Methods of isolating amyloid-inhibiting compounds and use of compounds isolated from Uncaria tomentosa and related plants
View Patent ↗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.
1. A method for isolating amyloid inhibitory components from Uncaria tomentosa , the method comprising the steps:
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 pouring off the supernatant from a remaining residue;
c) adding a second volume of methanol to the residue and repeating step b;
d) combining the supernatants from step b and c and evaporating until reduced in volume to about 4-5% of the volume of the supernatents using a rotary evaporator at 50° C.;
e) taking the reduced volume, washing 4 times with 300 ml of petroleum ether, and discarding the ether layer;
f) evaporating the methanol to dryness to form a solid material 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 from step g and then lypohilizing using a freeze-dryer;
i) dissolving the resulting lypohilized extract into about 500 ml of distilled water, and applying 50-100 ml portions of the dissolved extract to a 400 ml LH-20 column equilibrated
j) eluting the LH-20 column with ˜3 column volumes of distilled water and discarding the amber/yellow eluate;
k) eluting the LH-20 column with ˜3 column volumes of methanol, collecting the eluate and evaporating it to dryness using a rotary evaporator at 50° C;
l) dissolving the product of step k in water to a concentration of ˜80 mg/ml and applying 5 ml at a time to a 10 gm disposable C18 SPE column equilibrated in solvent A, where 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, where solvent B is 95% acentronitrile/5% water/0.1% TFA, and lyophilizing the eluate;
o) injecting 50 mg portions of the lyophilized eluate of step n 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% solvent B for minutes 0 to 20, 10-100% solvent B gradient for minutes 20 to 30, and 100-10% solvent B gradient for minutes 30-31; and
q) seperating and collecting at least one fraction selected from the following fractions: fraction G (˜13-14 minutes), fraction F (˜15-16 minutes), fraction H (˜17-20 minutes), fraction I (˜21 minutes), fraction J (˜22-23 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).
2. A method for isolating amyloid inhibitory components from Uncaria tomentosa , the method comprising the steps:
a) dissolving a quantity of Uncaria tomentosa in one or more volumes of methanol;
b) seperating and combining the supernatant from each volume, and reducing it in volume through evaporation;
c) taking the reduced volume, washing it with petroleum ether and discarding any ether layer, and evaporating any remaining methanol to dryness to form a solid material;
d) extracting the solid material with a plurality of volumes of distilled water and combining the supernatant volumes, and then lyophilizing using a freeze-dryer;
e) dissolving the resulting lyophilized extract into about 500 ml of distilled water, and applying 50-100 ml portions of the dissolved extract to a 400 ml LH-20 column equilibrated with distilled water;
f) eluting the LH-20 column with ˜3 column volumes of distilled water and discarding the amber/yellow eluate;
g) eluting the LH-20 column with ˜3 column volumes of methanol, collecting the eluate and evaporating it to dryness;
h) dissolving the product of step g in water to a concentration of ˜80 mg/ml and applying 5 ml at a time to 10 gm disposable C18 SPE column equilibrated in solvent A, where solvent A is 95% water/5% acetonitrile/0.1% TFA;
i) washing the C18 SPE column with 3 volumes of solvent A and discarding the eluate;
j) eluting the column with 3 volumes of solvent A containing 12.5% solvent B, where solvent B is 95% acentonitrile/5% water/0.1% TFA, and lyophilizing the eluate;
k) injecting 50 mg portions of the lyophilized eluate of step j into a Hewlett-Packard 1100 Series HPLC instrument, fitted with a Vydac 218TP1022 C18 reverse-phase column or the like, and maintained at about 25° C. and at a flow rate of 5 ml/min;
l) eluting the sample with the following solvent profile, 10% solvent B for minutes 0 to 20, 10-100% solvent B gradient for minutes 20 to 30, and 100-10% solvent B gradient for minutes 30-31; and
m) seperating and collecting at least one fraction selected from the following fractions: fraction G (˜13-14 minutes), fraction F (˜15-16 minutes), fraction H (˜17-20 minutes), fraction I (˜21 minutes), fraction J (˜22-23 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).