2.1 ELISA method

Principle of the Assay: The microtiter plate provided in this kit has been pre-coated with an antibody specific to Hcy. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific for Hcy and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB (3,3',5,5' tetramethylbenzidine) substrate solution is added to each well. Only those wells that contain Hcy, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm +/- 2 nm. The concentration of Hcy in the samples is then determined by comparing the O.D. of the samples to the standard curve. (the copy from MyBioSource co.)

3.1 The effect of green tea leaves on MMSE score and HA in blood

It is well-known that green tea can prevent Alzheimer disease (1), then we are interested in the healing-like effect of green tea on the cognition. 6 Alzheimer’s dementia patients in one care house agreed the ingestion of green tea leaves for every meal. They took this green tea leaves for 2 months. Before and after 2 months, their MMSE score and HA in blood measured.

6 Alzheimer’s patients were all female. Their ages were 77, 82, 86,87, 91, 91. Green tea powder induced MMSE change and their blood HA change. The results were shown in Figure 1. 3 patients showed the decrease change in blood HA (number 1,2,5), but two patients showed the increase (number 3,4) and 1 patient showed no change (number 6).

Figure 1: The effect of green tea leaves on MMSE score and HA in blood.

On the other hand, their MMSE score change were as follow: Number 1 :6 to 9, Number 2: 8 to 10, Number 3: 16 to 14, Number 4: 20 to 16 Number 5: 19 to 22, Number 6: 24 to 26.

The relationship between the blood HA change and MMSE score change. The result is shown in Figure 2. Clearly shown in Figure 2, the relationship between blood HA change (percent change) and MMSE score change is strong negative statistical significant (r=-0.96, p=0.00018, n=6).

Figure 2: The relationship between MMSE score change and HA in blood change Blood HA level change is shown as (after-before)/before x 100, MMSE scorechange is shown as after-before.

3.2 Open-labeled clinical trial of 91 AD patients

From 1 experiment, we understood that HA in blood was related to MMSE score. In other words, HA in blood is suggested to control MMSE score, or HA in blood may control the cognitive ability. If we can develop some supplement to decrease HA in blood, the cognitive recovery can be gotten in AD patients. First we observed the effect of our supplement on HA in blood. First 8 male volunteers who are suffering from memory problem and whose ages were 65-75 years old took our supplement for 2 months. Their memory problem were all disappeared and their blood HA were measured. The results are shown in Figure 3 Clearly shown in Figure 3, their blood HA were decreased after 2 months ingestion of our supplement.

Figure 3: Effect of HBF on blood homocysteic acid level. HA in blood was measured by ELISAmethod.

From this observation, we conducted the open-labeled clinical trial of 91 AD patients. The results are shown in Figure 4. 100% all patients increased their NM scores. These all date are summarized with statistically analysis. The result is shown in Table 1 (supplementary files). The results were really amazing. Even final stage of patients showed the recovery process as such. However they were still suffering from memory problem. But they could understand the conversation, they could greet the caregivers. They did not recognize their families, but after taking supplement, they could recognize their families. Some middle stage of AD patients, especially case 4, his cognitive recovery was perfect and he completely recovered to normal.

Figure 4: Open-labeled clinical trial of 91 ADpatients.

Figure 5 shows the MMSE score of all patients. Final stage patients did not increase their MMSE score. All final patients showed 0 score.

Figure 5: MMSE change induced by HBF.

4.1 The effect of green tea leaves on MMSE score and HA inblood

We were interested in the healing-like effect of green tea on the cognition of AD. Unfortunately this green tea healing-effect was transient. This healing- effect could not observed constantly. That is, after 3 months, this healing- effect was disappeared. Maybe green tea induced the drug-metabolizing enzyme.

We observed the relationship between blood HA level and MMSE score. And Figure 2 shows the very strong perfect relationship between blood HA level change and MMSE score change. From Figure 2 result, we can say that

blood HA can control the MMSE score change. That is, blood HA can control the cognitive ability in Alzheimer’s patient.

Why can the blood HA control cognitive ability inpatients?

