CN109966470B - Application of cyclic dipeptide in wound repair medicine - Google Patents
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Abstract
The invention relates to application of any one of cyclic preserved fruit-silk dipeptide, cyclic preserved fruit-asparagine dipeptide, cyclic preserved fruit-leu dipeptide and cyclic preserved fruit-isoleuc dipeptide in wound repair. The animal modeling experiment proves that the four cyclic dipeptides have excellent wound repair and treatment effects, such as high inhibition rate on acute and chronic gastric ulcers, high healing speed of skin ulcer wound surfaces, good anti-inflammatory effect and the like.
Description
Technical Field
The invention relates to the field of biological pharmacy, in particular to application of cyclic dipeptide in wound repair medicines.
Background
Cyclic dipeptide, also known as 2, 5-dioxopiperazine, formed by the cyclization of two amino acids through peptide bonds is the smallest cyclic peptide in nature. Many of the cyclic dipeptides are naturally occurring in animals and plants, and some are formed by fermentation or during food processing. Current studies on the biological activity of cyclic dipeptides include bacteriostasis, anticancer, action on the cardiovascular system, protection of neurons, improvement of brain function, withdrawal and detoxification. For example, it has been reported in the literature that cyclic (pro-silk) dipeptides have an antibacterial effect, in particular a more pronounced inhibitory effect on staphylococcus aureus; the cyclic (bright-preserved) dipeptide has the effects of inhibiting breast cancer, cervical cancer and colon cancer, simultaneously has the effects of immunocompetence, hormone regulation and energy generation regulation, has the activities of resisting filtration pathogens and antibiotics besides the antitumor activity, and has obvious inhibiting effect on staphylococcus aureus; the cyclic (pro-isoleucyl) dipeptide has tumor inhibiting and anti-MRSA activities.
There are several technical solutions for wound repair and peptic ulcer using cyclic dipeptide prepared by synthetic methods disclosed in the present patent document, for example, CN105288585a and CN105294830 disclose the use of cyclic pro-propylene dipeptide in the preparation of a medicament for treating skin wounds, CN105311619 discloses the use of cyclic pro-propylene dipeptide in the preparation of a medicament for treating peptic ulcer, CN105622716 discloses the use of cyclic pro-propylene dipeptide and cyclic pro-leupeptic dipeptide for treating acute and chronic gastric ulcers. CN105622717 discloses the use of cyclic pro-phenylpropanedipheptides, cyclic pro-prolyldipeptides, cyclic pro-valdipeptides, cyclic pro-group dipeptides, cyclic pro-tyrosin-dipeptides and cyclic pro-glycyldipeptides for wound treatment. CN105622718 discloses the therapeutic use of cyclic casein-leucinde for gastric ulcers. There is no document currently disclosing that cyclic dipeptide, cyclic-silk dipeptide, cyclic-asparagine dipeptide, cyclic-leu dipeptide, cyclic-isoleuc dipeptide can be used for wound repair, in particular for effective treatment of gastric ulcers and skin ulcers.
Gastric ulcers are common and frequently occurring diseases of the digestive system, are best seen in the lesser curvature of the stomach, especially in the vicinity of the lesser curvature of the antrum and the lateral aspect of the stomach, and are typically manifested as chronic, periodic, rhythmic postprandial timed pain in the middle-upper abdomen, with varying pain properties, with dull or dull pain. About 10% of patients may have no abdominal pain, but they may have hematemesis or black manure as their first symptoms, and may also have other digestive tract symptoms such as belch, acid regurgitation, nausea, vomiting, hunger discomfort, etc. Modern medicine considers that gastric ulcers occur due to an imbalance between gastric mucosal invasive factors and gastric mucosal self-defense/repair factors. H2-R or PPI is often used for clinical treatment, but the recurrence rate after drug withdrawal is high.
Skin ulcers are clinically common and frequently occurring diseases, including varicose ulcers, diabetic ulcers, pressure ulcers and the like, and the lower limbs, feet, buttocks and hands are good sites. The main causes are burn, chemical burn, skin injury infection, long-term compression and the like. Clinically, the traditional Chinese medicine composition is usually treated by adopting physical therapy such as laser, microwave, infrared rays and the like, biological therapy, external auxiliary materials and the like. Although a certain effect is obtained, the effect of healing the wound of the partial refractory skin ulcer is poor.
Six kinds of cyclic dipeptide are obtained through extraction and separation from periplaneta americana, wherein the cyclic proline-silk dipeptide, the cyclic proline-asparagine dipeptide, the cyclic proline-leu dipeptide and the cyclic proline-isoleuc dipeptide have excellent effects on the treatment of wound repair.
