CN115607492A - Radix ceratostigmae callus extract and preparation method and application thereof - Google Patents
Radix ceratostigmae callus extract and preparation method and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9783—Angiosperms [Magnoliophyta]
- A61K8/9789—Magnoliopsida [dicotyledons]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/805—Corresponding aspects not provided for by any of codes A61K2800/81 - A61K2800/95
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- Gerontology & Geriatric Medicine (AREA)
- Dermatology (AREA)
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- Rheumatology (AREA)
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- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
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- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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Abstract
The invention relates to the technical field of plant tissue extraction, and particularly provides a calliopsis chinensis callus extract, and a preparation method and application thereof, so as to research the soothing and anti-inflammatory effects of the calliopsis chinensis callus extract and develop the application of the calliopsis chinensis callus extract in the field of cosmetics. The saussurea involucrate callus extract provided by the invention is prepared by taking saussurea involucrate callus cell powder as a raw material and performing enzymolysis after alcohol pretreatment, can promote the elimination of DPPH and ABTS free radicals and inhibit the expression of cyclooxygenase COX-2, and has an obvious inhibiting effect on the secretion of HaCaT cell model inflammatory factors induced by propionibacterium acnes.
Description
Technical Field
The invention belongs to the technical field of plant tissue extraction, and relates to a radix ceratostigmatis callus extract and a preparation method and application thereof.
Background
Saussurea involucrate is a succulent plant of Mao Feng of Compositae, and is a perennial herb of Compositae. Saussurea involucrate contains abundant cells, especially stem cells. Plant stem cells are present in a special structure called meristem, with a very surprising regenerative capacity. These allow plants to grow continuously for hundreds of years and to generate entirely new organs.
The saussurea involucrate contains vitamin C, which can soften blood vessel, improve microcirculation, resist aging, and moisten skin, whiten skin, and reduce weight.
Saussurea involucrate is also widely used in hospitals in clinic. The results of the method for treating the patients with diabetic peripheral neuropathy by combining long needle deep puncture, electric needle and snow lotus acupoint injection show that the blood viscosity can be reduced, the blood flow variable can be improved, the effective perfusion of microcirculation can be increased, the blood and oxygen supply of peripheral nerve tissues can be increased, the disturbance of glycolipid metabolism of the patients can be partially corrected, and the nerve conduction velocity of the patients can be well improved. The saussurea involucrate injection plus lidocaine and planetary ganglion block is used for treating migraine to obtain satisfactory effect, and the mechanism of the injection is related to the functions of activating blood circulation, dredging channels, relieving pain, resisting inflammation and regulating vegetative nerve of saussurea involucrate besides the function of lidocaine. The snow lotus injection is respectively injected at the Quchi point and the Ashi point of the pressure pain point, has obvious curative effect on migraine, has short treatment course, does not contain hormone, and has high thorough cure rate. The acupoint injection greatly enhances the stimulation to the acupoints, promotes local metabolism, increases the blood flow speed and flow at the focus part, improves the blood circulation, and thus has the effects of eliminating inflammation and relieving pain.
The research aims to extract and prepare the calli of the radix ceratostigmae, research the relieving and anti-inflammatory effects of the calli, and develop the application of the calli in the field of cosmetics.
Disclosure of Invention
In order to solve the technical problems, the invention provides a calliopsis extract and a preparation method and application thereof, and the extracts are used for researching the antioxidant and anti-inflammatory effects of the calliopsis extract while establishing an effective extraction method, and developing the application of the extracts in the field of cosmetics.
In order to realize the purpose of the invention, the technical scheme adopted by the invention is as follows:
on one hand, the invention requests to protect the calli of the radix ceratostigmae, takes calli of the radix ceratostigmae as raw materials, and is prepared by enzymolysis and hot reflux after alcohol pretreatment.
The preparation method comprises the following steps:
step S1: accurately weighing the calliopsis chinensis callus cell powder, and adding alcohol for pretreatment to obtain a treatment solution;
step S2: adding enzyme into the treatment solution for enzymolysis to obtain an enzymolysis solution;
and step S3: and (3) carrying out hot reflux extraction on the enzymatic hydrolysate, cooling, then carrying out fine filtration, and collecting filtrate to obtain the calli of the snow lotus.
Preferably, the alcohol in the step S1 is 1,3-butanediol with the mass concentration of 10% -70%.
Further preferably, the alcohol in step S1 is 1,3-butanediol with a mass concentration of 30%.
Preferably, the feed-liquid ratio of the snowdrop callus cell powder to the alcohol in the step S1 is 1.
