Discovery and identification of Uncaria tomentosa (cat’s claw) as a potent natural plant extract inhibitor/disrupter of beta-amyloid protein “plaques”

Uncaria tomentosa, also known as “Uňa de Gato” (in Spanish) or cat’s claw (in English) is a woody vine that grows slowly in the Amazon rain forest. The vine is referred to as “cat’s claw” because of its distinctive claw-like thorns which project from the base of its leaves (Fig. 1a,b). The vine can take 20 years to reach maturity and can grow over 100 feet in length as it attaches and wraps itself around the native trees. The cat’s claw bark is harvested for extraction purposes (Fig. 1c) and sold in the Peruvian marketplace as bark bundles. It is found abundantly in the foothills in the Amazon rain forest at elevations of 2,000 to 8,000 feet. There are about 34 species of Uncaria, with Uncaria tomentosa being the most common species33,34.

Figure 1 PTI-00703 cat’s claw: a specific plant extract obtained from the amazon rain forest woody vine, Uncaria tomentosa (i.e. cat’s claw). (a,b) Uncaria tomentosa (cat’s claw) with its distinctive curved claw-like thorns which project from the base of its leaves. (c) Cat’s claw bark is used for extraction purposes and bark bundles are sold in the Peruvian market place. Cognitive Clarity Inc. has copyright license for digital and print use for the images of (a) from Getty Images, Chicago, IL, USA; (b) from Amazon-Images MBSI/Alamy Stock Photos; and (c) from imageBROKER/Alamy Stock Photo. Full size image

In the following set of studies, assay-guided fractionation and sophisticated structure elucidation techniques identified specific polyphenolic and proanthocyanidin ingredients within PTI-00703 cat’s claw (i.e. Uncaria tomentosa from a specific Peruvian source) that possess a natural ability to reduce and inhibit both beta-amyloid protein “plaques” and tau protein “tangles”, the two hallmarks of brain aging, and the pathological hallmarks of Alzheimer’s disease.

PTI-00703 cat’s claw is a potent inhibitor of beta-amyloid protein (Aβ) 1–40 fibril formation

In one study, Aβ 1–40 was incubated in the absence or presence of PTI-00703 cat’s claw for 3 days with aliquots analyzed at 0, 1, and 3 days. Thioflavin T fluorometry demonstrated that Aβ 1–40 fibril formation occurred within 1 day by an increase in fluorescence units from nearly 0 (at day 0) to 3500 fluorescence units within 1 day (Fig. 2a). Thioflavin S fluorescence of Aβ 1–40 fibrils at 1 day demonstrated a positive green fluorescence as viewed under fluorescent light indicative of fibril formation (Fig. 2b).This was confirmed by a positive red/green birefringence following Congo red staining as viewed under polarized light (not shown). Electron microscopy demonstrated abundant Aβ 1–40 fibrils by 1 day, with a fibril diameter of 10–20 nm (Fig. 2d). On the other hand, PTI-00703 cat’s claw significantly inhibited Aβ 1–40 fibril formation as demonstrated by a dose-dependent reduction in Thioflavin T fluorometry at 1 and 3 days (Fig. 2a), and a marked decrease in Thioflavin S fluorescence (Fig. 2c) and Congo red staining (not shown). Electron microscopy additionally demonstrated that in the presence of PTI-00703 cat’s claw, the majority of the Aβ 1–40 was inhibited from forming fibrils and only formed amorphous non-fibrillar Aβ (Fig. 2d). This study demonstrated that PTI-00703 cat’s claw markedly inhibited Aβ 1–40 fibril formation.

Figure 2 PTI-00703 cat’s claw is a potent inhibitor of Aβ 1–40 fibril formation. (a) PTI-00703 cat’s claw caused a dose-dependent inhibition of Aβ 1–40 fibril formation as assessed by Thioflavin T fluorometry. **p < 0.05, ***p < 0.001, by Student’s t-test, Bars represent mean +/− SEM. n = 5. (b) Aβ1–40 at 1 day shows positive Thioflavin S fluorescence (indicative of amyloid fibrils) under fluorescent light. Scale bar = 25 µm. (c) PTI-00703 cat’s claw inhibited Aβ fibril formation as shown by a marked reduction in Thioflavin S fluorescence (Aβ: PTI-00703 cat’s claw 1:1 wt/wt). Scale bar = 25 µm. (d) Electron microscopy at 1 day demonstrated abundant Aβ 1–40 amyloid fibrils with a characteristic fibril diameter of 10-20 nm. Scale bar = 0.2 µm. (e) Aβ1–40 in the presence of PTI-00703 cat’s claw demonstrated only formation of amorphous non-fibrillar material (Aβ: PTI-00703 cat’s claw 1:1 wt/wt). Scale bar = 200 nm. Full size image

PTI-00703 cat’s claw also disrupts/disaggregates pre-formed Aβ 1–42 fibrils nearly instantly

In another study, the dose-dependent effects of PTI-00703 cat’s claw on disruption/disaggregation of pre-formed Aβ 1–42 fibrils was determined. Aβ 1–42 was first incubated at 37 °C to observe instant amyloid fibril by Thioflavin T fluorometry at day 0 (Fig. 3a, day 0), positive Congo red staining [(i.e. red-green birefringence as viewed under polarized light)(Fig. 3b) and positive Thioflavin S fluorescence (Fig. 3d). Aβ 1–42 also demonstrated amyloid fibrils at day 0, as shown by negative stain electron microscopy (Fig. 3f). In the presence of PTI-00703 cat’s claw, Aβ 1–42 fibrils were near instantly (within a few minutes of mixing) disaggregated and/or dissolved as shown by a marked decrease in Thioflavin T fluorometry (Fig. 3a, Day 0, 1 and 3), Congo red staining (Fig. 3c) and Thioflavin S fluorescence (Fig. 3e). Electron microscopy revealed that PTI-00703 cat’s claw disaggregated and reduced pre-formed Aβ 1–42 fibrils into mostly amorphous non-fibrillar material (Fig. 3g). This study demonstrated that PTI-00703 cat’s claw was also a potent disaggregator/dissolver of pre-formed Aβ 1–42 fibrils and appeared to do so within minutes of the interaction with Aβ 1–42 fibrils.

