Angelica dahurica-ligusticum ethanol extract, preparation method, quality detection method and application in preparation of malassezia furfur-resistant drugs or wash supplies

Abstract

The invention provides an ethanol extract of radix angelicae and rhizoma ligustici sinensis, which is prepared by taking the same amount of radix angelicae and rhizoma ligustici sinensis as raw materials and adding 80% ethanol for extraction. The invention also provides a preparation method and application of the extract. The invention also provides a shampoo or hair conditioner additive with anti-dandruff washing and care effects, and the shampoo or hair conditioner additive contains the radix angelicae and ligusticum ethanol extract. The invention also provides a quality detection method of the angelica dahurica-ligusticum ethanol extract. The radix angelicae and rhizoma ligustici sinensis ethanol extract disclosed by the invention is high in dissolution rate of effective components, small in dosage, convenient to use and good in curative effect, and particularly, the 80% ethanol reflux extract has the strongest inhibition effect on a biofilm. The invention also establishes a quality detection method of the angelica dahurica-ligusticum ethanol extract, and the quality is controllable.

Description

Technical Field

[0001] The invention relates to an ethanol extract of Angelica dahurica and Ligusticum chuanxiong, a preparation method, a quality detection method and use of the extract in preparing medicines or toiletries for resisting Malassezia furfur. Background Art

[0002] Dandruff is a small flake of bran-like scales that continuously fall off the scalp. It is a product of the metabolism of the head. There are many reasons for the formation of dandruff, mainly including: excessive dryness of the scalp, excessive oil secretion, inappropriate shampoo, scalp allergy, fungal infection, excessive cleaning, lack of nutrition, environmental factors, mental stress, etc. Among them, fungal infection on the scalp, especially Malassezia furfur, can cause dandruff. Malassezia is a lipophilic yeast-like fungus that feeds on the triglyceride sebum of the scalp hair follicles, produces saturated fatty acids, consumes saturated fatty acids to help itself proliferate, and leaves unsaturated fatty acids. Unsaturated fatty acids penetrate into the stratum corneum and cause itching of the scalp, and lead to the proliferation and abnormal keratinization of the stratum corneum cells, causing the production of flaky scales on the scalp.

[0003] At present, the drugs used to fight against Malassezia furfur mainly include ketoconazole, itraconazole, selenium disulfide, terbinafine, fluconazole, clotrimazole, etc. Some antifungal drugs may cause side effects such as skin irritation, itching, dryness, and hair loss. Long-term use of certain antifungal drugs may lead to the development of drug resistance.

[0004] There are reports that Ligusticum chuanxiong and Angelica dahurica powder can remove dandruff. Use equal amounts of Ligusticum chuanxiong and Angelica dahurica, grind them into fine powder, sprinkle them on the scalp before going to bed at night, and comb them off in the morning. It is inconvenient to use and difficult to clean. The active ingredients of powdered drugs are not easily absorbed, the dissolution is limited, and the dosage is large. Summary of the invention

[0005] The technical scheme of the present invention is to provide an ethanol extract of Angelica dahurica and Ligusticum chuanxiong. The present invention also provides a preparation method and a quality detection method of the extract and the use of the extract in preparing medicines or toiletries for resisting Malassezia furfur.

[0006] The invention provides an ethanol extract of angelica dahurica and rhizoma ligusticum. The ethanol extract is prepared by taking equal amounts of angelica dahurica and rhizoma ligusticum as raw materials and adding 80% ethanol for extraction to obtain the extract.

[0007] Preferably, the ethanol extraction method is 85°C reflux extraction.

[0008] The present invention also provides a method for preparing the ethanol extract of Angelica dahuricae Ligusticum chuanxiong, which comprises the following steps:

[0009] a. Take equal amounts of Angelica dahurica and Ligusticum chuanxiong, grind them into coarse powder and mix them;

[0010] b. Add 80% ethanol to soak. After soaking, extract under reflux at 85℃. Concentrate the filtrate and freeze-dry to obtain the ethanol extract of Angelica dahurica Ligusticum.

[0011] The invention provides use of the ethanol extract of Angelica dahurica Ligusticum in preparing a medicine having an anti-Malassezia furfur effect.

[0012] The invention provides use of the angelica dahurica ligusticum ethanol extract in preparing medicines or daily chemicals with anti-dandruff effects.

