Table 1 PTI-777 isolation protocol. Step by step isolation protocol to isolate and identify the Aβ amyloid fibril inhibiting/reducing ingredients in PTI-00703 cat’s claw (Uncaria tomentosa) as shown in Fig. 6a.

Step 1

1 kilogram of Uncaria tomentosa bark powder + 4000 ml methanol (mix)

Step 2

Centrifuge for 30 minutes at 2,500 × g

Step 3

Collect supernatant – repeat centrifugation and supernatant collection steps – 4 times

Step 4

Evaporate to dryness or until volume is 500 ml at 50 °C

Step 5

Wash with 300 ml of petroleum ether and discard ether layer (repeat 4 times)

Step 6

Evaporate to dryness at 50 °C (100 grams or ~10% of starting materials). Extract with 150 ml of distilled water, followed by centrifugation for 30 minutes at 2,500 × g – repeat 5 times.

Step 7

Lyophilize water extract (yield ~50 grams = ~5% of starting material)

Step 8

Dissolve 50 grams lyophilized water extract in 500 ml distilled water and apply 50–100 ml at a time on a 400 ml LH-20 equilibrated with distilled water

Step 9

Elute with 1100 ml of distilled water and discard

Step 10

Elute with 1100 ml of methanol and collect fractions (these fractions contain mostly f, g, m and n fractions) (yield ~6 grams = ~0.6% of starting material)

Step 11

Elute with 1100 ml of methanol and collect fractions (these fractions contain mostly h, j, k1, k2, l and other more hydrophobic fractions (yield ~6 grams = ~0.6% of starting material; these fractions are the most active against Aβ fibrils)

Step 12

Clean up fractions obtained from step 10 and 11 as follows: Dissolve in distilled water (80 mg/ml) and apply 5 ml at a time to a 10gram disposable C18 SPE column equilibrated in solvent A and washed with 3 volumes of solvent A and discard the eluate. Elute the clean fraction with 3 volumes of solvent A containing 12.5% solvent B. Lyophilize the corresponding fractions (~5 grams each obtained from steps 10 and 11). Where, solvent A = 95% water/5% acetonitrile/0.1% TFA, and solvent B = 955 acetonitrile/5% water/0.1% TFA.

Step 13

Fractionate fractions from step 12 (which consist of two separate fractions) on HPLC using 90 ml C18 reverse-phase HPLC column to isolate individual components.