U.S. patent application number 16/683419 was filed with the patent office on 2021-04-01 for non-pharmaceutical bactericidal composition against helicobacter pylori.
This patent application is currently assigned to ECO-GEO BIO-TECHNOLOGY COMPANY LIMITED.. The applicant listed for this patent is ECO-GEO BIO-TECHNOLOGY COMPANY LIMITED.. Invention is credited to Fu-An Chen, Ta-Lu Shen.
Application Number | 20210093595 16/683419 |
Document ID | / |
Family ID | 1000004472270 |
Filed Date | 2021-04-01 |
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United States Patent
Application |
20210093595 |
Kind Code |
A1 |
Shen; Ta-Lu ; et
al. |
April 1, 2021 |
NON-PHARMACEUTICAL BACTERICIDAL COMPOSITION AGAINST HELICOBACTER
PYLORI
Abstract
The present invention provides a non-pharmaceutical bactericidal
composition against a Helicobacter pylori, including a first daily
edible component and a second daily edible component, wherein the
first daily edible component has a molecular state and a
dissociated state while in an aqueous solution state, the first
daily edible component of the molecular state is used for
performing functions of killing the Helicobacter pylori and
inhibiting an ascorbate oxidase activity of the Helicobacter pylori
in an environment with a pH value below 5, and the first daily
edible component is a citric acid, a malic acid or a combination
thereof, and wherein the second daily edible component is a salt
used to form a buffer combination with the first daily edible
component, and used to neutralize a bicarbonate, so that the first
daily edible component can continue its killing of the Helicobacter
pylori effectively and inhibiting the ascorbate oxidase activity of
the Helicobacter pylori, and the salt is a salt of the citric acid,
a salt of the malic acid or a combination thereof.
Inventors: |
Shen; Ta-Lu; (Taipei City,
TW) ; Chen; Fu-An; (Taipei City, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ECO-GEO BIO-TECHNOLOGY COMPANY LIMITED. |
Taipei City |
|
TW |
|
|
Assignee: |
ECO-GEO BIO-TECHNOLOGY COMPANY
LIMITED.
Taipei City
TW
|
Family ID: |
1000004472270 |
Appl. No.: |
16/683419 |
Filed: |
November 14, 2019 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/194 20130101;
A61K 31/375 20130101; A61K 9/0056 20130101 |
International
Class: |
A61K 31/194 20060101
A61K031/194; A61K 9/00 20060101 A61K009/00; A61K 31/375 20060101
A61K031/375 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 27, 2019 |
TW |
108135302 |
Claims
1. A non-pharmaceutical bactericidal composition against a
Helicobacter pylori, the non-pharmaceutical bactericidal
composition comprising: a first daily edible component being malic
acid; a used to form a buffer combination with the malic acid
wherein the salt is sodium malate; and a second daily edible
component being ascorbic acid.
2. The non-pharmaceutical bactericidal composition as claimed in
claim 1, wherein the ascorbic acid has a concentration being in a
range from 3000 ppm to 10000 ppm.
3. The non-pharmaceutical bactericidal composition as claimed in
claim 1, wherein the ascorbic acid has a concentration of 9000
ppm.
4. (canceled)
5. A non-pharmaceutical bactericidal composition against a
Helicobacter pylori, the non-pharmaceutical bactericidal
composition comprising: a first daily edible component being a
buffer combination consisting of an organic acid and a salt of the
organic acid, wherein the organic acid is malic acid and a second
daily edible being ascorbic acid.
6. The non-pharmaceutical bactericidal composition as claimed in
claim 5, wherein the non-pharmaceutical bactericidal composition
has a pH value below 5.
7. The non-pharmaceutical bactericidal composition as claimed in
claim 5, wherein the non-pharmaceutical bactericidal composition
has a pH value being in a range from 3 to 5.
8. The non-pharmaceutical bactericidal composition as claimed in
claim 5, wherein the non-pharmaceutical bactericidal composition
has a pH value of 3.
9. A non-pharmaceutical bactericidal composition against a
Helicobacter pylori, the non-pharmaceutical bactericidal
composition comprising: a first daily edible component being malic
acid; and a second daily edible component, being a salt, used to
form a buffer combination with the first daily edible component,
malic acid, wherein the non-pharmaceutical bactericidal composition
has a pH value of 3.
10. (canceled)
11. The non-pharmaceutical bactericidal composition as claimed in
claim 9, wherein the salt is sodium malate.
12. The non-pharmaceutical bactericidal composition as claimed in
claim 9, further comprising an ascorbic acid for killing the
Helicobacter pylori more thoroughly in presence of the first daily
edible component and the second daily edible component.
13. A method for treating a Helicobacter pylori infection, the
method comprising steps of: providing a non-pharmaceutical
bactericidal composition including a first daily edible component
and a second daily edible component, wherein the first daily edible
component has a molecular state and a dissociated state while in an
aqueous solution state, the first daily edible component of the
molecular state is used for performing functions of killing a
Helicobacter pylori and inhibiting an ascorbate oxidase activity of
the Helicobacter pylori in an environment with a pH value below 5,
the first daily edible component is a citric acid, a malic acid or
a combination thereof, and the second daily edible component is a
salt, is used to form a buffer combination with the first daily
edible component, and is used to neutralize a bicarbonate, so that
the first daily edible component can continue its killing of the
Helicobacter pylori effectively and inhibiting the ascorbate
oxidase activity of the Helicobacter pylori, wherein the salt is a
salt of the citric acid, a salt of the malic acid or a combination
thereof; and administering the non-pharmaceutical bactericidal
composition to a mammal.
14. The method as claimed in claim 13, wherein the ascorbic acid
has a concentration being in a range from 3000 ppm to 10000
ppm.
15. The method as claimed in claim 13, wherein the ascorbic acid
has a concentration of 9000 ppm.
16. The method as claimed in claim 13, wherein the
non-pharmaceutical bactericidal composition has a pH value below
5.
17. The method as claimed in claim 13, wherein the
non-pharmaceutical bactericidal composition has a pH value being in
a range from 3 to 5.
18. The method as claimed in claim 13, wherein the
non-pharmaceutical bactericidal composition has a pH value of
3.
19. The method as claimed in claim 13, wherein the
non-pharmaceutical bactericidal composition further comprises an
ascorbic acid for killing the Helicobacter pylori more thoroughly
in the presence of the first daily edible component and the second
daily edible component.
20. The method as claimed in claim 13, wherein the mammal is a
human being.
Description
CROSS-REFERENCED TO RELATED APPLICATION AND CLAIM OF PRIORITY
[0001] The application claims the benefit of Taiwan Patent
Application No. 108135302, filed on Sep. 27, 2019, at the Taiwan
Intellectual Property Office, the disclosures of which are
incorporated herein in their entirety by reference.
FIELD OF THE INVENTION
[0002] The present invention refers to a non-pharmaceutical
bactericidal composition against Helicobacter pylori, specifically
to a non-pharmaceutical bactericidal composition against
Helicobacter pylori for performing the functions of killing the
Helicobacter pylori and inhibiting the ascorbate oxidase activity
of the Helicobacter pylori in an environment below pH5.
BACKGROUND OF THE INVENTION
[0003] Global Helicobacter pylori infection rate exceeds 50% of the
global population, and 10%-15% of those infections result in the
development of peptic ulcer diseases. Helicobacter pylori is
associated with 95% of duodenal ulcers and 80% of gastric ulcers.
1%-3% of infected individuals develop gastric cancer. Therefore,
Helicobacter pylori is classified by the World Health Organization
(WHO) as a primary carcinogen (please refer to D(1)).
[0004] In recent years, studies of the survival and pathogenesis of
Helicobacter pylori in gastric mucus are the foci of the medical
community to observe the microbial pathogenesis (chronic
infections) in mucus in general. The medical community hopes to
work out a solution to eliminate other toxic pathogenic
microorganisms in mucus in general via eliminating Helicobacter
pylori effectively in the mucus, as intestinal dysbiosis is often
the cause of some chronic diseases such as obesity, diabetes and
inflammatory diseases (please refer to D(1)).
[0005] Helicobacter pylori is one of the most interesting
microorganisms in the human digestive tract, and is associated in
particular with active inflammatory changes within the
gastroduodenal mucus (about 50-65% with the gastric ulcer and about
95% with the duodenal ulcer). According to research, Helicobacter
pylori infected persons should eliminate Helicobacter pylori
completely to avoid ulcer recurrence and ulcer complications
(please refer to D(2)).
