at a time to a 10 gm disposable C18 SPE column equilibrated in solvent A (solvent A is 95% water/5% acetonitrile/0.1% TFA); m) washing the column with 3 volumes of solvent A and discarding the eluate; n) eluting the column with 3 volumes of solvent A containing 12.5% solvent B (solvent B is 95% acetonitrile/5% water/0.1% TFA) and lyophilizing the eluate; o) taking 50 mg of the lyophilized eluate of step (n) and injecting multiple times into a Hewlett-Packard 1100 Series HPLC instrument with diode array detector, fitted with a 2.2 cm×
25 cm Vydac 218TP1022 C18 reverse-phase column maintained at 25°
C. and at a flow rate of 5 ml/min;p) eluting the sample with the following solvent profile, 10% B for 0 to 20 minutes, 10-100% B gradient for minutes 20 to 30, and 100-10% B gradient for minutes 30-31, where B is 95% acetonitrile/5% water/0.1% TFA; q) and separating and collecting the fractions G (13-14 minutes), H (17-20 minutes), I (21 minutes), K1 (24 minutes), K2 (25 minutes), L (26-27 minutes), M (27-28 minutes), and N (28-29 minutes) whereby the Uncaria extract composition comprises one or more fractions. - View Dependent Claims (16, 17)
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15. An Uncaria extract composition made according to a method comprising the step of:
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a) preparing a polar solvent extract of Uncaria plant matter, b) 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, c) washing the first column first with distilled water, then with a dilute polar alcohol/water solution, d) eluting the first column with a polar alcohol/water solution, and lyophilizing the eluate, e) applying an aqueous solution of the lyophilized eluate of step (d) to a second column, f) eluting the second 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, g) collecting, combining and lyophilizing the eluate, h) HPLC purifying a solution of the lyophilized eluate of step (g) prepared in a solvent comprising about 80% water/20% methanol and about 0.1% TFA and applied at about 150 mg/run with detection at 280 and 300 nm, gradient conditions being 0 to 3 min for 20% to 25% methanol and about 0.1% TFA, 3 to 9 min for 25 to 45% methanol and about 0.1% TFA, all at a flow rate of about 20 ml/min, and i) collecting a fraction eluting between 7-8 minutes from start of the HPLC purification.
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Specification