Disclosure of Invention
The invention provides application of any one of cycloproteins-silk dipeptide, cycloproteins-asparagine dipeptide, cycloproteins-leu dipeptide and cycloproteins-isoleuc dipeptide in wound repair.
The invention provides an application of any one of cyclic preserved fruit-silk dipeptide, cyclic preserved fruit-asparagine dipeptide, cyclic preserved fruit-leupeptin and cyclic preserved fruit-isoleucwhere dipeptide in medicines for treating acute and chronic gastric ulcer and gastric mucosa injury or in medicines for treating skin injury such as skin ulcer, skin injury and skin inflammation.
The invention also provides a method for preparing any one of cyclic dipeptide (Pro-Ser), cyclic (Pro-asparagine) dipeptide (Pro-Asn), cyclic (Pro-Leu) dipeptide (Pro-Leu), cyclic (Pro-isoleuc) dipeptide (Pro-Lso), cyclic (Pro-propyl) dipeptide (Pro-Ala) and cyclic (Pro-Val) dipeptide (Pro-Val) by extracting and separating periplaneta americana:
step 1: leaching periplaneta americana coarse powder with ethanol and concentrating to obtain an extract;
step 2: subjecting the extract to macroporous resin column chromatography, eluting with water, and concentrating the eluate to obtain water sample;
step 3: subjecting the water-taking part sample to MCI column chromatography, eluting with water to obtain a component A; dividing the component A into an A1 component containing more amino acids and an A2 component containing less amino acids;
step 4: and (3) separating and detecting the A2 component by LC-MS liquid chromatography-mass spectrometry to obtain the compounds cycloprolyl-silk dipeptide, cycloprolyl-asparagine dipeptide, cycloprolyl-propylene dipeptide, cycloprolyl-valdipeptide, cycloprolyl-isoleucyl dipeptide and cycloprolyl-leucyl dipeptide.
In one embodiment of the invention, the macroporous resin column in step 2 is an HP-20 macroporous resin column.
In one embodiment of the invention, the packing type of the MCI column in step 3 is MCI GEL CHP P.
In one embodiment of the present invention, in step 1, the periplaneta americana coarse powder is extracted with ethanol and then concentrated under reduced pressure to obtain an extract.
In one embodiment of the present invention, in step 2, the extract is spin-dried with ethanol, dissolved with deionized water, and then applied to a macroporous resin column chromatography column.
In one embodiment of the invention, in step 2, the eluent is concentrated and lyophilized to obtain a water fraction sample.
In one embodiment of the present invention, in step 3, in combination with TLC detection, component a is divided into A1 component containing more amino acids and A2 component containing less amino acids according to the number of TLC spots by ninhydrin as a color developer; wherein component A having a TLC spot number of 5 or less is taken as component A2, and the remainder are component A1; preferably, component A having a TLC spot number of 3 or less is used as the A2 component, and the remainder are the A1 component.
In one embodiment of the present invention, in step 3, the developing agent for TLC detection is n-butanol/glacial acetic acid/water=4:1:1.
In one embodiment of the present invention, in step 4, the LC liquid chromatography conditions employ a chromatography column selected from the group consisting of: capcell Pak C 18 MGll，Boston Luna Clone C 18 ，Diamonsil C 18 Boston Luna Clone; preferably, the LC liquid chromatography conditions employ a chromatography column of Boston Luna Clone.
In one embodiment of the present invention, in step 4, mobile phase a and mobile phase B are used as mobile phase in the LC liquid chromatography condition; the mobile phase A is 0.06-0.3% formic acid aqueous solution or 0.06-0.3% phosphoric acid aqueous solution; preferably, the mobile phase A is 0.06-0.1% formic acid water solution; the mobile phase B is acetonitrile or an alcohol solvent; preferably, the mobile phase B is methanol.
In one embodiment of the present invention, in step 4, the LC liquid chromatography condition is an isocratic or gradient elution mode; preferably, the LC liquid chromatography condition adopts a gradient elution mode, wherein the volume ratio of the mobile phase a to the mobile phase B during the gradient elution is 90:10-5:95.