[0006] Helicobacter pylori is a smart bacterium that can coexist
peacefully with the human host once it resides in the stomach of a
human body. Clinically, it was found that 80% to 90% of persons
infected with Helicobacter pylori have chronic gastritis, but only
20% of them show symptoms; 15% to 20% of the infected persons
develop gastric ulcer or duodenal ulcer, 1% to 3% of carriers of
Helicobacter pylori develop gastric cancer, and one in one thousand
to one in ten thousand of them have gastric lymphoma problems.
Compared with someone uninfected by Helicobacter pylori, the risk
of developing stomach cancer of the infected persons is increased
by 6 times (please refer to D(3)).
[0007] Recent evidence suggests that prolonged colonization of the
gastric mucus by Helicobacter pylori may lead to chronic atrophic
gastritis and subsequently adenocarcinoma (please refer to
D(2)).
[0008] Future perspectives for therapies of Helicobacter pylori can
be discussed in the following aspects (1) to (3) (please refer to
D(4)): [0009] (1) The bottlenecks of today's therapy in general:
[0010] A rising antibiotic resistance of Helicobacter pylori [0011]
Health concerns over long-term use (a course of 10-14 days, 3 to 4
times per day) [0012] High single dosage required due to antibiotic
resistance (500 to 1000 mg of a single antibiotic treatment) [0013]
A rising recurrence [0014] Other complications induced by
antibiotics include:
[0015] a. obesity
[0016] b. fatty liver disease
[0017] c. Type 2 diabetes
[0018] The expected primary effect of antibiotics is significantly
affected and reduced by the presence of the gastric mucus, which
results in enhanced side effects such as the liver, kidney and
stomach damages due to the increase of dosage to compensate for the
reduction of the intended primary effect.
[0019] The adverse effects of antibiotics may lead to discomfort of
the patients. *The patients having cancer attributable solely to
Helicobacter pylori infection (780,000 cases) account for 6.2%, a
very high proportion, of all cancers yearly worldwide.
[0020] (2) Some advanced versions of traditional "triple therapy"
nowadays:
[0021] Bismuth is sometimes added to become "quadruple therapy" and
to increase the therapeutic effect, and the main effect of bismuth
is to inhibit the urease activity.
[0022] N-acetylcysteine (NAC) replaces bismuth to be added into the
triple therapy, or to be used alone.
[0023] Main side effects of the NAC:
[0024] a. It has a high toxicity and needs to be used in a high
dosage.
[0025] b. It reduces the viscosity of the gastric mucus effectively
but tends to cause ulcer deterioration as well.
[0026] (3) Latest research and development directions for the
therapy of Helicobacter pylori infection:
[0027] narrow-spectrum
[0028] non-antibiotic
[0029] Helicobacter pylori mainly colonizes the human antrum mucosa
(please refer to FIG. 1 in D(1)). The pH values in the gastric
mucus naturally form a pH-gradient. The main cause is that the
apical portion of the epithelial cells secretes bicarbonate
(HCO.sub.3-) into the mucus to fight against the acid attack from
the gastric lumen (please refer to the abstract in D(5)).
Therefore, the composition of the present invention must be able to
fight against alkali (i.e. bicarbonate (HCO.sub.3-)) in the gastric
mucus and sterilize bacteria in a low pH mucus-microenvironment
created by this composition. Furthermore, gastric juice contains
urea, and Helicobacter pylori will hydrolyze the urea there to
raise the pH value of the microenvironment while the ambient pH is
low. Generally, antibiotics are active between pH 5-8, so they need
to be taken in combination with an antacid or a Proton Pump
Inhibitor (PPI) to neutralize the gastric acid or inhibit gastric
acid secretion to avoid low pH in the stomach which causes
deactivation of antibiotics (please refer to FIG. 3 in D(6)). In
summary, the bicarbonate (HCO.sub.3-) in the gastric mucus is vital
for the colonization of Helicobacter pylori. In addition, the
urease there is also another crucial factor that helps Helicobacter
pylori colonize the gastric mucus.
[0030] The "bicarbonate barrier" in the gastric antral mucus gel
layer is described in detail as follows:
[0031] The colonization of the Helicobacter pylori in the gastric
antral mucus gel layer is shown in FIG. 1 in D(1).
[0032] Regarding the pH-gradient in the gastric antral mucus gel
layer, please refer to the abstract in D(5). According to D(5), the
characteristics of the "bicarbonate barrier" of the gastric mucosa
include a pH-gradient (the pH value varies from low to high from
the gastric lumen to the apical portion of the epithelial cells), a
resistance to acid, and a resistance to pepsin. In the in vivo
experiment in rats on page C6, FIG. 2 in D(5), group A used
pentagastrin to stimulate gastric parietal cells to secrete gastric
juice, and group B used ranitidine to inhibit gastric acid
secretion. Regarding the pH changes in the mucoepithelial
interface, a rapid decrease in pH from 7 to 2-3,when the gastric
acid secretion is inhibited temporarily, and a bounce back to 6 -7
after about 20 minutes were observed in group B. On Page C6, right
Column, Lines 4-20 in D(5), it clearly stated that the substances
having molecular weight (Da) less than 400, include ions like
hydrogen ion and small-sized molecules, are proved by in vivo
experiments that they are free to pass through the mucus.
Therefore, the main interfering factor which provides the
prevention of acid attack in the mucus-microenvironment should be
bicarbonate (HCO.sub.3-).
[0033] Therefore, for the effective elimination of Helicobacter
pylori in the stomach, it is crucial to find a composition that is
able to fight against bicarbonate (HCO.sub.3-) in the gastric mucus
and sterilize bacteria at the same time in such a low pH
mucus-microenvironment created by this composition (please refer to
FIG. 1 and page 4/11, right Column, Lines 8-20 in D(7) regarding
the organic acid kills Gram-negative bacteria including
Helicobacter pylori).
SUMMARY OF THE INVENTION
[0034] One aspect of the present invention is to provide a
non-pharmaceutical bactericidal composition against a Helicobacter
pylori, the non-pharmaceutical bactericidal composition includes a
first daily edible component, a second daily edible component, a
salt and a third daily edible component, wherein the first daily
edible component has a molecular state and a dissociated state
while in an aqueous solution state, the first daily edible
component of the molecular state is used for performing functions
of killing the Helicobacter pylori and inhibiting an ascorbate
oxidase activity of the Helicobacter pylori in an environment with
a pH value below 5, and the first daily edible component is a
citric acid, a malic acid or a combination thereof, wherein the
second daily edible component is used for neutralizing a
bicarbonate and being a citric acid, a malic acid or a combination
thereof, wherein the salt is used to form a buffer combination with
the citric acid, the malic acid or the combination thereof, the
salt is a salt of the citric acid, a salt of the malic acid or a
combination thereof, and wherein the third daily edible component
is used for killing the Helicobacter pylori more thoroughly in
presence of the first daily edible component, the second daily
edible component and the salt, and the third daily edible component
is an ascorbic acid.
[0035] Another aspect of the present invention is to provide a
non-pharmaceutical bactericidal composition against a Helicobacter
pylori, the non-pharmaceutical bactericidal composition includes a
first daily edible component and a second daily edible component,
wherein the first daily edible component is a buffer combination
consisting of an organic acid and a salt of the organic acid, the
first daily edible component is used for neutralizing a bicarbonate
and performing functions of killing the Helicobacter pylori and
inhibiting an ascorbate oxidase activity of the Helicobacter pylori
in an environment with a pH value below 5, and the organic acid is
a citric acid, a malic acid or a combination thereof, and wherein
the second daily edible component is used for killing the
Helicobacter pylori more thoroughly in presence of the first daily
edible component, and the second daily edible component is an
ascorbic acid.
[0036] A further aspect of the present invention is to provide a
non-pharmaceutical bactericidal composition against a Helicobacter
pylori, the non-pharmaceutical bactericidal composition includes a
first daily edible component and a second daily edible component,
wherein the first daily edible component has a molecular state and
a dissociated state while in an aqueous solution state, the first
daily edible component of the molecular state is used for
performing functions of killing the Helicobacter pylori and
inhibiting an ascorbate oxidase activity of the Helicobacter pylori
in an environment with a pH value below 5, and the first daily
edible component is a citric acid, a malic acid or a combination
thereof, and wherein the second daily edible component is a salt
used to form a buffer combination with the first daily edible
component, and used to neutralize a bicarbonate, so that the first
daily edible component can continue its killing of the Helicobacter
pylori effectively and inhibiting the ascorbate oxidase activity of
the Helicobacter pylori, and the salt is a salt of the citric acid,
a salt of the malic acid or a combination thereof.
