Trimer of Glu-His-Gly, its preparation and application in enhancing immunity

Abstract

The present invention discloses a trimer of Glu-His-Gly, namely [Glu-His-Gly]<subgt;3< / subgt;, and discloses its preparation method and application in enhancing immunity. Experiments have proved that [Glu-His-Gly]<subgt;3< / subgt; of the present invention not only has a good immune-enhancing effect, but also the immune-enhancing effect is significantly stronger than that of Glu-His-Gly. Therefore, it is proposed that the present invention provides an effective technical means for enhancing immunity.

Description

Technical Field

[0001] The present invention relates to a trimer of Glu-His-Gly, i.e., [Glu-His-Gly]3, to its preparation method and to its use in the treatment of inflammatory diseases. Experiments have shown that the [Glu-His-Gly]3 of the present invention not only has a good immune-enhancing effect, but also has a significantly stronger immune-enhancing effect than that of Glu-His-Gly. The present invention belongs to the field of biomedicine. Background Art

[0002] Cynomorium songaricum Rupr. is a perennial fleshy parasitic herb without chlorophyll, the whole plant is reddish-brown, most of the whole herb is buried in the sand, and Cynomorium songaricum Rupr. buds of different sizes are parasitized on the roots. The Cynomorium songaricum Rupr. buds gradually mature from the initial spherical shape to the later oval or long cylindrical shape. Most roots of Cynomorium songaricum Rupr. have fibrous roots and shed scale leaves. The stem of Cynomorium songaricum Rupr. is cylindrical, erect, brownish. The base of the stem of Cynomorium songaricum Rupr. is slightly thickened or swollen, and there are deciduous scale leaves arranged spirally and becoming sparser upwards on the stem. The scale leaves are ovate-triangular. The spadix of Cynomorium songaricum Rupr. is born at the top of the stem, protruding above the ground, club-shaped, 5-16 cm long and 2-6 cm in diameter. There are very dense small flowers on the spadix. Male flowers, female flowers and bisexual flowers are mixed together, with a fragrance. The flowering period is from May to July. Cynomorium songaricum Rupr. produces small nuts, nearly spherical or oval, the pericarp is white, and there is a persistent light yellow style at the top. The fruiting period is from June to July. The seeds of Cynomorium songaricum Rupr. are nearly spherical, dark red, and the seed coat is hard and thick. Cynomorium songaricum Rupr. grows in desert steppes, desert steppe deserts and deserts along rivers and lakes. Cynomorium songaricum Rupr. can tonify the kidney, benefit essence and moisten dryness, and is mainly used for treating impotence and spermatorrhea, weakness of the waist and knees, constipation due to intestinal dryness, and has a certain effect on paralysis and improving sexual function weakness. It is worth pointing out that when the inventor evaluated the immune-enhancing effect of Cynomorium songaricum Rupr., Glu-His-Gly was found in the spleen of mice. The inventor knows that aggregates of Glu-His-Gly often show stronger anti-inflammatory effects. So the inventor prepared a trimer of Glu-His-Gly, that is, [Glu-His-Gly]3 was prepared. Experiments have shown that the [Glu-His-Gly]3 of the present invention not only has a good immune-enhancing effect, but also has a significantly stronger immune-enhancing effect than that of Glu-His-Gly. Based on these findings, the inventor proposed the present invention. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to identify a trimer of Glu-His-Gly, i.e., [Glu-His-Gly]3. And further confirm that the said [Glu-His-Gly]3 has a better immune-enhancing effect than Glu-His-Gly. To achieve the above object, the present invention adopts the following three technical means.

[0004] The first technical means is to propose a trimer of Glu-His-Gly of the present invention, namely [Glu-His-Gly]3. The said [Glu-His-Gly]3 is composed of three Glu-His-Gly molecules through non-covalent bonds.

[0005] The second technical means is to propose a method for preparing a trimer of Glu-His-Gly, namely [Glu-His-Gly]3. This method includes the following 5 steps:

[0006] 1) Prepare Boc-His(Boc)-Gly-OBzl;

[0007] 2) Prepare His-Gly-OBzl;

[0008] 3) Prepare Boc-Glu(OBzl)-His-Gly-OBzl;

[0009] 4) Prepare Glu-His-Gly;

[0010] 5) Prepare [Glu-His-Gly]3.

[0011] The third technical means is to confirm the immune enhancement effect of the trimer of Glu-His-Gly, namely [Glu-His-Gly]3.

