The water extractable compounds (GKW) represented approximately 10% of the dry weight of CA herb. There was no difference in water consumption between control animals and animals receiving water containing GKW.
Open-field testing () showed that GKW treatment caused an improvement in behavioral abnormalities seen in Tg2576 mice. Wild-type mice, both GKW treated and untreated, were less active on the second trial of each day, presumably due to habituation. Untreated Tg2576 mice, in contrast, failed to habituate to the surroundings. However, GKW-treated Tg2576 mice explored in a manner similar to wild-type mice, with their data overlapping the two wild-type groups (P
= 0.02 for difference between untreated and GKW-treated Tg2576 mice by ANOVA). “Normalization” of open-field behavior in Tg2576 mice has also been reported with an intervention that suppresses soluble Aβ
Figure 1 Open-field assay: effect of GKW on total distance travelled. Wild-type mice, both treated (triangle) and untreated (filled diamond), are less active on the second trial of each day, presumably due to habituation. Untreated Tg2576 mice (open diamond), (more ...)
In the Morris water maze paradigm (), GKW treatment improved the impaired learning ability evident in Tg2576 mice. Wild-type animals exhibit a “learning curve” requiring less time and less distance to find the hidden platform with repeated trials, while untreated Tg2576 mice require equivalent time and distance despite repeated exposures. In contrast, the GKW-treated Tg2576 mice learn in a manner similar to the wild-type animals, with latency and distance traveled to find the platform declining with repeated exposures. On day 4, time to find the platform was significantly greater for untreated Tg2576 mice than for wild-type (P = 0.002) or GKW-treated Tg2576 mice (P = 0.004). Distance traveled to find the platform was also significantly greater on day 4 for untreated Tg2576 mice compared to wild-type (P = 0.006) or GKW-treated Tg2576 mice (P = 0.003). There was no significant difference between groups in the “visible platform” control for sensorimotor function (data not shown).
Figure 2 GKW effects in the Morris water maze. Mean ± SEM of (a) escape latency and (b) distance traveled to find platform is shown for each day of testing. Wild-type animals exhibit a “learning curve” requiring less time and less distance (more ...)
Since treatment with GKW ameliorated a spatial memory impairment in Tg2576 mice, which is specifically associated with the appearance of Aβ
plaques, without producing any change in wild-type mouse memory, it would appear that the observed effect of GKW is specific to Aβ
. However, shows that there were no significant differences between levels of any of the forms of Aβ
in treated and untreated Tg2576 mice. This is in contrast to results obtained in PSAPP mice, a model for Alzheimer's disease (AD) where mice express both amyloid precursor protein and presenilin 1 mutations, in the long term (8 months). In these mice, administration of CA extract displayed in vitro
antioxidant effects and also reduced beta amyloid plaque burden [26
]. The PSAPP mice develop amyloid plaque pathology at an earlier age than Tg2576 [27
], permitting more rapid completion of antiamyloid experiments. However, loss of the age- and region-dependence of pathology diminishes the fidelity of this strain to some extent. Since GKW treatment attenuated the neurologic consequences of abnormal Aβ
deposition in Tg 2576 mice without changing Aβ
levels per se
, the ability of GKW to modulate the toxic effects of Aβ
were pursued in vitro
, with an emphasis on mechanisms which are either independent of or “downstream” from Aβ
Soluble and insoluble Aβ in cortical tissue from treated and untreated mice. Mean values ± SEM are shown. Treated and untreated Tg2576 mice did not differ significantly in levels of any of the measured isoforms of Aβ.
In preliminary experiments, GKW showed a moderate protective effect against toxicity due to exogenously added Aβ in SH-SY5Y human neuroblastoma cells in vitro (). Lactate dehydrogenase (LDH) release from these cells, which is inversely related to cell viability, was reduced in the presence of GKW (). The effect of GKW on toxicity due to endogenously generated Aβ was investigated in MC65 human neuroblastoma cells. GKW added to the cell culture medium prevented MC65 cell death following tetracycline withdrawal, in a dose-dependent manner (). Evidence from Western blots indicated that GKW may prevent the aggregation of Aβ in these cells. In a related study (data not shown), GKW inhibited Aβ-induced nitric oxide (NO) production in the RAW 264.7 macrophage cell line. Interestingly, GKW inhibited NO production induced by Aβ but did not influence LPS-induced NO levels in these cells. Taken together, these data indicated that components in GKW are able to modulate the toxic effects of Aβ.
Figure 4 (a) GKW at 100 and 200μg/mL shows a modest protective effect on SH-SY5Y cells from Aβ toxicity (beta amyloid 25–35) in vitro. The % of live cells is decreased on treatment with Aβ, an effect which is attenuated (more ...)
Figure 5 Effect of GKW on survival of MC65 cells following tetracycline withdrawal. Cell viability is expressed as a % of the cell growth obtained in control cultures containing tetracycline, TET(+). On withdrawal of tetracycline from the media, TET(−), (more ...)