[0037] A further aspect of the present invention is to provide a
method for treating a Helicobacter pylori infection, the method
including steps of providing a non-pharmaceutical bactericidal
composition including a first daily edible component and a second
daily edible component, and administering the non-pharmaceutical
bactericidal composition to a mammal, wherein the first daily
edible component has a molecular state and a dissociated state
while in an aqueous solution state, the first daily edible
component of the molecular state is used for performing functions
of killing a Helicobacter pylori and inhibiting an ascorbate
oxidase activity of the Helicobacter pylori in an environment with
a pH value below 5, the first daily edible component is a citric
acid, a malic acid or a combination thereof, and the second daily
edible component is a salt, is used to form a buffer combination
with the first daily edible component, and is used to neutralize a
bicarbonate, so that the first daily edible component can continue
its killing of the Helicobacter pylori effectively and inhibiting
the ascorbate oxidase activity of the Helicobacter pylori, and the
salt is a salt of the citric acid, a salt of the malic acid or a
combination thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] Other objectives, advantages and efficacies of the present
invention will be described in detail below taken from the
preferred embodiments with reference to the accompanying
drawings.
[0039] FIG. 1 is a histogram showing the results of an
antibacterial experiment against Helicobacter pylori of the citrate
buffer combination (with or without the ascorbic acid) and a
phosphate buffer combination (with or without the ascorbic acid) at
pH 3.0, 4.0 and 5.0.
[0040] FIG. 2 is a histogram showing the results of an
antibacterial experiment against Helicobacter pylori of the citrate
buffer combination with the ascorbic acid at pH 3.0.
[0041] FIG. 3 is a histogram showing the results of an
antibacterial experiment against Helicobacter pylori of the malate
buffer combination, a lactate buffer combination, the citrate
buffer combination and the phosphate buffer combination at pH 3.0,
4.0 and 5.0.
[0042] FIG. 4 is a histogram showing the results of an
antibacterial experiment against Helicobacter pylori of the malate
buffer combination with the ascorbic acid, the lactate buffer
combination with the ascorbic acid, the citrate buffer combination
with the ascorbic acid and the phosphate buffer combination with
the ascorbic acid at pH 3.0, 4.0 and 5.0.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0043] The following are embodiments of the present invention.
[0044] I. Experimental Instruments [0045] Autoclave (FD5OR,
Zealway, USA) [0046] Microcentrifuge (Spectrafuge.TM. 24D, Labnet,
USA) [0047] Electronic Microbalance (XR 205SM-DR, Precisa,
Switzerland) [0048] Electronic Balance (PB1502-S, Mettler Toledo,
USA) [0049] pH Meter (Delta 320, Mettler Toledo, USA) [0050]
Orbital Shaker Incubator (721 SR, HIPOINT, Taiwan) [0051]
Biological Safety Cabinets (Ever Win Technology, Taiwan) [0052]
Microplate reader (SPECTROstar.RTM. Nano, BMG Labtech, Germany)
[0053] Water Purification system (Polycon DIU-3, Scienpak
Enterprise, Taiwan) [0054] Pipette 10-100 .mu.L (Acura .RTM. manual
825, Socorex, Switzerland) [0055] Pipette 20-200 .mu.L (Acura .RTM.
manual 825, Socorex, Switzerland) [0056] Pipette 100-1000 .mu.L
(Acura .RTM. manual 825, Socorex, Switzerland) [0057] Serum Bottle
1000 mL (Schott, Germany) [0058] Serum Bottle 500 mL (Schott,
Germany) [0059] Serum Bottle 50 mL (Schott, Germany) [0060] Beaker
1000 mL (Kimax, USA) [0061] Beaker 500 mL (Kimax, USA) [0062]
Beaker 100 mL (Kimax, USA) [0063] Stirrer (Dogger, Taiwan) [0064]
Glass Triangle-headed Cell Spreader (Dogger, Taiwan) [0065] Alcohol
Burner (Dogger, Taiwan) [0066] AnaeroPack Jar (MGC AnaeroPack.RTM.,
Mitsubishi Gas Chemical, Japan)
[0067] II. Experimental Consumables [0068] Pipetmen tips 10-200
.mu.L (Vertex.RTM., USA) [0069] Pipetmen tips 100-1000 .mu.L
(Vertex.RTM., USA) [0070] Microcentrifuge Tubes 1.5 mL (Jet
Biofil.RTM., China) [0071] Centrifuge Tubes 15 mL (Jet Biofil.RTM.,
China) [0072] Centrifuge Tubes 50 mL (Jet Biofil.RTM., China)
[0073] Cell and Tissue Culture Dishes 9.0 cm (Jet Biofil.RTM.,
China) [0074] Cell and Tissue Culture Plates 48 well (Jet
Biofil.RTM., China) [0075] Cell and Tissue Culture Plates 96 well
(Jet Biofil.RTM., China) [0076] Syringe Driven Filter 0.22 .mu.m
(PVDF membrane, Jet Biofil.RTM., China) [0077] Disposable Syringe 1
mL (Terumo, Japan) [0078] Genbox Microaer (Ref. 96125, bioMerieux,
France)
[0079] III. Experimental Drugs [0080] Urea (Nihon Shiyaku, Taiwan)
[0081] Citric Acid (Nihon Shiyaku, Taiwan) [0082] Sodium Citrate
(Nihon Shiyaku, Taiwan) [0083] Potassium Dihydrogen Phosphate(Nihon
Shiyaku, Taiwan) [0084] Sodium Hydrogen Phosphate (Nihon Shiyaku,
Taiwan) [0085] DL-Malic acid (Shun Ching Raw Material, Taiwan)
[0086] Sodium DL-Malate (Gemfont, Taiwan) [0087] Lactic acid (Alfa
Aesar, USA) [0088] Sodium Lactate (Hsin Eing, Taiwan) [0089] Sodium
hydrogen carbonate (Shimakyu yakuhin, Japan) [0090] Sodium Chloride
(Nihon Shiyaku, Taiwan) [0091] Hydrogen Chloride (Nihon Shiyaku,
Taiwan) [0092] Sodium Hydroxide (Nihon Shiyaku, Taiwan) [0093]
BBL.TM. Brucella broth (BD Biosciences, USA) [0094] Bacto.TM. Agar
(BD Biosciences, USA) [0095] Donor Equine Serum (Hyclone, USA)
[0096] L-Ascorbic acid (Sigma, USA)
[0097] IV. Experimental Methods
Embodiment 1
[0098] The antibacterial experiment against Helicobacter pylori of
the citrate buffer combination (with or without the ascorbic acid)
and a phosphate buffer combination (with or without the ascorbic
acid)(viability check-point:0/20/40/60 minutes, triple
repetition).
[0099] Bacteria Strain and Culturing Method
[0100] The bacteria strain used in the experiment was Helicobacter
pylori strain 26695 (ATCC700392), and the bacteria were subcultured
on Brucella solid medium (Brucella broth 28 g/L, Agar 15 g/L and
10% (v/v) Horse serum, pH 7.0). After the bacteria were seeded by
streaking method, they were cultured in a humidified anaerobic
chamber at 37.degree. C. for 72 hours using a microaerobic bag
(Genbox microaer, BioMerieux, 96125).
[0101] Each culture medium was filtered through a 0.22 .mu.m
injection type filter for use. Brucella broth containing 10 mg/mL
of NaHCO.sub.3 was added to the Helicobacter pylori subcultured for
72 hours, bacterial concentration was adjusted to 10.sup.7-10.sup.8
CFU/mL as a bacterial solution, the bacterial solution was mixed
with each culture solution in a 1:1 ratio, and the mixture was
incubated at 37.degree. C. in a humidified anaerobic chamber using
a micro-aerobic bag (Genbox microaer, BioMerieux, 96125).
[0102] The citrate buffer solutions were prepared by mixing the
citric acid and sodium citrate in different ratios according to
their pH values, and the unmodulable portion was prepared by HCl
and NaOH to reach a constant pH value. The phosphate buffer
solutions were prepared by mixing KH.sub.2PO.sub.4 and
Na.sub.2HPO.sub.4 in different ratios according to their pH values,
and the unmodulable portion was prepared by HCl and NaOH to reach a
constant pH value. The pH of the medium was adjusted with HCl and
NaOH. After each culture medium was adjusted to a constant pH value
using the method above, a constant concentration of ascorbic acid
was additionally added to each culture medium. The components of
the bacterial solution and each culture medium were as follows:
[0103] Bacteria solution: (Brucella broth 28 g/L, NaHCO.sub.3 10
g/L and 10 mM urea).
[0104] Group 1: N3 (Brucella broth 28 g/L and 10 mM urea, pH
3.0).
[0105] Group 2: N3V3000 (Brucella broth 28 g/L and 10 mM urea, pH
3.0)+(6000 mg/L Vitamin C).