Figure 3 PTI-00703 cat’s claw was also a potent disaggregator/disrupter of Aβ 1–42 fibrils almost instantly. (a) PTI-00703 cats claw caused a dose-dependent disruption/disaggregation of Aβ 1–42 fibrils as assessed by Thioflavin T fluorometry. A dose dependent disaggregation/disruption of Aβ 1–42 fibrils was observed nearly immediately (upon mixing). **p < 0.05, ***p < 0.001 by Student’s t-test. Bars represent mean +/− SEM. n = 5. (b) Congo red staining of Aβ 1–42 fibrils by day 0 demonstrated a robust red/green birefringence under polarized light indicative of amyloid fibrils. Scale bar = 25 µm. (c) Aβ1–42 in the presence of PTI-00703 cat’s claw near completely diminished Congo red staining indicative of a disruption/disaggregation of Aβ 1–42 fibrils (Aβ: PTI-00703 cat’s claw 1:1 wt/wt). Scale bar = 25 µm. (d) Aβ 1–42 by day 0 also showed positive Thioflavin S fluorescence under fluorescent light. Scale bar = 25 µm. (e) PTI-00703 cat’s claw disaggregated/disrupted Aβ 1–42 fibrils as shown by a marked reduction in Thioflavin S fluorescence (Aβ: PTI-00703 cat’s claw 1:1 wt/wt). Scale bar = 25 µm. (f) Electron microscopy at day 0 demonstrated abundant Aβ 1–42 amyloid fibrils with a characteristic fibril diameter of 10-20 nm. Scale bar = 100 nm (g) Aβ 1–42 fibrils in the presence of PTI-00703 cat’s claw demonstrated primarily the formation of amorphous non-fibrillar material, nearly immediately upon mixing (Aβ: PTI-00703 cat’s claw 1:1 wt/wt). Scale bar = 100 nm. Full size image

PTI-00703 cat’s claw is also a potent inhibitor/disrupter of “tangles” as demonstrated by in vitro assays

The human tau 4-repeat domain (generated as recombinant protein in human E. coli) that overlaps with amino acids Q244-E372 of tau-441 was expressed in E. coli (Fig. 4a,b). It formed paired helical filaments in the presence of heparin identical to brain paired helical filaments in “tangles” as shown by electron microscopy (Fig. 4c,d). The tau 4-repeat domain and full-length tau protein was used to determine if PTI-00703 cat’s claw was also an inhibitor/reducer of tau protein paired helical filaments. In one assay, a Thioflavin S binding assay determined the effects of PTI-00703 cat’s claw on inhibition of tau protein filament/fibril formation. As shown in Fig. 4e, PTI-00703 cat’s claw inhibited tau protein filament/fibril formation with an IC 50 of 29 µg/ml with a PTI-00703 cat’s claw: tau protein ratio of ~0.2:1. This study demonstrated that PTI-00703 cat’s claw can inhibit tau protein from forming paired helical filaments/tau fibrils.

Figure 4 PTI-00703 cat’s claw also inhibited tau protein tangles and filament formation, and reduced preformed tau fibrils. (a) Tau protein isoforms and a purified tau 4-repeat domain. Diagram of six tau isoforms aligned with a “new” tau 4-repeat domain that was generated (top of image). (b) SDS-PAGE and silver staining showed purified tau 4-repeat domain protein at ~15 kDa (250 ng of protein loaded). (c,d) Electron microscopy showed paired helical filaments (i.e. tangles) generated from in vitro aggregated tau 4-repeat domain (10 µM) in the presence of heparin. Scale bars = 50 nm. (e) Inhibition of tau protein fibril formation by PTI-00703 cat’s claw using a Thioflavin S binding assay. n = 4. (f) PTI-00703 cat’s claw also disrupted/disaggregated pre-formed tau protein filaments and fibrils using a Thioflavin S binding assay. n = 4 (g) PTI-00703 cat’s claw inhibited tau protein fibril formation in a dose-dependent manner as determined by CD spectroscopy. Tau protein alone (black bar) showed a characteristic beta-sheet CD spectra with a minima at a wavelength of 218 nm. Increasing concentrations of PTI-00703 cat’s claw demonstrated a dose-dependent reduction of the tau protein beta-sheet secondary folding as shown by a dose-dependent flattening of the 218 nm minima. n = 3. (h) PTI-00703 cat’s claw inhibited tau protein fibril formation in a dose-dependent manner as shown by EM. Increasing concentrations of PTI-00703 cat’s claw (i.e. 10 µg/ml, 50 µg/ml and 200 µg/ml) demonstrated reduction and disaggregation of tau protein filaments and fibrils into primarily amorphous non-fibrillar material. Scale bars = 200 nm. Full size image

In another study, PTI-00703 cat’s claw also disrupted/reduced pre-formed tau protein filaments/fibrils as determined using a Thioflavin S binding assay. The IC 50 of PTI-00703 cat’s claw in this study was 16 µg/ml with a PTI-00703 cat’s claw: tau protein ratio of ~0.11:1 (Fig. 4f). Circular dichroism (CD) spectroscopy further demonstrated that in 24 hours tau protein in the presence of heparin formed a β-sheet secondary folding confirmation (indicative of tau protein filaments/fibrils) with a minima at 218 nm (Fig. 4g, tau alone). Incubation of tau protein + heparin with increasing concentrations of PTI-00703 cat’s claw demonstrated a dose-dependent reduction of tau protein β-sheet formation observed by a flattening of the curve at 218 nm shown by CD spectroscopy (Fig. 4g). This demonstrated that PTI-00703 cat’s claw causes tau protein tangle disruption by a reduction of the β-sheet secondary folding of tau protein tangles.

Electron microscopy (Fig. 4h) confirmed the data observed by other methods as described above. Abundant tau filaments/fibrils were observed by EM within 3-days of incubation at 37 °C (Fig. 4h, control). In the presence of increasing concentrations of PTI-00703 cat’s claw, less and less tau protein filaments/fibrils were formed within a 3-day co-incubation period (Fig. 4h, 50 µg/ml). At the highest concentration tested, PTI-00703 cat’s claw completely abolished the formation of tau protein filaments and fibrils (Fig. 4h, 200 µg/ml). This study demonstrated that PTI-00703 cat’s claw was also effective as an inhibitor and a disrupter of tau tangles (i.e. paired helical filaments) as well. Thus, PTI-00703 cat’s claw was identified as a natural inhibitor and reducer of both Aβ fibrils and tau protein filaments/fibrils.

