An active peptide having ace-inhibitory activity, a complex comprising the same, and use thereof

Abstract

The present application relates to an active peptide with ACE inhibitory activity, a complex comprising the same and uses thereof. The amino acid sequence of the active peptide is FSQYPPLGR. The active peptide FSQYPPLGR provided in the present application has high ACE inhibitory activity, and also has significant DPPH scavenging effect.

Description

Technical Field

[0001] This invention belongs to the field of food processing, specifically relating to an ACE-inhibiting active peptide, a complex containing the peptide, and its uses. Background Technology

[0002] Hypertension is a major risk factor for cardiovascular and cerebrovascular diseases and kidney failure, affecting more than 1.3 billion people worldwide. The renin-angiotensin-aldosterone system (RAAS) plays a central role in blood pressure regulation, with angiotensin-converting enzyme (ACE) catalyzing the conversion of angiotensin I into the potent vasoconstrictor angiotensin II, a key step in raising blood pressure. Therefore, inhibiting ACE activity is an important strategy for treating hypertension.

[0003] Synthetic ACE inhibitors (such as captopril and enalapril) are widely used in clinical practice and have a clear antihypertensive effect. However, long-term use is often accompanied by adverse reactions such as persistent dry cough, hypotension, and hyperkalemia. In particular, the incidence of dry cough can reach 10% to 30%, which seriously affects patient compliance. Developing safe, effective, and low-side-effect natural ACE inhibitors is of great clinical significance.

[0004] In recent years, the enzymatic preparation of ACE-inhibiting peptides from natural food proteins has become a research hotspot. Food-derived ACE-inhibiting peptides consist of 2-20 amino acids, have small molecular weights, are easily absorbed, have high safety, and no obvious drug side effects. Previous studies have successfully obtained peptides with ACE-inhibiting activity from various raw materials such as milk protein, egg white, fish, duck meat, and plant protein. For example, yak milk casein, after fermentation and hydrolysis, can produce polypeptides with good ACE-inhibiting activity; the TLVP peptide obtained from duck meat protein through alkaline protease hydrolysis shows significant inhibitory activity. These studies indicate that animal and plant proteins are excellent sources for obtaining natural ACE-inhibiting peptides.

[0005] Although existing technologies have disclosed various ACE-inhibiting peptides, novel peptides derived from common meat proteins with high ACE-inhibiting activity remain relatively limited, and their activity levels and yields still need improvement. Therefore, developing a novel bioactive peptide derived from high-quality animal proteins with high ACE-inhibiting activity has significant research value and application prospects for enriching the library of functional foods and antihypertensive drug candidates, and for meeting the needs of safe and effective adjuvant therapy for hypertensive patients. Summary of the Invention

[0006] In view of the shortcomings of the prior art, one of the objectives of the present invention is to provide an active peptide with ACE inhibitory activity, wherein the amino acid sequence of the active peptide is FSQYPPLGR.

[0007] The active peptide provided in this application has significant ACE inhibitory activity, achieving an ACE inhibition rate of 84.68% at a concentration of 1 mmol / L, while also exhibiting a DPPH scavenging rate of 21.7%.

[0008] The bioactive peptide described in this application is derived from the enzymatic hydrolysate of frozen pork chops, a food source with high safety. Furthermore, bioinformatics copolymerization (ToxinPred and AllerCatPro 2.0) has been used to predict that the bioactive peptide with ACE inhibition is non-toxic and non-allergenic.

[0009] In the amino acid sequence of the active peptide, F is phenylalanine, S is serine, Q is glutamine, Y is tyrosine, P is proline, L is leucine, G is glycine, and R is arginine.

[0010] The second objective of this application is to provide the use of an ACE-inhibiting active peptide, wherein the active peptide is used to prepare any one or a combination of at least two of the following: a drug for lowering blood pressure and a food for assisting in lowering blood pressure.

[0011] Preferably, the dosage form of the drug or food is selected from at least one of fermented milk, solid beverage, compressed candy, gel candy, lyophilized polypeptide powder, capsule, tablet, granule, and oral liquid.

[0012] Preferably, the concentration of the active peptide is 55 μmol / L to 1032 μmol / L, more preferably 55 μmol / L to 280 μmol / L.