[0106] Group 3: N4 (Brucella broth 28 g/L and 10 mM urea, pH
4.0).
[0107] Group 4: N4V3000 (Brucella broth 28 g/L and 10 mM urea, pH
4.0)+(6000 mg/L Vitamin C).
[0108] Group 5: N5 (Brucella broth 28 g/L and 10 mM urea, pH
5.0).
[0109] Group 6: N5V3000 (Brucella broth 28 g/L and 10 mM urea, pH
5.0) +(6000 mg/L Vitamin C).
[0110] Group 7: P3 (Brucella broth 28 g/L, 0.1 M phosphate buffered
saline (PBS) and 10 mM urea, pH 3.0).
[0111] Group 8: P3V3000 (Brucella broth 28g/L, 0.1 M phosphate
buffered saline (PBS) and 10 mM urea, pH 3.0)+(6000 mg/L Vitamin
C).
[0112] Group 9: P4 (Brucella broth 28 g/L, 0.1 M phosphate buffered
saline (PBS) and 10 mM urea, pH 4.0).
[0113] Group 10: P4V3000 (Brucella broth 28 g/L, 0.1 M phosphate
buffered saline (PBS) and 10 mM urea, pH 4.0)+(6000 mg/L Vitamin
C).
[0114] Group 11: P5 (Brucella broth 28 g/L, 0.1 M phosphate
buffered saline (PBS) and 10 mM urea, pH 5.0).
[0115] Group 12: P5V3000 (Brucella broth 28 g/L, 0.1 M Phosphate
buffered saline (PBS) and 10 mM urea, pH 5.0)+(6000 mg/L Vitamin
C).
[0116] Group 13: C3 (Brucella broth 28 g/L, 0.1 M citrate buffer
and 10 mM urea, pH 3.0).
[0117] Group 14: C3V3000 (Brucella broth 28 g/L, 0.1 M citrate
buffer and 10 mM urea, pH 3.0)+(6000 mg/L Vitamin C).
[0118] Group 15: C4 (Brucella broth 28 g/L, 0.1 M citrate buffer
and 10 mM urea, pH 4.0).
[0119] Group 16: C4V3000 (Brucella broth 28 g/L, 0.1 M citrate
buffer and 10 mM urea, pH 4.0)+(6000 mg/L Vitamin C).
[0120] Group 17: C5 (Brucella broth 28 g/L, 0.1 M citrate buffer
and 10 mM urea, pH 5.0).
[0121] Group 18: C5V3000 (Brucella broth 28 g/L, 0.1 M citrate
buffer and 10 mM urea, pH 5.0)+(6000 mg/L Vitamin C).
[0122] After each culture medium incubated according to the
viability check-point (0/20/40/60 minutes) was serially diluted 10
times with physiological saline, the bacteria solutions diluted at
each magnification was applied to the Brucella solid medium for
subculture by a coating method, and the coated plates were
incubated at 37.degree. C. for 72 hours in a humidified anaerobic
chamber using a micro-aerobic package (Genbox microaer, BioMerieux,
96125). Finally, the viable count of each culture medium was
calculated.
Embodiment 2
[0123] The antibacterial experiment against Helicobacter pylori of
the citrate buffer combination with the ascorbic acid (viability
check-point: 0/20/40/60 minutes, triple repetition).
[0124] Bacteria Strain and Culturing Method
[0125] The bacteria strain used in the experiment was Helicobacter
pylori strain 26695 (ATCC700392), and the bacteria were subcultured
on Brucella solid medium (Brucella broth 28 g/L, Agar 15 g/L and
10% (v/v) Horse serum, pH 7.0). After the bacteria were seeded by
streaking method, they were cultured in a humidified anaerobic
chamber at 37.degree. C. for 72 hours using a microaerobic bag
(Genbox microaer, BioMerieux, 96125).
[0126] Each culture medium was filtered through a 0.22 .mu.m
injection type filter for use. Brucella broth containing 10 mg/mL
of NaHCO.sub.3 was added to the Helicobacter pylori subcultured for
72 hours, bacterial concentration was adjusted to 10.sup.7-10.sup.8
CFU/mL as a bacterial solution, the bacterial solution was mixed
with each culture solution in a 1:1 ratio, and the mixture was
incubated at 37.degree. C. in a humidified anaerobic chamber using
a micro-aerobic bag (Genbox microaer, BioMerieux, 96125).
[0127] The citrate buffer solutions were prepared by mixing the
citric acid and sodium citrate in different ratios according to
their pH values, and the unmodulable portion was prepared by HCl
and NaOH to reach a constant pH value. The phosphate buffer
solutions were prepared by mixing KH.sub.2PO.sub.4 and
Na.sub.2HPO.sub.4 in different ratios according to their pH values,
and the unmodulable portion was prepared by HCl and NaOH to reach a
constant pH value. The pH of the medium was adjusted with HCl and
NaOH. After each culture medium was adjusted to a constant pH value
using the method above, a constant concentration of ascorbic acid
was additionally added to each culture medium.
The components of the bacterial solution and each culture medium
were as follows:
[0128] Group 1: C3V3000 (Brucella broth 28 g/L, 0.1 M citrate
buffer and 10 mM urea, pH 3.0)+(6000 mg/L Vitamin C).
[0129] Group 2: C3V6000 (Brucella broth 28 g/L, 0.1 M citrate
buffer and 10 mM urea, pH 3.0)+(12000 mg/L Vitamin C).
[0130] Group 3: C3V9000 (Brucella broth 28 g/L, 0.1 M citrate
buffer and 10 mM urea, pH 3.0)+(18000 mg/L Vitamin C).
[0131] After each culture medium incubated according to the
viability check-point (0/20/40/60 minutes) was serially diluted 10
times with physiological saline, the bacteria solutions diluted at
each magnification was applied to the Brucella solid medium for
subculture by a coating method, and the coated plates were
incubated at 37.degree. C. for 72 hours in a humidified anaerobic
chamber using a micro-aerobic package (Genbox microaer, BioMerieux,
96125). Finally, the viable count of each culture medium was
calculated.
[0132] Regarding the details for the addition of urea, please refer
to references D(8) and D(9), and regarding the details for the
addition of NaHCO.sub.3, please refer to references D(6), D(10) and
D(11).
Embodiment 3
[0133] The antibacterial experiment against Helicobacter pylori of
the malate buffer combination, the lactate buffer combination, the
citrate buffer combination and the phosphate buffer combination
(viability check-point: 0/20/40/60 minutes, triple repetition).
[0134] Bacteria Strain and Culturing Method
[0135] The bacteria strain used in the experiment was Helicobacter
pylori strain 26695 (ATCC700392), and the bacteria were subcultured
on Brucella solid medium (Brucella broth 28 g/L, Agar 15 g/L and
10% (v/v) Horse serum, pH 7.0). After the bacteria were seeded by
streaking method, they were cultured in a humidified anaerobic
chamber at 37.degree. C. for 72 hours using a microaerobic bag
(Genbox microaer, BioMerieux, 96125).
[0136] Each culture medium was filtered through a 0.22 .mu.m
injection type filter for use. Brucella broth containing 10 mg/mL
of NaHCO.sub.3 was added to the Helicobacter pylori subcultured for
72 hours, bacterial concentration was adjusted to 10.sup.7-10.sup.8
CFU/mL as a bacterial solution, the bacterial solution was mixed
with each culture solution in a 1:1 ratio, and the mixture was
incubated at 37.degree. C. in a humidified anaerobic chamber using
a micro-aerobic bag (Genbox microaer, BioMerieux, 96125).
[0137] The citrate buffer solutions were prepared by mixing the
citric acid and sodium citrate in different ratios according to
their pH values, and the unmodulable portion was prepared by HCl
and NaOH to reach a constant pH value. The malate buffer
combination and the lactate buffer combination were prepared by
mixing malic acid and sodium malate, and lactic acid and sodium
lactate, respectively, in the same way. The phosphate buffer
solutions were prepared by mixing KH.sub.2PO.sub.4 and
Na.sub.2HPO.sub.4 in different ratios according to their pH values,
and the unmodulable portion was prepared by HCl and NaOH to reach a
constant pH value. The pH of the medium was adjusted with HCl and
NaOH. The components of the bacterial solution and each culture
medium were as follows:
[0138] Bacteria solution: (Brucella broth 28 g/L, NaHCO.sub.3 10
g/L and 10 mM urea).
[0139] Group 1: M3 (Brucella broth 28 g/L, 0.1 M malate buffer and
10 mM urea, pH 3.0).
[0140] Group 2: M4 (Brucella broth 28 g/L, 0.1 M malate buffer and
10 mM urea, pH 4.0).