The invention also provides a method for preparing any one of cyclic dipeptide (Pro-Ser), cyclic (Pro-asparagine) dipeptide (Pro-Asn), cyclic (Pro-Leu) dipeptide (Pro-Leu), cyclic (Pro-isoleuc) dipeptide (Pro-Lso), cyclic (Pro-propyl) dipeptide (Pro-Ala) and cyclic (Pro-Val) dipeptide (Pro-Val) by extracting and separating periplaneta americana:
step 1: leaching periplaneta americana coarse powder with ethanol, and concentrating under reduced pressure to obtain extract;
step 2: spin-drying the extract with ethanol, dissolving with deionized water, loading onto macroporous resin column chromatography column, eluting with water, concentrating the eluate, and freeze-drying to obtain water sample;
step 3: subjecting the water-taking part sample to MCI column chromatography, eluting with water to obtain a component A; separating the component A into a part A1 component with more amino acid and a part A2 component with less amino acid by combining TLC detection through ninhydrin as a color reagent; wherein, component A with TLC spots less than or equal to 3 is taken as A2 component, and the rest is A1 component, wherein, developing agent for TLC detection is n-butanol, glacial acetic acid and water=4:1:1;
step 4: and (3) separating and detecting the A2 component by LC-MS to obtain the compounds of cycloprolyl-silk dipeptide, cycloprolyl-asparagine dipeptide, cycloprolyl-leupeptin, cycloprolyl-isoleucwhere dipeptide, cycloprolyl-propylene dipeptide and cycloprolyl-valdipeptide respectively.
In one embodiment of the present invention, in the above step 4,
LC liquid chromatography conditions were:
chromatographic column: boston Luna Clone;
the mobile phase was subjected to the gradient elution procedure of table a;
wherein, mobile phase A:0.1% formic acid in water, mobile phase B: acetonitrile
Table A
Wherein the retention time of the cycloproteins-silk dipeptide is 6.17+/-0.02 min, the retention time of the cycloproteins-asparagine dipeptide is 8.25+/-0.02 min, the retention time of the cycloproteins-propylene dipeptide is 10.38+/-0.02 min, the retention time of the cycloproteins-valadipeptides is 20.23+/-0.02 min, the retention time of the cycloproteins-isoleucin dipeptide is 25.58+/-0.02 min, and the retention time of the cycloproteins-leucins dipeptide is 27.38+/-0.02 min.
The cyclic dipeptide of the present invention can be produced by the production method of the present invention or by the synthesis method of the published literature.
On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.
The reagents and materials used in the present invention are commercially available.
The invention has the positive progress effects that: the treatment effect of animal model experiments proves that the cyclic (pro-silk) dipeptide, the cyclic (pro-asparagine) dipeptide, the cyclic (pro-bright) dipeptide and the cyclic (pro-iso-bright) dipeptide have excellent treatment effect on wound repair such as skin injury and gastric ulcer, and are superior to those of the cyclic (pro-vall) dipeptide group, the cyclic (pro-vall) dipeptide group and the positive control group.
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
Example 1 example of the simultaneous preparation of six cyclic dipeptides from periplaneta americana
1) 400ml (about 218 g) of American cockroach ethanol extract is taken and is dried by spin, dissolved by deionized water and concentrated to a proper concentration under reduced pressure, and then is directly loaded on an HP-20 macroporous resin column with the volume of 3.2L without filtering for chromatography, eluted by water, and the eluent is concentrated under reduced pressure and then is subjected to freeze drying treatment to obtain a water part (156 g).
2) 35g of the eluted portion of the American cockroach obtained above was subjected to MCI column chromatography (packing: mitsubishi chemical MCI GEL CHP P) having a column volume of 380ml, and eluted with water, followed by obtaining a water flow component A (32 g) in each of the conical flasks. Each bottle of the component A was examined by TLC, the developing agent was n-butanol/glacial acetic acid/water=4:1:1, and ninhydrin was developed, and the developing agent was separated into an A1 component (30 g) containing more amino acids and an A2 component (2 g) containing less amino acids. Wherein, component A having a TLC spot number of 3 or less is taken as the A2 component, and the remainder is the A1 component. And (5) combining the two parts separately. Concentrating under reduced pressure.
3) The A2 fraction (2 g) obtained above was combined with MS mass spectrometry (LC-MS) under the following chromatographic conditions to obtain compound 1-ring (pro-silk) dipeptide, compound 2-ring (pro-asparagine) dipeptide, compound 3-ring (pro-propyl) dipeptide, compound 4-ring (pro-val) dipeptide, compound 5-ring (pro-isoleucyl) dipeptide, and compound 6-ring (pro-leu) dipeptide, respectively, according to polarity size.
Wherein LC chromatographic conditions are as follows:
chromatographic column: boston Luna Clone (4.6 mm. Times.250 mm,5 μm);
mobile phase a 0.1% formic acid, mobile phase B: the concentration of acetonitrile in the aqueous solution is,
gradient elution procedure was used, see table a;
the flow rate is 0.4ml/min; column temperature 40 ℃; the sample injection amount is 2 μl;
table A
Wherein, the retention time of the compound 1 cycloprolyl-silk dipeptide is 6.17+/-0.02 min, the retention time of the compound 2 cycloprolyl-asparagine dipeptide is 8.25+/-0.02 min, the retention time of the compound 3 cycloprolyl-valdipeptide is 10.38+/-0.02 min, the retention time of the compound 4 cycloprolyl-valdipeptide is 20.23+/-0.02 min, the retention time of the compound 5 cycloprolyl-isoleucyl dipeptide is 25.58+/-0.02 min, and the retention time of the compound 6 cycloprolyl-leucyl dipeptide is 27.38+/-0.02 min.