Further preferably, the feed-liquid ratio of the calliopsis chinensis callus cell powder and alcohol in the step S1 is 1.
Preferably, the enzymes described in step S2 are cellulase and pectinase.
Further preferably, the addition amount of the cellulase is 0.5-2.5% of the mass of the calliopsis chinensis callus cell powder; the addition amount of the pectinase is 0.5-2.5% of the mass of the radix ceratostigmatis japonici callus cell powder.
More preferably, the addition amount of the cellulase is 1 percent of the mass of the calliopsis chinensis callus cell powder; the addition amount of the pectinase is 1% of the mass of the radix ceratostigmae callus cell powder.
Preferably, the conditions of the enzymolysis in step S2 are as follows: the temperature is 40-60 ℃; the pH is 4-5; the time is 1.5-2.5h.
Further preferably, the enzymolysis conditions in step S2 are: the temperature is 50 ℃; the pH was 4.5; the time is 2h.
Preferably, the conditions of the thermal reflow described in step S3 are: the extraction temperature is 80-120 deg.C, and the extraction time is 2-3h.
Further preferably, the conditions of the thermal reflow described in step S3 are: the extraction temperature is 100 ℃, and the extraction time is 2.5h.
In another aspect, the invention also provides an application of the radix ceratostigmatis callis callus extract in preparing an antioxidant and/or anti-inflammatory cosmetic.
In another aspect, the invention also provides an antioxidant cosmetic, which comprises the extract of the calli of the snowdrop.
In another aspect, the invention also provides an anti-inflammatory cosmetic, which comprises the extract of the calli of the snowdrop.
Compared with the prior art, the invention has the following beneficial effects:
1. the method adopts a multi-step method to prepare the calli of the zixuelia and improves the cell permeability further through the synergistic effect of two enzymes, and improves the yield of the calli of the zixuelia;
2. the calliopsis chinensis callus extract extracted by the method has good DPPH and ABTS free radical removing effects and strong oxidation resistance;
3. the callicarpa bodinieri callus extract has good anti-inflammatory effect, can effectively inhibit the expression of COX-2 and inhibit the secretion of inflammatory factors TNF-a in cells;
drawings
FIG. 1 shows the contents of flavone and polyphenol in the calli extract of snow lotus obtained in examples 1-5 and comparative examples 1-4;
FIG. 2 is a graph of experimental data of the removal of DPPH free radicals by the extract of the calli of the snowdrop;
FIG. 3 is a graph of data of the experiment of eliminating ABTS free radicals by the extract of the callus of the ice cream plant.
Detailed Description
The following non-limiting examples are presented to enable those of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way. The following is merely an exemplary illustration of the scope of the invention as claimed, and various changes and modifications of the invention of the present application may be made by those skilled in the art based on the disclosure, which also fall within the scope of the invention as claimed.
The present invention will be further described below by way of specific examples. The various chemicals used in the examples of the present invention were obtained by conventional commercial routes unless otherwise specified.
In the following examples, the calliopsis callicarpa callus cell powder was purchased from anseibo biotechnology limited; 1,3 butanediol is available from OXEA, usa; cellulase was purchased from Biotopped, cat # C6270; pectinase was purchased from Biotopped under the accession number P6280; the DPPH kit is purchased from Taixi (Shanghai) chemical industry development Limited company, and has a product number of D4313; ABTS kit purchased from Biotopped, cat # S016; the COX-2 kit is purchased from Shanghai Bin Yuntian biotechnology limited company, and has a product number of S0168; the TNF-a kit is purchased from Shanghai Weiao Biotechnology Co., ltd, and has a product number of EH0313.
Example 1
Radix Ceratostigmatis Willemottiani callus extract and its preparation method
Accurately weighing 5g of the calli of the zixuelia, adding 250mL of 30 percent of 1, 3-butanediol; after the alcohol treatment, 1.0% of cellulase and 1.0% of pectinase are added, the pH is adjusted to 4.5 by using citric acid and NaOH at the temperature of 50 ℃, and the enzymolysis treatment is carried out for 2 hours; performing enzymolysis, extracting at 100 deg.C under reflux for 2.5 hr, cooling, fine filtering with 0.45 μm filter plate, and collecting filtrate to obtain radix Hemsleyae Macrospermae callus extract.