[0012] The trimer of the present invention, namely [Glu-His-Gly]3, which is composed of three Glu-His-Gly molecules through non-covalent bonds. Experiments have shown that the [Glu-His-Gly]3 of the present invention not only has a good immune enhancement effect, but also the immune enhancement effect is significantly stronger than that of Glu-His-Gly. Therefore, it is proposed that the present invention provides an effective technical means in the preparation of drugs for enhancing immunity. Description of the Drawings

[0013] Figure 1 It is the synthetic route diagram of Glu-His-Gly: i) HOBt, DCC, NMM, THF; ii) ethyl acetate solution of hydrogen chloride with a concentration of 4N; iii) Pd / C, hydrogen, CH3OH.

[0014] Figure 2 It is the FT-ICR-MS spectrum of [Glu-His-Gly]3 and the qCID spectrum of [Glu-His-Gly]3. Detailed Embodiments

[0015] In order to further illustrate the present invention, a series of embodiments are given below. These embodiments are completely illustrative and are only used to specifically describe the present invention and should not be construed as a limitation of the present invention.

[0016] Example 1 Preparation of Glu-His-Gly

[0017] Example 1.1 Preparation of Boc-His(Boc)-Gly-OBzl

[0018] At 0 °C, Boc-His(Boc) (3.82 g, 7.11 mmol) and N-hydroxybenzotriazole (HOBt, 0.96 g, 7.10 mmol) were dissolved in anhydrous tetrahydrofuran. To this solution was added a solution of dicyclohexylcarbodiimide (DCC, 1.47 g, 7.10 mmol) in anhydrous tetrahydrofuran. The two solutions were stirred well for 20 minutes to obtain activated Boc-His(Boc) / HOBt. At 0 °C, HCl·Gly-OBzl (2.40 g, 7.11 mmol) was added to the activated Boc-His(Boc) / HOBt to obtain a reaction mixture. At 0 °C, the pH value of the reaction mixture was adjusted to 9 with N-methylmorpholine (NMM) and stirred for 30 minutes, then stirred at room temperature for 12 hours. TLC (dichloromethane / methanol = 20 / 1) showed the disappearance of HCl·Gly-OBzl. The insoluble matter in the reaction mixture was filtered off, and the filtrate was concentrated to dryness under reduced pressure. The residue was dissolved in 300 mL of ethyl acetate. The obtained ethyl acetate solution was washed successively with saturated aqueous NaHCO3 (200 mL × 3), saturated aqueous NaCl (200 mL × 3), 5% aqueous KHSO4 (200 mL × 3), saturated aqueous NaCl (200 mL × 3), saturated aqueous NaHCO3 (200 mL × 3) and saturated aqueous NaCl (200 mL × 3). The separated ethyl acetate solution was dried over anhydrous Na2SO4 for 12 hours, filtered, and the filtrate was concentrated under reduced pressure. The residue was triturated repeatedly with ether to obtain 2.87 g (96%) of the title compound as a pale yellow solid, which was directly used in the next step. ESI-MS (m / e): 503 [M+H] + 。

[0019] Example 1.2 Preparation of His-Gly-OBzl

[0020] Dissolve Boc-His(Boc)-Gly-OBzl (2.00 g, 4.98 mmol) in 15 mL of anhydrous ethyl acetate, and add 20 mL of a 4 N hydrogen chloride solution in anhydrous ethyl acetate while cooling in an ice-salt bath. The reaction mixture was stirred for 120 minutes while cooling in an ice-salt bath. TLC (dichloromethane / methanol = 20 / 1) showed the disappearance of Boc-His(Boc)-Gly-OBzl. The reaction mixture was concentrated under reduced pressure to remove free hydrogen chloride gas. The residue was redissolved in anhydrous ethyl acetate and concentrated under reduced pressure to remove free hydrogen chloride gas. This operation was repeated three times to completely remove free hydrogen chloride gas. The residue was triturated with petroleum ether three times to obtain 1.84 g (92%) of the title compound as a colorless solid, which was directly used in the next reaction. ESI-MS (m / e): 403 [M+H] + 。