[0141] Group 3: M5 (Brucella broth 28 g/L, 0.1 M malate buffer and
10 mM urea, pH 5.0).
[0142] Group 4: L3 (Brucella broth 28 g/L, 0.1 M lactate buffer and
10 mM urea, pH 3.0).
[0143] Group 5: L4 (Brucella broth 28 g/L, 0.1 M lactate buffer and
10 mM urea, pH 4.0).
[0144] Group 6: L5 (Brucella broth 28 g/L, 0.1 M lactate buffer and
10 mM urea, pH 5.0).
[0145] Group 7: P3 (Brucella broth 28 g/L, 0.1 M phosphate buffered
saline (PBS) and 10 mM urea, pH 3.0).
[0146] Group 8: P4 (Brucella broth 28 g/L, 0.1 M phosphate buffered
saline (PBS) and 10 mM urea, pH 4.0).
[0147] Group 9: P5 (Brucella broth 28 g/L, 0.1 M phosphate buffered
saline (PBS) and 10 mM urea, pH 5.0).
[0148] Group 10: C3 (Brucella broth 28 g/L, 0.1 M citrate buffer
and 10 mM urea, pH 3.0).
[0149] Group 11: C4 (Brucella broth 28 g/L, 0.1 M citrate buffer
and 10 mM urea, pH 4.0).
[0150] Group 12: C5 (Brucella broth 28 g/L, 0.1 M citrate buffer
and 10 mM urea, pH 5.0).
[0151] After each culture medium incubated according to the
viability check-point (0/20/40/60 minutes) was serially diluted 10
times with physiological saline, the bacteria solutions diluted at
each magnification was applied to the Brucella solid medium for
subculture by a coating method, and the coated plates were
incubated at 37.degree. C. for 72 hours in a humidified anaerobic
chamber using a micro-aerobic package (Genbox microaer, BioMerieux,
96125). Finally, the viable count of each culture medium was
calculated.
Embodiment 4
[0152] The antibacterial experiment against Helicobacter pylori of
the malate buffer combination with the ascorbic acid, the lactate
buffer combination with the ascorbic acid, the citrate buffer
combination with the ascorbic acid, and the phosphate buffer
combination with the ascorbic acid (viability check-point:
0/20/40/60 minutes, triple repetition).
[0153] Bacteria Strain and Culturing Method
[0154] The bacteria strain used in the experiment was Helicobacter
pylori strain 26695 (ATCC700392), and the bacteria were subcultured
on Brucella solid medium (Brucella broth 28 g/L, Agar 15 g/L and
10% (v/v) Horse serum, pH 7.0). After the bacteria were seeded by
streaking method, they were cultured in a humidified anaerobic
chamber at 37.degree. C. for 72 hours using a microaerobic bag
(Genbox microaer, BioMerieux, 96125).
[0155] Each culture medium was filtered through a 0.22 .mu.m
injection type filter for use. Brucella broth containing 10 mg/mL
of NaHCO.sub.3 was added to the Helicobacter pylori subcultured for
72 hours, bacterial concentration was adjusted to 10.sup.7-10.sup.8
CFU/mL as a bacterial solution, the bacterial solution was mixed
with each culture solution in a 1:1 ratio, and the mixture was
incubated at 37.degree. C. in a humidified anaerobic chamber using
a micro-aerobic bag (Genbox microaer, BioMerieux, 96125).
[0156] The citrate buffer solutions were prepared by mixing the
citric acid and sodium citrate in different ratios according to
their pH values, and the unmodulable portion was prepared by HCl
and NaOH to reach a constant pH value. The malate buffer
combination and the lactate buffer combination were prepared by
mixing malic acid and sodium malate, and lactic acid and sodium
lactate, respectively, in the same way. The phosphate buffer
solutions were prepared by mixing KH.sub.2PO.sub.4 and
Na.sub.2HPO.sub.4 in different ratios according to their pH values,
and the unmodulable portion was prepared by HCl and NaOH to reach a
constant pH value. The pH of the pure medium was adjusted with HCl
and NaOH. After each culture medium was adjusted to a constant pH
value using the method above, a constant concentration of ascorbic
acid was additionally added to each culture medium. The components
of the bacterial solution and each culture medium were as
follows:
[0157] Bacteria solution: (Brucella broth 28 g/L, NaHCO.sub.3 10
g/L and 10 mM urea).
[0158] Group 1: M3V3000 (Brucella broth 28 g/L, 0.1 M malate buffer
and 10 mM urea, pH 3.0)+(6000 mg/L Vitamin C).
[0159] Group 2: M4V3000 (Brucella broth 28 g/L, 0.1 M malate buffer
and 10 mM urea, pH 4.0)+(6000 mg/L Vitamin C).
[0160] Group 3: M5V3000 (Brucella broth 28 g/L, 0.1 M malate buffer
and 10 mM urea, pH 5.0)+(6000 mg/L Vitamin C).
[0161] Group 4: L3V3000 (Brucella broth 28 g/L, 0.1 M lactate
buffer and 10 mM urea, pH 3.0)+(6000 mg/L Vitamin C).
[0162] Group 5: L4V3000 (Brucella broth 28 g/L, 0.1 M lactate
buffer and 10 mM urea, pH 4.0)+(6000 mg/L Vitamin C).
[0163] Group 6: L5V3000 (Brucella broth 28 g/L, 0.1 M lactate
buffer and 10 mM urea, pH 5.0)+(6000 mg/L Vitamin C).
[0164] Group 7: P3V3000 (Brucella broth 28 g/L, 0.1 M phosphate
buffered saline (PBS) and 10 mM urea, pH 3.0)+(6000 mg/L Vitamin
C).
[0165] Group 8: P4V3000 (Brucella broth 28 g/L, 0.1 M phosphate
buffered saline (PBS) and 10 mM urea, pH 4.0)+(6000 mg/L Vitamin
C).
[0166] Group 9: P5V3000 (Brucella broth 28 g/L, 0.1 M phosphate
buffered saline (PBS) and 10 mM urea, pH 5.0)+(6000 mg/L Vitamin
C).
[0167] Group 10: C3V3000 (Brucella broth 28 g/L, 0.1 M citrate
buffer and 10 mM urea, pH 3.0)+(6000 mg/L Vitamin C).
[0168] Group 11: C4V3000 (Brucella broth 28 g/L, 0.1 M citrate
buffer and 10 mM urea, pH 4.0)+(6000 mg/L Vitamin C).
[0169] Group 12: C5V3000 (Brucella broth 28 g/L, 0.1 M citrate
buffer and 10 mM urea, pH 5.0)+(6000mg/L Vitamin C).
[0170] After each culture medium incubated according to the
viability check-point (0/20/40/60 minutes) was serially diluted 10
times with physiological saline, the bacteria solutions diluted at
each magnification was applied to the Brucella solid medium for
subculture by a coating method, and the coated plates were
incubated at 37.degree. C. for 72 hours in a humidified anaerobic
chamber using a micro-aerobic package (Genbox microaer, BioMerieux,
96125). Finally, the viable count of each culture medium was
calculated.
[0171] V. Experimental results
Embodiment 1
[0172] The results of Embodiment 1 are shown in Table 1 and FIG.
1.
TABLE-US-00001 TABLE 1 pH value Time (min) measured Buffer Sample 0
20 40 60 after 60 solution number Viable count (Log.sub.10 CFU/mL)
minutes N N3 7.18 .+-. 0.11 7.21 .+-. 0.13 7.25 .+-. 0.21 7.29 .+-.
0.2 6.01 (Water) N3V3000 7.34 .+-. 0.08 7.11 .+-. 0.09 6.92 .+-.
0.06 6.8 .+-. 0.07 5.13 N4 7.29 .+-. 0.12 7.19 .+-. 0.14 7.23 .+-.
0.23 7.27 .+-. 0.24 6.57 N4V3000 7.35 .+-. 0.08 7.05 .+-. 0.08 7
.+-. 0.09 7.01 .+-. 0.05 5.13 N5 7.31 .+-. 0.1 7.19 .+-. 0.13 7.47
.+-. 0.42 7.38 .+-. 0.35 7.45 N5V3000 7.34 .+-. 0.12 7.07 .+-. 0.12
7.15 .+-. 0.04 7 .+-. 0.02 6.79 P P3 7.22 .+-. 0.09 7.16 .+-. 0.12
7.15 .+-. 0.12 7.32 .+-. 0.16 5.51 (PBS) P3V3000 7.35 .+-. 0.08
6.99 .+-. 0.09 6.93 .+-. 0.08 5.63 .+-. 0.06 4.92 P4 7.22 .+-. 0.15
7.16 .+-. 0.15 7.28 .+-. 0.15 7.4 .+-. 0.22 6.08 P4V3000 7.35 .+-.