Wherein compound 1 is a white powder. ESI-MS m/z of Compound 1 positive ion mode 185[ M+H ]] + . The molecular formula: c (C) 8 H 12 N 2 O 3 Molecular weight: 184.1H-NMR (D2O, 400 MHz) δ:1.04 (3H, D, J=7.0 Hz, H-5), 2.28 (1H, m, H-3), 3.61 (1H, D, J=4.4 Hz, H-2), 4.51 (1H, m, ser- αH). 13C-NMR (D2O, 100 MHz) delta 172.0 (Pro-CO), 165.3 (Ser-CO), 60.0 (Ser- βCH2), 59.0 (Pro- αCH), 56.9 (Ser- αCH), 45.2 (Pro- δCH2), 28.0 (Pro- βCH2), 21.6 (Pro- γCH2). The above spectral data were compared with literature to identify compound 1 as a cyclic (pro-silk) dipeptide.
Wherein the compound 2 is white powder. ESI-MS m/z of Compound 2 positive ion mode 212[ M+H ]] + ,234[M+Na] + . The molecular formula: c (C) 9 H 13 N 3 O 3 Molecular weight: 211.1H-NMR (D2O, 400 MHz) delta 4.51 (1H, m, ser-alpha H), 1.04 (3H, D, J=7.0 Hz, H-5), 2.28 (1H, m, H-3), 3.61 (1H, D, J=4.4 Hz, H-2). 13C-NMR (D2O, 100 MHz) delta 174.2 (NH 2-CO), 171.6 (Pro-CO), 166.6 (Asn-CO), 59.1 (Pro-alpha CH), 51.8 (Asn-alpha CH), 45.5 (Pro-delta CH 2), 34.4 (Asn-beta CH 2), 27.6 (Pro-beta CH 2), 21.7 (Pro-gamma CH 2). The above spectral data were compared with literature to identify compound 2 as a cyclic (pro-asparagine) dipeptide.
Wherein compound 3 is a white powder. ESI-MS m/z of Compound 3 positive ion mode 169[ M+H ]] + . The molecular formula: c (C) 8 H 12 N 2 O 2 Molecular weight: 168.1H-NMR (D2O, 400 MHz) delta: 1.40 (3H, D, J=6.9 Hz, H-10), 1.94 (1H, m, H-5 b), 2.03 (2H, m, H-4a, H-5 a), 2.33 (1H, m, H-4 b), 3.53 (2H, m, H-3), 4.21 (1H, m, H-6), 4.27 (1H, m, H-9). 13C-NMR (D2O, 100 MHz) delta 171.2 (C-7), 167.7 (C-1), 59.1 (C-6), 50.7 (C-9), 45.0 (C-3), 27.8 (C-5), 22.2 (C-4), 14.3 (C-10). The above spectral data were compared with literature to identify compound 3 as a cyclic (pro-pro) dipeptide.
Wherein compound 4 is a white powder. ESI-MS m/z of Compound 4 positive ion mode 197[ M+H ]] + . The molecular formula: c (C) 10 H 16 N 2 O 2 Molecular weight: 196.1H-NMR (D2O, 400 MHz) delta 0.95 (3H, D, J=6.9 Hz, H-12), 1.11 (3H, D, J=7.2 Hz, H-11), 1.95 (2H, m, H-4, 10), 2.01 (1H, m, H-4), 2.34 (1H, m, H-5), 2.51 (1H, m, H-5), 3.55 (2H, m, H-3), 4.06 (1H, s, H-9), 4.23 (1H, t, J=7.2 Hz, H-6). 13C-NMR (D2O, 100 MHz) delta 171.2 (C-1), 166.2 (C-7), 60.1 (C-6), 58.6 (C-9), 44.8 (C-3), 28.5 (C-10), 28.1 (C-5), 21.9 (C-4), 17.5 (C-11), 15.3 (C-12). The above spectral data were compared with literature to identify compound 4 as a cyclic (pro-val) dipeptide.