[0021] Example 1.3 Preparation of Boc-Glu(OBzl)-His-Gly-OBzl

[0022] The crude product obtained from Boc-Glu(OBzl) (1.88 g, 5.56 mmol) and His-Gly-OBzl (1.68 g, 5.56 mmol) according to the procedure of Example 1.1 was purified by silica gel column chromatography to obtain 2.54 g (88%) of the title compound as a colorless solid. ESI-MS (m / e): 622 [M+H] + ; 1 1H NMR (300 MHz, DMSO-d6): δ / ppm = 8.41 (s, 1H), 8.12 (d, J = 8.0 Hz, 1H), 7.97 (d, J = 25.5 Hz, 1H), 7.36 (m, 10H), 7.06 (d, J = 7.6 Hz, 1H), 6.97 (d, J = 14.4 Hz, 1H), 5.10 (d, J = 9.3 Hz, 4H), 4.54 (m, 2H), 3.96 (m, 4H), 2.90 (dd, J1 = 21.4 Hz, J2 = 13.3 Hz, 2H), 2.35 (t, J = 7.9 Hz, 2H), 1.81 (m, 2H), 1.36 (s, 9H); 13 13C NMR (75 MHz, DMSO-d6): δ / ppm = 172.74, 171.86, 171.21, 169.96, 155.96, 136.69, 136.33, 134.70, 132.49, 128.89, 128.55, 128.50, 128.43, 128.40, 128.29, 126.88, 78.85, 66.33, 65.87, 54.12, 52.54, 41.26, 30.48, 29.18, 28.63, 27.37。

[0023] Example 1.4 Preparation of Glu-His-Gly

[0024] Dissolve Boc-Glu(OBzl)-His-Gly-OBzl (500 mg, 0.81 mmol) in methanol, add 75 mg of Pd / C, and stir to make it homogeneous. First, evacuate the air in the reaction flask under reduced pressure, and then introduce hydrogen gas into the reaction flask for a 48-hour debenzylation reaction. TLC (dichloromethane / methanol = 20 / 1) shows the disappearance of Boc-Glu(OBzl)-His-Gly-OBzl. Filter the reaction mixture to remove Pd / C, and concentrate the filtrate under reduced pressure to dryness. Wash the residue with diethyl ether (30 mL × 3) to obtain Boc-Glu-His-Gly as a colorless powder. Dissolve the Boc-Glu-His-Gly powder in 2 mL of anhydrous ethyl acetate solution at 0 °C, and mix the resulting ethyl acetate solution with 5 mL of 4N hydrogen chloride in anhydrous ethyl acetate solution. Stir the reaction compound for 2 hours, and TLC (ethyl acetate / acetic acid / water = 2 / 1 / 1) shows the disappearance of Boc-Glu-His-Gly. Concentrate the reaction mixture under reduced pressure to dryness. Dissolve the residue in 5 mL of anhydrous ethyl acetate and concentrate under reduced pressure to remove the free hydrogen chloride gas. Repeat this operation three times to completely remove the free hydrogen chloride gas. Purify the residue by C18 column chromatography to obtain 237 mg (88%) of the title compound. M.p. 160 - 161 °C; ESI-MS (m / e): 342 [M+H] + ; 1 1H-NMR (300 MHz, DMSO-d6): δ / ppm = 8.98 (m, 2H), 8.64 (t, J = 5.9 Hz, 1H), 7.46 (d, J = 1.3 Hz, 1H), 4.68 (dt, J1 = 7.5 Hz, J2 = 4.7 Hz, 1H), 3.89 (m, 2H), 3.74 (dd, J1 = 17.6 Hz, J2 = 5.6 Hz, 1H), 3.13 (dq, J1 = 15.3 Hz, J2 = 6.2 Hz, 3H), 2.39 (m, 2H), 1.94 (m, J = 7.0 Hz, 2H); 13 13C NMR (75 MHz, DMSO-d6): δ / ppm = 173.84, 171.51, 170.11, 168.72, 133.83, 129.26, 117.55, 52.55, 51.84, 41.20, 29.59, 27.33, 26.73。

[0025] Example 2 Preparation of the trimer of Glu-His-Gly

[0026] Dissolve 100 mg of Glu-His-Gly powder in 5 mL of ultrapure water. The resulting solution is vortexed for 15 minutes first and then sonicated in an ultrasonic bath for 4 hours until the solution becomes completely colorless and transparent. The colorless and transparent solution is concentrated to dryness under reduced pressure to obtain the Glu-His-Gly trimer, namely [Glu-His-Gly]3. The structure of [Glu-His-Gly]3 is confirmed by ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Figure 2 The FT-ICR-MS spectrum of Figure 2 gives the peak of [Glu-His-Gly]+H at 342.14447 (theoretical value: 342.14081), the peak of [Glu-His-Gly]2+H at 683.28529 (theoretical value: 683.27434), and the peak of [Glu-His-Gly]3+H at 1024.43122 (theoretical value: 1024.40788).

