CN110946848A - Compound bactericide with broad-spectrum bactericidal effect - Google Patents
Compound bactericide with broad-spectrum bactericidal effect Download PDFInfo
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- CN110946848A CN110946848A CN201811130115.7A CN201811130115A CN110946848A CN 110946848 A CN110946848 A CN 110946848A CN 201811130115 A CN201811130115 A CN 201811130115A CN 110946848 A CN110946848 A CN 110946848A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/01—Hydrocarbons
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/11—Aldehydes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K31/00—Medicinal preparations containing organic active ingredients
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/02—Local antiseptics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention provides a compound bactericide with bacteriostatic or bactericidal activity, which comprises, by weight, 40% -50% of linalool, 18% -25% of terpineol, 8% -15% of geraniol, 2% -4% of β -cyclocitral, 2.5% -5% of nerol, 1% -3% of myrcene, 1% -3% of ocimene, 3% -6% of ionone and 5% -8% of 2H- β -ionone.
Description
Technical Field
The invention belongs to the field of manufacture of daily chemical products, and particularly relates to a compound bactericide with a broad-spectrum bactericidal effect.
Background
Infectious diseases caused by pathogenic microorganisms such as bacteria, fungi and viruses still seriously threaten human health so far due to the influence of factors such as variation of microorganisms, change of human living environment, and change of certain hosts, and some of them are difficult to completely control. Statistically, about 1600 million people die of microbial infections annually worldwide, accounting for about 1/3 of the total number of patients who die. Bacteria and fungi are ubiquitous in nature, most of which are directly or indirectly beneficial to humans, but few species are also harmful to the environment, humans and animals. In recent years, with the improvement of living standard of people, companion animals such as pets gradually come into the lives of people, the number of companion animals is greatly increased, and the companion animals also pose the threat of people and livestock suffering from diseases while enjoying the people. According to statistics of relevant data, more than half of over 200 animal infectious diseases which are proved worldwide at present can be transmitted to human beings, including bacterial diseases, fungal diseases, viral diseases, rickettsia, protozoal diseases, parasitic diseases and the like, and about 90 animal diseases which are proved to exist and prevail in China are known. With the development of pet career, the relationship between pets and human beings is increasingly close, and the physical health and social public health conditions of human beings are seriously threatened by zoonosis. At present, although few antibiotics and chemically synthesized antibacterial drugs are used in clinic, drug-resistant strains are increasing, and due to the fact that allergy is easy to cause or adverse reactions are large, the clinical application of some drugs is limited, and meanwhile, some antibiotics for resisting bacterial infection cannot effectively inhibit fungi. Since 1987, no new antibiotic species have been discovered in the last thirty years. In order to resist microbial infection, a high-efficiency and low-toxicity bactericide is urgently needed to be found for human beings.
Chemical agents can inhibit or kill microorganisms and are thus used for the control of microbial growth. Chemical agents can be divided into bactericides and bacteriostats depending on the nature of action. Bactericides are chemical agents that destroy the metabolic functions of bacteria and have a lethal effect. The bacteriostatic agent does not destroy the protoplasm of the bacteria, but only suppresses the synthesis of new cell substances, so that the bacteria cannot proliferate, such as sulfonamides, antibiotics and the like. Biocides are used primarily to inhibit or kill microorganisms on surfaces, appliances, excretions and the surrounding environment. Bacteriostatic agents are commonly used on body surfaces, such as skin, mucous membrane, wound, etc., to prevent infection, and also used for antisepsis of food, beverage, and medicine.
The ideal chemical bactericide and bacteriostat should have the characteristics of quick action, high efficacy, small damage to tissues, strong penetrability but small corrosion, convenient and stable preparation, low price and the like. But very few chemicals are really fully satisfactory for the above requirements. Research for more than 10 years shows that part of plant essential oil has strong characteristics of inhibiting or killing microorganisms such as bacteria, fungi and the like. The plant essential oil belongs to natural, green and residue-free substances, and has the characteristics of wide source, wide antimicrobial spectrum, rapid action, difficult generation of drug resistance and the like. The plant essential oil can be used as a novel bactericidal substance to be developed for research. However, the natural essential oil has high production cost and cannot be applied to disinfection products. We find that part of the components of the plant essential oil have the functions of bacteriostasis and sterilization by researching the plant essential oil, so that the compound bactericide with broad-spectrum sterilization function is invented.