0.07 7.13 .+-. 0.12 7.12 .+-. 0.06 7.02 .+-. 0.04 5.75 P5 7.29 .+-.
0.06 7.23 .+-. 0.12 7.39 .+-. 0.31 7.48 .+-. 0.36 6.83 P5V3000 7.36
.+-. 0.06 7.12 .+-. 0.19 7.15 .+-. 0.07 7.05 .+-. 0.07 6.35 C C3
7.29 .+-. 0.09 5.94 .+-. 0.28 5.28 .+-. 0.09 4.35 .+-. 0.05 4.37
(Citrate C3V3000 7.37 .+-. 0.09 4.79 .+-. 0.03 3.86 .+-. 0.06 2.08
.+-. 0.43 4.27 buffer) C4 7.25 .+-. 0.09 7 .+-. 0.12 6.76 .+-. 0.36
6.7 .+-. 0.34 5.06 C4V3000 7.34 .+-. 0.1 6.38 .+-. 0.04 6.08 .+-.
0.04 5.14 .+-. 0.05 4.83 C5 7.23 .+-. 0.08 7.21 .+-. 0.22 7.26 .+-.
0.1 7.21 .+-. 0.22 6.09 C5V3000 7.31 .+-. 0.1 7.28 .+-. 0.1 7.31
.+-. 0.04 7.19 .+-. 0.04 5.87
Embodiment 2
[0173] The results of Embodiment 2 are shown in Table 2 and FIG.
2.
TABLE-US-00002 TABLE 2 pH value Time (min) measured Buffer Sample 0
20 40 60 after 60 solution number Viable count (Log.sub.10 CFU/mL)
minutes C C3V3000 7.75 .+-. 0.07 5.77 .+-. 0.18 4.7 .+-. 0.18 3.56
.+-. 0.08 4.51 (Citrate C3V6000 7.59 .+-. 0.04 4.56 .+-. 0.33 3.01
.+-. 0.51 2.2 .+-. 0.28 4.33 buffer) C3V9000 7.66 .+-. 0.04 2.5
.+-. 0.28 2.15 .+-. 0.21 0.0 .+-. 0.0 4.26
Embodiment 3
[0174] The results of Embodiment 3 are shown in Table 3 and FIG.
3.
TABLE-US-00003 TABLE 3 pH value Time (min) measured Buffer Sample 0
20 40 60 after 60 solution number Viable count (Log.sub.10 CFU/mL)
minutes M M3 7.56 .+-. 0.06 7.42 .+-. 0.05 6.23 .+-. 0.08 5.21 .+-.
0.1 4.91 .+-. 0.3 (Malate M4 7.61 .+-. 0.08 7.59 .+-. 0.16 7.64
.+-. 0.08 7.67 .+-. 0.08 6.24 .+-. 0.32 buffer) M5 7.56 .+-. 0.09
7.58 .+-. 0.1 7.58 .+-. 0.04 7.65 .+-. 0.11 7.05 .+-. 0.32 L L3
7.58 .+-. 0.05 7.63 .+-. 0.16 7.67 .+-. 0.07 7.62 .+-. 0.07 6.11
.+-. 0.42 (Lactate L4 7.59 .+-. 0.06 7.63 .+-. 0.04 7.65 .+-. 0.09
7.64 .+-. 0.11 6.87 .+-. 0.48 buffer) L5 7.56 .+-. 0.1 7.56 .+-.
0.13 7.67 .+-. 0.07 7.64 .+-. 0.09 7.35 .+-. 0.32 P P3 7.59 .+-.
0.13 7.64 .+-. 0.05 7.65 .+-. 0.04 7.62 .+-. 0.09 6.27 .+-. 0.33
(PBS) P4 7.61 .+-. 0.13 7.66 .+-. 0.05 7.66 .+-. 0.08 7.62 .+-.
0.06 6.51 .+-. 0.3 P5 7.63 .+-. 0.05 7.65 .+-. 0.08 7.62 .+-. 0.11
7.66 .+-. 0.06 6.64 .+-. 0.29 C C3 7.66 .+-. 0.05 6.74 .+-. 0.22
5.98 .+-. 0.1 4.68 .+-. 0.13 4.69 .+-. 0.27 (Citrate C4 7.66 .+-.
0.12 7.67 .+-. 0.09 7.2 .+-. 0.18 6.85 .+-. 0.23 5.61 .+-. 0.19
buffer) C5 7.61 .+-. 0.13 7.65 .+-. 0.07 7.67 .+-. 0.05 7.64 .+-.
0.09 6.29 .+-. 0.22
Embodiment 4
[0175] The results of Embodiment 4 are shown in Table 4 and FIG.
4.
TABLE-US-00004 TABLE 4 pH value Time (min) measured Buffer Sample 0
20 40 60 after 60 solution number Viable count (Log.sub.10 CFU/mL)
minutes M M3V3000 7.23 5.08 4.90 3.27 4.60 (Malate M4V3000 7.22
7.40 7.26 7.12 5.79 buffer) M5V3000 7.28 7.38 7.42 7.43 6.87 L
L3V3000 7.36 7.16 7.07 6.86 5.24 (Lactate L4V3000 7.41 7.55 7.23
7.45 6.60 buffer) L5V3000 7.48 7.34 7.29 7.51 6.95 P P3V3000 7.28
7.13 7.08 7.24 6.15 (PBS) P4V3000 7.39 7.42 7.34 7.38 6.31 P5V3000
7.36 7.44 7.37 7.40 6.55 C C3V3000 7.27 4.85 4.00 2.60 4.50
(Citrate C4V3000 7.38 7.28 7.20 7.08 5.21 buffer) C5V3000 7.34 7.31
7.30 7.38 6.22
[0176] It can be seen from the results of Embodiment 1 that, the
citrate buffer combination having a pH value below 5 of the present
invention has a significant bactericidal effect in a bicarbonate
environment. Normally, we take it for granted that the citric acid
not only shows no bactericidal effect against Helicobacter pylori,
but also has a diametrically opposite trend (please refer to Page
1766, left Column, Lines 26-35 and FIG. 1 in D(12), which discloses
that the citric acid significantly enhances urease activity of the
Helicobacter pylori, while the environmental pH is not a critical
issue). In the present invention, the citrate buffer combination
having a pH value below 5 is used as a combination component of a
bactericide against Helicobacter pylori, which is the first time
that the citric acid is used as a component for killing
Helicobacter pylori in a bicarbonate environment with a pH value
below 5, and does show a bactericidal effect, and thus the present
invention displays novelty and inventiveness.
[0177] The citrate buffer combination with the ascorbic acid having
a pH value below 5 demonstrates a significant "dose-dependent"
bactericidal effect in the bicarbonate environment. In addition, by
comparing C3 with C3V3000, and C4 with C4V3000, it can be observed
that the ascorbic acid shows a significant "synergistic effect" on
the sterilization of the citrate buffer combination.
[0178] Conversely, the phosphate buffer combination or water does
not demonstrate bactericidal efficacy in the presence of
bicarbonate. Moreover, there is neither the bactericidal effect nor
the synergistic effect even when the ascorbic acid is added to the
above two systems.
[0179] According to the experimental results of the Embodiment 1,
the antibacterial experiment of the citrate buffer combination at
pH 3 with various concentrations of the ascorbic acid was carried
out in the Embodiment 2 to verify the bactericidal and the
synergistic effect of the various concentrations of the ascorbic
acid, and to verify the optimum concentration of bactericidal
activity within 20 minutes.
[0180] Bactericidal Activity is generally defined as a 99.9%
reduction in the number of colony-forming units relative to the
inoculum density at a specified incubation time, which is usually
20 to 24 hours (please refer to D(13)).
[0181] It should be noted that the viable count of Embodiment 2 in
FIG. 2 decreased from 7 Logio(CFU/mL) to 3 Logio(CFU/mL), which
achieves a bactericidal effect of 99.9%, in other words. Regarding
the bactericidal synergistic effect of various concentrations of
the ascorbic acid at pH 3, C3 (without the ascorbic acid) cannot
effectively kill the bacteria within 60 minutes. On the other hand,
C3V3000 (with 3000 mg/L of the ascorbic acid) killed the bacteria
effectively within 60 minutes, C3V6000 (with 6000 mg/L of the
ascorbic acid) killed the bacteria effectively within 40 minutes,
and C3V9000 (with 9000 mg/L of the ascorbic acid) killed the
bacteria effectively within 20 minutes as well and essentially
eliminated all the bacteria within 60 minutes in fact!! The
concentration of the ascorbic acid in the bactericidal composition
of the present invention may be even higher as long as it meets the
recommended upper limit of daily intake for the human body (up to
about 2 g per day). Because the standard capacity of the buffer
solution in the present invention is 1000 mL, 3000 ppm of the
ascorbic acid represents 3 g of the ascorbic acid in 1000 mL of the
buffer solution. Consequentially, If the actual application
capacity is 200 mL, there is 0.6 g of the ascorbic acid in 200 mL
of the buffer solution when the concentration of the ascorbic acid
is 3000 ppm, and there is 2 g of the ascorbic acid in 200 mL of the
buffer solution when the concentration of the ascorbic acid is
10,000 ppm.
