CN113402460A - Gardenia blue pigment with mental disease resistance and preparation method and application thereof - Google Patents
Gardenia blue pigment with mental disease resistance and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a gardenia blue pigment compound A, D, E, G, H with MAO-B inhibitory activity and provides a preparation method of the compound, and the invention obtains the best gardenia aglycone and 6 beta-hydroxyl genipin aglycone through a large amount of experimental screening and synthesizes the gardenia blue pigment A, D, E, G, H compound with high yield and purity and obvious biological activity with amino acid. Experimental results show that the blue pigment compound obtained by the invention can be used for preparing medicaments for resisting mental diseases such as senile dementia, depression and the like.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to a gardenia blue pigment compound with an MAO-B inhibition effect, and a preparation method and application thereof.
Background
Gardenia blue pigment (gardenia blue pigment) is a pigment product prepared by taking iridoid components in gardenia fruits as raw materials and reacting hydrolyzed aglycon with amino acid. The gardenia blue is added as a food additive in 1990 in China, is used for dyeing foods such as protein, sugar, starch and the like, particularly in the production of compound wine, is an indispensable component in compound pigments such as pomegranate red and the like, has the advantages of high safety, good tinting strength and the like, and has a wide application range in food processing. Based on literature research and market research, the application of gardenia blue pigment products still has two problems at present: firstly, the structure is not clear, although the forming mechanism of gardenia blue is partially researched at present, the specific structure of the gardenia blue is not determined, and the gardenia blue is considered to be a polymer with the molecular weight of about 500kDa in a view point, but the polymerization mode is unknown; secondly, the product has single use, and the gardenia blue pigment is mostly used for dyeing foods such as protein, sugar, starch and the like, so the economic benefit is limited.
Monoamine oxidase (MAO), A flavoprotein enzyme bound to the outer membrane of mitochondriA, catalyzes the oxidative deamination of various endogenous or exogenous monoamine species in the body, and has two subtypes MAO-A and MAO-B. MAO-A is mainly distributed in catecholaminergic neurons, and can be used for treating anxiety and depression by inhibiting MAO-A activity and increasing 5-HT concentration in nerve. MAO-B is distributed mainly in glial cells and histaminergic neurons, and inhibiting MAO-B activity increases Dopamine (DA) concentration in the basal nerve center, and can be used for treating Parkinson's disease and Alzheimer's disease.
Modern pharmacological research and clinical experience show that the gardenia iridoid component has various biological activities of protecting liver, resisting depression, resisting senile dementia, resisting inflammation and the like, and the action range of the gardenia iridoid component relates to various aspects of digestive system, nervous central system, cardiovascular system and cerebrovascular system and the like. However, related researches on the gardenia blue pigment serving as a derivative are few, and the nerve activity of the gardenia blue pigment compound is optimized by evaluating the inhibitory activity of monoamine oxidase B (MAO-B) on the gardenia blue pigment, so that the application range of the gardenia blue pigment can be expanded, and the method has important significance for improving the comprehensive utilization level of gardenia resources.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to prepare the gardenia blue pigment compound with MAO-B inhibitory activity by hydrolyzing gardenoside and 6 beta-hydroxyl geniposide enzyme and then reacting with amino acid. Another object of the present invention is to provide a method for preparing gardenia blue pigment compounds.
The technical scheme is as follows: in order to realize the purpose, the invention adopts the technical scheme that:
a gardenia blue pigment with mental disease resistance is prepared from the following compounds:
as a further preferable scheme of the invention, the preparation method of the gardenia blue pigment comprises the following steps:
(1) respectively weighing geniposide and 6 beta-hydroxy geniposide, respectively adding water to dissolve, adding cellulase to perform enzymatic hydrolysis reaction, and concentrating under reduced pressure after hydrolysis to obtain the extractum after enzymolysis of the geniposide and 6 beta-hydroxy geniposide;
(2) taking an extract obtained after enzymolysis of geniposide, dissolving the extract by using a phosphate buffer solution, dividing into 3 parts, respectively adding a proper amount of glycine, L-tryptophan and L-cysteine, heating, stirring and reacting, taking out a reaction solution, performing D101 macroporous resin column chromatography, eluting by using pure water, eluting by using a 50% ethanol aqueous solution, collecting blue components, and further purifying each component by using a high performance liquid preparative chromatography to respectively obtain a gardenia blue pigment A, a gardenia blue pigment D and a gardenia blue pigment E;
(3) taking the extract of 6 beta-hydroxyl geniposide after enzymolysis, dissolving the extract by using phosphate buffer solution, dividing the extract into two parts, respectively adding a proper amount of L-threonine and L-tryptophan, and heating and stirring the two parts for reaction; and taking out the reaction liquid, performing D101 macroporous resin column chromatography, eluting with pure water, eluting with ethanol water solution, collecting blue components, and further purifying the components by high performance liquid chromatography to obtain gardenia blue G and gardenia blue H.