[0013] The IC of the ACE-inhibiting active peptide provided in this application 50 The concentration corresponding to an ACE inhibition rate of 50% is 55.01 μmol / L. The active peptide described in this application achieves an ACE inhibition rate of approximately 50% or higher at a concentration of 55 μmol / L or higher. Furthermore, when the concentration of the active peptide reaches 1031.2 μmol / L, the ACE inhibition rate can reach 90%. Therefore, the maximum concentration of the active peptide provided in this application is limited to 1032 μmol / L. However, when the concentration of the active peptide reaches 280 μmol / L, a detection threshold is reached; that is, the active peptide can be tasted at concentrations exceeding 280 μmol / L, and is generally not tasted at concentrations below 280 μmol / L. Therefore, it is preferable that the concentration of the active peptide is below 280 μmol / L.

[0014] The third objective of this application is to provide an active peptide complex with ACE inhibitory activity, the active peptide complex comprising the active peptide described in the first objective and a first peptide; the sequence of the first peptide is LFL.

[0015] Preferably, the molar ratio of the active peptide to the first peptide is 1:2 to 2:1, more preferably 1:1 to 2:1.

[0016] In the ACE-inhibiting active peptide complex, the first peptide LFL has an ACE inhibition rate of 68.25% and a DPPH clearance rate of 69.72% at a concentration of 1 mmol / L. Although its ACE inhibition rate is lower than that of the ACE-inhibiting active peptide FSQYPPLGR, the combination of the two yields an ACE inhibition rate higher than either of them at the same concentration. That is, the ACE inhibition rate of the active peptide FSQYPPLGR and the first peptide LFL at a concentration of 1 mmol / L is higher than that of either of them.

[0017] With a preferred compounding ratio (the molar ratio of the active peptide to the first peptide is 1:2 to 2:1), the ACE inhibition rate is 87.11% to 92.39%. With a further preferred compounding ratio (the molar ratio of the active peptide to the first peptide is 1:1 to 2:1), the ACE inhibition rate is 90.01% to 92.39%, achieving unexpected technical effects.

[0018] Furthermore, the complex of the active peptide and the first peptide achieved a higher DPPH clearance rate than when the active peptide was used alone, with the complex having a DPPH clearance rate of 26.48% to 30.42%.

[0019] That is, the complex of the active peptide and the first peptide has a higher ACE inhibition rate and a higher DPPH clearance rate than the active peptide alone, resulting in unexpected technical effects.

[0020] The fourth objective of this application is to provide a peptide, wherein the amino acid sequence of the peptide is LFL.

[0021] The LFL (first peptide) described in this application is also derived from the enzymatic hydrolysate of frozen pork chops, a food source with high safety. Furthermore, bioinformatics copolymerization (ToxinPred and AllerCatPro 2.0) predicts that the ACE-inhibiting active peptide is non-toxic and non-allergenic.

[0022] Similarly, in the sequence of LFL (first peptide), L represents leucine and F represents phenylalanine.

[0023] The fifth objective of this application is to provide an active peptide complex with ACE inhibitory activity, the active peptide complex comprising the active peptide of claim 1 and a second peptide; the sequence of the second peptide is LLF.

[0024] Preferably, the molar ratio of the active peptide to the second peptide is 1:2 to 2:1, more preferably 1:1 to 2:1.

[0025] In the ACE-inhibiting active peptide complex, the second peptide LLF has an ACE inhibition rate of 70.73% and a DPPH clearance rate of 36.07% at a concentration of 1 mmol / L. Although its ACE inhibition rate is lower than that of the ACE-inhibiting active peptide FSQYPPLGR, the combination of the two peptides yields an ACE inhibition rate higher than either of them at the same concentration. That is, the ACE inhibition rate of the active peptide FSQYPPLGR and the second peptide LLF at a concentration of 1 mmol / L is higher than that of either of them.

[0026] With a preferred compounding ratio (the molar ratio of the active peptide and the second peptide LLF is 1:2 to 2:1), the ACE inhibition rate is 85.70% to 91.16%. With a further preferred compounding ratio (the molar ratio of the active peptide and the second peptide LLF is 1:1 to 2:1), the ACE inhibition rate is 86.88% to 91.16%, achieving unexpected technical effects.

