AU2015101912A4 - Uses of multipolar microkinetic drinking water in preparing drink, healthcare product or medicament used for combating fatigue - Google Patents
Uses of multipolar microkinetic drinking water in preparing drink, healthcare product or medicament used for combating fatigue Download PDFInfo
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Abstract
Uses of a multipolar microkinetic drinking water in preparing a drink, healthcare product or medicament used for combating fatigue. The multipolar microkinetic drinking water is prepared from raw drinking water being passed through an electromagnetic wave without contact. Compared with the raw drinking water, the ultraviolet absorption peak is offset by 25 nm to 40 nm in the direction of shortwaves, where the preferred offset is 28 nm to 30 nm in the direction of the shortwaves, and the optimal offset is either 30 nm or 28 nm in the direction of the shortwaves.
Description
USES OF MULTIPOLAR MICRO KINETIC DRINKING WATER IN PREPARING DRINK, HEALTHCARE PRODUCT OR MEDICAMENT
USED FOR COMBATING FATIGUE
TECHNICAL FIELD
The present invention relates to the field of water treatment technology and more particularly to uses of multipolar microkinetic drinking water in the preparation of beverages, health care products or medicaments for anti-fatigue.
BACKGROUND OF THE INVENTION
Water (H2O) is an inorganic substance consisting of two elements of hydrogen and oxygen, and is a colorless, odorless and transparent liquid at normal temperature and pressure. Water is one of the most common substances and it is an important resource for the survival of all life, including human beings and it is also the most important part of the organism. Water plays an important role in the evolution of life. Humans began to generate awareness on the water very early, in the ancient East-West simple material concept, water was regarded as a basic constituent element.
Modem people have the following cognitions on water from a scientific angle:
Water molecules have memory: when scientists melted and then refreezed up individual snow crystals with different shapes and weights, it was found that the shape and weight of the re-frozen snow crystals were exactly the same as that before melting and therefore, scientists come to the conclusion that the water itself has memory.
Since the water molecule itself has its own special structural shape, it is made up of two hydrogens and one oxygen ion at an angle of 104.5. It is a kind of ionization system with electronic magnetic polarity. Water molecule at zero and
2015101912 20 Mar 2015 below exhibits the state of solid crystal, and we call it ice. However water is not completely liquid at a temperature of 0 to 60 °C, but in the form of a liquid crystal. But if the temperature is higher than 60 °C, this liquid crystal molecule will be destroyed.
Due to the special angle of the water molecule, the body of the diluted solute molecule can be contained in its liquid crystal, which is the general understanding of the dissolution process. At present, new finding is that water molecules are still able to keep the shape” memory on the solute molecules when the solute molecules are separated from the water molecules by some means (such as by repeated dilution and shock). When the body cells are exposed to such water molecules, due to the shape memory on the solute molecule they will be treated as true solute molecules, this is the cornerstone of homeopathic therapy for more than 200 years.
Water is composed of two elements of hydrogen and oxygen, it does not exsit in the form of individual water molecules, but in the form of water molecules group (H2O)n (i.e. the liquid crystal described above), which is formed by gathering many water molecules in nature. The water molecules group may be chain-like, ring-like, slug-like or grape-like, and have at least five, or as many as more than ten, dozens to hundreds of water molecules.
There are already some methods for treating water molecules to increase the energy level of water molecules or disinfect or prevent and remove dirts by using electromagnetic waves. However, all of these methods can not adequately control the treatment effect, and the treated water by such methods is mostly used as the drinking water without other additional beneficial effects.
SUMMARY OF THE INVENTION
In order to overcome the drawbacks of the prior art, the present invention provides a multi-polar micro-kinetic energy drinking water, which was obtained by using the electromagnetic wave in non-contact mode and preparation method
2015101912 20 Mar 2015 for same and use thereof.
It is an object of the present invention to provide a multi-polar micro-kinetic energy drinking water. It is another object of the present invention to provide a preparation method of the above multi-polar micro-kinetic energy drinking water. It is yet another object of the present invention to provide a use of the above multi-polar micro-kinetic energy drinking water.
The above objects of the present invention are achieved by the following technical solution.
In one aspect, the present invention provides a multi-polar micro-kinetic energy drinking water, which is prepared by treating raw water of drinking water using the electromagnetic wave in non-contact mode, the ultraviolet absorption peak of the multi-polar micro-kinetic energy drinking water is shifted to the short-wave direction by 25 nm to 40 nm, preferably 28 nm to 30 nm, and most preferably 30 nm or 28 nm when compared with the raw water of drinking water.
Compared with the raw water of drinking water, the intensity of the fluorescence emission spectrum of the multi-polar micro-kinetic energy drinking water is greatly improved in the spectral range between 300nm and 400nm. The results from many repeated experiments on the same experimental sample show that the experiment has good reproducibility
The experiments show that the multi-polar micro-kinetic energy drinking water of the present invention has more extranuclear electrons in the non-radiative high energy level.
Preferably, the raw water of drinking water is ordinary tap water or various mineral water or purified water such as Evian Natural Mineral Water, Tibet Glacier Mineral Water, NongFu Spring, Wahaha and so on.
It is best to drink or use the multi-polar micro-kinetic energy drinking water of the present invention within 72 hours after preparation.
In another aspect, the present invention provides a preparation method of the above-mentioned multi-polar micro-kinetic energy drinking water, which
2015101912 20 Mar 2015 comprises treating raw water of drinking water using three different types of electromagnetic waves: low, medium and high frequency electromagnetic waves, in non-contact mode, wherein the frequency range of the low frequency electromagnetic wave is 30-100 kHz, the frequency range of the medium frequency electromagnetic wave is 550-720 kHz, the frequency range of the high frequency electromagnetic wave is 300-725 MHz.
Preferably, the raw water of drinking water is ordinary tap water or various mineral water or purified water such as Evian Natural Mineral Water, Tibet Glacier Mineral Water, NongFu Spring, Wahaha and so on.
Preferably, the frequency range of the low frequency electromagnetic wave is 45-75 kHz.
Preferably, the frequency range of the medium frequency electromagnetic wave is 600-720 kHz.
Preferably, the frequency range of the high frequency electromagnetic wave is 300-425 MHz.