Example 2
Accurately weighing 5g of the calli of the zixuelia, adding 250mL of 10 percent of 1, 3-butanediol; after the alcohol treatment, 1.0% of cellulase and 1.0% of pectinase are added, the pH is adjusted to 4.5 by using citric acid and NaOH at the temperature of 50 ℃, and the enzymolysis treatment is carried out for 2 hours; performing enzymolysis, performing heat reflux extraction at 100 deg.C for 2.5 hr, cooling, fine filtering with 0.45 μm filter plate, and collecting filtrate to obtain radix Ceratostigmatis Willemottiani callus extract.
Example 3
Accurately weighing 5g of the calli of the zixuelia, adding 250mL of 70 percent of 1, 3-butanediol; after the alcohol treatment, 1.0% of cellulase and 1.0% of pectinase are added, the pH is adjusted to 4.5 by using citric acid and NaOH at the temperature of 50 ℃, and the enzymolysis treatment is carried out for 2 hours; performing enzymolysis, extracting at 100 deg.C under reflux for 2.5 hr, cooling, fine filtering with 0.45 μm filter plate, and collecting filtrate to obtain radix Hemsleyae Macrospermae callus extract.
Example 4
Accurately weighing 5g of the calli of the zixuelia, adding 250mL of 30 percent of 1, 3-butanediol; adding 1.3% of cellulase and 0.7% of pectinase after alcohol treatment, adjusting the pH value to 4 at 40 ℃ by using citric acid and NaOH, and performing enzymolysis for 1.5h; performing enzymolysis, extracting at 80 deg.C under reflux for 2 hr, cooling, fine filtering with 0.45 μm filter plate, and collecting filtrate to obtain radix Hemsleyae Macrospermae callus extract.
Example 5
Accurately weighing 5g of the calli of the zixuelia, adding 250mL of 30 percent of 1, 3-butanediol; adding 1.5% of cellulase and 0.5% of pectinase after alcohol treatment, adjusting the pH value to 5 by using citric acid and NaOH at the temperature of 60 ℃, and performing enzymolysis for 2.5h; performing enzymolysis, extracting at 120 deg.C under reflux for 3 hr, cooling, fine filtering with 0.45 μm filter plate, and collecting filtrate to obtain radix Hemsleyae Macrospermae callus extract.
Comparative example 1
Accurately weighing 5g of the calli of the zixuelia, adding 250mL of 30 percent of 1, 3-butanediol; extracting at 100 deg.C under reflux for 2.5 hr, cooling, fine filtering with 0.45 μm filter plate, and collecting filtrate to obtain extract.
Comparative example 2
Accurately weighing 5g of the calliopsis chinensis callus cell powder, adding 250mL of 30%1, 3-butanediol, adding 1.0% cellulase and 1.0% pectinase after alcohol treatment, adjusting the pH value to 4.5 by using citric acid and NaOH at the temperature of 50 ℃, and carrying out enzymolysis pretreatment for 2h; after enzymolysis, fine filtering with a 0.45 μm filter plate, and collecting the filtrate to obtain the extract.
Comparative example 3
Accurately weighing 5g of the calli of the zixuelia, adding 150mL of 30 percent of 1, 3-butanediol; adding 1.0% of cellulase and 1.0% of pectinase after alcohol treatment, adjusting the pH value to 4.5 by using citric acid and NaOH at the temperature of 50 ℃, and carrying out enzymolysis pretreatment for 2 hours; after enzymolysis, fine-filtering with a 0.45 μm filter plate, and collecting the filtrate to obtain the extract.
Comparative example 4
Accurately weighing 5g of the calli of the zixuelia, adding 250mL of 90 percent of 1, 3-butanediol; after the alcohol treatment, 1.0% of cellulase and 1.0% of pectinase are added, the pH is adjusted to 4.5 by using citric acid and NaOH at the temperature of 50 ℃, and the enzymolysis treatment is carried out for 2 hours; performing enzymolysis, extracting at 100 deg.C under reflux for 2.5 hr, cooling, fine filtering with 0.45 μm filter plate, and collecting filtrate to obtain radix Hemsleyae Macrospermae callus extract.
In the examples and the comparative examples, the contents of flavone and polyphenol in the examples 1 to 5 are obviously higher than those in the comparative examples 1 to 4, and the specific results are shown in figure 1.
Effect test
Preparing a solution to be detected: the calli of the snowdrop obtained in examples 1-5 and comparative examples 1-4 were respectively prepared into solutions to be tested with concentrations of 0.5%, 1.0%, 2.0% and 5.0% by using absolute ethanol.