Wherein compound 5 is a white powder. ESI-MS m/z of Compound 5 positive ion Pattern 211[ M+H ]] + . The molecular formula: c (C) 11 H 18 N 2 O 2 Molecular weight: 210.1H-NMR (D2O, 400 MHz) delta 0.95 (3H, t, J=7.4 Hz, H-12), 1.09 (3H, D, J=7.1 Hz, H-12), 1.33 (1H, m, H-11), 1.45 (1H, m, H-11), 1.94 (2H, m, H-4), 2.05 (1H, m, H-5 a), 2.19 (1H, m, H-5 b), 2.34 (1H, m, H-10), 3.54 (2H, m, H-3), 4.09 (1H, s, H-9), 4.22 (1H, m, H-6). 13C-NMR (D2O, 1)00 MHz) delta 170.7 (C-7), 166.1 (C-1), 59.9 (C-6), 58.6 (C-9), 44.8 (C-3), 36.6 (C-10), 28.1 (C-5), 24.0 (C-11), 21.8 (C-4), 15.9 (C-13), 11.1 (C-12). The above spectral data were compared with literature to identify compound 5 as a cyclic (pro-isoleucyl) dipeptide.
Wherein compound 6 is a white powder. ESI-MS m/z of Compound 6 positive ion Pattern 211[ M+H ]] + . The molecular formula: c (C) 11 H 18 N 2 O 2 Molecular weight: 210.1H-NMR (D2O, 400 MHz) delta 0.98 (6H, dd, J=2.5, 6.4Hz, H-12, H-13), 1.54 (1H, m, H-11), 1.91 (2H, m, H-4), 2.03 (2H, m, H-10), 2.32 (1H, m, H-5), 3.54 (2H, m, H-3), 4.15 (1H, m, H-9), 4.28 (1H, t, J=8.0 Hz, H-6). 13C-NMR (D2O, 100 MHz) delta 171.4 (C-7), 167.5 (C-1), 58.8 (C-6), 53.2 (C-9), 45.1 (C-3), 38.0 (C-10), 27.7 (C-5), 24.4 (C-11), 22.2 (C-12), 21.9 (C-4), 20.8 (C-13). The above spectral data were compared with literature to identify compound 6 as a cyclic (pro-leu) dipeptide.
Example 2 Effect of treating gastric ulcer
1. Experimental materials:
1.1 sample: the examples 1 were cyclopro-silk, cyclopro-asparagine, cyclopro-bright, cyclopro-isoleuc, cyclopro-pro-valdipeptide
1.2 experimental animals: SD rats, body weight: 180-200g, male and female halves, source: shanghai Siepper-BiKai laboratory animal Co., ltd; kunming mice, body weight: 18-22g, male and female halves, source: shanghai Siepper-BiKai laboratory animal Co., ltd.
1.3 test reagents: sucralfate (Nanjing white & Jingyu pharmaceutical liability Co., ltd.), ranitidine hydrochloride (Suzhou third pharmaceutical Co., ltd.), absolute ethyl alcohol, glacial acetic acid, pentobarbital sodium (national pharmaceutical Co., ltd.).
2. Experimental method
2.1 action on mouse gastric ulcer caused by absolute ethanol
The KM mice were randomly divided into a blank group, a model group, and a cyclic-prolyl-silk, cyclic-prolyl-asparagine, cyclic-prolyl-bright, cyclic-prolyl-isoleucyl, cyclic-prolyl-propyl, cyclic-valdipeptide group, positive control group (ranitidine hydrochloride) (see Table 1 for specific cases), each of which was given the corresponding drug, each group of mice was given for 10 days, each of the cyclic dipeptide administration groups of example 1 was given a dose of 15mg/kg, and ranitidine hydrochloride group was given a dose of 75mg/kg. Fasted 24h after the administration on day 9, and drinking water is free. Mice in each group were gavaged 3 hours after the last administration, all mice were given 0.2ml absolute ethanol, animals were sacrificed 1 hour later, pylorus ligated, 1% formaldehyde fixed, and ulcer index and inhibition were observed and assessed. And measuring the maximum diameter of the ulcer and the maximum diameter perpendicular to the maximum diameter by using a vernier caliper, wherein the product of the maximum diameter and the maximum diameter is the ulcer index. Ulcer inhibition = (average model group ulcer index-average experimental group ulcer index)/average model group ulcer index x 100%.