Preferably, the low frequency electromagnetic wave and the medium frequency electromagnetic wave are sine wave, square wave, sharp wave, sawtooth wave or trapezoidal wave.
Preferably, the waveform of the low frequency electromagnetic wave and the medium frequency electromagnetic wave may be the same or different, and are preferably the same.
Preferably, the low frequency electromagnetic wave and the medium frequency electromagnetic wave are propagated in the same direction.
Preferably, the high frequency electromagnetic wave is triangular wave, and its propagation direction is perpendicular to that of the low frequency electromagnetic wave and the medium frequency electromagnetic wave.
Preferably, when treating raw water of drinking water using low, medium and high frequency electromagnetic waves, which are three different types of electromagnetic waves, in non-contact mode, firstly, the low-frequency
2015101912 20 Mar 2015 electromagnetic wave and the high frequency electromagnetic wave are used simultaneously and then the medium electromagnetic wave and high frequency electromagnetic wave are used simultaneously
Preferably, the treating time with the low frequency electromagnetic wave and the high frequency electromagnetic wave is 10 to 30 minutes, preferably 10 to 20 minutes; and the treating time with the medium frequency electromagnetic wave and the high frequency electromagnetic wave is 10 to 30 minutes, preferably 10 to 20 minutes.
Preferably, when compared with the raw water of drinking water, the ultraviolet absorption peak of the obtained multi-polar micro-kinetic energy drinking water is shifted to the short-wave direction by 25 nm to 40 nm, preferably 28 nm to 30 nm, and most preferably 30 nm or 28 nm.
Compared with raw water of drinking water, the intensity of the fluorescence emission spectrum of the multi-polar micro-kinetic energy drinking water of the present invention is greatly improved in the spectral range between 300nm and 400nm. The results from many repeated experiments on the same experimental sample show that the experiment has good reproducibility.
The experiments show that the multi-polar micro-kinetic energy drinking water of the present invention has more extranuclear electrons in the non-radiative high energy level.
In the present invention, the method and apparatus for generating the electromagnetic waves are conventional technical means in the art.
In yet another aspect, the present invention provides a use of the multi-polar micro-kinetic energy drinking water described above for the preparation of various mineral water, purified water, beverages, health care products or medicaments.
Preferably, the beverage is a functional beverage and can be used for inhibiting fatigue, losing weight, defaecating, lowering blood pressure, lowering blood glucose, lowering blood uric acid and lowering blood urea.
2015101912 20 Mar 2015
Preferably, the health care products or medicaments can be used for inhibiting fatigue, losing weight, defaecating, lowering blood pressure, lowering blood glucose, lowering blood uric acid and lowering blood urea.
Compared with the prior art, the present invention treats water using the electromagnetic wave non-contact treatment water without adding any additives, the preparation method of the present invention is simple and has high production efficiency and low cost.
Compared with the existing water, the ultraviolet absorption peak of the multi-polar micro-kinetic energy drinking water of the present invention is shifted obviously to the short-wave direction relative to the untreated raw water of drinking water, and the intensity of the fluorescence emission spectrum of the multi-polar micro-kinetic energy drinking water is greatly improved in the spectral range between 300nm and 400nm, and more extranuclear electrons are in the non-radiative high energy level. Animal experiments have proven that the multi-polar micro-kinetic energy drinking water of the present invention has the following effects: inhibiting fatigue, losing weight, defaecating, lowering blood pressure, lowering blood glucose, lowering blood uric acid and lowering blood urea.
BRIEF DESCRIPTION OF THE DRAWINGS
Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings, wherein:
Figure 1 shows the effect of the water from different treatment groups on DNA damage induced by peroxide;
Figure 2 shows the effect of the culture medium from different treatment groups on cells;
Figure 3 shows the absorption spectrum of raw water of ordinary drinking water and the multi-polar micro-kinetic energy water according to the present invention obtained from the raw water of ordinary drinking water.
2015101912 20 Mar 2015
Figure 4 shows the absorption spectrum distribution of Evian Natural Mineral Water (raw water) and the multi-polar micro-kinetic energy water according to the present invention obtained from the Evian Natural Mineral Water.
Figure 5 shows the absorption spectrum distribution of Tibet Glacier Mineral Water (raw water) and the multi-polar micro-kinetic energy water according to the present invention obtained from the Tibet Glacier Mineral Water.
Figure 6 shows the fluorescence steady-state and transient result of ordinary drinking water (raw water) and the multi-polar micro-kinetic energy drinking water obtained from the ordinary drinking water;
Figure 7 shows the fluorescence steady-state and transient result of Evian Natural Mineral Water (raw water) and the multi-polar micro-kinetic energy drinking water obtained from the Evian Natural Mineral Water;
Figure 8 shows the fluorescence steady-state and transient result of Tibet Glacier Mineral Water (raw water) and the multi-polar micro-kinetic energy drinking water obtained from the Tibet Glacier Mineral Water;
Figure 9 shows the results of repeatability tests of the multi-polar micro-kinetic energy drinking water prepared from Tibet Glacier Mineral Water as the raw water in triplicate;
Figure 10 shows the effect of the kinetic energy water of the present invention on the weight-bearing swimming time in mice.
DETAILED DESCRIPTION OF THE EMBODIMENTS
The invention will now be described in further detail with reference to specific embodiments thereof, and the invention is given by way of illustration only and is not intended to limit the scope of the invention.
The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
2015101912 20 Mar 2015
Example 1 A preparation method of the multi-polar micro-kinetic energy drinking water of the present invention
Three different frequencies of electromagnetic waves: low, medium and high frequency electromagnetic waves, were applied at the outside of the pipe of raw water of ordinary drinking water in non-contact mode, wherein the frequency of the low frequency electromagnetic wave was 30 kHz, the frequency of the medium frequency electromagnetic wave was 550 kHz, and the frequency of the high frequency electromagnetic wave was 300 MHz.