1. Inhibiting activity on DPPH free radical
(1) Preparing a DPPH ethanol solution:
weighing 20mg of DPPH, adding absolute ethyl alcohol to dissolve, fixing the volume in a 250mL volumetric flask, and preparing the DPPH concentration into 2 multiplied by 10 -4 mol/L; can be stored at 0-4 deg.C in dark place, and is effective within 4 h. Vitamin C is selected as positive control, and the concentration is 0.5mg/mL.
(2) The reagents were added as in Table 1, and after 30min of dark reaction, the absorbance of A, B, C tubes was measured at 517 nm.
TABLE 1
| Numbering | DPPH solution | Solvent(s) | Liquid to be tested | Total volume |
| A | 1mL | - | 1mL | 2mL |
| B | 1mL | 1mL | - | 2mL |
| C | - | 1mL | 1mL | 2mL |
(3) The DPPH free radical inhibition rate calculation formula is as follows: DPPH inhibition ratio (%) = (B + C-A)/B
The results of the experiment are shown in FIG. 2.
2. ABTS free radical inhibition assay
(1) Preparing an ABTS aqueous solution: precisely weighing 0.03841g ABTS, dissolving and dissolving in 10mL water;
(2)K 2 S 2 O 8 preparing an aqueous solution: precision weighing 0.0662g K 2 S 2 O 8 Dissolving and fixing in 100mL of water;
(3) Mother liquor preparation: mixing aqueous ABTS solution and K 2 S 2 O 8 Mixing the aqueous solution in equal volume, and reacting at low temperature in a dark place for 12-16h;
(4) ABTS working solution: diluting the mother liquor with anhydrous ethanol until OD value at 734nm is 0.7 + -0.02, and preparing for use;
(5) Vitamin C with concentration of 0.5mg/mL is selected as positive control, and is preserved at 0-4 deg.C in dark place;
(6) The specific experimental steps are as follows:
adding the reagents according to the table 2, reacting for 30min in a dark place at room temperature, and measuring the light absorption value of the reagents at 734nm by using an enzyme-linked immunosorbent assay (ELISA) instrument;
TABLE 2
| Numbering | ABTS working solution | Solvent(s) | Liquid to be tested |
| Experimental group (A) | 0.8mL | - | 0.2mL |
| Blank group (A0) | 0.8mL | 0.2mL | - |
(7) ABTS free radical inhibition rate calculation formula: ABTS inhibition (%) = [ (A0-a)/A0 ] × 100%.
The results of the experiment are shown in FIG. 3.
3. Inhibiting activity on COX-2
The kit is used for detecting the human COX-2 inhibitor, and the specific steps are carried out according to the instructions. The concrete steps are briefly explained as follows:
(1) The reaction solutions were added to a black 96-well plate, and the sample to be tested and various solvents were sequentially added in the order of addition shown in Table 3, and the positive control inhibitor Celecoxib concentration provided by the kit was 100. Mu. Mol/L, and was formulated in DMSO.
(2) After the sample application was completed, care was taken not to allow the liquid to splash out of the plate, and incubation was carried out at 37 ℃ for 10min. After incubation was complete, 5. Mu.L of COX-2 Probe was added to each well.
(3) Then 5 mu L of Substrate working solution is rapidly added into each hole, the mixture is evenly mixed, and the reaction is started after COX-2Substrate working solution is added.
(4) And (4) carrying out fluorescence measurement after incubation for 5min at 37 ℃ in the dark. The excitation wavelength was 560nm and the emission wavelength was 590nm.
The percentage of COX-2 inhibition was calculated for each group. Calculating the formula: inhibition (%) = (RFU 100% enzyme activity control-RFU sample)/(RFU 100% enzyme activity control-RFU blank) × 100%.
TABLE 3
Note: the sample solvent is the solvent used to formulate and dilute the inhibitor to be tested.
(5) The percentage COX-2 inhibition was calculated for each group. Calculating the formula: inhibition (%) = (RFU 100% enzyme activity control-RFU sample)/(RFU 100% enzyme activity control-RFU blank) × 100%.
The results are shown in Table 4.
4. Inhibition effect on propionibacterium acnes-induced HaCaT cell model inflammatory factor
HaCaT cells were cultured at 2X 10 5 cells/mL were plated in 96-well plates at a density of 200. Mu.L per well. At 37 deg.C, 5% CO 2 After culturing for 12h under the condition, the induction sample loading of the propionibacterium acnes is carried out.