Initial isolation and testing of the active ingredients within PTI-00703 cat’s claw responsible for Aβ fibril inhibitory and reducing activity

Assay-guided affinity fractionation and high-pressure liquid chromatography (HPLC) separated and purified the major Aβ fibril inhibitory and reducing active components present in PTI-00703 cat’s claw. Aliquots from fractions 1–22 (i.e. 4 to 84 minutes) were incubated with pre-formed Aβ 1–40 (for 3 days) (Fig. 5) or Aβ 1–42 fibrils (instantly formed), for 2 hours (at a wt/wt ratio of 1:1) and tested for their ability to reduce/disassemble pre-formed Aβ fibrils using a Thioflavin T fluorometry assay as previously described35. As shown in Fig. 5, fibrillar Aβ 1–40 alone demonstrated a fluorescence of 836 +/− 61 fluorescent units. Fractions 13–18 (i.e. 52–72 minutes) demonstrated the greatest ability (from 60–75%) to reduce/disassemble pre-formed Aβ 1–40 fibrils, as indicated by a marked lowering of fluorescence units (Fig. 5). Similar results were obtained with pre-formed Aβ 1–42 fibrils (not shown). This study suggested that the most active Aβ amyloid fibril inhibitory components within PTI-00703 cat’s claw (Uncaria tomentosa) were located within fractions 13–18.

Figure 5 A Thioflavin T fluorometry assay identified water-soluble fractions of cat’s claw (Uncaria tomentosa) that possessed Aβ fibril reducing/disaggregation activity. Fractions 13–18 (52–72 minutes) contain components that reduce/dissolve pre-formed Aβ 1–40 fibrils by 60–75%. Bars represent mean +/− SEM. n = 4. Full size image

Purification of the major Aβ amyloid inhibitory components in the water-soluble fraction of Uncaria tomentosa (PTI-777 isolation protocol)

For the isolation of PTI-777 the methodology outlined in Table 1 was used. Under these conditions, PTI-777 separated into 11–13 major components (Fig. 6a), as revealed by uv/vis detection (diode array). These fractions were collected and designated as follows: 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) (Fig. 6a).

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. Full size table

Figure 6 PTI-777 profile (major Aβ inhibitory ingredients in PTI-00703 cat’s claw). (a) Preparative HPLC Profile of PTI-777 that demonstrated the major water-soluble Aβ inhibitory components isolated from Uncaria tomentosa (cat’s claw), noted (from left to right) as Fractions g, f, h, i, j, k 1 , k 2 , l, m/n and o. (b) Identification of each fraction using a variety of techniques including PTI-00703 cat’s claw, PTI-777, and individual fractions of PTI-777 were assessed by Thioflavin T fluorometry. PTI-00703 cat’s claw, PTI-777, and individual fractions f-n were all significant effective disrupters/reducers of Aβ 1–42 fibrils. On the other hand, the cat’s claw alkaloids isopteropodine, pteropodine, isomatraphyline and mitraphylline had no significant effects on disaggregation/reduction of pre-formed Aβ 1–42 fibrils. Bars represent mean +/− SEM. n = 5. Full size image

In vitro testing of individual fractions within PTI-777 for Aβ amyloid fibril inhibitory/reducing activity

The bioactivities of PTI-777 and its isolated individual fractions (i.e. fractions f through o) were evaluated in a number of different in vitro assays. Testing included the use of Thioflavin T fluorometry, Congo red staining assays, and negative stain EM. In most experiments, individual isolated fractions of PTI-777 were directly compared to (PTI-00703 cat’s claw, PTI-777 and the major oxindole alkaloids isolated from Uncaria tomentosa and thought to contain important bioactivity as described in 2 US Patents36,37 (Fig. 6b).

In one set of studies, Thioflavin T fluorometry compared the ability of PTI-00703 cat’s claw, PTI-777 (major components in cat’s claw), PTI-777 individual fractions (including fractions f, g, h, j, k, l, m and n), and alkaloids isolated from Uncaria tomentosa (including isopteropodine, pteropodine, isomitraphylline and mitraphylline), to disrupt/reduce pre-formed Aβ 1–42 fibrils. The results of 5 different Thioflavin T fluorometry experiments indicated that PTI-00703 cat’s claw caused a significant (p < 0.001) 53 +/− 2.5% disruption/reduction of pre-formed Aβ 1–42 fibrils (Fig. 6b). On the other hand, individual PTI-777 fractions including fraction f (64.0 +/− 1.7% inhibition), fraction g (62.3 +/− 8.5% inhibition), fraction h (which consisted of both h1 and h2; 56.3 +/− 2.1%), fraction j (68.7 +/− 2.0%), fraction k (which consisted of both k1 and k2; 58.0 +/− 4.6% inhibition), fraction l (68.3 +/− 2.3% inhibition), fraction m (64.0 +/− 1.5% inhibition), and fraction n (63.0 +/− 1.0% inhibition) were all similarly quite effective in causing a significant disruption/reduction of pre-formed Aβ 1–42 fibrils. Surprisingly, the oxindole alkaloids (isopteropodine, pteropodine, isomitraphylline, and mitraphylline) had no significant effects in disruption/disaggregation of pre-formed Aβ 1–42 fibrils (Fig. 6b).This study also showed that PTI-777 (i.e. mixture of fractions f through o) was a significantly more effective disrupter of Aβ of 1–42 fibrils (i.e. 87.3 +/− 3.0%) than any of the individual fractions tested. These studies indicated that PTI-777 and its individual fractions (fractions f, g, h, j, k, l, m, and n) were effective Aβ 1–42 fibril disaggregators and reducers. In addition, it was evident that the combination of fractions as observed with PTI-777 (a mixture of ~11–13 major components) were even more active than any of the individual PTI-777 fractions alone, suggesting a possible synergistic effect between different PTI-777 components. Lastly, the fact that isolated alkaloids from Uncaria tomentosa were basically ineffective in the disruption of Aβ 1–42 fibrils, suggested that oxindole alkaloids were not responsible for the Aβ amyloid inhibitory effects exerted by PTI-777, the individual PTI-777 fractions tested above, or PTI-00703 cat’s claw.