[0027] To clarify the relationship among the peaks of [Glu-His-Gly]+H, [Glu-His-Gly]2+H, and [Glu-His-Gly]3+H, the qCID spectrum of [Glu-His-Gly]3+H was measured. Figure 2 The qCID spectrum of [Glu-His-Gly]3+H of Figure 2 gives the peak of [Glu-His-Gly]2+H at 683.27856 (theoretical value: 683.27434) and the peak of [Ala-Gln-Gly]+H at 42.14280 (theoretical value: 342.14081). That is to say, [Glu-His-Gly]3 is the only existing form of Glu-His-Gly.

[0028] Example 3: Evaluate the effect of [Glu-His-Gly]3 on the proliferation of splenocytes

[0029] The enhancing effect of [Glu-His-Gly]3 on the proliferation of splenocytes was determined by the MTT method. [Glu-His-Gly]3 was prepared into a solution with a final concentration of 0.5 mg / mL using RPMI-1640 medium. The positive control thymopentin was prepared into a solution with a final concentration of 10 μM using RPMI-1640 medium. Glu-His-Gly was prepared into a solution with a final concentration of 2 μM using RPMI-1640 medium. The blank control was RPMI-1640 medium.

[0030] ICR male mice weighing 22 g were anesthetized with ether and sacrificed by cervical dislocation. The spleens were taken under sterile conditions. The obtained spleens were ground with a 200-mesh steel mesh and a glass syringe plunger. The resulting spleen homogenate was washed twice with HANK’S solution. Centrifuge at 1500 rpm for 10 minutes, count, and prepare into 5×106 After preparing a cell suspension of spleen cells at 5 5×10 cells / mL of culture medium, inoculate it into a 96-well plate, and add 100 μL to each well (each well contains 5×10 spleen cells, and add 100 μL of PBS solution to seal the outer wells of the 96-well plate). PBS is prepared by dissolving 8.00 g of NaCl, 0.20 g of KCl, 1.56 g of Na2HPO4·12H2O, and 0.20 g of KH2PO4 in triple-distilled water to 1000 mL and sterilizing it in an autoclave. Incubate the 96-well plate in an incubator with 5% CO2 at 37 °C for 4 hours, then add 25 μL of RPMI-1640 culture solution containing [Glu-His-Gly]3 or 25 μL of RPMI-1640 medium solution containing thymopentin or 25 μL of RPMI-1640 medium solution containing Glu-His-Gly to each well, and gently shake to mix evenly (set an RPMI-1640 medium control for each plate). After incubating for 48 hours, add 25 μL of MTT solution (5 mg / mL) to each well, continue to incubate for 4 hours, then aspirate the supernatant, add 100 μL of DMSO to each well, and shake for 15 minutes to fully dissolve the formed blue formazan crystals in DMSO. Measure the optical density value of formazan in each well at a wavelength of 490 nm using an enzyme-linked immunosorbent assay reader within 5 minutes. This value is converted to the percentage of the promotion of mouse spleen lymphocyte proliferation using a standard formula.

[0031] The data in Table 1 show that the percentage enhancement rate of the RPMI-1640 solution containing [Glu-His-Gly]3 at a concentration of 0.5 mg / mL on the proliferation of mouse spleen lymphocytes is significantly higher than that of the RPMI-1640 medium on the proliferation of mouse spleen lymphocytes (P < 0.01 compared with the RPMI-1640 medium). The data in Table 1 also show that the percentage enhancement rate of the RPMI-1640 solution containing [Glu-His-Gly]3 at a concentration of 0.5 mg / mL on the proliferation of mouse spleen lymphocytes is significantly greater than that of the RPMI-1640 solution containing thymopentin on the proliferation of mouse spleen lymphocytes (P < 0.01 compared with thymopentin). [Glu-His-Gly]3 has prominent technical features. The data in Table 1 further illustrate that the percentage enhancement rate of the RPMI-1640 solution containing [Glu-His-Gly]3 at a concentration of 0.5 mg / mL on the proliferation of mouse spleen lymphocytes is significantly greater than that of the RPMI-1640 solution containing Glu-His-Gly on the proliferation of mouse spleen lymphocytes (P < 0.01 compared with Glu-His-Gly). [Glu-His-Gly]3 has unexpected technical effects.