[0182] Embodiment 1 and Embodiment 2 demonstrate that the citrate
buffer combination with the ascorbic acid having a pH value below 5
shows a bactericidal effect in a bicarbonate environment, the
bactericidal effect exhibits a "dose-dependent" relationship, and
the ascorbic acid shows a significant "synergistic effect" on the
bactericidal effect of the citrate buffer combination. Moreover,
the bactericidal compositions of the present invention have a
bigger buffer capacity for the absorption of bicarbonate (HCO.sub.3
-) at pH 3 (please refer to D(14)) hence more effective in terms of
sterilization.
[0183] It can be seen from the results of Embodiment 3 and
Embodiment 4 that the malate buffer combination having a pH value
below 5 of the present invention has a bactericidal effect in a
bicarbonate environment as well. Normally, one tends to take it for
granted that the malic acid not only exhibits no bactericidal
effect against Helicobacter pylori, but also has an entirely
reverse effect that both the citric acid and the malic acid enhance
the urease activity of the Helicobacter pylori in an acid
environment (please refer to Paragraph "Conclusions" in Summary and
FIGS.1-2 in D(15)). In the present invention, however, the citrate
or the malate buffer combination having a pH value below 5 is used
as a combination component of a bactericide against Helicobacter
pylori, which is the first time that the citric acid or the malic
acid is used as a component for killing Helicobacter pylori in a
bicarbonate environment with a pH value below 5, and indeed it
exhibits a significant bactericidal effect. Thus the present
invention does show novelty and inventiveness.
[0184] In addition, by comparing Embodiment 3 and Embodiment 4, it
can be observed that the ascorbic acid demonstrates a significant
"synergistic effect" on the sterilization effect of the citrate or
the malate buffer combination.
[0185] Conversely, the phosphate buffer combination or water does
not show the bactericidal efficacy in the presence of bicarbonate,
and there is neither bactericidal effect nor synergistic effect
even when the ascorbic acid is added.
[0186] The citrate or the malate buffer combination having a pH
value below 5 has a significant "dose-dependent" bactericidal
effect associated with the ascorbic acid added in the bicarbonate
environment.
[0187] The results of C3, C3V3000, C4V3000, M3, M3V3000, and
M4V3000 in the embodiments demonstrate the pH value and
sterilization results of the buffer combination having a pH value
below 5 measured after 60 minutes can be observed.
[0188] Referring to Page 292, FIG. 3 in D(16), which shows the
correlation between pH value and the ascorbate oxidase activity in
Helicobacter pylori, there are two ascorbate oxidases in the
Helicobacter pylori: interstitial ascorbate oxidase and
endomembranous ascorbate oxidase. This correlation indicates that
the two kinds of the ascorbate oxidase exhibit weak activity at pH
4-5 (please refer to FIG. 3 in D(16) mentioned above), which is
advantageous for the sterilization of ascorbic acid (please also
refer to paragraph [0084] and Embodiment 2 in Table 2 and FIG. 2,
where the pH values after 60 minutes are kept within this range as
well).
[0189] Since both the citric acid and the malic acid demonstrate an
inhibitory effect on the ascorbic acid oxidase activity at pH level
below 5, an advantageous environment is thus established where
ascorbic acid can exhibit the synergistic effect on the
disinfection significantly. On the other hand, both the phosphate
buffer combination and water control groups do not exhibit such an
inhibitory effect on ascorbate oxidase activity, even with the
addition of the ascorbic acid (please refer to FIGS. 1-4 and Tables
1-4 of Embodiments 1, 2, 3 and 4 showing the pH values and the
sterilization results measured after 60 minutes).
[0190] Helicobacter pylori is one of the few microorganisms that
have both the urease and the ascorbate oxidase (please refer to
Table 1 in D(16)), thus Helicobacter pylori does have a strong
capability of degrading ascorbic acid. The relationship between the
ascorbate oxidase of Helicobacter pylori and pH value is shown in
FIG. 3 in D(16).
[0191] The gastric mucus gel layer becomes unfavorable to the
mobility of Helicobacter pylori in the low pH
mucus-microenvironment (please refer to the abstract in D(17)),
whereas this low pH mucus-microenvironment is advantageous for the
buffer composition of the present invention to sterilize and
eradicate bacteria in the mucus gel layer.
[0192] While the pH value of the buffer combination of the present
invention is equal to 3 at first, it goes down to less than 3 in
the gastric antrum later on, which will terminate the gastric acid
secretion temporarily (please refer to Page 422 in D(18) regarding
pH changes), which leads to the termination of the secretion of the
chloride ion accordingly. The bicarbonate secreted by the
epithelial cells is subsequently inhibited temporarily as well due
to this temporary termination of the chloride ion secretion in the
stomach (please refer to FIG. 2 in D(19)). Thus it is even more
advantageous to sterilize and eradicate bacteria under the actual
condition mentioned above.
[0193] The actually obtainable pKa of the citric acid is 3.1 in
practice, and the standard pKa of the citric acid is 3.12 in
literature (please refer to D(14)). Therefore the citrate buffer
combination exhibits an even bigger buffer capacity for the
absorption of bicarbonate (HCO.sub.3-) at pH level equal to 3 as
compared with other buffer combinations we have prepared.
[0194] Therefore, it is crucial to set the pH value equal to 3 in
order to effectively sterilize and eradicate bacteria. The buffer
capacity presented at this pH condition is also critical. Moreover,
it is also crucial to inhibit ascorbate oxidase activity
effectively at this pH condition! The addition of the ascorbic acid
at this pH condition is the most critical factor for rapid
sterilization (synergistic effect). For experiments conducted at pH
level equal to 3, although the original pH at the start of
Embodiments 1, 2, 3 and 4 are set to 3, the actual pH level of the
citrate or the malate buffer combination with ascorbic acid added
in the mucus-microenvironment tends to approach a level below 5
eventually, which proves to be beneficial for sterilizing and
eradicating bacteria.
[0195] The pH value of the citrate or the malate buffer combination
(pH=3) in the present invention in the gastric antrum can be
adjusted by the detailed composition of the buffer with a goal to
keep it less than 3 in practice in order to maintain the low pH
environment in the gastric antrum (please refer to FIG. 1 in
D(20)), which is beneficial in inhibiting the gastric acid
secretion (i.e. inhibiting chloride ion secretion). The gastric
acid secretion is inhibited when the pH value in the gastric antrum
is less than 3, which is similar to the functions of antacids or
PPI, but the pH change of the present invention is different from
that of the antacid or PPI because the present invention does not
lead to a significant increase of pH value above 6 (please refer to
reference D(18)), and the normally continuous secretion of
bicarbonate by the epithelial cells is temporarily inhibited as a
result. This is advantageous for the citrate or the malate buffer
composition in the present invention to sterilize and eradicate
bacteria in a low pH mucus-microenvironment.
[0196] The concentration of bicarbonate (HCO.sub.3-) secreted by
the epithelial cells in the gastric mucus gel layer is positively
correlated with the concentration of chloride ions in the stomach
(please refer to FIG. 4 in D(21)).
[0197] In the Embodiments 1, 2, 3, and 4, the key outcome to be
noted is "the pH value after 60 minutes", which indicates the pH
value variation of the mucoepithelial interface with time.
[0198] In the present invention, particularly in the Embodiments 2,
the citrate buffer combination alone and the citrate buffer
combination with the ascorbic acid added, are capable of
sterilizing and eradicating bacteria, with the former sterilizes
bacteria within 60 minutes, and the latter disinfects bacteria
within 20 minutes. The amount of sodium bicarbonate (NaHCO.sub.3)
added demonstrates the pH value in the mucoepithelial interface
after 60 minutes. Moreover, it also represents the required buffer
capacity of the present invention which can absorb bicarbonate
(HCO.sub.3-) effectively to keep the pH value of the
mucus-microenvironment below 5 in 60 minutes.