In another study, Thioflavin T fluorometry determined the dose-dependent effectiveness of PTI-777 (i.e. Aβ amyloid inhibitory components present in PTI-00703 cat’s claw) in disrupting/reducing pre-formed Aβ 1–42 fibrils. For example, PTI-777 at an Aβ:PTI-777 wt/wt ratio of 1:0.1 significantly (p < 0.001) reduced Aβ 1–42 fibrils by 73.2%; whereas at an Aβ:PTI-777 wt/wt/ratio of 1:1 significantly (p < 0.001) reduced Aβ 1–42 fibrils by 98.6% (Fig. 7a).

Figure 7 PTI-777 disrupts Aβ 1–42 fibrils. (a) Dose-dependent disruption/disaggregation of Aβ 1–42 fibrils by PTI-777 as assessed by Thioflavin T fluorometry. PTI-777 significantly (p < 0.001) disrupted/disaggregated Aβ 1–42 fibrils in a dose-dependent manner. **p < 0.01, ***p < 0.001, by Student’s t-test. Bars represent mean +/− SEM. n = 4. (b) Dose-dependent inhibition of Congo red binding to Aβ 1–42 fibrils by PTI-777. ***p < 0.001, by Student’s t-test. Bars represent mean +/− SEM. n = 4. (c–f) PTI-777 caused a marked disruption/disaggregation of β-sheet secondary structure for Aβ 1–42 fibrils, and prevented Aβ 1–40 fibril formation, as determined by CD spectroscopy. (c) PTI-777 at 3 days caused a decrease in β-sheet of Aβ 1–42 fibrils at a PTI-777:Aβ 1–42 (wt/wt) ratio of 0.1:1, as shown by a decrease in the in the ~218 nm minima curve. (d) By 7 days of co-incubation, PTI-777 caused an even greater disruption of the β-sheet secondary structure in Aβ 1–42 fibrils. (e) PTI-777 at 3 days completely prevented Aβ 1–40 fibril formation as demonstrated by a complete flattening of the characteristic β-sheet minima observed in Aβ 1–40 fibrils alone. (f) A similar inhibition of Aβ 1–40 β-sheet formation was observed with PTI-777 at 7 days. Full size image

A Congo red binding assay demonstrated similar results. As shown in Fig. 7b, PTI-777 effectively reduced pre-formed Aβ 1–42 fibrils in a dose-dependent manner. For example, PTI-777 significantly (p < 0.001) reduced Aβ 1–42 fibrils by 54.5% at a PTI-777:Aβ 1–42 wt/wt ratio of 1:0.1:, and by 71.3% (p < 0.001) at a PTI-777: Aβ wt/wt ratio of 1:1. A reduction of Congo red staining on glass slides also demonstrated a marked reduction in the red/green birefringence (not shown) indicative that PTI-777 significantly disrupts pre-formed Aβ -42 fibrils.

Circular dichroism (CD) spectroscopy confirmed that PTI-777 (i.e. Aβ amyloid inhibitory components present in PTI-00703 cat’s claw) not only inhibited Aβ 1–40 fibril formation, but also disrupted/reduced the β-sheet secondary folding of Aβ 1–42 fibrils. In one study, 50 µM of pre-formed Aβ 1–42 fibrils was incubated with PTI-777 at a PTI-777: Aβ 1–42 wt/wt ratio of 0.1:1. As shown in Fig. 7c, Aβ 1–42 (red closed circles) at 3 and 7 days demonstrated a characteristic pattern of extensive β-sheet secondary structure as shown by the curve and the minima at ~218 nm. In the presence of PTI-777 (blue closed circles) at both 3 days (Fig. 7c) and at 7 days (Fig. 7d) there was a marked disruption/reduction of pre-formed Aβ 1–42 fibrils. At 7 days, there was approximately 85–90% disruption/reduction of β-pleated sheet secondary folding by PTI-777, as shown by a flattening/smoothing of the curve especially at ~218 nm wavelength (Fig. 7d).

In another study, 50 µM of Aβ 1–40 was incubated at 37 °C for 1 week in phosphate-buffered saline (pH 7.4) either alone or in the presence of PTI-777 at a PTI-777: Aβ 1–40 wt/wt ratio of 0.1:1. PTI-777 caused a complete reduction of the β-sheet secondary folding curve (i.e. minima at 218 nm) within 3 days of co-incubation with Aβ 1–40 (Fig. 7e, blue closed circles). A similar inhibition of Aβ 1–40 fibril formation with PTI-777 was observed following 7 days of incubation (Fig. 7f, blue closed circles).

Marked reduction of brain amyloid plaque load by PTI-777 (major Aβ amyloid inhibitory ingredients of PTI-00703 cat’s claw) in a transgenic mouse model of Alzheimer’s disease

In the next set of studies, we determined whether PTI-777 may have direct effects on brain amyloid plaque load when directly infused into the cortex of 8-month TASD-41 APP double transgenic mice (i.e. London and Swedish mutations under Thy-1 promoter)38. As shown in Fig. 8a, representative Bielchowsky silver-stained sections of hippocampus and cortex from 8-month old plaque-producing transgenic mice are shown. Numerous amyloid plaques (white arrowheads) were observed. Following a 14-day treatment with PTI-777, there was a marked reduction in the number of amyloid plaques in hippocampus and cortex with only a few plaques remaining (Fig. 8b, arrows). Quantitation of % Aβ amyloid load and plaque number (per square mm) indicated that PTI-777 infusion significantly (p < 0.01) reduced % Aβ load by 59%, and plaque number (p < 0.001) by 78%, following only after 14-days of treatment with PTI-777 (Fig. 8c). The infusion site was not seen in these photomicrographs indicating that PTI-777 treatment was effective across the brain, even relatively far away from the infusion site.