[0013] The invention provides a shampoo or conditioner additive with anti-dandruff washing and conditioning functions, which contains the angelica dahurica and ligusticum ethanol extract.

[0014] The present invention provides a quality detection method for the ethanol extract of Angelica dahurica Ligusticum chuanxiong, which uses high performance liquid chromatography to detect the contents of ferulic acid, bergamotolactone, imperatorin, oxidized imperatorin and isoimperatorin in the ethanol extract of Angelica dahurica Ligusticum chuanxiong, and the chromatographic conditions are:

[0015] The chromatographic column was an ACQUITY UPLC BEH C18 (2.1×100nm, 1.7μm) column; the mobile phase was acetonitrile (B)-0.1% formic acid water (D), gradient elution (0-6min, 15% B; 6-9min, 15-45% B, 9-19min, 45% B, 19-23min, 45-85% B, 23-26min, 85% B, 26-29min, 85-15% B), the column temperature was 35°C; the flow rate was 0.25mL / min; the detection wavelength was 302nm; and the injection volume was 10μL.

[0016] Among them, it includes the following steps:

[0017] a. Preparation of test solution:

[0018] Accurately weigh the freeze-dried powder of Angelica dahurica Ligusticum ethanol extract, dilute it with 50% methanol to make a sample solution, and filter it with a 0.22μm microporous filter membrane to obtain the test solution;

[0019] b. Preparation of reference solution

[0020] Accurately weigh 0.57, 0.47, 1.16, 3.55, and 0.78 mg of ferulic acid, bergamot lactone, imperatorin, oxidized imperatorin, and isoimperatorin reference substances into a 10 mL volumetric flask, add methanol to prepare reference substance mother solutions with concentrations of 0.057, 0.047, 0.116, 0.355, and 0.078 mg / mL for ferulic acid, bergamot lactone, imperatorin, oxidized imperatorin, and isoimperatorin, respectively; accurately pipette 1, 2, 5, 1, and 1 mL of the 5 reference substance mother solutions into a 10 mL volumetric flask, respectively, and shake well to obtain a mixed reference substance solution;

[0021] c. Detection using the chromatographic conditions described.

[0022] Among them, the contents of ferulic acid, bergamot lactone, imperatorin, oxidized imperatorin and isoimperatorin are: ferulic acid 4.1420, bergamot lactone 0.5748, imperatorin 0.5847, oxidized imperatorin 7.9873, isoimperatorin 25.8118, and the content range fluctuates by 10%; the relative retention times are: ferulic acid 4.939min, bergamot lactone 11.174min, oxidized imperatorin 12.076min, imperatorin 14.442min, isoimperatorin 16.410min, and the relative retention times fluctuate by 0.5min.

[0023] The ethanol extract of Angelica dahurica Ligusticum chuanxiong of the present invention has high effective component dissolution, small dosage, convenient use and good curative effect, especially the 80% ethanol reflux extract has the strongest biofilm inhibition effect. The present invention also establishes a quality detection method for the ethanol extract of Angelica dahurica Ligusticum chuanxiong, and the quality is controllable. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Alcohol extraction of Angelica dahurica and Ligusticum chuanxiong (acetonitrile-0.1% formic acid water; condition 1) (A), alcohol extraction of Angelica dahurica and Ligusticum chuanxiong (acetonitrile-water) (B), alcohol extraction of Angelica dahurica and Ligusticum chuanxiong (methanol-0.1% formic acid water) (C), alcohol extraction of Angelica dahurica and Ligusticum chuanxiong (acetonitrile-0.1% formic acid water; condition 2) (D) chromatogram (including 1-ferulic acid 2-bergamotolactone 3-oxidized peucedanum 4-imperatorin 5-isoimperatorin);

[0025] Figure 2 Chromatograms of the alcohol extract sample (A), water extract sample (B), and percolation sample (C) of Angelica dahurica (including 1-ferulic acid 2-bergamotolactone 3-oxidized peucedanum 4-imperatorin 5-isoimperatorin);

[0026] Figure 3 Time-killing curve of Angelica dahurica Ligusticum extract on Malassezia furfur (Note: A. Time-killing curve of Angelica dahurica Ligusticum ethanol extract on Malassezia furfur, B. Time-killing curve of Angelica dahurica Ligusticum water extract on Malassezia furfur, C. Time-killing curve of Angelica dahurica Ligusticum percolation extract on Malassezia furfur);