[0027] The sixth objective of this application is to provide a peptide, wherein the amino acid sequence of the peptide is LLF.

[0028] The LLF (second peptide) described in this application is also derived from the enzymatic hydrolysate of frozen pork chops, a food source with high safety. Furthermore, bioinformatics copolymerization (ToxinPred and AllerCatPro 2.0) predicts that the ACE-inhibiting active peptide is non-toxic and non-allergenic.

[0029] Similarly, in the sequence of the LLF (second peptide), L represents leucine and F represents phenylalanine.

[0030] It should be noted that the active peptides FSQYPPLGR, LFL (first peptide), and LLF (second peptide) provided in this application are all derived from the enzymatic hydrolysate of frozen pork chops. They can also be synthesized using standard methods or several synthetic routes well known to those skilled in the art of peptide synthesis to prepare the short-chain peptides of this invention.

[0031] For example, the active peptide FSQYPPLGR, the first peptide LFL, and the second peptide LLF can be synthesized using the methods described below, as well as conventional techniques known to those skilled in the art of peptide synthesis or variations thereof understood by those skilled in the art.

[0032] For example, when using solid-phase synthesis of active peptides FSQYPPLGR, LFL (first peptide), or LLF (second peptide), a resin is used as the solid-phase carrier. The resin is soaked and swollen, then washed. Fmoc-protected proline and a condensing agent are added according to the sequence of the target peptide to carry out deprotection and condensation reactions of the amino groups. The deprotection-condensation process is repeated until all amino acids are linked. The target peptide is then obtained by cutting the resin.

[0033] For example, the active peptides are all synthesized via the following solid-phase synthesis steps:

[0034] (1) Based on the first amino acid at the C-terminus of the polypeptide sequence, select 0.5 mmol of the corresponding Fmoc protected amino acid-Wang resin, add it to the solid phase reactor, add DCM to swell the resin for 30 min, dry it, wash it three times with DMF, add a 20% hexahydropyridine DMF solution, react for 5 min, add another 20% hexahydropyridine DMF solution and react for 10 min, wash with DMF once in the middle, dry it after the reaction is finished, and wash it with DMF 3 times.

[0035] (2) Following the sequence of the peptide from C-terminus to N-terminus, condensation reaction and Fmoc removal reaction were carried out alternately in a reaction amount of 1.5 mmol to condense all the remaining amino acids of the peptide onto the resin. After the last amino acid was condensed, a 20% hexahydropyridine DMF solution was added and reacted for 5 min. Then, another 20% hexahydropyridine DMF solution was added and reacted for 10 min. DMF was washed once in the middle. After the reaction was completed, the solution was dried and washed with DMF 3 times.

[0036] (3) Add 1 mmol of FITC and N-methylmorpholine to the reactor and react for 5-10 minutes. Use ninhydrin to check whether the reaction is complete. After the reaction is complete, wash three times with DMF and DMC alternately, and then wash with methanol to shrink and obtain dried polypeptide-resin.

[0037] (4) Place the polypeptide-resin into a round-bottom flask and slowly add the prepared lysis buffer at 0°C (the volume ratio of the reagent formula used in the lysis buffer is TFA: benzyl sulfide: phenol: triisopropylsilane: water = 82.5: 7.5: 5: 3: 2). Stir slowly and react at low temperature for 0.5 hours, then at room temperature for 2 hours. Filter to obtain the lysis buffer. Slowly add the lysis buffer to anhydrous ice-cold ether and stir. Filter to separate the crude polypeptide. Wash with ice-cold ether 3 times to obtain the crude peptide.

[0038] (5) Use mass spectrometry to detect whether the molecular weight of the crude product is correct. If it is correct, use high performance liquid chromatography to purify and separate it, freeze dry it, and then obtain the pure polypeptide.

[0039] The seventh objective of this application is to provide a use of the active peptide complex as described in objective five or objective six, wherein the active peptide complex is used to prepare any one or a combination of at least two of the following: a drug for lowering blood pressure, a food for assisting in lowering blood pressure, and / or a food for anti-aging or assisting in anti-aging.

[0040] Preferably, the dosage form of the drug or food is selected from at least one of fermented milk, solid beverage, polypeptide freeze-dried powder, capsules, tablets, granules, and oral liquid.