Figure 8 PTI-777 markedly reduced amyloid plaques in brains of 8-month old TASD-41 APP transgenic mice following direct infusion into hippocampus. (a) Bielchowsky silver stain showing abundant amyloid plaques in hippocampus and cortex of TASD-41 APP transgenic mouse infused with saline for 14 days. Scale bar = 100 µm. (b) PTI-777 treatment (100 µl at 8 mg/ml; 14-days infusion) demonstrated a marked reduction in amyloid plaques (arrows) in 8-month old TASD-41 APP transgenic mice. Scale bar = 100 µm. (c, d) Marked reduction in % beta-amyloid protein load and plaque number (per sq. mm.) following PTI-777 infusion. **p < 0.01; ***p < 0.001 by Student’s t-test. Bars represent mean +/− SEM. n = 4. Full size image

PTI-777 crosses the blood-brain-barrier and enters the brain parenchyma within 2 minutes of being in the blood

The HPLC profile of unlabeled PTI-777 is shown in Fig. 9a, which was nearly identical to the HPLC profile of 3H-PTI-777 (Fig. 9b), indicating that the radiolabeling with tritium did little to alter the structure of PTI-777. The distribution of radioactivity measured in 0.5 ml fractions that were eluted from the HPLC column and collected beginning at 16.5 minutes is shown in Fig. 9c. This latter figure demonstrated that the HPLC peaks of PTI-777 also had the greatest radioactivity. In order to determine the potential ability of 3H-PTI-777 to cross the blood-brain-barrier and enter into brain tissue, male and female Sprague-Dawley rats were administered with a single i.v. injection of 3H-PTI-777. Two animals per group were then sacrificed at 2, 5, 10, 20 minutes, 1, 6 and 24 hours following administration, and the amount of 3H-PTI-777 in brain tissue was determined. As shown in Fig. 9d, ~18,000 dpm/g tissue of 3H-PTI-777 in brain was found within 5 minutes following intravenous administration. By 1 hour, this had decreased to ~10,100 dpm/g. Even at 24 hours, the brain tissue still contained about ~7,000 dpm/gram of 3H-PTI-777, indicating ~41% of the initial dose found to enter the brain parenchyma was retained in the brain tissue even over a 24-hour period.

Figure 9 3H-PTI-777 crossed the blood-brain-barrier and entered the brain parenchyma within 2 minutes following peripheral administration. 0.9 mCi of 3H-PTI-777 with specific activity of 1.25 mCi/mg (at a concentration of 1 mCi/ml) was prepared and used for animal studies. (a) Figure demonstrates the HPLC profile of unlabeled PTI-777 which is nearly identical to the HPLC profile of (b) 3H-PTI-777. Fractions g, f, h, i, j, k and m/n are shown. (c) The distribution of radioactivity measured in 0.5 ml fractions that was eluted from the HPLC column and collected beginning at 16.5 minutes. This graph demonstrates that the HPLC peaks of PTI-777 also coincide with the greatest radioactivity. (d) Penetration of 3H-PTI-777 into rat brain, with radioactivity present in brain tissue in 2 minutes and lasted over a 24-hour period. n = 14 (7 females and 7 male Sprague-Dawley rats) (e) Penetration of 3H-PTI-777 into mouse brain tissue (CD-1 mice) within 2 minutes after being in the blood. n = 16. Bars represent mean +/− SEM. Full size image

In another study, groups of CD-1 mice (n = 4 per group) were injected intravenously with 3H-PTI-777 (25,000 cpm/µl) and 99mTC-albumin (200 µl of each). Following capillary depletion methods, a high brain/serum ratio of radiolabeled 3H-PTI-777 was present in brain parenchyma and cortex, with minimal amounts in the capillary fraction (Fig. 9e). Within 2 minutes of administration, 3H-PTI-777 (following capillary depletion methods) was found in brain parenchyma and cortex, and was maintained in brain tissue following 5, 10 and 20 minutes of administration (Fig. 9e). This data demonstrated that PTI-777 can cross the blood-brain-barrier and enter the brain parenchyma, even within 2 minutes of being in the blood.

Marked reduction of brain plaque load by PTI-777 within 30 days of peripheral administration

PTI-777 (25 mg/kg) or saline was injected intraperitoneally daily for 30 days into 6-month old TASD-41 transgenic mice. Following the 30-day treatment, the results indicated that PTI-777 treatment caused a significant (p < 0.01) 33.2% reduction in % Aβ amyloid load, and a significant (p < 0.001) 56.6% reduction in amyloid plaque number (per square mm) (Fig. 10). These studies demonstrate the ability of the major components of Uncaria tomentosa (cat’s claw) to enter the brain and markedly reduce brain plaque load within 30 days following peripheral administration.

Figure 10 Marked reduction of brain plaque load in TASD-41 APP transgenic mice following 30-days of intraperitoneal injections of PTI-777. 6 month old TASD-41 APP transgenic mice were injected daily with saline or PTI-777. Within 30 days, PTI-777 reduced % Aβ plaque load by 33.2% (p < 0.05) and plaque number (per sq. mm) by 55.6% (p < 0.01). **p < 0.05; ***p < 0.001, by Student’s t-test. Bars represent mean +/− SEM. n = 8 per group. Full size image

Identification of the Major Components within PTI-00703 cat’s claw and PTI-777 responsible for the observed Aβ amyloid inhibitory activity

The preparative HPLC work on PTI-777 described here resulted in 11–13 major water-soluble active fractions, each of which contained primarily one major compound. Composition and structural studies demanded that the major individual compounds within each fraction to be purified to homogeneity in sufficient quantity for NMR and other spectroscopy studies (about 30–50 mg for each compound). Although each of the fractions of PTI-777 showed significant activity in the in vitro assays, fractions f, j, h1, h2, k1 and k2 were initially singled out for initial final purification based on their starting purity as determined by analytical HPLC, and the amount of material available. These fractions were passed through the preparative HPLC column until they were deemed pure.

PTI-777 fraction f identified as chlorogenic acid

A variety of methods were used to isolate and identify the major compound within PTI-777 fraction f. HPLC, mass spectroscopy, NMR spectroscopy (1H-NMR and 13C-NMR), correlation spectroscopy (COSY) and ultraviolet (UV) spectroscopy were all used. As shown in Supplementary Material Fig. S1a,S1b, PTI-777 fraction f compound was identified as chlorogenic acid (C 16 H 18 O 9 ; MW 354.31) with a structure as shown in Fig. 11a.