Wherein, the low frequency electromagnetic wave and the medium frequency electromagnetic wave were both sine wave and they were propagated in the same direction. The high frequency electromagnetic wave was triangular wave, and its propagation direction was perpendicular to that of the low frequency electromagnetic wave and the medium frequency electromagnetic wave. When treatment, firstly, the low frequency electromagnetic wave and the high frequency electromagnetic wave were used simultaneously for 20 minutes, and then the medium electromagnetic wave and the high frequency electromagnetic wave were used simultaneously for 20 minutes. After the treatment was completed, the multi-polar micro-kinetic energy drinking water of the present invention was obtained.
Example 2 A preparation method of the multi-polar micro-kinetic energy drinking water of the present invention
Three different frequencies of electromagnetic waves: low, medium and high frequency electromagnetic waves, were applied at the outside of the pipe of raw water of ordinary drinking water in non-contact mode, wherein the frequency of the low frequency electromagnetic wave was 100 kHz, the frequency of the medium frequency electromagnetic wave was 720 kHz, and the frequency of the high frequency electromagnetic wave was 725 MHz.
2015101912 20 Mar 2015
Wherein, the low frequency electromagnetic wave and the medium frequency electromagnetic wave were both sine wave and they were propagated in the same direction. The high frequency electromagnetic wave was triangular wave, and its propagation direction was perpendicular to that of the low frequency electromagnetic wave and the medium frequency electromagnetic wave. When treatment, firstly, the low frequency electromagnetic wave and the high frequency electromagnetic wave were used simultaneously for 10 minutes, and then the medium electromagnetic wave and the high frequency electromagnetic wave were used simultaneously for 10 minutes. After the treatment was completed, the multi-polar micro-kinetic energy drinking water of the present invention was obtained.
Example 3 A preparation method of the multi-polar micro-kinetic energy drinking water of the present invention
Three different frequencies of electromagnetic waves: low, medium and high frequency electromagnetic waves, were applied at the outside of the pipe of raw water of ordinary drinking water in non-contact mode, wherein the frequency of the low frequency electromagnetic wave was 45 kHz, the frequency of the medium frequency electromagnetic wave was 600 kHz, and the frequency of the high frequency electromagnetic wave was 300 MHz.
Wherein, the low frequency electromagnetic wave and the medium frequency electromagnetic wave were both sine wave and they were propagated in the same direction. The high frequency electromagnetic wave was triangular wave, and its propagation direction was perpendicular to that of the low frequency electromagnetic wave and the medium frequency electromagnetic wave. When treatment, firstly, the low frequency electromagnetic wave and the high frequency electromagnetic wave were used simultaneously for 15 minutes, and then the medium electromagnetic wave and the high frequency electromagnetic wave were used simultaneously for 15 minutes. After the
2015101912 20 Mar 2015 treatment was completed, the multi-polar micro-kinetic energy drinking water of the present invention was obtained.
Example 4 A preparation method of the multi-polar micro-kinetic energy drinking water of the present invention
Three different frequencies of electromagnetic waves: low, medium and high frequency electromagnetic waves, were applied at the outside of the pipe of raw water of ordinary drinking water in non-contact mode, wherein the frequency of the low frequency electromagnetic wave was 75 kHz, the frequency of the medium frequency electromagnetic wave was 720 kHz, and the frequency of the high frequency electromagnetic wave was 425 MHz.
Wherein, the low frequency electromagnetic wave and the medium frequency electromagnetic wave were both sine wave and they were propagated in the same direction. The high frequency electromagnetic wave was triangular wave, and its propagation direction was perpendicular to that of the low frequency electromagnetic wave and the medium frequency electromagnetic wave. When treatment, firstly, the low frequency electromagnetic wave and the high frequency electromagnetic wave were used simultaneously for 20 minutes, and then the medium electromagnetic wave and the high frequency electromagnetic wave were used simultaneously for 20 minutes. After the treatment was completed, the multi-polar micro-kinetic energy drinking water of the present invention was obtained.
Example 5 Physical and chemical properties of the multi-polar micro-kinetic energy drinking water of the present invention
In accordance with the Standard examination methods for drinking water (GB/T 5750-2006), the multi-polar micro-kinetic drinking water according to examples 1 to 4 of the present invention was tested and the testing results were basically the same. The results are shown in Table 1.
io
2015101912 20 Mar 2015
Table 1 physical and chemical properties of the multi-polar micro-kinetic energy drinking water of the present invention
| Nos. | Testing items | Units | National standards | Testing results | Conclusions |
| 1 | Chroma | Degree | <15 | <5 | Qualified |
| 2 | Turbidity | NTU | <1 | 0.39 | Qualified |
| 3 | Smell and taste | Description | none | none | Qualified |
| 4 | Visible material to the naked eye | Description | none | none | Qualified |
| 5 | pH | 6.5-8.5 | 8.00 | Qualified | |
| 6 | Total hardness | mg/L | <450 | 164 | Qualified |
| 7 | Aluminum (Al) | mg/L | <0.2 | <0.01 | Qualified |
| 8 | Iron (Fe) | mg/L | <0.3 | 0.089 | Qualified |
| 9 | Manganese (Mn) | mg/L | <0.1 | 0.0082 | Qualified |
| 10 | Copper (Cu) | mg/L | <1 | <0.01 | Qualified |
| 11 | Zinc (Zn) | mg/L | <1 | 0.108 | Qualified |
| 12 | Sulfates | mg/L | <250 | 87.4 | Qualified |
| 13 | Chlorides | mg/L | <250 | 47.3 | Qualified |
| 14 | Total dissolved solids | mg/L | <1000 | 396 | Qualified |
| 15 | Oxygen consumption | mg/L | <3 | 0.68 | Qualified |
| 16 | Volatile phenols | mg/L | <0.002 | <0.002 | Qualified |
| 17 | Anionic synthetic detergents | mg/L | <0.3 | <0.10 | Qualified |
| 18 | Cyanides | mg/L | <0.05 | <0.002 | Qualified |
| 19 | Fluorides | mg/L | <1.0 | 0.50 | Qualified |
| 20 | Arsenic (As) | mg/L | <0.01 | <0.0005 | Qualified |
| 21 | Selenium (Se) | mg/L | <0.01 | 0.0014 | Qualified |
| 22 | Mercury (Hg) | mg/L | <0.001 | <0.00005 | Qualified |
| 23 | Cadmium (Cd) | mg/L | <0.005 | <0.0001 | Qualified |
2015101912 20 Mar 2015
| 24 | Chromium (hexavalent) (Cr(VI)) | mg/L | <0.05 | <0.004 | Qualified |
| 25 | Nitrate nitrogen | mg/L | <10 | 0.08 | Qualified |
| 26 | Lead(Pb) | mg/L | <0.01 | <0.0005 | Qualified |
| 27 | Trichloromethane | mg/L | <60 | <0.5 | Qualified |
| 28 | Carbon Tetrachloride | mg/L | <2 | <0.1 | Qualified |
According to the above table, it can be seen that the micro-kinetic energy drinking water of the present invention meet the requirements of Standard examination methods for drinking water (GB/T 5750-2006).