[0041] Compared with the prior art, this application has the following beneficial effects:

[0042] (1) The active peptide FSQYPPLGR provided in this application has high ACE inhibitory activity and significant DPPH scavenging effect;

[0043] (2) The first peptide LFL and the active peptide FSQYPPLGR provided in this application can synergistically enhance the ACE inhibitory activity and DPPH scavenging effect.

[0044] (3) The second peptide LLF and the active peptide FSQYPPLGR provided in this application can synergistically enhance the ACE inhibitory activity. Attached Figure Description

[0045] Figure 1 The HPLC chromatogram of the bioactive peptide FSQYPPLGR is shown below.

[0046] Figure 2 This is a secondary mass spectrometry analysis chromatogram of the bioactive peptide FSQYPPLGR;

[0047] Figure 3 The HPLC chromatogram of the bioactive peptide LFL is shown below.

[0048] Figure 4 This is a secondary mass spectrometry analysis chromatogram of the bioactive peptide LFL;

[0049] Figure 5 The HPLC chromatogram of the bioactive peptide LLF is shown below.

[0050] Figure 6 This is a secondary mass spectrometry analysis of the bioactive peptide LLF. Detailed Implementation

[0051] The technical solution of the present invention will be further explained and described below with reference to specific embodiments. However, it should be noted that the specific embodiments are only a specific implementation and explanation of the essence of the technical solution of the present invention, and should not be construed as a limitation on the scope of protection of the present invention.

[0052] The reagents and instruments used in the examples are all commercially available, and the detection methods are conventional methods well known in the art.

[0053] Synthesis example 1

[0054] Nanjing Peptide Research Biotechnology Co., Ltd. was commissioned to synthesize the active peptide FSQYPPLGR.

[0055] The purity of the synthesized bioactive peptide FSQYPPLGR was determined using HPLC (Waters 2695). Figure 1(Figure 1 shows the HPLC chromatogram of the active peptide FSQYPPLGR), with a purity of 98.27%.

[0056] The molecular weight and structure of the bioactive peptide FSQYPPLGR were determined using HPLC-MS (Waters ZQ-2000). Figure 2 ( Figure 2 The secondary mass spectrometry analysis of the bioactive peptide FSQYPPLGR shows that its molecular weight is 1064.19 Da and its structure is H-Phe-Ser-Gln-Tyr-Pro-Pro-Leu-Gly-Arg-OH.

[0057] Synthesis example 2

[0058] Nanjing Peptide Research Biotechnology Co., Ltd. was commissioned to synthesize the active peptide LFL.

[0059] The purity of the synthesized bioactive peptide LFL was determined using HPLC (Waters 2695). Figure 3 ( Figure 3 The HPLC chromatogram of the active peptide LFL is shown in the figure, with a purity of 99.24%.

[0060] The molecular weight and structure of the bioactive peptide LFL were determined using HPLC-MS (Waters ZQ-2000). Figure 4 ( Figure 4 The secondary mass spectrometry analysis of the bioactive peptide LFL shows that its molecular weight is 391.50 Da and its structure is H-Leu-Phe-Leu-OH.

[0061] Synthesis example 3

[0062] Nanjing Peptide Research Biotechnology Co., Ltd. was commissioned to synthesize the active peptide LLF.

[0063] The purity of the synthesized bioactive peptide LLF was determined using HPLC (Waters 2695). Figure 5 ( Figure 5 The HPLC chromatogram of the active peptide LLF is shown in the figure, with a purity of 98.90%.

[0064] The molecular weight and structure of the bioactive peptide LLF were determined using HPLC-MS (Waters ZQ-2000 instrument). Figure 6 ( Figure 6 The secondary mass spectrometry analysis of the active peptide LLF shows that its molecular weight is 391.50 Da and its structure is H-Leu-Leu-Phe-OH.

[0065] Example 1

[0066] A substance with ACE inhibitory activity, consisting of a 1 mmol / L active peptide FSQYPPLGR.

[0067] Example 2

[0068] A substance with ACE inhibitory activity consists of 0.5 mmol / L of the active peptide FSQYPPLGR and 0.5 mmol / L of the first peptide LFL (in a 1:1 ratio).