[0199] Based on the Embodiments 1, 2 and 4 above, it appears that
the ascorbic acid has the functions of repairing and protecting the
gastric mucus gel layer (please refer to the abstract in D(22)) as
well as the mucolytic property (please refer to the abstract in
D(23)) in the present invention. Moreover, the ascorbic acid
demonstrates a significant effect of rapid sterilization and a
significant synergistic effect on the citrate or the malate buffer
combination alone. The gastric antrum inhibits gastric acid
secretion when pH below 3 (please refer to Page 422 in D(18)
regarding pH changes), which is advantageous to sterilize and
eradicate bacteria. C3V9000 (the citrate buffer combination with
the ascorbic acid at pH 3) can eradicate the Helicobacter pylori
completely within 60 minutes, which is a preferred sterilization
combination.
[0200] Regarding the increasing antibiotic resistance of the
Helicobacter pylori, the buffer composition of the present
invention (the ascorbic acid combined with the citrate or the
malate buffer combination) is a bactericidal combination from
nature rather than synthesized artificially. The antibiotic
resistance of the Helicobacter pylori is due to the ability of
Helicobacter pylori to develop antibiotic resistance via gene
exchange or chromosomal mutation (please refer to D(24) and D(25)).
The Helicobacter pylori naturally undergoes gene mutation during
replication. If antibiotics are used during this process, it tends
to generate antibiotic-resistant mutants (please refer to D(24) and
D(25)). The Helicobacter pylori will divide and multiply in the
environment of pH 6-8, this environment is also the optimum pH
environment for antibiotics to kill the bacteria effectively
(please refer to (D5)), and thus it seems to be the main cause of
the increasing antibiotic resistance of Helicobacter pylori. The
buffer composition of the present invention (the ascorbic acid
combined with the citrate or the malate buffer combination)
sterilizes bacteria mainly in the environment at pH of 3, hence the
Helicobacter pylori does not divide and multiply in this low pH
environment in general, therefore it is less likely to generate an
antibiotic-resistance mutant.
[0201] As the non-pharmaceutical bactericidal composition of the
present invention does not contain any antibiotic component, there
should be no concerns of drug resistance and food safety in
principle when used as a medicine for human beings.
[0202] It is understood, that the present invention is not limited
to the particular embodiments disclosed, but is intended to cover
all modifications which are within the spirit and scope of the
invention as defined by the appended claims, the above description,
and/or shown in the attached drawings.
[0203] Embodiments:
[0204] 1. A non-pharmaceutical bactericidal composition against a
Helicobacter pylori including a first daily edible component, a
second daily edible component, a salt and a third daily edible
component, wherein the first daily edible component has a molecular
state and a dissociated state while in an aqueous solution state,
the first daily edible component of the molecular state is used for
performing functions of killing the
[0205] Helicobacter pylori and inhibiting an ascorbate oxidase
activity of the Helicobacter pylori in an environment with a pH
value below 5, and the first daily edible component is a citric
acid, a malic acid or a combination thereof, wherein the second
daily edible component is used for neutralizing a bicarbonate and
being a citric acid, a malic acid or a combination thereof, wherein
the salt is used to form a buffer combination with the citric acid,
the malic acid or the combination thereof, the salt is a salt of
the citric acid, a salt of the malic acid or a combination thereof,
and wherein the third daily edible component is used for killing
the Helicobacter pylori more thoroughly in the presence of the
first daily edible component, the second daily edible component and
the salt, and the third daily edible component is an ascorbic
acid.
[0206] 2. The non-pharmaceutical bactericidal composition according
to Embodiment 1, wherein the ascorbic acid has a concentration
being in a range from 3000 ppm to 10000 ppm.
[0207] 3. The non-pharmaceutical bactericidal composition according
to one of Embodiment 1 and 2, wherein the ascorbic acid has a
concentration of 9000 ppm.
[0208] 4. The non-pharmaceutical bactericidal composition according
to any one of Embodiments 1 to 3, wherein the salt is a sodium
citrate, a sodium malate or a combination thereof.
[0209] 5. A non-pharmaceutical bactericidal composition against a
Helicobacter pylori, including a first daily edible component and a
second daily edible component, wherein the first daily edible
component is a buffer combination consisting of an organic acid and
a salt of the organic acid, the first daily edible component is
used for neutralizing a bicarbonate and performing functions of
killing the Helicobacter pylori and inhibiting an ascorbate oxidase
activity of the Helicobacter pylori in an environment with a pH
value below 5, and the organic acid is a citric acid, a malic acid
or a combination thereof, and wherein the second daily edible
component is used for killing the Helicobacter pylori more
thoroughly in presence of the first daily edible component, and the
second daily edible component is an ascorbic acid.
[0210] 6. The non-pharmaceutical bactericidal composition according
to Embodiments 5, wherein the non-pharmaceutical bactericidal
composition has a pH value below 5.
[0211] 7. The non-pharmaceutical bactericidal composition according
to one of Embodiments 5 and 6, wherein the non-pharmaceutical
bactericidal composition has a pH value being in a range from 3 to
5.
[0212] 8. The non-pharmaceutical bactericidal composition according
to any one of Embodiments 5 to 7, wherein the non-pharmaceutical
bactericidal composition has a pH value of 3.
[0213] 9. A non-pharmaceutical bactericidal composition against a
Helicobacter pylori including a first daily edible component and a
second daily edible component, wherein the first daily edible
component has a molecular state and a dissociated state while in an
aqueous solution state, the first daily edible component of the
molecular state is used for performing functions of killing the
Helicobacter pylori and inhibiting an ascorbate oxidase activity of
the Helicobacter pylori in an environment with a pH value below 5,
and the first daily edible component is a citric acid, a malic acid
or a combination thereof, and wherein the second daily edible
component is a salt used to form a buffer combination with the
first daily edible component, and used to neutralize a bicarbonate,
so that the first daily edible component can continue its killing
of the Helicobacter pylori effectively and inhibiting the ascorbate
oxidase activity of the Helicobacter pylori, and the salt is a salt
of the citric acid, a salt of the malic acid or a combination
thereof.
[0214] 10. The non-pharmaceutical bactericidal composition
according to Embodiments 9, wherein the non-pharmaceutical
bactericidal composition has a pH value of 3.
[0215] 11. The non-pharmaceutical bactericidal composition
according to one of Embodiments 9 and 10, wherein the salt is a
sodium citrate, a sodium malate or a combination thereof.
[0216] 12. The non-pharmaceutical bactericidal composition
according to any one of Embodiments 9 to 11, further including an
ascorbic acid for killing the Helicobacter pylori more thoroughly
in presence of the first daily edible component and the second
daily edible component.
[0217] 13. A method for treating a Helicobacter pylori infection,
including steps of providing a non-pharmaceutical bactericidal
composition including a first daily edible component and a second
daily edible component, and administering the non-pharmaceutical
bactericidal composition to a mammal, wherein the first daily
edible component has a molecular state and a dissociated state
while in an aqueous solution state, the first daily edible
component of the molecular state is used for performing functions
of killing a Helicobacter pylori and inhibiting an ascorbate
oxidase activity of the Helicobacter pylori in an environment with
a pH value below 5, the first daily edible component is a citric
acid, a malic acid or a combination thereof, and the second daily
edible component is a salt, is used to form a buffer combination
with the first daily edible component, and is used to neutralize a
bicarbonate, so that the first daily edible component can continue
its killing of the Helicobacter pylori effectively and inhibiting
the ascorbate oxidase activity of the Helicobacter pylori, and the
salt is a salt of the citric acid, a salt of the malic acid or a
combination thereof.
[0218] 14. The method according to Embodiment 13, wherein the
ascorbic acid has a concentration being in a range from 3000 ppm to
10000 ppm.
[0219] 15. The method according to one of Embodiments 13 and 14,
wherein the ascorbic acid has a concentration of 9000 ppm.
[0220] 16. The method according to any one of Embodiments 13 to 15,
wherein the non-pharmaceutical bactericidal composition has a pH
value below 5.
[0221] 17. The method according to any one of Embodiments 13 to 16,
wherein the non-pharmaceutical bactericidal composition has a pH
value being in a range from 3 to 5.
[0222] 18. The method according to any one of Embodiments 13 to 17,
wherein the non-pharmaceutical bactericidal composition has a pH
value of 3.
[0223] 19. The method according to any one of Embodiments 13 to 18,
wherein the non-pharmaceutical bactericidal composition further
includes an ascorbic acid for killing the Helicobacter pylori more
thoroughly in presence of the first daily edible component and the
second daily edible component.
[0224] 20. The method according to any one of Embodiments 13 to 19,
wherein the mammal is a human being.
* * * * *