[0027] Figure 4 The inhibitory effect of Angelica dahurica Ligusticum extract on the biofilm of Malassezia furfur (Note: A. CVSA crystal violet staining method was used to detect the inhibitory effect of different extracts on biofilm, B. XTT analysis method was used to detect the inhibitory effect of different extracts on biofilm, C. Microscopic examination of the inhibitory effect of alcohol extract on biofilm);

[0028] Figure 5The eradication effect of Angelica dahurica Ligusticum extract on Malassezia furfur biofilm (Note: A. CVSA crystal violet staining method was used to detect the eradication effect of different extracts on biofilm, B. XTT analysis method was used to detect the eradication effect of different extracts on biofilm, C. Microscopic examination of the eradication effect of alcohol extract on biofilm);

[0029] Figure 6 Toxicity of Angelica dahurica Ligusticum chuanxiong extract on rat skin (Note: Figure A is the blank group, and Figure B is the drug-treated group). DETAILED DESCRIPTION

[0030] Example 1 Preparation and quality testing method of the Angelica dahurica extract of the present invention

[0031] 1. Preparation of ethanol extract, water extract and percolation extract of Angelica dahurica Ligusticum

[0032] 1.1 Alcohol extract and water extract

[0033] Grind the Angelica dahuricae Ligusticum into coarse powder, accurately weigh 25g of each, mix, add 3 times the amount of 80% ethanol or water, and soak for 12 hours. After soaking, reflux extract at 85℃ for 2 hours, 3 times, and combine the filtrates of the three times. Concentrate and freeze-dry, and place in a refrigerator at 4 degrees for later use.

[0034] The results showed that the crude drug content of the freeze-dried powder of the alcohol extract of Angelica dahurica and Ligusticum chuanxiong was 3.0756 g / g; the crude drug content of the freeze-dried powder of the water extract of Angelica dahurica and Ligusticum chuanxiong was 4.1586 g / g.

[0035] 1.2 Percolation extract

[0036] Grind Angelica dahurica and Ligusticum chuanxiong into coarse powder, accurately weigh 25g of each, mix, add one amount of 80% ethanol to moisten, put into a tube, add 8 times the amount of 80% ethanol to soak for 12h, then start percolation at a percolation rate of 3ml / min.

[0037] The crude drug content of Angelica dahurica and Ligusticum chuanxiong in the freeze-dried powder of the diafiltration extract was determined to be 3.035 g / g.

[0038] 2. Determination of components of different extracts

[0039] 2.1 Screening of chromatographic conditions

[0040] In this experiment, the mobile phase was investigated. Figure 1 As shown, acetonitrile-0.1% formic acid water was selected as the mobile phase for elution. The peak shapes of ferulic acid, bergamot glycosides, oxidized peucedanum, imperatorin, and isoimperatorin were better, and the baseline was relatively stable. When acetonitrile-water was used as the mobile phase for elution, the baseline moved forward as a whole at a detection wavelength of 302nm. When methanol-0.1% formic acid water was used as the mobile phase for elution, the baseline was not flat. Therefore, acetonitrile-0.1% formic acid water was selected as the mobile phase for subsequent experiments (see Figure 1 AC).

[0041] In this experiment, the elution conditions were investigated. Condition 1: acetonitrile (B)-0.1% formic acid water (D), gradient elution (0-6 min, 15% B; 6-9 min, 15-45% B, 9-19 min, 45% B, 19-

[0042] 23min, 45~85%B, 23~26min, 85%B, 26~29min, 85~15%B); Condition 2: (0~6min, 15%B; 6~9min, 15~45%B, 9~19min, 45%B, 19~23min, 45~

[0043] 75% B, 23-26min, 75-15% B). When condition 1 was used for elution, the baseline was relatively stable. When condition 2 was used for elution, the baseline was relatively unstable. Therefore, condition 1 was selected as the elution procedure for subsequent experiments (see Figure 1 A, D).