First HA is a glutamate neurotransmitter and HA itself can induce neurodegeneration at a higher level with no amyloid (4), and the HA toxicity induces a calcium influx (5) or a mitochondrial inhibition (6) or oxidative stress (4) to bring the neuronal disfunction.

NMDA receptor in blood brain barrier (BBB) can control its permeability [7,8]. Then the blood HA can increase the permeability of BBB and HA can pass through BBB and enters into hippocampus, because hippocampus’s BBB is very thin [9]. And entered HA can interact with already accumulated amyloid beta and HA increases its neurotoxicity, because amyloid beta increases HA toxicity (unpublished observation).

In a study published in Nature [10,11], Stanford University School of Medicine scientists have found substances in the blood of old mice that makes young brains act older. These substances, whose levels rise with increasing age, appear to inhibit the brain’s ability to produce new nerve cells critical to memory and learning.

The findings raise the question of whether it might be possible to shield the brain from aging by eliminating or mitigating the effects of these apparently detrimental blood-borne substances.

APOE4 is the gene involved in the mechanism of development of AD. APOE4 increases the permeability of Blood Brain Barrier (BBB) [12], which allows the pathogen in the peripheral blood to pass BBB and disturb the brain functions. This report indicates the possibility of APOE4 early-onset gene.

Finally, the third report [13] describes an actual early patient of AD showing the destruction of hippocampal BBB. However, these reports did not describe which pathogen in the blood is involved and what resulted in an increased BBB permeability in aged hippocampus.

It has been reported that homocysteic acid (HA) is the probable pathogen of AD in blood [14-17], and it increases BBB permeability by NMDA receptor activation [18]. Especially it is interesting that HA can induce the amyloid β-42 which accumulated into neuronal cell [17]. HA is known as a glutamate receptor agonist [19]. JAD (14) study reported a significant negative relationship between MMSE scores and HA levels in blood. Thus, HA can be one of the pathogens in blood.

4.2 Open-labeled clinical trial

First we should give comments about our supplement.

Our HBF supplement contained Ferulic acid (FA). This FA chemical structure is very similar as that of HA, which can compete HA at NMDA receptor of BBB. In fact the uptake competition between HA and FA of 10 uM in cultured cell of the mouse fetus brain was observed (unpublished observation). Also HBF produced the hydrogen gas in blood which could reduce the sulfite group of HA to homocysteine. In fact the some patients showed the decrease in HA was observed parallel to the increase in homocysteine in blood.

Second we should give comment for double-blind clinical trial for the final stage of AD patients. They cannot understand what happened in their world. In other words, I am sorry to say so, they are almost experimental animal, which can not induce the placebo effect. Also caregivers and doctors also observed the completely their qualitative change of behavior. For example, one patients showed all day comma, but after taking our HBF, they showed awake and said something to neighbors. So in case of final stage of patients, they needed not double-blind test.

Now we can present the clinical trial of brain food which can destroy blood HA on the recovery of cognitive impairment ofAD patients. The results are very impressive. Even the final stage of patient could recover their cognitive behavior, could understand his or her families, and finally could understand the conversations with others. However they still suffered from memory problem. In fact all final stage of patients showed no increasing of MMSE score. All final patients showed 0 score before and after the HBF ingestion. Because the hippocampal structure of a final stage of patients would be destroyed and HBF supplement could not recover the hippocampal structure. From Figure 5, it is clear that MMSE scale measurement is not better than NM scale, because at even MMSE score 0, patients showed the recovery process of NM scale. Because the some cognitive behavior is not dependent on the hippocampal function [20].

However middle stage of patients increased their MMSE score, because their hippocampal activities could be recovered and consequently their MMSE score increased compared with before one.

Our clinical trial can offer the some hopes in AD field. That is, the cognitive impairment of AD can be recovered by the adequate treatments such as brain food or drugs so on. These adequate treatments should destroy the possible pathogens in blood such as HA or beta2-microglobulin. Then this adequate treatment can recover the cognitive reserve abilities of AD patients. Because it knows that the peoples with even Alzheimer’s pathological structure’s brain can show the normal brain works, and it says that this is cognitive reserve ability.

So we present the hope that the adequate treatments for AD can bringback their cognitive reserve abilities.