Disclosure of Invention
The invention aims to provide a compound bactericide.
The compound bactericide provided by the invention is prepared from the raw materials comprising linalool, terpineol, geraniol, β -cyclocitral, nerol, myrcene, ocimene, ionone and 2H- β -ionone.
The compound bactericide comprises, by weight, 40-50% of linalool, 18-25% of terpineol, 8-15% of geraniol, 2-4% of β -cyclocitral, 2.5-5% of nerol, 1-3% of myrcene, 1-3% of ocimene, 3-6% of ionone and 5-8% of 2H- β -ionone.
Specifically, the compound bactericide comprises, by weight, 45% of linalool, 18% of terpineol, 8% of geraniol, β -cyclocitral 3%, 3% of nerol, 1% of myrcene, 1% of ocinene, 4% of ionone and 6% of 2H- β -ionone.
The application of the compound bactericide in inhibiting and killing bacteria and fungi or the application of the compound bactericide in preparing products for inhibiting and killing bacteria and fungi also belongs to the protection scope of the invention.
The bacteria may specifically be: staphylococcus epidermidis, Bacillus subtilis, Staphylococcus aureus and Escherichia coli, preferably Staphylococcus epidermidis and Bacillus subtilis.
The fungi may specifically be: candida albicans, Saccharomyces cerevisiae, Trichophyton rubrum and Trichophyton schenckii, preferably Candida albicans, Saccharomyces cerevisiae and Trichophyton rubrum.
The invention also provides a reagent for killing zoonosis and other pathogenic bacteria infected by human or animals, wherein the reagent is the bactericide or contains the bactericide.
The invention prepares a compound bactericide according to effective bactericidal components in essential oil. Experiments show that the bactericide has broad-spectrum sterilization and bacteriostasis effects, has obvious bacteriostasis and sterilization effects on bacteria (escherichia coli, bacillus subtilis, staphylococcus aureus, staphylococcus epidermidis and the like) and fungi (candida albicans, trichosporon rubrum, trichophyton rubrum and saccharomyces cerevisiae and the like), and has obvious effects on common bacteria of people and livestock (trichosporon rubrum, trichophyton rubrum, candida albicans and the like). The bactericide can be used for development and application of disinfectant and zoonosis reagent.
Drawings
Fig. 1 is a schematic diagram of the inhibition zone of the compound bactericide of the invention to eight bacteria. A, escherichia coli; b, bacillus subtilis; staphylococcus aureus; staphylococcus epidermidis; c, Candida albicans; f, saccharomyces cerevisiae; g, Sporothrix schenckii; h, Trichophyton rubrum. The starting concentration of ampicillin is 100 mg/ml; the initial concentration of amphotericin B is 100 mg/ml; (1:60) represents a 60-fold dilution; (1:5) represents a 5-fold dilution; (1:2000) represents a 2000-fold dilution; (0) represents no dilution; the diameter of the filter paper sheet is 6 mm; the diameter of the inhibition zone is 7-9mm, and the low sensitivity is obtained; the medium sensitivity is 10-15 mm; a high sensitivity of >15 mm; <6mm is not sensitive.
FIG. 2 shows the absorbance values of Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Staphylococcus epidermidis, Candida albicans and Saccharomyces cerevisiae at a wavelength of 600nm after treatment with the compound bactericide of the present invention. Indicates extreme significance; indicates significance; "Amp" represents ampicillin; "Amphotericin B" stands for Amphotericin B.
FIG. 3 is a Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) chart of the compound bactericide of the present invention against Sporothrix schenckii. Wherein A is DMSO treatment; b is 0.005 percent of compound bactericide treatment; c is 0.025 percent of compound bactericide treatment; d is 0.05 percent of compound bactericide treatment; e is 0.1 percent of compound bactericide treatment; f is treated by 0.125 percent of compound bactericide; g is 0.25 percent of compound bactericide treatment; h is treated by 0.4 percent of compound bactericide.