[0044] 2.2 Chromatographic conditions

[0045] The chromatographic column was an ACQUITY UPLC BEH C18 (2.1×100nm, 1.7μm) column; the mobile phase was acetonitrile (B)-0.1% formic acid water (D), gradient elution (0-6min, 15% B; 6-9min, 15-45% B, 9-19min, 45% B, 19-23min, 45-85% B, 23-26min, 85% B, 26-29min, 85-15% B), the column temperature was 35°C; the flow rate was 0.25mL / min; the detection wavelength was 302nm; and the injection volume was 10μL.

[0046] 2.3 Preparation of reference solution

[0047] Accurately weigh 0.57, 0.47, 1.16, 3.55, and 0.78 mg of ferulic acid, bergamot lactone, imperatorin, oxidized imperatorin, and isoimperatorin reference substances into a 10 mL volumetric flask, add methanol to make reference substance mother solutions with concentrations of 0.057, 0.047, 0.116, 0.355, and 0.078 mg / mL, respectively, of ferulic acid, bergamot lactone, imperatorin, oxidized imperatorin, and isoimperatorin. Accurately pipette 1, 2, 5, 1, and 1 mL of the 5 reference substance mother solutions into a 10 mL volumetric flask, respectively, shake well to obtain a mixed reference substance solution, and store in a 4°C refrigerator.

[0048] 2.4 Preparation of test solution

[0049] Accurately weigh 21.56, 13.39, and 17.7 mg of freeze-dried powder of Angelica dahurica ethanol extract, water extract, and percolation extract, respectively, and dilute them with 50% methanol to prepare sample solutions with concentrations of 0.5390, 0.6695, and 0.5531 mg / mL, respectively. Filter through a 0.22 μm microporous filter membrane to obtain the test solution.

[0050] 2.5 Investigation of linear relationship

[0051] Accurately pipette the mixed reference solution, dilute it with 50% methanol in proportion to 2, 4, 8, 16, 32, and 64 times, inject the sample for measurement in sequence, record the peak area, draw a standard curve with the concentration of the reference solution as the horizontal axis and the peak area as the vertical axis, calculate the regression equation, and the results are shown in Table 1.

[0052] Table 1 Results of linear relationship investigation of five chemical components

[0053]

[0054]

[0055] 2.6 Sample content determination

[0056] Take three sample powders, prepare the test solution according to the method in 1.3, and measure the sample under the chromatographic conditions in "2.1". The results are shown in Figure 2 The peak areas of the five components were measured and the mass fractions of the five components in each sample were calculated according to the linear regression equation. The results are shown in Table 2.

[0057] Table 2 Determination results of five chemical components in Angelica dahurica Ligusticum samples under different extraction methods

[0058]

[0059] 3 Determination of antibacterial activity of different extracts

[0060] 3.1 Determination of in vitro antibacterial activity against Malassezia furfur

[0061] 3.1.1 Determination of MIC for Malassezia furfur

[0062] The three kinds of Angelica dahurica extracts all had an inhibitory effect on Malassezia furfur. The MIC of the Angelica dahurica ethanol extract was 5 mg / ml, and that of the water extract and the percolation extract was 10 mg / ml, indicating that the alcohol extract was significantly better than the water extract and the percolation extract in inhibiting Malassezia furfur (Table 3).

[0063] Table 3 MIC of Angelica dahurica extract against Malassezia

[0064]

[0065] Note: 0 - optically clear, 1 - slightly hazy, 2 - significantly reduced haze (50%), 3 - slightly reduced haze, 4 - no reduction in haze

[0066] The beneficial effects of the present invention are demonstrated by the following efficacy tests.

[0067] Test Example 1 Time-killing curve of the extract of the present invention against Malassezia furfur

[0068] The time-kill curve can reflect the antibacterial or bactericidal effect of Angelica dahurica Ligustrum on Malassezia furfur at different time points (2, 4, 8, 12 and 24 hours). In this experiment, the number of monoclonal colonies of Malassezia furfur on the plate was counted, and the original CFU was calculated according to the dilution multiple. The results showed that the Angelica dahurica Ligustrum alcohol extract at a concentration of 8×MIC maintained an effective bactericidal effect on Malassezia furfur, and the Angelica dahurica Ligustrum alcohol extract at concentrations of 1×MIC and 4×MIC had a certain antibacterial effect, but the Angelica dahurica Ligustrum alcohol extract at a concentration of 1×MIC had a weaker antibacterial effect. In addition, the Angelica dahurica Ligustrum water extract and the Angelica dahurica Ligustrum percolation extract at a concentration of 8×MIC also had a certain antibacterial effect, but the Angelica dahurica Ligustrum water extract and the Angelica dahurica Ligustrum percolation extract at a concentration of 1×MIC had no obvious inhibitory effect, see. Figure 3 .