Example 6 Toxicology experiment of the multi-polar micro-kinetic energy drinking water of the present invention
I. Acute oral toxicity test
1. Materials and Methods
1.1 Test substance: the micro-kinetic drinking water prepared in Example 3
1.2 Animals: Kunming mice, which were obtained from Institute of Laboratory Animal Resources, National Institute for Food and Drug Control (certificate number: SCXK (Beijing) 2009-0017), SPF level, weight 18.0-22.0 g; before testing, the animals were fasted overnight and allowed to intake water freely.
1.3 Groups according to dose: the dose of the test substance was 20000 mg/kg body weight, 10 female animals and 10 male animals in each group. They were caged by sex to feed. They were infected by a single oral gavage at a dose of O.lml/lOg body weight.
1.4 Observations: after infection, the animal’s general situation, infected symptoms and death were observed. The observation period was two weeks. At the end of the trial, all animals were dissected to record the general pathological changes. Acute toxicity was graded according to the acute toxicity rating criteria.
Test results of acute oral toxicity test in mice
2015101912 20 Mar 2015
2. Test results
| Sex | Dose (mg/kg) | Number of Animals | Number of Dead Animals | Mortality Rate (%) |
| Female | 20000 | 10 | 0 | 0 |
| Male | 20000 | 10 | 0 | 0 |
3. Conclusion
After infecntion, the animals do not show any infected symptoms and have no death and no significant pathological changes, and on the contrary, the weight of the animals showed an increasing trend. Therefore, the LD50 of the test substance on female and male mice by oral was greater than 10000 mg/kg body weight, so the test substance belongs to the actual non-toxic level.
II. Effect on DNA damage induced by peroxide
1. Experimental materials and equipment
1.1 5mM Vitamin C solution: it was prepared by dissolving it in three distilled water and it needs to be prepared before use. Vitamin C powder was obtained from Sigma-Aldrich;
1.2 5mM CuCl2 solution: it was prepared by dissolving it in three distilled water and it needs to be prepared before use. CuCl2 powder was obtained from Sinopharm Chemical Reagent Co. Ltd.;
1.3 Plasmid DNA: it was obtained by amplifing and extracting the pET28a plasmid from DH5a Escherichia coli (the plasmid extraction kit was purchased from Promega);
1.4 5mMEDTA solution, pH8.0: EDTA was obtained from Sinopharm Chemical Reagent Co. Ltd.;
1.5 lOxPBS buffer: 80g NaCl, 2g KC1, 14.4g Na2HPO4 and 2.4g KH2PO4 were dissolved in distilled water, the pH was adjusted to 7.4 and the volume was adjusted to IL. The required reagents were purchased from Sinopharm Chemical
2015101912 20 Mar 2015
Reagent Co. Ltd.;
1.6 Agarose: obtained from Bio west;
1.7 Goldview nucleic acid dyes were obtained from Biotium;
1.8 Gel Imager: obtained from Sage Creation Science And Technology Co., Ltd;
1.9 Electrophoresis apparatus: LiuYi instrument factory;
1.10 Test substance: the micro-kinetic drinking water of Examples 3 and 4
2. Experimental methods
2.1 Group of Experiments
Group I: untreated group, the untreated sterile water was applied to the reaction system;
Group II: the micro-kinetic drinking water of example 3;
Group III: the micro-kinetic drinking water of example 4.
The reaction substances were added according to the following order, the final volume of the reaction system was 10 pL:
lOxPBS buffer 1 pL;
Plasmid DNA (2 pg) 2 pL;
mM Vitamin C 1 pL;
mM CuCl2 1 pL;
supplemented with the untreated water or treated water to 10 pL.
2.2 The reaction was terminated by adding lpL of 5 mM EDTA and 2.2 pL of loading buffer at 0.5 hour and 1 hour respectively;
2.3 0.8% agarose gel (containing Goldview dye), electrophoresis 1 h;
2.4 Gel imaging.
3. Experimental results
Vitamin C and divalent copper ions occur redox reaction to form peroxide, which will cause plasmid DNA damage and fracture, this embodies in the weakening of the electrophoretic bands. The experimental results are shown in figure 1, the experiment is divided into three groups, the grouping method was as
2015101912 20 Mar 2015 described above, each group is reacted at two time points: 0.5 hours and 1 hour, the brightness of DNA represents DNA content. According to the experimental results, the degradation rate of plasmid DNA by redox reaction was basically the same in group II and III compared with untreated group I, and the micro-kinetic drinking water does not prevent or accelerate DNA damage and fracture.
4. Analysis on the results
Vitamin C and divalent copper ions occur redox reaction to form peroxide, which will cause plasmid DNA damage and fracture, this embodies in the weakening of the electrophoretic bands. The experimental results showed the degradation rate of plasmid DNA by redox reaction was basically the same in group II and III compared with untreated group I, and the micro-kinetic drinking water did not prevent or accelerate DNA damage and fracture. It is shown that the multi-polar micro-kinetic energy water of the present invention has no effect on DNA damage induced by peroxide.