2.2 Effect on acetic acid-induced rat gastric ulcer model
SD rats were randomly divided into a blank group, a model group and a ring-silk, ring-asparagine, ring-light, ring-heterolight, ring-propyl, ring-valdipeptide 1-6 groups, positive control group (sucralfate) (see Table 2 in detail), after the rats of each test group except the blank group were fasted for 12 hours, skin was conventionally sterilized under ether anesthesia, the back was fixed to a rat plate, the stomach was opened by laparotomy, a glass tube holder with an inner diameter of 7.0mm and a length of 3.0cm was placed on the stomach of the spleen, glacial acetic acid 80. Mu.L was injected into the glass tube, the glacial acetic acid was sucked for 1min, and the burned site was covered with physiological saline, and the stomach was slowly fed into the abdomen, and was sutured in layers, followed by careful feeding. Each dosing group was given the corresponding drug 1 time per day for 10 days post-surgery. Each of the cyclic dipeptide administration groups of example 1 was administered at a dose of 10mg/kg, and the sucralfate administration group was administered at a dose of 100mg/kg. After last day, animals were sacrificed, the abdominal cavity was opened to ligate the pylorus and the cardia, the whole stomach was removed, and the ulcer index and the ulcer inhibition were calculated.
Ulcer index = ulcer maximum diameter x maximum diameter perpendicular to maximum diameter;
ulcer inhibition = (average model group ulcer index-average experimental group ulcer index)/average model group ulcer index x 100%.
3. Experimental results
3.1 Effect on damage to mouse gastric mucosa by absolute ethanol
Table 1 shows that each administration group can significantly reduce the ulcer index (P < 0.05) of the gastric mucosa injury of mice caused by absolute ethanol compared with the model group, wherein the inhibition effect of the cycloprolyl-asparagus is optimal, and the inhibition rate is 71.67% with extremely significant difference (P < 0.01). After 10 days of administration, the ulcer index and the inhibition rate of each cyclic dipeptide group are obviously better than those of a positive drug ranitidine hydrochloride Ding Zu, and the treatment effect is obviously better than that of a positive control group. It can also be seen from Table 1 that the 4 cyclic dipeptides cyclic pro-silk dipeptide, cyclic pro-asparagine dipeptide, cyclic pro-leu dipeptide, cyclic pro-isoleuc dipeptide of the present invention have a more pronounced protective effect on chronic gastric mucosal lesions caused by absolute ethanol than the cyclic pro-valdipeptide of comparative example 1 and the cyclic pro-propan dipeptide of comparative example 2.
TABLE 1 influence on absolute ethanol induced gastric ulcers in mice
Note that: comparison to model set: * p<0.05；**p<0.01；
comparison to the positive control group: # p<0.05， ## p<0.01
3.2 Effect on acetic acid-induced gastric mucosal injury in mice
Table 2 shows that each of the administration groups significantly reduced the ulceration index of acetic acid-induced gastric mucosal lesions in mice compared to the model group, with significant differences in the effects of cyclic pro-aspdipeptide, cyclic pro-silk dipeptide, cyclic pro-leupeptin (P < 0.01). The ulcer index and inhibition rate of each cyclic dipeptide group were superior to those of the positive control group sucralfate after 10 days of administration. Table 2 shows that the 4 cyclic dipeptides cyclic pro-silk dipeptide, cyclic pro-asparagine dipeptide, cyclic pro-leu dipeptide, cyclic pro-isoleuc dipeptide of the invention have a better protective effect against acetic acid-induced acute gastric mucosal injury than the cyclic pro-valdipeptide of comparative example 1 and the cyclic pro-propanedipeptide of comparative example 2.
TABLE 2 influence on acetic acid-induced gastric ulcers in mice
Note that: comparison to model set: * p<0.05； ** p<0.01
comparison to the positive control group: # p<0.05
the above study results show that: the cycloproteins-silk dipeptide, cycloproteins-asparagine dipeptide, cycloproteins-leupeptin and cycloproteins-isoleuclidins have better mucous membrane repairing effects on chronic gastric ulcer caused by ethanol and acute gastric ulcer models caused by acetic acid, and compared with the common medicines of a positive control group and the cycloproteins-valdi-peptides and cycloproteins-malondipeptides of a comparison example, the cycloproteins-asparagine dipeptide and cycloproteins-isoleuclidins have high ulcer inhibiting rate and more obvious effect.
Example 3 Effect of treating skin injury
1. Experimental materials:
1.1 sample: the ring-silk, ring-asparagine, ring-bright, ring-isoleuch, ring-propyl, ring-valdipeptide of example 1.
1.2 experimental animals: wistar rats, body weight: 180-200g, male and female halves, source: shanghai Siepper-BiKai laboratory animal Co., ltd; kunming mice, body weight: 18-22g, male and female halves, source: shanghai Siepper-BiKai laboratory animal Co., ltd.
1.3 test reagents: wet burn cream (Shanzhao Mibao pharmaceutical Co., ltd.), carrageenan (Shanghai Lan Hua chemical technology Co., ltd.), xylene (Jingzuo trade Co., ltd.), dexamethasone acetate (Huarun Sanjiu medical Co., ltd.)