Figure 11 Purification and identity of the major components in PTI-777. (a) Structure of fraction f as chlorogenic acid (b) Structure of fraction j as epicatechin. (c) Structure of fraction h2 as epicatechin-4β-8-epicatechin (known as proanthocyanidin B2). (d) Structure of fraction h1 as catechin-4α-8-epicatechin (known as proanthocyanidin B4). (e) Structure of fraction k2 as epicatechin-4β-8-epicatechin-4β-8-epicatechin (known as proanthocyanidin C1). (f) Structure of fraction k1 as epiafzelechin-4β-8-epicatechin. See Supplement Results and Figures for structural elucidation and identification studies. Full size image

PTI-777 fraction j identified as epicatechin

Fraction j was the second material to be purified in a quantity sufficient for structural elucidation work. Following the isolation and purification of PTI-777-compound j, mass spectroscopy, NMR spectroscopy, correlation spectroscopy (COSY), and UV spectroscopy were all used on the fraction j compound and its pentaacetate derivative. As shown in Supplementary Material Fig. S2a–S2w, PTI-777 fraction j compound was identified as epicatechin (C 15 H 14 O 6 ; MW 290.27), with a structure as shown in Fig. 11b.

PTI-777 fraction h2 identified as epicatechin-4β → 8-epicatechin (a specific epicatechin dimer known as proanthocyanidin B2)

Fraction h2 was the third material to be purified in a quantity sufficient for structural elucidation work. Following the isolation and purification of PTI-777-compound h2, HPLC, -ve ion electrospray mass spectroscopy, NMR spectroscopy (1H NMR and 13C NMR), correlation spectroscopy (COSY) and ultraviolet (UV) spectroscopy were all used. The main component of fraction h from the PTI-777 extract was also a major component in Uncaria tomentosa (cat’s claw) responsible for the Aβ (plaque) and tau protein (tangle) inhibitory and disaggregation activity (see Fig. 6a). As shown in Supplementary Material Fig. S3a–S3m, PTI-777 fraction h2 compound was identified as epicatechin-4β-8-epicatechin, also known as proanthocyanidin B2 (C 30 H 26 O 12 ; MW 577), with a structure as shown in Fig. 11c.

PTI-777 fraction h1 identified as catechin-4α → 8-epicatechin (proanthocyanidin B4)

Fraction h1 was the fourth material to be purified in a quantity sufficient for structural elucidation work. Following the isolation and purification of PTI-777-compound h1, HPLC, -ve ion electrospray mass spectroscopy. NMR spectroscopy (1H-NMR and 13C-NMR), correlation spectroscopy (COSY), constant time inverse detection gradient accordion rescaled (CIGAR) spectroscopy, and ultraviolet (UV) spectroscopy were all used. As shown in Supplementary Material Fig. S4a–S4j, PTI-777 fraction h1 compound was identified as catechin-4α-8-epicatechin (Fig. 11d), also known as proanthocyanidin B4 (C 30 H 26 O 12 ; MW 577).

PTI-777 fraction k2 identified as epicatechin-4β → 8-epicatechin-4β → 8-epicatechin or proanthocyanidin C1

Fraction k2 was the fifth material to be purified in a quantity sufficient for structural elucidation work. Following the isolation and purification of PTI-777-compound k2, HPLC, -ve ion electrospray mass spectroscopy. NMR spectroscopy (1H NMR and 13C NMR), correlation spectroscopy (COSY), constant time inverse detection gradient accordion rescaled (CIGAR) spectroscopy, and ultraviolet (UV) spectroscopy were all used. As shown in Supplementary Material Fig. S5a–S5j, PTI-777 fraction k2 compound was identified as epicatechin-4β → 8-epicatechin-4β → 8-epicatechin (Fig. 11e), also known as proanthocyanidin C1 (C 45 H 38 O 18 ; MW 866).

PTI-777 fraction k1 identified as epiafzelechin-4β → 8-epicatechin

Fraction k1 was the sixth material to be purified in a quantity sufficient for structural elucidation work. Following the isolation and purification of PTI-777-compound k1, HPLC, -ve ion electrospray mass spectroscopy, NMR spectroscopy (1H NMR and 13C NMR), correlation spectroscopy (COSY), constant time inverse detection gradient accordion rescaled (CIGAR) spectroscopy, and ultraviolet (UV) spectroscopy were used. As shown in Supplementary Material Fig. S6a–S6j, PTI-777 fraction k1 compound was identified as epiafzelechin-4β → 8-epicatechin (Fig. 11f) (C 30 H 26 O 11 ; MW 562).

PTI-777 fraction l identified as epicatechin-4β → 8-epicatechin-4β → 8-epicatechin-4β → 8- epicatechin (an epicatechin tetramer)

Fraction l was the seventh material to be purified in a quantity sufficient for structural elucidation work. Following the isolation and purification of PTI-777-compound l, HPLC, -ve ion electrospray mass spectroscopy, NMR spectroscopy (1H NMR and 13C NMR), correlation spectroscopy (COSY), constant time inverse detection gradient accordion rescaled (CIGAR) spectroscopy, and ultraviolet (UV) spectroscopy were used. As shown in Supplementary Material Fig. S7a, PTI-777 fraction l compound was identified epicatechin-4β → 8-epicatechin-4β → 8-epicatechin-4β → 8-epicatechin (an epicatechin tetramer) (C 80 H 50 O 24 ; MW 1153).

PTI-00703 cat’s claw proanthocyanidins are potent disaggregators/reducers of Aβ 1–42 fibrils

In the next study, Thioflavin T fluorometry determined the effects of compounds h2, f, k1, k2 and mitraphylline (major alkaloid in cat’s claw) on disassembly/disaggregation of pre-formed Aβ 1–42 fibrils. In this study, 25 µM of pre-fibrillized Aβ 1–42 was incubated at 37 °C for 1 week alone or in the presence of compound h2, f, k1, k2 and mitraphylline at Aβ: test compound weight ratios of 1:1; 1:0.1; 1:0.01; 1:0.001 and 1:0.0001. The results of day 7 incubations are presented in Fig. 12a, but similar results were observed as early as 3 days (not shown). As shown in Fig. 12a, whereas the cat’s claw alkaloid mitraphylline caused no significant disruption of Aβ 1–42 fibrils at all concentrations tested, compound h2 (epicatechin-4β → 8-epicatechin; proanthocyanidin B2) caused a dose-dependent disruption/reduction of pre-formed Aβ 1–42 fibrils, with a significant (p < 0.001) 94% reduction when used at an Aβ:h2 wt/wt ratio of 1:1, and a significant (p < 0.01) 63% reduction when used at a Aβ:h2 wt/wt ratio of 1:0.1 (~1:1 molar ratio). Similarly, compound f (chlorogenic acid), compound k2 (epicatechin trimer; proanthocyanidin C1) and compound k1 (epiafzelechin -4β → 8-epicatechin) all caused a marked disruption/reduction of preformed Aβ 1–42 fibrils when used at a 1:1 wt/wt ratio (Fig. 12a). The most effective compound of these tested for disassembly/disaggregation of Aβ 1–42 fibrils was compound h2 (epicatechin-4β → 8-epicatechin; proanthocyanidin B2).