III. Effect on cell membrane integrity of HepG2 cultured in vitro
1. Experimental materials and equipment
1.1 Human hepatocellular carcinoma cells: HepG2;
1.2 Cell culture medium: DMEM culture medium+10%FBS;
1.3 FAM-aptamer: purchased from Sangon Biotech (Shanghai) Co., Ltd.;
1.4 Fluorescent EZ micromirrors: OLYMPUS;
1.5 96-well cell culture plate;
2. Experimental methods
2.1 A proper amount of pancreatic enzyme was added to digest the cells in logarithmic growth phase. The cell concentration was adjusted, and the cell was seeded in 96-well plates at 4000 cells per well and cultured overnight;
2.2 5 ml of DMEM culture medium containing 10% FBS (culture medium A) was treated in the same manner as in Example 3 for 40 minutes (treatment group);
2015101912 20 Mar 2015
2.3 5 ml of DMEM culture medium containing 10% FBS (culture medium B) was treated for 40 minutes at the position which was more than 2m away from the wave source of example 3 (untreated group);
2.4 4 qg of FAM-labeled C6-8 aptamer was added to 100 pL of culture medium A and culture medium B respectively and mixed uniformly;
2.5 The cell fluid adherent cultured in the 96-well plate were replaced with culture medium A and culture medium B containing FAM-C6-8 aptamer, incubated at 37 °C for 30 min, and then washed once with PBS and observed under the fluorescence microscope.
3. Experimental results
The results were shown in figure 2, there was no difference in cell morphology between the treated and untreated groups, the cells in the two groups showed autofluorescence and had intact cell membranes, and no apparent FAM-labeled C6-8 aptamer green fluorescence was observed in the cells.
4. Results and analysis
If the cells are perforated, the FAM-labeled C6-8 aptamer can enter the cell and bind with the protein in the cell and emit green fluorescence upon excitation at 530nm wavelengths. The results show that there is no difference between the cells in the treatment group and that in the untreated group, the cells in the two groups showed autofluorescence, which indicates that the cell membrane is intact. The multi-polar micro-kinetic energy drinking water of the present invention has no effect on cell morphology and cell membrane integrity.
Example 7 Wide spectral absorptance and UV absorptance of the multi-polar micro-kinetic energy drinking water of the present invention
1. Experimental materials and equipment
1.1 The micro-kinetic energy drinking water prepared in Example 3
1.2 The raw water of ordinary drinking water used in Example 3;
1.3 The drinking water prepared from Evian Natural Mineral Water, which
2015101912 20 Mar 2015 was treated in the same manner as in Example 3;
1.4 Evian Natural Mineral Water: purchased from Carrefour supermarket;
1.5 The drinking water prepared from Tibet Glacier Mineral Water, which was treated in the same manner as in Example 3;
1.6 Tibet Glacier Mineral Water: purchased from Carrefour supermarket;
1.7 Ultra-pure water: prepared by the German Sartorius ultra-pure water preparation system;
1.8 Fiber optic spectrometer Ava-Spec3648, the measuring range is 200-1100 nm, the uncertainty/accuracy is 0.5nm;
1.9 Oscilloscope MS04104, equipment factory number: C001163, measurement accuracy: 1 GHz;
1.10 Photodetector Thorlab DET10A/M, equipment factory number: JGZX-ZXZC-012, measurement accuracy: 200 nm-1100 nm;
1.11 Test conditions: the temperature is 21 °C, the humidity is 38%.
2. Experimental methods
2.1 Wide spectrum absorptance (1) Rinsing the experimental vessels by using about 200 ml ultra-pure water till the physical and chemical indexes were stable.
(2) Placing the raw water of ordinary drinking water used in Example 3 and the micro-kinetic energy drinking water prepared in Example 3 in an experimental vessel, and scanning the spectral absorption of the standard light source of sample at 200 nm to 800 nm, and the experimental results were recorded.
(3) Rinsing the experimental vessels by using about 200 ml ultra-pure water till the physical and chemical indexes restored the original value and reached stability.
(4) Placing the Evian Natural Mineral Water and the drinking water prepared from the Evian Natural Mineral Water by using the same manner as Example 3 in the experimental vessels, repeating the step (2), and then recording the changes in
2015101912 20 Mar 2015
UV intensity during the experimental period.
(5) Rinsing the experimental vessels by using about 200 ml ultra-pure water till the physical and chemical indexes restored the original value and reached stability (6) Placing the Tibet Glacier Natural Mineral Water and the drinking water prepared from the Tibet Glacier Natural Mineral Water by using the same manner as Example 3 in the experimental vessels, repeating the step (2), and then recording the changes in UV intensity during the experimental period.
(7) Rinsing the experimental vessels by using about 200 ml ultra-pure water till the physical and chemical indexes restored the original value and reached stability
2.2 UV absorptance (1) Rinsing the experimental vessels by using about 200 ml ultra-pure water till the physical and chemical indexes were stable.
(2) Placing the micro-kinetic energy drinking water prepared in example 3 in an ultraviolet intensity detection system, irradiatting the water to be measured using the ultraviolet light source, observaing the ultraviolet intensity value of the water to be measured through the oscilloscope MS04104 and the photodetector Thorlab DET10A/M for 72 hours continuously, and recording the changes in UV intensity during the experimental period.
(3) Rinsing the experimental vessels by using about 200 ml ultra-pure water till the physical and chemical indexes restored the original value and reached stability (4) Placing the Evian Natural Mineral Water and the drinking water prepared from the Evian Natural Mineral Water by using the same manner as Example 3 in the ultraviolet intensity detection system, repeating the step (2), and then recording the changes in UV intensity during the experimental period.
(5) Rinsing the experimental vessels by using about 200 ml ultra-pure water till the physical and chemical indexes restored the original value and reached
2015101912 20 Mar 2015 stability.
(6) Placing the Tibet Glacier Natural Mineral Water and the drinking water prepared from the Tibet Glacier Natural Mineral Water by using the same manner as Example 3 in the the ultraviolet intensity detection system, repeating the step (2), and then recording the changes in UV intensity during the experimental period.
(7) Rinsing the experimental vessels by using about 200 ml ultra-pure water till the physical and chemical indexes restored the original value and reached stability
3. Experimental results
3.1 Wide spectrum absorptance test
The results are shown in Figure 3-5.