Fig. 4 is a graph of the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of the compound bactericide of the present invention against trichophyton rubrum. Wherein A is DMSO treatment; b is 0.005 percent of compound bactericide treatment; c is 0.025 percent of compound bactericide treatment; d is 0.05 percent of compound bactericide treatment; e is 0.1 percent of compound bactericide treatment; f is treated by 0.125 percent of compound bactericide; g is 0.25 percent of compound bactericide treatment; h is treated by 0.4 percent of compound bactericide.
Detailed Description
The present invention will be described below with reference to specific examples, but the present invention is not limited thereto.
The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, biomaterials, etc. used in the following examples are commercially available unless otherwise specified.
Example 1 study of antibacterial Activity of Compound Fungicide
Step 1, formula of compound bactericide
A series of compound bactericides are prepared according to main components of plant essential oil. Through antibacterial and bacteriostatic drug sensitivity tests, the best formula of the compound bactericide is determined.
The compound bactericide comprises, by weight, 40% -50% of linalool, 18% -25% of terpineol, 8% -15% of geraniol, 2% -4% of β -cyclocitral, 2.5% -5% of nerol, 1% -3% of myrcene, 1% -3% of ocimene, 3% -6% of ionone and 5% -8% of 2H- β -ionone, and the specific components are shown in Table 1.
TABLE 1 composition of the Compound Fungicide
Step 2, determining the bacteriostatic activity of the compound bactericide
2.1 preparation of the culture Medium
The bacterial culture adopts LB culture medium: per liter of culture medium, 950ml of deionized water should be added: tryptone 10g, yeast extract 5g, sodium chloride 10g, shake the container until the solutes are dissolved. The pH value is adjusted to 7.0 by using 1mol/L NaOH, and the volume is adjusted to 1000 ml. Subpackaging, and sterilizing with high pressure steam at 121 deg.C for 20 min. Solid medium: adding 15g of agar powder based on liquid culture medium, subpackaging, and sterilizing with high pressure steam at 121 deg.C for 20 min.
YPD culture medium is adopted for fungus culture: dissolving yeast extract 10g, peptone 20g in 900ml water, and adding agar powder 20g if preparing plate. Packaging, sterilizing with high pressure steam at 121 deg.C for 20min, and adding 100ml of 20% glucose solution.
Bottom layer agar: melting the prepared solid culture medium of the corresponding bacteria, pouring the melted solid culture medium into sterile 10cm plates, wherein each plate is about 20-25 ml, and waiting for solidification.
Top agar layer: melting the prepared solid culture medium containing 0.7% agar powder, placing 3ml into a 5ml EP tube when the temperature is reduced to no scald, placing 1ml of bacterial suspension into the EP tube, mixing uniformly and pouring into a bottom layer agar plate.
Preparing a drug-containing agar plate: according to the experimental design, antibacterial drugs with different concentrations and diluted in multiple times are respectively added into agar which is heated and dissolved, the agar is balanced in a water bath at 45-50 ℃, the mixture is fully mixed and poured into a sterilization plate, and the thickness of the agar is 3-4 mm. Drug agar plates are usually formulated in a 1: 9 ratio, with drug concentration ranges being selected as desired.
2.2 preparation of the bacterial suspension
Activating bacteria stored in a refrigerator at 4 deg.C with solid slant culture medium, transferring the activated strain to nutrient liquid culture medium, further culturing, diluting the bacterial liquid to 0.5 McLee standard concentration of about 1.5 × 108CFU/mL is ready for use.
2.3 preparation of Compound Fungicide
Linalool 45%, terpineol 18%, geraniol 8%, β -cyclocitral 3%, nerol 3%, myrcene 1%, ocimene 1%, ionone 4%, 2H- β -ionone 6%.
The compound bactericide is prepared by compounding and modulating a plurality of different chemical reagents according to natural essential oil components (the bactericide is the compound bactericide prepared in 2.3).
DMSO (dimethyl sulfoxide) solution was used as a negative control, and antibiotics (ampicillin, amphotericin B) were used as a positive control.