Other potential mechanisms by which GKW may have improved cognitive function in the Tg2576 mice were also investigated but yielded negative results. No direct inhibitory effect of GKW (2.5 to 250μ
g/mL) on cholinesterase activity in vitro
was observed, whereas robust inhibition was observed using the positive control neostigmine. Effects of GKW on glutamate toxicity to rat cortical neurons were investigated. GKW (100 or 200μ
g/mL) was not directly toxic to rat cortical neurons nor did it protect the cells from toxicity induced by 250 and 1000μ
M glutamate (cell viability 30% and 25% of control, resp.). To examine potential antioxidant effects of GKW, SHSY5Y neuronal cells were exposed to H2
, which showed dose-dependent toxicity to SH-SY5Y cells over the range 125–500μ
M. GKW (50–200μ
g/mL), while not toxic to the cells, did not protect against toxicity at any of the peroxide concentrations tested. Thus, GKW does not appear to possess antioxidant effects.
Five drugs are currently FDA-approved for the symptomatic treatment of AD, targeting mechanisms of unclear relationship to the primary neurodegenerative process. The first four drugs (tacrine, donepezil, rivastigmine, and galantamine) are acetylcholinesterase inhibitors, which act by augmenting cholinergic neurotransmission [28
]. Each of these drugs has shown improved cognitive outcomes in treated AD patients compared to placebo-treated subjects, and the efficacy across drugs makes the case that cholinesterase inhibition is a viable treatment strategy for AD [28
]. The fifth and most recent antidementia drug to receive FDA approval is memantine. Memantine is a noncholinergic drug, acting instead at the NMDA class of glutamate receptor. In addition to showing clinical efficacy in human subjects with AD [29
], memantine has also been shown to improve cognition in murine models of cerebral amyloidosis. NMDA antagonism should therefore be considered among the possible mechanisms of action of treatments producing cognitive improvement in murine models of AD. Our in vitro
experiments showed no evidence that CA acts by way of these established therapeutic targets since there was no effect on cholinesterase activity or glutamate neurotoxicity.
In addition to the established therapies just described, strategies aimed at preventing the accumulation of, or promoting the clearance of, Aβ
are under study. Inhibitors of amyloid synthesis and immunization against Aβ
have diminished brain pathology and yielded cognitive and behavioral improvements in murine models of AD. Although amyloid synthesis inhibitors such as Lilly semagacestat have yielded negative clinical results [31
], antiamyloid immunotherapy (bapineuzumab) remains under development with some promising initial results [32
]. “Antiamyloid” strategies, therefore, represent another potential mechanism of cognition-enhancing therapies in AD. Our results do not show an effect of CA on Aβ
levels per se
but suggest that CA may protect neurons from Aβ
-induced neurotoxicity without actually changing brain levels of Aβ
. Most current clinical trials are focused on suppression of Aβ
levels, thus the neuroprotectant effect of CA described here represents a novel mechanism, potentially complementary to the drugs in development.
CA may also be a source of a novel chemical class for the treatment of AD. HPLC analysis of GKW revealed a complex mixture of substances (). This did not include asiatic acid or madecassic acid, well-known triterpene components of CA [33
], which were, however, extractable from the same plant material using ethanol (). The absence of asiatic acid in GKW is notable since asiatic acid has been previously associated with neuroprotective and neurotropic effects [35
]. However, an aqueous extract lacking asiatic acid produced robust behavioral effects in this study. lists the spectral characteristics of the major peaks found in GKW using LC-UV and LC-MS. UV spectra with maxima over 300
nm are indicative of a highly conjugated system, characteristic of flavonoids. CA is reported to be a rich source of quercetin [39
]. Flavonoids isolated to date in CA include 3-glucosylkaempferol, 3-glucosylquercetin and diosmin [40
]. The molecular weights listed in did not correspond to any of these 3 compounds, nor any other compounds isolated hitherto from CA (online chemical database, SciFinder). These findings imply the presence of potentially novel neuroactive ingredients in GKW which are yet to be fully characterized.
Figure 6 HPLC comparison of CA water and ethanol extracts made from the same batch of plant material. Asiatic acid (AA) and madecassic acid (MA) are detected in the ethanolic extract, but not in the water extract (GKW). The water extract GKW contains mostly very (more ...)
Table 1 Molecular weight and UV data obtained for GKW components using LC-MS and LC-UV. Reversed phase gradient HPLC chromatography with UV and negative ion mass spectral detection was performed as described in Section 2.
Compared to the wealth of animal data described earlier, there have been fewer studies on cognitive effects of CA in humans. In one study, 30 mentally retarded children aged 7–18 years showed improvement in their general abilities after receiving 500
mg daily of dried CA herb for 3 months [42
]. A more recent study [43
] showed that an extract of CA (250–750
mg daily for 2 months) improved cognitive performance in healthy, elderly volunteers. In a placebo-controlled study, administration of CA herb (0.5
g/kg body weight) to healthy, middle-aged volunteers for 2 months resulted in improvements in several tests of cognitive function [44
]. A study in elderly subjects with mild cognitive impairment found improvements in their cognitive test results, including the mini mental state examination, following administration of 500
mg dried CA twice a day for a 6-month period [45
The traditional use of CA as an enhancer of cognitive function is therefore well supported by in vitro, in vivo, and small-scale human studies conducted so far. The ultimate goal of these studies is to develop evidence for the clinical use of CA, or compounds derived from CA, in the treatment or prevention of AD. The combination of data from in vitro and animal studies in the present work supports the impression that CA has the potential for clinical benefit in AD by way of a novel mechanism of action. Well-designed, controlled clinical trials of CA in AD and other forms of cognitive impairment are clearly warranted. The characterization of the active components of CA and elucidation of their mechanism of action would support these clinical studies.