3.2 UV absorptance test
The multi-polar micro-kinetic energy drinking water prepared from ordinary water:
UV absorptance 0.9135 -> 0.9037
The multi-polar micro-kinetic energy drinking water prepared from Evian Natural Mineral Water:
UV absorptance
The multi-polar micro-kinetic
Glacier Mineral Water:
0.9244 ->0.9188 energy drinking water prepared from Tibet
0.9157->0.9079
UV absorptance
4. Experimental conclusions and analysis
Through the above experiments, the following conclusions are obtained:
(1) Compared with the raw water, the absorption peak of the absorption spectrum of the multi-polar micro-kinetic energy drinking water of the present invention is shifted to the short wave by about 30 nm;
(2) The absorbance of the multi-polar micro-energy drinking water of the present invention decreased by 1% after testing for 72 hours.
2015101912 20 Mar 2015
Example 8 The stimulated fluorescence spectrum change of the multi-polar micro-kinetic energy drinking water of the present invention
1. Experimental materials and equipment
1.1 The micro-kinetic energy drinking water prepared in Example 3
1.2 The raw water of ordinary drinking water used in Example 3;
1.3 The drinking water prepared from Evian Natural Mineral Water, which was treated in the same manner as in Example 3;
1.4 Evian Natural Mineral Water: purchased from Carrefour supermarket;
1.5 The drinking water prepared from Tibet Glacier Mineral Water, which was treated in the same manner as in Example 3;
1.6 Tibet Glacier Mineral Water: purchased from Carrefour supermarket;
1.7 Ultra-pure water: prepared by the German Sartorius ultra-pure water preparation system;
1.8 Fiber optic spectrometer Ava-Spec3648, equipment factory number is 1101230U1, the measuring range is 200-1100 nm, and the uncertainty/accuracy is 0.5nm;
1.9 Test conditions: the temperature is 22 °C, and the humidity is 37 %.
2. Experimental methods (1) Rinsing the experimental vessels by using about 200 ml ultra-pure water till the physical and chemical indexes were stable.
(2) Placing the micro-kinetic energy drinking water prepared in Example 3 in a fiber-optic spectrometer sample pool, enforcing an excitation pulse on the sample with a 266 nm pulsed ultraviolet laser, and measuring the emission spectrum of the samples using the scanning function on the fiber-optic spectrometer and recording the fluorescence emission spectra of the samples.
(3) Rinsing the experimental vessels by using about 200 ml ultra-pure water till the physical and chemical indexes restored the original value and reached stability
2015101912 20 Mar 2015 (4) Placing the raw water of ordinary drinking water used in Example 3 in the fiber-optic spectrometer sample pool, repeating the step (2), and then recording the changes in UV intensity during the experimental period.
(5) Rinsing the experimental vessels by using about 200 ml ultra-pure water till the physical and chemical indexes restored the original value and reached stability.
(6) Placing the drinking water prepared from Evian Natural Mineral Water by using the same manner as in Example 3 in the fiber-optic spectrometer sample pool, repeating the steps (2) and (3), and then recording the experimental results.
(7) Placing the Evian Natural Mineral Water in the fiber-optic spectrometer sample pool, repeating the steps (2) and (3), and then recording the experimental results.
(8) Placing the drinking water prepared from Tibet Glacier Natural Mineral Water by using the same manner as in Example 3 in the fiber-optic spectrometer sample pool, repeating the steps (2) and (3), and then recording the experimental results.
(9) Placing the Tibet Glacier Natural Mineral Water in the fiber-optic spectrometer sample pool, repeating the steps (2) and (3), and then recording the experimental results.
(10) Placing the drinking water prepared from Tibet Glacier Natural Mineral Water in triplicate by using the same manner as in Example 3 in the fiber-optic spectrometer sample pool, repeating the steps (2) and (3), and then recording the experimental results.
3. Experimental results
3.1 Fluorescence steady-state transient test
The fluorescence steady-state transient test results of ordinary drinking water and the multi-polar micro-kinetic energy drinking water prepared from the same are shown in Figure 6. The fluorescence steady-state transient test results of Evian Natural Mineral Water and the multi-polar micro-kinetic energy drinking
2015101912 20 Mar 2015 water prepared from the same are shown in Figure 7. The fluorescence steady-state transient test results of Tibet Glacier Mineral Water and the multi-polar micro-kinetic energy drinking water prepared from the same are shown in Figure 8. The repeatability test results of the drinking water prepared from Tibet Glacier Natural Mineral Water in triplicate are shown in Figure 9.
4. Experimental conclusions and analysis
From the above experimental results, it can be seen that compared with raw water of drinking water, the intensity of the fluorescence emission spectrum of the multi-polar micro-kinetic energy drinking water is greatly improved in the spectral range between 300nm and 400nm. The results from many repeated experiments on the same experimental sample show that the experiment has good reproducibility.
The experiments show that the multi-polar micro-kinetic energy drinking water of the present invention has more extranuclear electrons in the non-radiative high energy level.
Example 9 Anti-fatigue function of the multi-polar micro-kinetic energy drinking water of the present invention: effect on the content of lactate in serum of mice and weight-bearing swimming time in mice
1. Materials and Method
El Samples:
The multi-polar micro-kinetic energy drinking water (the kinetic energy water) prepared in Example 3, which is a colorless, tasteless and transparent water-like liquid, stored at room temperature and should be used within 72 hours after being prepared.
Ang Li American Ginseng Capsule, which was obtained from Shanghai Jiaoda Onlly Co., Ltd., the batch number is C2102001.
1.2 Experimental animals: ICR mice were purchased from Beijing Huafu Kang Biotechnology Co. Ltd., the production license number is SCXK (Beijing)
2015101912 20 Mar 2015
2007-0001, the laboratory animal quality certificate number is 11401300009361. There are 40 in all, all of them are males, 10 in each group, and 4 groups in total, the ordering weight is 18-20 g.
Feeding conditions: the laboratory animal facilities continue to maintain the barrier environmental standards. The main environmental indicators are as follows: the room temperature is 20 - 26 °C, the daily temperature difference is <4 °C. The relative humidity is 40 to 70%. The minimum ventilation time is 15 times per hour. Lighting: Dark = 12 h: 12 h. Animals were housed in PC mice rearing cages and the specifications of cages were 545x395x200 mm , 5 in each cage, and their space was in accordance with the provisions of GB 14925-2010 of the People's Republic of China on the minimum required space for experimental animals. All animals are fed and managed by trained and qualified personnel. The padding and cage are replaced once a week. And the special feed for mice is added every day for animal consumption and the animal diet activities are kept free during the whole feeding process.