2.4 determination of the bacteriostatic Activity
Adopting a filter paper sheet agar plate diffusion method: taking a round small filter paper with the diameter of 6mm, respectively dripping 6 mu l of compound bactericide and control solution (DMSO is a negative control and antibiotics is a positive control) into the center of the small filter paper, standing for a few minutes, and after the filter paper is completely wetted, placing the filter paper on a prepared bacteria-carrying flat plate at equal intervals. The zone diameter was measured with a vernier caliper using a cross method and repeated three times for each treatment. The schematic diagram of the inhibition zone is shown in fig. 1, and the specific numerical values are shown in table 2.
TABLE 2 diameter (mm) of zone of inhibition of the compound bactericide against test bacteria
The starting concentration of ampicillin is 100 mg/ml; the initial concentration of amphotericin B is 100 mg/ml; (1:60) represents a 60-fold dilution; (1:5) represents a 5-fold dilution; (1:2000) represents a 2000-fold dilution; (0) represents no dilution; "-" indicates that no control was made therewith; the diameter of the filter paper sheet is 6 mm; the diameter of the inhibition zone is 7-9mm, and the low sensitivity is obtained; the medium sensitivity is 10-15 mm; a high sensitivity of >15 mm; <6mm is not sensitive.
The results show that: from the diameter of the inhibition zone of bacteria, it can be found that staphylococcus epidermidis is highly sensitive to the bactericide, bacillus subtilis and staphylococcus aureus are moderately sensitive to the bactericide, and escherichia coli is less sensitive to the bactericide. From the diameter of the zone of inhibition of the fungus, it can be found that Candida albicans, Saccharomyces cerevisiae, and Trichophyton rubrum are highly sensitive to the bactericide, and Trichophyton schenckii is moderately sensitive to the bactericide.
Step 4, determining the minimum bacteriostatic concentration and the minimum bactericidal concentration of the compound bactericide
4.1 determination of the Minimum Inhibitory Concentration (MIC)
(1) Principle of experiment
Under certain pH and temperature conditions, the bacteria can grow and become cloudy in the nutrient broth. If the bacteriostatic agent is added into the nutrient broth, the bacteriostatic agent can inhibit the growth of bacteria, so that the nutrient broth is clear and transparent.
(2) Method of producing a composite material
Within a certain range, the concentration of bacteria in the suspension of bacteria is directly proportional to the turbidity, i.e. to the optical density, the more bacteria, the higher the optical density. Therefore, the Minimum Inhibitory Concentration (MIC) was further determined by analyzing the optical density of bacteria treated at various concentrations (0.4%, 0.25%, 0.125%, 0.1%, 0.05%, 0.025%, 0.005% μ l/ml) by a full-wavelength microplate reader at 600nm as a standard for the growth of the concentration of bacteria.
4.2 Minimum Bactericidal Concentration (MBC)
The method for measuring the minimum bactericidal concentration comprises the steps of utilizing the culture of each test tube with the Minimum Inhibitory Concentration (MIC) above, respectively sucking 0.1ml, transplanting the culture onto an agar culture medium without medicines, incubating overnight, respectively counting bacterial colonies, and obtaining MBC with the medicine concentration of less than 5 bacterial colonies.
FIG. 2 shows the absorbance values of Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Staphylococcus epidermidis, Candida albicans and Saccharomyces cerevisiae at a wavelength of 600nm after treatment with the compound bactericide of the present invention. Indicates extreme significance; indicates significance; "Amp" represents ampicillin; "Amphotericin B" stands for Amphotericin B.
Since the culture of Trichophyton rubrum and Trichophyton schenckii in liquid medium is difficult, the method of medicated agar plate was used for these two bacteria. And respectively adding the compound bactericide which is diluted in multiple times and has different concentrations into the agar which is heated and dissolved, balancing the agar in a water bath at 45-50 ℃, fully mixing uniformly, pouring into a sterilization plate, and enabling the thickness of the agar to be 3-4 mm. Drug agar plates are usually formulated in a 1: 9 ratio, with drug concentration ranges being selected as desired. MIC, MBC values were determined by colony counts. As shown in fig. 3 and 4.
Table 3 shows the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the compound bactericide.