[0069] Test Example 2 Determination of the Anti-Malassezia furfur Biofilm Activity of the Extract of the Present Invention

[0070] 1. Inhibition of Malassezia furfur biofilm

[0071] The inhibitory effects of different extracts of Angelica dahurica Ligustrum on the biofilm of Malassezia furfur were tested by CVSA crystal violet staining and XTT analysis. The 0.5×MIC alcohol extracts could inhibit the biofilm, while the 0.5×MIC water extracts and percolation extracts were ineffective. The 1×MIC alcohol extract showed an inhibitory effect similar to that of amphotericin B, and the 1×MIC water extracts and percolation extracts could also inhibit the formation of biofilm. It was suggested that the alcohol extract had the strongest inhibitory effect on biofilm, and the alcohol extract under microscopic examination had a significant inhibitory effect on the biofilm of Malassezia furfur.

[0072] 2. Eradication of mature biofilm of Malassezia furfur

[0073] Crystal violet staining was used to quantitatively detect the effects of different extracts from Angelica dahurica on mature Malassezia furfur biofilms. Figure 5It can be observed that the biomass of the biofilm of Malassezia furfur in the untreated group was higher than that of the biofilm of Malassezia furfur after treatment with different extracts of Angelica dahurica and Ligusticum chuanxiong. Since mature biofilms require higher concentrations of drugs to eradicate, the biofilm of Malassezia furfur gradually decreases with the increase in the concentration of Angelica dahurica and Ligusticum chuanxiong extract, and the most obvious decrease in biofilm biomass was observed after treatment with 8×MIC Angelica dahurica and Ligusticum chuanxiong alcohol extract. The results of XTT metabolic activity analysis also showed the same results. After treatment with 8×MIC Angelica dahurica and Ligusticum chuanxiong alcohol extract, the metabolic activity of Malassezia furfur decreased most significantly.

[0074] Based on the results of the quantitative analysis above, the removal effect of high concentration of Angelica dahurica ethanol extract on the biofilm of Malassezia furfur was observed by using an optical microscope. Figure 4 As shown in the figure, without the addition of Angelica dahurica Ligusticum ethanol extract, Malassezia furfur can form a dense biofilm. After treatment with 1×MIC of Angelica dahurica Ligusticum ethanol extract, the biofilm did not change significantly. In contrast, after adding 8×MIC of Angelica dahurica Ligusticum ethanol extract, the biomass of the biofilm decreased significantly, indicating that the amount of cells and their matrix in the biofilm decreased with the increase of the concentration of the drug solution.

[0075] Test Example 3 Toxicity test of the Angelica dahurica extract on rat skin

[0076] Eight rats were randomly divided into a drug administration group (Angelica Ligusticum chuanxiong extract group) and a control group (warm water) by random number table method, half male and half female. The same part of the back of each rat was prepared, and the preparation area was 4cm×7cm (≥10% of body surface area). After applying the drug solution, it was applied to the prepared skin of each rat, and sterilized gauze and mesh nylon bandage were fixed for 4 hours. The control group was applied with warm water. After 24 hours of drug administration, the skin was washed with warm water. The presence of erythema and edema in the application site was observed and recorded with the naked eye at 1, 24, 48, and 72 hours after the removal of the drug.

[0077] 1. Evaluation criteria

[0078] In the skin toxicity experiment, the skin irritation reaction score and irritation intensity evaluation were performed according to Tables 4 and 5. The skin irritation intensity evaluation score = (total erythema score + total edema score) / total number of animals.

[0079] Table 4 Skin irritation response scoring table

[0080]

[0081] Table 5 Evaluation criteria for skin irritation intensity

[0082]

[0083] 2. Results:

[0084] No animals died during the experiment. The animals showed no physiological abnormalities during the drug administration. Their fur was smooth, and their eating and defecation were normal. Within 72 hours after drug administration, the rats showed no edema or erythema on their skin. Figure 6 The irritation evaluation is shown in Table 6. According to the irritation intensity evaluation standard in Table 5, the ethanol extract of Angelica dahurica and Ligusticum chuanxiong is non-toxic and non-irritating to rat skin.