Figure 12 PTI-00703 cat’s claw proanthocyanidins are potent disrupters of Aβ 1–42 fibrils. (a) Disruption/disassembly of Aβ 1–42 fibrils by specific PTI-00703 cat’s claw proanthocyanidins. Compound h2 (epicatechin-4β → 8-epicatechin; proanthocyanidin B2) caused a dose-dependent disruption/disaggregation of pre-formed Aβ 1–42 fibrils. Similarly, compound f (chlorogenic acid), compound k1 (epicatechin trimer; proanthocyanidin C1) and compound k2 (epifzeledin-4β-8-epicatechin) all caused a marked disruption/reduction of preformed Aβ 1–42 fibrils when used at a 1:1 wt/wt ratio. The major cat’s claw alkaloid mitraphylline was found to be totally ineffective. **p < 0.01, ***p < 0.001, by Student’s t-test. Bars represent mean +/− SEM. n = 5. (b,c) Disruption/disaggregation of pre-formed Aβ 1–42 and 1–40 fibrils by epicatechin-4β-8-epicatechin (i.e. proanthocyanidin B2; compound h2) as assessed by CD spectroscopy. Full size image

PTI-00703 cat’s claw proanthocyanidin B2 (epicatechin-4β-8-epicatechin) is a potent disaggregator/reducer of pre-formed Aβ 1–42 and Aβ 1–40 fibrils as shown by circular dichroism spectroscopy

Circular dichroism (CD) spectroscopy determined the effects of test compounds to disrupt/reduce pre-formed amyloid fibrils. In one study, CD spectroscopy determined the effects of pure compound h2 (i.e. epicatechin-4β-8-epicatechin) on disruption of β-pleated sheet secondary structure of pre-formed Aβ 1–40 or Aβ 1–42 fibrils. As shown in Fig. 12b, Aβ 1–42 alone in 10% TFE buffer showed the typical CD spectra of an amyloid protein with significant β-structure, as demonstrated by the sharp minima observed at 218 nm. However, in the presence of the h2 compound (i.e. epicatechin-4β-8-epicatechin; proanthocyanidin B2) at a 1:1 molar ratio, a marked disruption of β-sheet structure in Aβ 1–42 fibrils was evident as shown by the flattening out of the minima observed at 218 nm (compared to Aβ 1–42 alone) (Fig. 12b). This was observed at both 3 days (not shown) and 7 days (Fig. 12b) following co-incubation of Aβ 1–42 fibrils with compound h2. This study clearly demonstrated that compound h2 (epicatechin-4β-8-epicatechin; proanthocyanidin B2) had the ability to disrupt/disassemble the beta-pleated sheet secondary structure characteristic of Aβ 1–42 fibrils.

As shown in Fig. 12c, Aβ 1–40 alone in 10% TFE PBS buffer also showed the typical CD spectra of an amyloid protein with significant β-sheet structure, as demonstrated by the sharp minima at 218 nm. However, in the presence of compound h2 (at a 1:1 molar ratio), a nearly complete disruption/disassembly of β-sheet structure in Aβ 1–40 fibrils was evident (with a significant increase in random coil or α-helix) as shown by the complete flattening out of the minima observed at 218 nm (Fig. 12c). This was observed at both 3 days (Fig. 12c) and 7 days (not shown) following co-incubation of Aβ 1–40 fibrils with compound h2. This study clearly demonstrated that proanthocyanidin B2 (i.e. compound h2) disrupted/disassembled the beta-pleated sheet secondary structure of Aβ 1–40 fibrils as well.

Proanthocyanidin B2 (epicatechin-4β-8-epicatechin; compound h2) reduces brain plaque load and improves memory in a “plaque-producing” transgenic mouse model

As shown in Fig. 13, brain (i.e. cortex) levels of Aβ 42 and Aβ 40 soluble and insoluble levels in younger 4-month old TASD-41 APP transgenic mice were significantly (p < 0.01) reduced following administration of proanthocyanidin B2 (compound h2; epicatechin-4β-8-epicatechin) at 50 mg/kg/day following 90-days of treatment (i.e. 7-months old at sacrifice) as determined using ELISAs for both Aβ 1–42 and 1–40. Proanthocyanidin B2 treatment significantly (p < 0.05) reduced brain Aβ 42 and Aβ 40 insoluble levels by 18.5% (Fig. 13a), and 23.9% (Fig. 13b), respectively. Proanthocyanidin B2 treatment also significantly (p < 0.01) reduced Aβ 42 and Aβ 40 soluble levels by 70.4% (Fig. 13c), and 58.9% (Fig. 13d), respectively, as well.

Figure 13 Reduction of brain plaque load in younger (a–h) and older (i–j) TASD-41 APP transgenic mice by proanthocyanidin B2 (compound h2). (a–h) Reduction of brain Aβ 1–42 and Aβ 1–40 load by proanthocyanidin B2 (i.e. compound h2) in younger (4 months old at start; 7 months old at sacrifice) APP transgenic mice. n = 10 per group. (a) Proanthocyanidin B2 caused a significant (p < 0.05) 18.5% reduction of insoluble Aβ 1–42, and (c) a significant (p < 0.05) 70.4% reduction of soluble Aβ 1–42, as determined by Aβ ELISAs. (b) Compound h2 also caused a significant 23.9% reduction of insoluble Aβ 1–40, and (d) a significant 58.9% reduction in soluble Aβ 1–40 in brains of TASD-41 transgenic mice. n = 4 per group. (e–h) Reduction of brain amyloid load and plaque number by proanthocyanidin B2 in younger (4 months old at start; 7 months old at sacrifice) TASD-41 transgenic mice. n = 10 per group. (e) Image analysis of Thioflavin-S stained sections from cortex revealed that proanthocyanidin B2 (h2 treatment) caused a significant 74.2% reduction in Thioflavin S amyloid load, and (f) a significant 74.9% reduction in plaque number (per sq. mm). Similarly, Congo red staining revealed a significant 82.9% in % amyloid load (g), and an 80.8% reduction in plaque number (h). Thioflavin S-stained sections from cortex of older TASD-41 APP transgenic mice (6 months old at start; 9 month old at sacrifice) revealed that proanthocyanidin B2 caused a significant 58.2% reduction in Thioflavin S amyloid load (i), and a significant 51.9% reduction in plaque number (j). *p < 0.05, **p < 0.01, ***p < 0.001, by Student’s t-test. Bars represent mean +/− SEM. Full size image