Setting a blank group, a model group and a sample group, culturing cells for 12h until the cells adhere to the wall, sucking out a culture medium, and respectively adding the culture medium according to the following groups:
inoculating 200 μ L of serum-free culture medium into the blank group; the model group was inoculated into 200. Mu.L of serum-free medium and cultured for 4h at a concentration of (2X 10) 8 CFU/mL) propionibacterium acnes stimulator 20 μ L; the sample group was inoculated into 200. Mu.L of sample medium and cultured for 4h, adding the sample at a concentration of (2X 10) 8 CFU/mL) propionibacterium acnes stimulus 20 μ L.
The 96-well plate was placed in an incubator at 37 ℃ with 5% CO 2 Culturing for 24h in the environment, collecting cell supernatant, and detecting the content of TNF-a according to the kit instruction.
The results are shown in Table 5.
TABLE 4
TABLE 5
Note: all experiments were repeated 3 times and the results are expressed as Mean ± standard deviation (Mean ± SD).
As can be seen from FIGS. 2 to 3, the calli of the snowdrop extracts extracted in examples 1 to 5 have more excellent DPPH and ABTS free radical scavenging promoting effects than those extracted in comparative examples 1 to 4, which indicates that the calli of the snowdrop extracts provided by the present application can promote the scavenging of oxygen free radicals and have antioxidant effects.
COX-2 in vitro inhibition experiment results (see Table 4) show that the radix Hemsleyae Macrospermae callus extract has the effect of inhibiting the expression of inflammatory factor COX-2, and compared with comparative examples 1-4, the inhibition effect of the extract obtained in examples 1-5 is obviously higher. By detecting the content of TNF-a by adopting a propionibacterium acnes induced HaCaT cell model inflammatory factor high-expression model, as can be seen from Table 5, the extracts obtained in examples 1-5 can obviously inhibit the secretion of inflammatory factors TNF-a. The results show that the radix ceratostigmae callus extract has the effects of relieving and resisting inflammation.
Finally, it should be noted that the above-mentioned contents are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, and that the simple modifications or equivalent substitutions of the technical solutions of the present invention by those of ordinary skill in the art can be made without departing from the spirit and scope of the technical solutions of the present invention.
Claims (13)
1. The calli extract is characterized by being prepared by taking calli cell powder as a raw material, performing alcohol pretreatment, performing enzymolysis and performing heat reflux.
2. The extract of the calliopsis bodinieri callus tissue as claimed in claim 1, wherein the preparation comprises the following steps:
step S1: accurately weighing the calli ceratostigma callus cell powder, and adding alcohol for pretreatment to obtain a treatment solution;
step S2: adding enzyme into the treated liquid for enzymolysis to obtain an enzymolysis liquid;
and step S3: and (3) carrying out hot reflux extraction on the enzymatic hydrolysate, cooling, then carrying out fine filtration, and collecting filtrate to obtain the calli of the snow lotus.
3. The snowdrop callus extract according to claim 2, wherein the alcohol in step S1 is 1,3-butanediol with a mass concentration of 10% -70%.
4. The snowdrop callus extract according to claim 2, wherein the feed-liquid ratio of the snowdrop callus cell powder to the alcohol in step S1 is 1.
5. The radix ceratostigmae callus extract according to claim 4, wherein the feed-liquid ratio of the radix ceratostigmae callus cell powder to the alcohol in step S1 is 1.
6. The snowdrop callus extract according to claim 2, wherein the enzymes in step S2 are cellulase and pectinase.
7. The radix ceratostigmae callus extract as claimed in claim 6, wherein the addition amount of the cellulase is 0.5% -2.5% of the mass of the radix ceratostigmae callus cell powder; the addition amount of the pectinase is 0.5-2.5% of the mass of the radix ceratostigmatis japonici callus cell powder.
8. The radix ceratostigmae callus extract as claimed in claim 7, wherein the addition amount of the cellulase is 1% of the mass of the radix ceratostigmae callus cell powder; the addition amount of the pectinase is 1% of the mass of the radix ceratostigmae callus cell powder.
9. The radix ceratostigmae callus extract according to claim 2, wherein the conditions of enzymolysis in step S2 are as follows: the temperature is 40-60 ℃; the pH is 4-5; the time is 1.5-2.5h.
10. The durian callus extract according to claim 2, wherein the heat reflux conditions in step S3 are: the extraction temperature is 80-120 deg.C, and the extraction time is 2-3h.
11. Use of the extract of the calli of any one of claims 1 to 10 for the preparation of an antioxidant and/or anti-inflammatory cosmetic.
12. An antioxidant cosmetic comprising the extract of the calli of snowdrop according to any one of claims 1 to 10.
13. An anti-inflammatory cosmetic comprising the extract of the calli of snowdrop according to any one of claims 1 to 10.
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