[0085] Table 6 Rat skin irritation evaluation table

[0086]

Claims

1. An ethanol extract of Angelica dahuricae Ligusticum chuanxiong, characterized in that: The extract is obtained by taking equal amounts of Angelica dahurica and Ligusticum chuanxiong as raw materials and adding 80% ethanol for extraction.

2. The ethanol extract of Angelica dahuricae Ligusticum chuanxiong according to claim 1, characterized in that: The ethanol extraction method is 85°C reflux extraction.

3. A method for preparing the ethanol extract of Angelica dahurica Ligusticum chuanxiong according to claim 1 or 2, characterized in that: It includes the following steps: a. Take equal amounts of Angelica dahurica and Ligusticum chuanxiong, grind them into coarse powder and mix them; b. Add 80% ethanol to soak. After soaking, extract under reflux at 85℃. Concentrate the filtrate and freeze-dry to obtain the ethanol extract of Angelica dahurica Ligusticum.

4. Use of the ethanol extract of Angelica dahurica Ligusticum according to claim 1 or 2 in the preparation of a drug having an anti-Malassezia furfur effect.

5. Use of the Angelica dahurica Ligusticum ethanol extract according to claim 1 or 2 in the preparation of medicines or daily chemical products with anti-dandruff effects.

6. A shampoo or conditioner additive with anti-dandruff cleaning and conditioning effects, characterized in that: The invention contains the ethanol extract of Angelica dahuricae Ligusticum chuanxiong according to claim 1 or 2.

7. A quality detection method for the Angelica dahurica Ligusticum ethanol extract according to claim 1 or 2, characterized in that: It uses high performance liquid chromatography to detect the contents of ferulic acid, bergamotolactone, imperatorin, oxidized imperatorin and isoimperatorin in the ethanol extract of Angelica dahurica. The chromatographic conditions are: The chromatographic column was an ACQUITY UPLC BEH C18 (2.1×100nm, 1.7μm) column; the mobile phase was acetonitrile (B)-0.1% formic acid water (D), gradient elution (0-6min, 15% B; 6-9min, 15-45% B, 9-19min, 45% B, 19-23min, 45-85% B, 23-26min, 85% B, 26-29min, 85-15% B), the column temperature was 35°C; the flow rate was 0.25mL / min; the detection wavelength was 302nm; and the injection volume was 10μL.

8. The quality detection method of the Angelica dahurica Ligusticum ethanol extract according to claim 7, characterized in that: It includes the following steps: a. Preparation of test solution: Accurately weigh the freeze-dried powder of Angelica dahurica Ligusticum ethanol extract, dilute it with 50% methanol to make a sample solution, and filter it with a 0.22μm microporous filter membrane to obtain the test solution; b. Preparation of reference solution Accurately weigh 0.57, 0.47, 1.16, 3.55, and 0.78 mg of ferulic acid, bergamot lactone, imperatorin, oxidized imperatorin, and isoimperatorin reference substances into a 10 mL volumetric flask, add methanol to prepare reference substance mother solutions with concentrations of 0.057, 0.047, 0.116, 0.355, and 0.078 mg / mL for ferulic acid, bergamot lactone, imperatorin, oxidized imperatorin, and isoimperatorin, respectively; accurately pipette 1, 2, 5, 1, and 1 mL of the 5 reference substance mother solutions into a 10 mL volumetric flask, respectively, and shake well to obtain a mixed reference substance solution; c. Use the chromatographic conditions described in claim 7 for detection.

9. The quality detection method of the Angelica dahurica Ligusticum ethanol extract according to claim 8, characterized in that: The contents of ferulic acid, bergamot lactone, imperatorin, oxidized imperatorin and isoimperatorin are: ferulic acid 4.1420, bergamot lactone 0.5748, imperatorin 0.5847, oxidized imperatorin 7.9873, isoimperatorin 25.8118, and the content range fluctuates by 10%; the relative retention times are: ferulic acid 4.939min, bergamot lactone 11.174min, oxidized imperatorin 12.076min, imperatorin 14.442min, isoimperatorin 16.410min, and the relative retention times fluctuate by 0.5min.

Images