[0032] Table 1 Effects of [Glu-His-Gly]3 on the proliferation of spleen cells

[0033] Therapeutic agent Concentration Percentage of splenic lymphocyte proliferation, mean ± SD% RPMI-1640 medium - 3.9±3.1 <![CDATA[[Glu-His-Gly]3]]> 0.5 mg / mL <![CDATA[57.9±2.1 a > Thymopentin 10 μM 25.3±2.1 Glu-His-Gly 2 μM <![CDATA[28.2±1.9 b >

[0034] a) Compared with RPMI-1640 medium, thymopentin and Glu-His-Gly, P<0.01; b) Compared with RPMI-1640 medium, P<0.01, compared with thymopentin, P>0.05; n = 9

[0035] Example 4 Evaluation of the Effect of [Glu-His-Gly]3 on the Phagocytosis Index of Macrophages

[0036] The effect of [Glu-His-Gly]3 on the phagocytosis index of macrophages was evaluated in a mouse carbon clearance model. Male ICR mice (20±2 g) were randomly divided into groups of 10 mice after 1 day of resting feeding. The mice were orally administered normal saline (dose: 0.1 mL / 10 g / day, once a day for 3 consecutive days) or intraperitoneally injected with a normal saline solution of thymopentin (dose: 2 μmol / kg / day, once a day for 3 consecutive days) or orally administered a normal saline solution of [Glu-His-Gly]3 (dose: 0.2 μmol / kg / day, once a day for 3 consecutive days) or orally administered a normal saline solution of Glu-His-Gly (dose: 2 μmol / kg / day, once a day for 3 consecutive days). Twenty-four hours after the last treatment, the mice were injected with Indian ink diluted 5-fold with normal saline via the tail vein. At two time points of 5 minutes and 15 minutes after injection, 10 μL of blood was taken from the tail, added to 2 mL of an aqueous solution of 0.1% NaHCO3, mixed well, and the absorbance of the sample was measured at a wavelength of 660 nm. After the mice were anesthetized and sacrificed, the liver and spleen were removed and weighed. According to the phagocytosis index = body weight / (liver weight + spleen weight) × [(logOD1 - logOD2) / (t2 - t1)] 1 / 3 Calculate the phagocytosis index of mononuclear macrophages in each group of mice.

[0037] The data in Table 2 showed that the phagocytosis index of mononuclear macrophages in mice orally administered [Glu-His-Gly]3 was significantly increased (P<0.01 compared with normal saline). The data in Table 2 also showed that the phagocytosis index of mononuclear macrophages in mice orally administered [Glu-His-Gly]3 at a dose of 0.2 μmol / kg / day was significantly greater than that in mice intraperitoneally injected with thymopentin at a dose of 2 μmol / kg / day (P<0.01 compared with thymopentin). [Glu-His-Gly]3 has outstanding technical features. The data in Table 2 further showed that the phagocytosis index of mononuclear macrophages in mice orally administered [Glu-His-Gly]3 at a dose of 0.2 μmol / kg / day was significantly greater than that in mice orally administered Glu-His-Gly at a dose of 0.2 μmol / kg / day (P<0.01 compared with Glu-His-Gly). [Glu-His-Gly]3 has unexpected technical effects.

[0038] Effect of [Glu-His-Gly]3 on phagocytic index of mononuclear macrophages

[0039] Therapeutic agent Dose Phagocytosis index, mean ± SD Normal saline 0.1 mL / 10 g / day 4.37±0.55 <![CDATA[[Glu-His-Gly]3]]> 0.2 μmol / kg / day <![CDATA[9.59±0.76 a > Thymopentin 2 μmol / kg / day 5.38±0.59 Glu-His-Gly 2 μmol / kg / day <![CDATA[5.32±0.57 b >

[0040] a) P < 0.01 compared with normal saline, thymopentin and Glu-His-Gly; b) P < 0.01 compared with normal saline, P > 0.05 compared with thymopentin; n = 10.

Claims

1. A trimer of Glu-His-Gly, characterized in that, The trimer described is [Glu-His-Gly]3, and the [Glu-His-Gly]3 is composed of three Glu-His-Gly molecules through non-covalent bonds.

2. A method for preparing the trimer of Glu-His-Gly according to claim 1, characterized in that, The method described includes the following steps: 1) Prepare Boc-His(Boc)-Gly-OBzl; 2) Prepare His-Gly-OBzl; 3) Prepare Boc-Glu(OBzl)-His-Gly-OBzl; 4) Prepare Glu-His-Gly; 5) Prepare [Glu-His-Gly]3.

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