As shown in Fig. 13e, quantitative image analysis of Thioflavin-S stained sections from cortex of younger TASD-41 APP transgenic mice (i.e. 4 months of age to start; 7-months old at sacrifice) revealed that proanthocyanidin B2 treatment caused a significant (p < 0.001) 74.2% reduction in Thioflavin-S amyloid load (Fig. 13e), and a significant (p < 0.001) 74.9% reduction in plaque number (Fig. 13f) compared to saline-treated APP mice. Quantitative image analysis of Congo red stained sections demonstrated that proanthocyanidin B2 also caused a significant (p < 0.001) 82.9% reduction in Congo red stained amyloid load (Fig. 13g) and a significant (p < 0.001) 80.8% reduction in plaque number (Fig. 13h) compared to saline-treated APP mice.

A similar reduction in brain amyloid load and plaque number was also found after 90 days of peripheral treatment with proanthocyanidin B2 in older (9 months at sacrifice) TASD-41 APP transgenic mice. In this latter study, proanthocyanidin B2-treated animals displayed a significant (p < 0.001) 58.2% reduction in beta-amyloid protein load (as assessed using an Aβ 6E10 antibody) (Fig. 13i) and a significant (p < 0.001) 51.9% reduction in plaque number (Fig. 13j) compared to saline-treated mice.

Examples of 2 TASD-41 APP transgenic mice treated with saline for 90 days and stained for amyloid plaques in cortex using Thioflavin S is shown in Fig. 14a (a, b, saline treatment). Proanthocyanidin B2 treatment (h2 treated) caused a marked reduction of brain amyloid load/plaque number in cortex as shown in 2 different TASD-41 APP mice using Thioflavin S fluorescence (Fig. 14a, h2 treatment as shown in c & d).

Figure 14 Improvement of memory and reduction of inflammation (astrocytosis and microgliosis) by proanthocyanidin B2. (a) Example of reduction of Thioflavin-S stained plaques in cortex of 2 saline-treated TASD-41 transgenic mice (Fig. a, upper panels) compared to two proanthocyanidin B2 (i.e. h2-treated TASD-41 transgenic mice (Fig. a, lower panels) Scale bar = 50 µm. (b) Morris water maze testing demonstrated that proanthocyanidin B2 treatment (Tg/h2) markedly and significantly (p < 0.001) improved short-term memory by 57.8% compared to saline-treated animals (Tg/Saline) as shown on day 4 of the invisible platform. A similar reduction was observed at day 5 of the invisible platform following procyanidin B2 treatment. p < 0.001 by student’s t-test and ANOVA. n = 12 per group. (c) Proanthocyanidin B2 was a potent reducer of astrocytosis in TASD-41 transgenic animals as demonstrated by a significant (p < 0.001) 69% reduction in astrocytosis (i.e. GFAP immunostained brain sections). Scale bar = 50 µm. (d) MHC-II immunostaining and image analysis also revealed that proanthocyanidin B2 treatment (significantly p < 0.001) reduced MHC-II (i.e. microgliosis) immunostaining by 80.3%. ***p < 0.001, by Student’s t-test. Bars represent mean +/− SEM. Full size image

Following 90 days of daily i.p. injections (50 mg/kg/day) with saline (Tg/saline) or proanthocyanidin h2 (Tg/h2), TASD-41 APP transgenic mice, and non-transgenic littermate controls (Non-Tg) were tested in a Morris water maze to determine effects on hippocampus-dependent memory (i.e. spatial acquisition). Proanthocyanidin h2 treatment caused marked improvements in hippocampus-dependent memory (by 57.8% on day 4 of the invisible platform; Fig. 14b) and by 57.3% on day 5 of the invisible platform (not shown). This was detected by both path length (distance in meters) (Fig. 14b) and in latency (m/sec)(not shown). No change in swimming speed (m/sec) between all groups were therefore found. Proanthocyanidin B2-treated TASD-41 APP mice had remarkable improvements in short-term memory approaching those levels observed in non-transgenic animals (Non-Tg) (Fig. 14b). On the last day of training, all groups found the visible platform demonstrating that all animals had no motor abnormalities. This study confirmed that the major component of PTI-00703 cat’s claw, proanthocyanidin B2, was a potent reducer of brain amyloid “plaques” that coincided with marked improvements in short-term memory.

The major component of PTI-00703 cat’s claw (proanthocyanidin B2) is a potent reducer of both brain astrocytosis and microgliosis

Both GFAP immunostaining (for astrocytes) and MHC-II immunostaining (for microglia) from saline-treated versus proanthocyanidin B2-treated APP transgenic mice were quantified using an image analysis program. As shown in Fig. 14c, proanthocyanidin B2 treatment (h2 treatment) caused a significant reduction in astrocytosis as shown by a marked decrease in GFAP-immunostained brain sections. Quantitative image analysis indicated that compound h2 caused a significant (p < 0.001) 69.0% reduction in astrocytosis. As shown in Fig. 14d, proanthocyanidin B2 treatment also caused a significant (p < 0.001) 80.3% reduction in microgliosis as indicated by a marked reduction in MHC-II immunostaining in brain sections. These studies further verified the potent anti-inflammatory activity of proanthocyanidin B2 by markedly reducing both brain astrocytosis and microgliosis in TASD-41 APP transgenic mice. Cat’s claw has previously been shown to be a potent anti-inflammatory agent39,40.