Animal welfare: the animal and related treatment used in this test should meet the requirements of animal welfare. Experiments were carried out after being reviewed and approved by the Laboratory's Institutional Animal Care and Use Committee (IACUC), Beijing Jianhao Pharmaceutical Technology Development Co. Ltd.. The specific considerations are as follows:
(1) During the course of the experiment, the animal's panic and pain should be minimized.
(2) Effective anesthesia must be performed in the operation and dissection of experimental animals. Analgesic, targeted care and diet conditioning should be performed based on the actual situation during postoperative recovery period.
(3) Animal disturbances, panic, pain and injure should be avoided in the experiments of fixation of animal. Fixation machine should have a reasonable structure, appropriate specification, should be durable, environmentally friendly and easy to operate. Without prejudice to the experiment, the mandatory
2015101912 20 Mar 2015 restrictions on the animal's body should be reduced to a minimum.
(4) Sample collection: the operation of the sample collection of experimental animals should be carried out in a safe and humane manner (to minimize stress and discomfort caused to animals).
(5) Dying animals, diseased animals and animals with severe toxicity should be promptly reported to the veterinarian and the person in charge to timely treat or euthanize. Without affecting the determination of the experiment results, “humane endpoint should be chosen to avoid prolonging the time when the animals suffer from pain. It should be euthanized in accordance with the humanitarian principles when the animals are sacrificed. At the scene of the execution, there should be no other animals present. The body can be properly disposed of after confirmation of the death of the animal.
(6) Occupational safety: personal protective measures such as gloves, gowns, masks, goggles and earplugs etc. should be taken during the experiment.
1.3 Dose selection and the administering manner of the test substances:
The experiment was divided into four groups: a model control group, a low-dose kinetic energy water group, a high-dose kinetic energy water group and an Ang Li American Ginseng capsule group. In the model control group, the animals drank the kinetic energy water daily (the animals was given normal drinking water by gavage once a day with a dose of 0.3 mL/10 g body weight); in the low-dose kinetic energy water group, the animals drank the kinetic energy water daily (the kinetic energy water was kept in a drinking bottle, and supplemented once a day); in addition to drinking the kinetic energy water daily, in the high dose kinetic energy water group, the animals were given the kinetic energy water by gavage once a day, the dose was 0.3 ml/10 g body weight; in the Ang Li American Ginseng capsule group, the administration dose was 0.6 g/kg (the daily dosage of Ang Li American Ginseng capsule per person is 4 grains, the net content of each grain is 0.28g, if the weight of a person is 60kg, the dosage on the person is equivalent to 0.01867g/kg, if the dosage used in mice is 30 times of
2015101912 20 Mar 2015 that used in the person, the dosage on the mice is equivalent to 0.56 g/kg, the administration volume is 30 mL/kg and the concentration of drugs is 0.0186 mg/ml, the concentration of reagents is 0.02g/ml, the actual administration dose is 0.6g/kg). No fasting water requirement during the experiment. The test of lactic acid was administered continuously for 33 days, after administering continuously for 2 days, the swimming test was performed, and then the drugs were administering continuously for 35 days.
1.4 Main instruments and reagents:
lactic acid analyzer, Lactate Scout+, Germany EKF; lactic acid analyzer dedicated test strip, batch number: 3312307, 3311351, 3312307, Germany EKF, timer.
1.5 Test methods:
Blood lactate test: after continuous administration of 33 days, the blood samples were taken at 30 min after the last administration and the blood lactate was measured before swimming; the blood lactate was measured immediately when the blood was collected after swimming in the water of 30 °C for 10 min without bearing any weight; and then another blood collection for determination was performed after rest for 20 min. The blood samples were taken by pricking the tail vein with a needle, and the volume of the collected blood was at least 0.5 pL.
Weight-bearing swimming experiment: the same batch of animals were administrated continuously for 2 days, there were 35 days of continuous administration in total, 30 min after the last administration, the mice with a lead sheath (5% of the mouse’s body weight) attached to the tail root were put in a swimming pool to swim. Water depth was not less than 30 cm, and the water temperature was 25°C±1.0°C. The time was recorded from beginning to swim to sinking into the water, i.e., the mice weight-bearing swimming time.
1.6 Statistics and analysis of the test data
A SPSS statistical software was used to process the results of body weight
2015101912 20 Mar 2015 and compare and analyze the administration and control group. Statistical analysis was done according to the following methods: the normal test was performed using Kolmogorov-Smirnov method and the test for homogeneity of variance was performed using Levene median method, if P>0.05, then one-way ANOVA method will be performed, and if the normal test and the test for homogeneity of variance failed (P<0.05), then a nonparametric Mann-Whitney test will be required.
1.7 Determination of results
The lactic acid determination data was as the measurement data, before the analysis of variance for data, the homogeneity test of variance should be performed according to the variance analysis procedure. If the variance was homogeneous, the F value was calculated. If the F value<0.05, the difference among the groups was not significant. If the F value > 0.05, P < 0.05, the pairwise comparison method on the means of the multiple experimental groups and the control group was used to statistics; an appropriate variable conversion of non-normal or unequal variance data was made to meet the normal or equal variance requirements, and then the converted data were used to statistics; if the converted variable still did not reach the normal or equal variance, the rank sum test will be performed for statistics.
If the lactate level of the test sample was significantly lower than that of the control group and the difference was significant, the result was judged as positive.
The swimming time was as the measurement data, before the analysis of variance for data, the homogeneity test of variance should be performed according to the variance analysis procedure. If the variance was homogeneous, the F value was calculated. If the F value <0.05, the difference among the groups was not significant. If the F value > 0.05, P < 0.05, the pairwise comparison method on the means of the multiple experimental groups and the control group was used to statistics; an appropriate variable conversion of non-normal or
2015101912 20 Mar 2015 unequal variance data was made to meet the normal or equal variance requirements, and then the converted data were used to statistics; if the converted variable still did not reach the normal or equal variance, the rank sum test will be performed for statistics.
If the weight-bearing swimming time was significantly longer than that of the control group and the difference was significant, the result was judged as positive. Otherwise, the results were analyzed according to the actual situation of the test substance.