2. The test method comprises the following steps:
2.1 Effect on rat traumatic skin ulcer model
Wistar rats were randomly divided into a blank group, a model group, a ring-silk, a ring-asparagine, a ring-bright, a ring-heterobright, a ring-propyl, a ring-valdipeptide group and a positive control group (moist burn cream) (see Table 3 in particular), and the rats in the test groups except the blank group were intraperitoneally injected with 10% chloral hydrate 0.3ml/100g, anesthetized rats, and back hairline (3 cm. Times.2 cm), and 1 circular ulcer (1.8 cm diameter, 2.54 cm. Times.2) was cut 1.5cm beside the right side of the spine in the back with a punch, subcutaneous tissue was removed to myofascial, and hemostatic was prepared. Each dosing group was started on the next day of modeling for a total of 10 days. Ulcer healing and wound surface area were observed on days 3, 7 and 10 after administration, respectively.
2.2 effects on Experimental rat granuloma formation with cotton ball
Wistar rats were randomly divided into model groups, ring-silk, ring-asparagine, ring-light, ring-isolumin, ring-propyl, ring-valdipeptide groups and positive control groups (dexamethasone acetate groups) of example 1 (see Table 4 for details). Rats of each test group were anesthetized with diethyl ether, a small incision was cut in the back, sterilized cotton balls, which had been weighed to 20mg, were subcutaneously implanted from the small incision with ophthalmic forceps, and then the skin was sutured, sterilized with iodine solution, and fed conventionally for use. Each dosing group was started on the next day of surgery and was continued for 10 days. After the administration, the cotton ball and the surrounding connective tissue are taken out together, the surrounding adipose tissue is removed, and the cotton ball is put into an oven for drying at 50 ℃ for 24 hours to weigh dry weight. Granuloma dry weight = post-experimental cotton dry weight-pre-experimental cotton weight.
2.3 Effect on mouse xylene-induced ear swelling model
The KM mice were randomly divided into model groups, ring-silk, ring-asparagine, ring-bright, ring-isoluming, ring-propyl, ring-valdipeptide groups and positive control groups (wet burn cream groups) of example 1 (see table 5 for specific cases). 100% xylene inflammatory fluid (0.02 ml/mouse) was applied to both front and back sides of the right ear of the mouse. After 30min, the corresponding groups of test drugs were applied to the swollen parts of the right ear of the mice, and the left ear was not treated as a control. After 4 hours the animals were sacrificed, the ears were cut off immediately, the same position ears were removed with an 8mm punch and weighed on an analytical balance. Swelling degree = right ear weight-left ear weight, swelling rate = (right ear weight-left ear weight)/left ear weight x 100%.
3. Experimental results
3.1 Effect on rat traumatic skin ulcer model
Table 3 shows that the ulcer area was significantly reduced on days 3, 7, and 10 of the administration of each of the administration groups compared to the model group, with statistical differences (p <0.05, p < 0.01); compared with the positive control group wet burn ointment, the four cyclic dipeptide cyclic prolyl-silk dipeptide, cyclic prolyl-asparagine dipeptide, cyclic prolyl-leu dipeptide and cyclic prolyl-isol dipeptide of the invention have the advantages of reduced skin ulcer area of each administration group, lighter wound scar color of rats and faster healing speed. Compared with the cyclic proline-valdipeptide of the comparative example 1 and the cyclic proline-propanedipeptide of the comparative example 2, the cyclic proline-silk dipeptide, the cyclic proline-asparagine dipeptide, the cyclic proline-leu dipeptide and the cyclic proline-isoleuc dipeptide of the invention have faster healing speed and better healing effect on traumatic skin ulcers.
TABLE 3 influence on traumatic skin ulcers in rats
Note that: comparison to model set: * p<0.05；**p<0.01
3.2 Effect on formation of granuloma on cotton ball in laboratory rats
Table 4 shows that each of the cyclic dipeptide and dexamethasone acetate groups reduced the dry weight of granuloma on the cotton balls compared to the model group. Among them, the group of ring-asparagus fern, ring-silk, ring-light and dexamethasone acetate had the most pronounced effect on lowering, with a very significant difference (p < 0.01). The research results show that: the four cyclic dipeptide cycloprop-silk dipeptide, cycloprop-asparagine dipeptide, cycloprop-leu dipeptide and cycloprop-isoleuc dipeptide can inhibit granulation tissue proliferation and have obvious anti-inflammatory effect. The 4 cyclic dipeptides cyclic pro-silk dipeptide, cyclic pro-asparagine dipeptide, cyclic pro-leu dipeptide, cyclic pro-isoleuc dipeptide of the present invention have more pronounced anti-inflammatory effects on granulation tissue proliferation than the cyclic pro-valdipeptide of comparative example 1 and the cyclic pro-propan dipeptide of comparative example 2.