[0069] Example 3

[0070] A substance with ACE inhibitory activity consists of 0.67 mmol / L of the active peptide FSQYPPLGR and 0.33 mmol / L of the first peptide LFL (in a 2:1 ratio).

[0071] Example 4

[0072] A substance with ACE inhibitory activity consists of 0.33 mmol / L of the active peptide FSQYPPLGR and 0.67 mmol / L of the first peptide LFL (ratio 1:2).

[0073] Example 5

[0074] A substance with ACE inhibitory activity, the first peptide LFL at a concentration of 1 mmol / L.

[0075] Example 6

[0076] A substance with ACE inhibitory activity consists of 0.5 mmol / L of the active peptide FSQYPPLGR and 0.5 mmol / L of the second peptide LLF (in a 1:1 ratio).

[0077] Example 7

[0078] A substance with ACE inhibitory activity consists of 0.67 mmol / L of the active peptide FSQYPPLGR and 0.33 mmol / L of the second peptide LLF (ratio 2:1).

[0079] Example 8

[0080] A substance with ACE inhibitory activity consists of 0.33 mmol / L of the active peptide FSQYPPLGR and 0.67 mmol / L of the second peptide LLF (ratio 1:2).

[0081] Example 9

[0082] A substance with ACE inhibitory activity, the second peptide LLF at a concentration of 1 mmol / L.

[0083] Comparative Example

[0084] FILR peptide, a synthetic peptide from sardines (Sardina pilchardus). The concentration at which ACE inhibition is 50% is 1.01 mg / mL, or 1.84 mmol / L (IC50). 50 At 1 mg / mL (i.e., 1.83 mmol / L), the DPPH clearance rate was 23.05% ± 0.37%.

[0085] ACE inhibition rate assay

[0086] Enzyme solution (0.1 U / mL) and substrate solution (1.5 mg / mL) were prepared by dissolving ACE and HHL (malyurel histidine leucine) separately in borate buffer (BBS) (0.1 mol / L, containing 0.03 mol / L NaCl). The ACE-inhibiting substance from the examples was dissolved in pure water to obtain the test solution. First, the test solution (20 μL) and HHL (100 μL) were mixed and incubated at 37 °C for 5 min. Then, 20 μL of ACE solution was added (at this point, the concentration of the ACE-inhibiting substance from the examples was 1 mmol / L), and incubation continued for 1 h. Finally, 100 μL of 1 mol / L HCl was added to terminate the reaction. The mixture was filtered through a 0.22 μm nylon filter. The content of HA (hippuric acid) in the samples was determined using an Agilent high-performance liquid chromatography system (1260 Infinity II Prime) equipped with a C18 (Venusil XBP C18, 4.62 × 250 mm, Å, 5 μm, Agela Technology). The injection volume of the sample was 10 μL. The control group did not contain the ACE-inhibiting substance from the examples, and the blank group did not contain ACE, but used BBS buffer instead of peptide solution and ACE. Mobile phase A was pure water (containing 0.1% TFA), and mobile phase B was acetonitrile (containing 0.1% TFA), with a mobile phase ratio of 75% A:25% B. The flow rate was 0.8 mL / min, and the elution time was 20 min. The peak area of ​​HA was detected at 228 nm using a UV detector. All experiments were repeated three times, and the ACE inhibitory activity was calculated using Equation 1:

[0087] ACE inhibition rate (%) = (BA) / (BC) × 100% Formula 1

[0088] Where B is the HA peak area of ​​the control group (without sample), A is the HA peak area of ​​the experimental group (with the ACE-inhibiting substance provided in the example), and C is the HA peak area of ​​the blank group (without ACE).

[0089] ACE-inhibited IC 50 Value determination

[0090] Different concentrations of test sample solutions were prepared by dissolving the ACE-inhibiting substances from Example 1 and the comparative example in pure water. After mixing the test sample solution (20 μL) and HHL (100 μL), solutions with concentrations of 2 mmol / L, 1 mmol / L, 0.5 mmol / L, 0.25 mmol / L, 0.125 mmol / L, 0.0625 mmol / L, and 0.03125 mmol / L were measured. The ACE inhibition rate was determined according to the described method. An ACE inhibition rate-concentration curve was fitted based on the measurement results, and the concentration at which the ACE inhibition rate was 50% was calculated and recorded as the IC50 of the ACE-inhibiting substance. 50 Value. The IC50 of the ACE-inhibiting substance provided in Example 1 was determined. 50 The value was 55.01 μmol / L.