2. 2. Results
The results were shown in Table 2, Table 3 and Figure 10.
Table 2 Effect of the multi-polar micro-kinetic energy drinking water of the present invention on the content of lactate in serum of mice (mmol/L, x±5,n=10)
| Measuring time | Low-dose | American ginseng group | ||
| Blank control group | kinetic energy water group | High-dose kinetic energy water group | ||
| Before | 2.0±1.0 | 1.9±0.5 | 1.5±0.4 | 1.4±0.2 |
| swimming Immediately after | 4.8±0.8 | 4.7±1.4 | 4.7±1.2 | 4.7±2.2 |
| swimming 20 min after | 5.3±1.9 | 5.2±1.2 | 5.H2.3 | 4.8±1.5 |
| swimming |
It can be seen from Table 2 that there was no significant difference in blood lactate value before swimming, immediately after swimming and 20 min after swimming among the low-dose kinetic energy water group, the high-dose kinetic energy water group and the blank control group, and the absolute values were consistent; there was no significant difference in blood lactate value before swimming, immediately after swimming and 20 min after swimming between the
2015101912 20 Mar 2015
American ginseng group and the blank control group, and the absolute values were consistent. This result suggested that the kinetic energy water of the present invention and the positive-drug American ginseng had no significant effect on the blood lactate before and after swimming in mice.
Table 3 Effect of the multi-polar micro-kinetic energy drinking water of the present invention on the weight-bearing swimming time of mice (min, x±5, n=I0)________________________________________________________________
| Measuring time | Blank control group | Low-dose kinetic energy water group | High-dose kinetic energy water group | American ginseng group |
| Administrati | ||||
| on for 35 | 9.54±4.64 | 11.62±5.40 | 13.08±6.94 | 14.21±6.28 |
| days |
It can be seen from Table 3 and Figure 10 that in the weight-bearing swimming experiment, the average swimming time of the mice in the blank control group was 9.54 ± 4.64 min, the average swimming time of the mice in the low-dose kinetic energy water group was 11.62 ± 5.40 min, the average swimming time of the mice in the high-dose kinetic energy water group was 13.08 ± 6.94 min, the average swimming time of the mice in the positive-drug American ginseng group was 14.21 ± 6.28 min. Although there was no statistical difference, the absolute value of the swimming time in low and high dose kinetic energy water group increased compared with that in the blank control group, and there was a certain dose-effect relationship. Although no statistical difference was found in the positive-drug group, the swimming time was also significantly increased. It is suggested that the kinetic energy water of the present invention can prolong the weight-bearing swimming time of mice.
3. Conclusion
3.1 Determination of blood lactate
2015101912 20 Mar 2015
Under the experimental conditions, the kinetic energy water of the present invention and the positive-drug American ginseng had no significant effect on the blood lactate before and after swimming in mice.
3.2 The weight-bearing swimming swimming time of mice
Under the experimental conditions, the kinetic energy water of the present invention can prolong the weight-bearing swimming time of mice.
Claims (10)
1. Use of a multi-polar micro-kinetic energy drinking water in the preparation of beverages, health care products or medicaments for anti-fatigue.
2. The use according to claim 1, wherein the multi-polar micro-kinetic energy drinking water was prepared by treating raw water of drinking water using three different types of electromagnetic waves: low, medium and high frequency electromagnetic waves, in non-contact mode, wherein the frequency range of the low frequency electromagnetic wave is 30-100 kHz, the frequency range of the medium frequency electromagnetic wave is 550-720 kHz, the frequency range of the high frequency electromagnetic wave is 300-725 MHz.
3. The use according to claim 1 or 2, wherein the raw water of drinking water is ordinary tap water or various mineral water or purified water such as Evian Natural Mineral Water, Tibet Glacier Mineral Water, NongFu Spring, Wahaha and so on.
4. The use according to any one of the claims 1 to 3, wherein wherein the frequency range of the low frequency electromagnetic wave is 45-75 kHz;
preferably, the frequency range of the medium frequency electromagnetic wave is 600-720 kHz;
preferably, the frequency range of the high frequency electromagnetic wave is 300-425 MHz.
5. The use according to any one of the claims 1 to 4, wherein the low frequency electromagnetic wave and the medium frequency electromagnetic wave are sine wave, square wave, sharp wave, sawtooth wave or trapezoidal wave;
2015101912 20 Mar 2015 preferably, the waveform of the low frequency electromagnetic wave and the medium frequency electromagnetic wave may be the same or different;
more preferably, the waveform of the low frequency electromagnetic wave and the medium frequency electromagnetic wave is the same.
6. The use according to any one of the claims 1 to 5, wherein the low frequency electromagnetic wave and the medium frequency electromagnetic wave are propagated in the same direction.
7. The use according to any one of the claims 1 to 6, wherein tthe high frequency electromagnetic wave is triangular wave, and its propagation direction is perpendicular to that of the low frequency electromagnetic wave and the medium frequency electromagnetic wave.
8. The use according to any one of the claims 1 to 7, wherein when treating raw water of drinking water using low, medium and high frequency electromagnetic waves, which are three different types of electromagnetic waves, in non-contact mode, firstly, the low-frequency electromagnetic wave and the high frequency electromagnetic wave are used simultaneously and then the medium electromagnetic wave and high frequency electromagnetic wave are used simultaneously;
preferably, the treating time with the low frequency electromagnetic wave and the high frequency electromagnetic wave is 10 to 30 minutes, preferably 10 to 20 minutes;
preferably, the treating time with the medium frequency electromagnetic wave and the high frequency electromagnetic wave is 10 to 30 minutes, preferably 10 to 20 minutes.
9. The use according to any one of the claims 1 to 8, wherein when
2015101912 20 Mar 2015 compared with the raw water of drinking water, the ultraviolet absorption peak of the obtained multi-polar micro-kinetic energy drinking water is shifted to the short-wave direction by 25 nm to 40 nm, preferably 28 nm to 30 nm, and most preferably 30 nm or 28 nm.
10. The use according to any one of the claims 1 to 9, wherein the beverage is a functional beverage and can be used for anti-fatigue;
preferably, the health care products or medicaments can be used for anti-fatigue.
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