TABLE 3 Minimal Inhibitory Concentration (MIC) and minimal inhibitory concentration (MBC) of the Compound Fungicide
Represents the growth of a small number of bacteria
The results show that: as shown in FIG. 2, FIG. 3, FIG. 4 and Table 3, the MIC and MBC of the compound bactericide for different test bacteria are different, but both have bacteriostatic effects. The compound bactericide has the best inhibiting effect on trichophyton rubrum, and the MIC and MBC are 0.025 percent and 0.05 percent. Secondly, the MIC and MBC of the Sporothrix schenckii are 0.10% and 0.125%. The inhibitory effects of Bacillus subtilis, Candida albicans and Saccharomyces cerevisiae were 0.25% different from that of the MBC of Sporothrix schenckii. The bacteriostatic effect on staphylococcus aureus and staphylococcus epidermidis is general, and the MIC is 0.125%. The compound bactericide has the worst bacteriostatic effect on escherichia coli. The MIC result is basically consistent with the inhibition zone result.
In conclusion, the compound bactericide has broad-spectrum inhibition effect on bacteria and fungi, has bacteriostasis and sterilization effects on various bacteria and fungi, and particularly aims at pathogenic bacteria of people and livestock co-morbid diseases such as candida albicans, trichosporon schenckii, trichophyton rubrum and the like. Therefore, the bactericide can be applied to the development of zoonosis infection and other disinfection products.
Comparative example
The following compositions were prepared, and the diameters of the zones were measured by a filter paper agar plate diffusion method, the results of which are shown in table 4:
the compound bactericide prepared in the step a.2.3 comprises 45 percent of linalool, 18 percent of terpineol, 8 percent of geraniol, β -cyclocitral 3 percent, 3 percent of nerol, 1 percent of myrcene, 1 percent of ocimene, 4 percent of ionone and 6 percent of 2H- β -ionone.
b. The component 1 comprises linalool 45%, terpineol 18%, geraniol 8%, β -cyclocitral 3%, nerol 3%, ocimene 2%, ionone 4%, and 2H- β -ionone 6%.
c. The component 2 comprises linalool 45%, terpineol 24%, β -cyclocitral 3%, nerol 3%, myrcene 1%, ocimene 1%, ionone 4%, and 2H- β -ionone 6%.
TABLE 4 diameter (mm) of zone of inhibition of compound bactericide, component 1 and component 2 to test bacteria
The diameter of the filter paper sheet is 6 mm; the diameter of the inhibition zone is 7-9mm, and the low sensitivity is obtained; the medium sensitivity is 10-15 mm; a high sensitivity of >15 mm; <6mm is not sensitive.
As can be seen from Table 4: when the amount of myrcene in the compound bactericide is reduced and the content of ocimene is changed to 2%, the antibacterial effect of the compound bactericide is researched, and compared with the component 1, the compound bactericide has a better antibacterial effect. Compared with the compound bactericide of the invention, geraniol is reduced, the content of terpineol is changed to 24%, and then the bacteriostasis effect is researched, compared with the component 2, the compound bactericide of the invention has better bacteriostasis effect. Therefore, we can see that the components of the compound bactericide of the invention are all none.
Claims (7)
1. A compound bactericide is prepared from linalool, terpineol, geraniol, β -cyclocitral, nerol, myrcene, ocimene, ionone, and 2H- β -ionone.
2. The compound bactericide as claimed in claim 1, wherein the compound bactericide comprises, by weight, 40% -50% of linalool, 18% -25% of terpineol, 8% -15% of geraniol, 2% -4% of β -cyclocitral, 2.5% -5% of nerol, 1% -3% of myrcene, 1% -3% of ocimene, 3% -6% of ionone, and 5% -8% of 2H- β -ionone.
3. The use of the compound bactericide of claim 1 or 2 for inhibiting and killing bacteria and fungi.
4. The use of a compound bactericide as claimed in claim 1 or 2 in the preparation of products for inhibiting and killing bacteria and fungi.
5. Use according to claim 3 or 4, characterized in that: the bacteria are: staphylococcus epidermidis, bacillus subtilis, staphylococcus aureus and escherichia coli.
6. Use according to claim 3 or 4, characterized in that: the fungi are: candida albicans, Saccharomyces cerevisiae, Trichophyton rubrum, and Trichosporon schenckii.
7. An agent for killing zoonosis and zoonosis, which is the compound bactericide of claim 1 or 2, or contains the compound bactericide of claim 1 or 2.
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