[0091] DPPH scavenging rate determination

[0092] Add 100 μL of freshly prepared 0.2 mmol / L DPPH methanol solution to each well of a 96-well microtiter plate. Then, add 100 μL of samples of different concentrations and components sequentially. Incubate at room temperature in the dark for 30 min, and measure the absorbance at 517 nm using a microplate reader. The DPPH free radical scavenging rate is calculated using Equation 2:

[0093] DPPH free radical scavenging rate (%) = [1 - (A1 - A2) / A0] × 100% Formula 2

[0094] In the formula, A0 is the absorbance of a 1:1 volume ratio of water and a 0.2 mmol / L DPPH methanol solution, A1 is the absorbance of a 1:1 volume ratio of the example sample and the DPPH methanol solution, and A2 is the absorbance of a 1:1 volume ratio of the example sample and methanol.

[0095] The measurement results are shown in Table 1.

[0096] Table 1

[0097]

[0098] As can be seen from Table 1, the IC50 of the active peptides provided in the comparative example 50 The value was 1.84 mmol / L, which is much higher than the IC50 of the ACE-inhibiting substance provided in Example 1. 50The value (55.01 μmol / L) indicates that the active peptide FSQYPPLGR provided in this application has higher ACE inhibition, and there is no significant difference in DPPH clearance. Furthermore, data from other examples in Table 1 show that the active peptide provided in this application and its compounded composition all exhibit ACE inhibition of over 50% at a concentration of 1 mmol / L, which is superior to the comparative example. The DPPH clearance rate shows a significant improvement in Examples 2, 3, 4, 6, and 8, especially in Example 4. Examples 2 and 3 show that the ACE inhibition rate is synergistically enhanced after compounding with the second peptide LLF. Example 7 shows that the ACE inhibition rate is synergistically enhanced after compounding with the first peptide LFL. The first peptide LFL exhibits a significantly excellent DPPH clearance rate.

[0099] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An active peptide with ACE inhibitory activity, characterized in that, The amino acid sequence of the active peptide is FSQYPPLGR.

2. The use of an ACE-inhibiting active peptide, characterized in that, The active peptide is used to prepare any one or a combination of at least two of the following: drugs for lowering blood pressure and foods that assist in lowering blood pressure. Preferably, the dosage form of the drug or food is selected from at least one of fermented milk, solid beverage, gel candy, lyophilized polypeptide powder, capsule, tablet, granule, and oral liquid.

3. The use as described in claim 2, characterized in that, The concentration of the active peptide used is 55 μmol / L to 1032 μmol / L, preferably 55 μmol / L to 280 μmol / L.

4. An active peptide complex with ACE inhibitory activity, characterized in that, The active peptide complex comprises the active peptide of claim 1 and the first peptide; The sequence of the first peptide is LFL.

5. The active peptide complex according to claim 4, characterized in that, The molar ratio of the active peptide to the first peptide is 1:2 to 2:1, preferably 1:1 to 2:

1.

6. A peptide, characterized in that, The amino acid sequence of the peptide is LFL.

7. An active peptide complex with ACE inhibitory activity, characterized in that, The active peptide complex includes the active peptide and the second peptide as described in claim 1; The sequence of the second peptide is LLF.

8. The active peptide complex according to claim 7, characterized in that, The molar ratio of the active peptide and the second peptide is 1:2 to 2:1, preferably 1:1 to 2:

1.

9. A peptide, characterized in that, The amino acid sequence of the peptide is LLF.

10. Use of the active peptide complex as described in claims 4-5 or 7-8, characterized in that, The active peptide complex is used to prepare any one or a combination of at least two of the following: drugs for lowering blood pressure, foods that assist in lowering blood pressure, and / or foods that have anti-aging or assist in anti-aging. Preferably, the dosage form of the drug or food is selected from at least one of fermented milk, solid beverage, polypeptide freeze-dried powder, capsules, tablets, granules, and oral liquid.

Images