TABLE 4 influence on formation of granuloma on cotton balls of rats
Note that: comparison to model set: * p<0.05；**p<0.01
3.3 Effect of paraxylene on mouse auricle swelling
Table 5 shows that, compared with the model group, the dose groups of the cyclic dipeptide and the wet burn cream group can obviously reduce the swelling degree of the right ear of the mice caused by the xylene, and have extremely significant difference (p < 0.01). The results show that: the four cyclic dipeptide cycloprop-silk dipeptide, cycloprop-asparagine dipeptide, cycloprop-leu dipeptide and cycloprop-isoleuc dipeptide can obviously reduce the auricle swelling rate of mice caused by dimethylbenzene, and has a certain anti-inflammatory effect. The 4 cyclic dipeptides cyclic pro-silk dipeptide, cyclic pro-asparagine dipeptide, cyclic pro-leu dipeptide, cyclic pro-isoleuc dipeptide of the present invention have a stronger anti-inflammatory effect on the extent of ear swelling than the cyclic pro-valdipeptide of comparative example 1 and the cyclic pro-propan dipeptide of comparative example 2.
TABLE 5 Effect of paraxylene on mouse auricle swelling
Note that: comparison to model set: * p<0.05；**p<0.01
by combining the results, the four cyclic dipeptide cyclic pro-silk dipeptide, cyclic pro-asparagine dipeptide, cyclic pro-leu dipeptide and cyclic pro-isoleuc dipeptide in the periplaneta americana extract have the effects of better promoting wound healing, promoting mucosal repair and resisting inflammation. Has better treatment effect on a chronic gastric ulcer model caused by ethanol, an acute gastric ulcer model caused by acetic acid and a rat traumatic skin ulcer model, can obviously inhibit granulation tissue proliferation and relieve auricle swelling degree of mice caused by dimethylbenzene, and has obvious anti-inflammatory effect. Among them, cyclic (pro-aspara) dipeptide, cyclic (pro-silk) dipeptide, cyclic (pro-leu) dipeptide are obvious in the treatment effect on acute and chronic gastric ulcers and skin lesions relative to the positive control group and the cyclic pro-valdipeptide of comparative example 1 and the cyclic pro-propan dipeptide of comparative example 2; among them, cyclic (pro-asparagi) dipeptide and cyclic (pro-silk) dipeptide groups are more effective.
Claims (2)
1. A method for preparing cyclic preserved fruit-asparagine dipeptide by extracting and separating periplaneta americana comprises the following steps:
step 1: leaching periplaneta americana coarse powder with ethanol and concentrating to obtain an extract;
in the step 2, the extractum is dried by spin, dissolved by deionized water and then put on a macroporous resin column chromatographic column;
in the step 2, the eluent is concentrated and then freeze-dried to obtain a water part sample;
step 3: subjecting the water-taking part sample to MCI column chromatography, eluting with water to obtain a component A; dividing the component A into an A1 component containing more amino acids and an A2 component containing less amino acids;
step 4: separating and detecting the A2 component by LC-MS liquid chromatography-mass spectrometry to obtain compounds of cycloprolyl-silk dipeptide, cycloprolyl-asparagine dipeptide, cycloprolyl-propylene dipeptide, cycloprolyl-valdipeptide, cycloprolyl-isoleucyl dipeptide and cycloprolyl-leucyl dipeptide;
the macroporous resin column in the step 2 is an HP-20 macroporous resin column;
the packing type of the MCI column in step 3 was MCI GEL CHP P;
in the step 3, combining TLC detection, using ninhydrin as a color reagent, and dividing the component A into an A1 component with more amino acids and an A2 component with less amino acids according to the number of TLC spots; wherein component A having a TLC spot number of 5 or less is taken as the A2 component, and the remaining component A is the A1 component;
in step 3, the developing agent for TLC detection is n-butanol, glacial acetic acid and water=4:1:1;
LC liquid chromatography conditions were:
chromatographic column: boston Luna Clone;
the mobile phase was subjected to the gradient elution procedure of table a;
wherein, mobile phase A:0.1% formic acid in water, mobile phase B: acetonitrile
Table A
Wherein the retention time of the cyclic preserved asparagine dipeptide is 8.25+/-0.02 min.
2. The method for preparing cyclic-preserved-asparagine dipeptide from periplaneta americana according to claim 1, wherein component a having a TLC spot number of 3 or less is used as A2 component and the remaining component a is A1 component.
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