As a further preferable scheme of the invention, the preparation method of the gardenia blue pigment comprises the following steps:
(1) respectively weighing geniposide and 6 beta-hydroxy geniposide, respectively adding water for dissolving, adding cellulase according to a weight ratio of 2:1, hydrolyzing at 40-50 ℃ for 24-48 h, ending the reaction, and concentrating under reduced pressure to obtain an extract obtained after enzymolysis of the geniposide and the 6 beta-hydroxy geniposide;
(2) dissolving geniposide-enzymolyzed extract with phosphate buffer solution with pH of 7.3, dividing into 3 parts, adding appropriate amount of glycine, L-tryptophan and L-cysteine, heating at 75 deg.C under stirring for 5 hr, taking out reaction solution, subjecting to D101 macroporous resin column chromatography, eluting with pure water for 3 column volumes, eluting with 50% ethanol water solution, collecting blue component, subjecting to high performance liquid chromatography, and collecting blue component18Semi-preparing a column; the mobile phase is methanol and water; purifying the components at a flow rate of 3ml/min to obtain gardenia blue pigment A, gardenia blue pigment D and gardenia blue pigment E respectively;
(3) dissolving the extract obtained after 6 beta-hydroxyl geniposide enzymolysis in phosphate buffer solution with pH of 7.3, dividing into two parts, respectively adding appropriate amount of L-threonine and L-tryptophan, heating and stirring at 75 deg.C for 5 hr; taking out reaction solution, performing D101 macroporous resin column chromatography, eluting with pure water for 3 column volumes, eluting with 50% ethanol water solution, collecting blue component, and performing high performance liquid chromatography, C18Semi-preparing a column; the mobile phase is methanol and water, the flow rate is 3ml/min, and the components are further purified to obtain gardenia blue pigment G and gardeniaBlue pigment H.
As a further preferable scheme of the invention, the gardenia blue pigment with the function of resisting mental diseases is applied to the preparation of the medicine for resisting mental diseases.
As a further preferred embodiment of the present invention, the use of gardenia blue pigment having an anti-psychotic disorder in the manufacture of a medicament for the treatment of anxiety, depression, Parkinson's disease and Alzheimer's disease.
Has the advantages that: the experimental result shows that the gardenia blue pigment A, the gardenia blue pigment D, the gardenia blue pigment E, the gardenia blue pigment G and the gardenia blue pigment H have obvious inhibitory activity to MAO-B and can play a role in treating mental diseases such as Parkinson's disease, Alzheimer's disease and the like.
The invention obtains the best hydrolysis conditions of the geniposide and the 6 beta-hydroxyl geniposide through a large number of experimental screens to obtain the genipin and the 6 beta-hydroxyl geniposide, and then the genipin and the specific amino acid are synthesized to obtain the genistein A, D, E, G, H compound with high yield and purity and remarkable biological activity.
Drawings
FIG. 1 shows the results of the MAO-B inhibitory activity of each compound.
Detailed Description
Example 1
1 Material
1.1 instruments
Sartorius BT125D electronic analytical balance (cyados, germany); clean bench (Thermo corporation, usa); HH-4 digital display constant temperature water bath (Changzhou national electric appliance Co., Ltd.); EPED ultra pure water system (south kyo easy podda easy science and technology development ltd); waters 2695 high performance liquid chromatograph (Waters corporation, usa); triple Tof high resolution Mass Spectrometry System (AB SCIEX Triple TOFTM 5600 system); preparative liquid chromatographs (Waters corporation, usa); brucker AV500 type nuclear magnetic resonance apparatus (Brucker, Germany).
1.2 reagents
Benzylamine hydrochloride available from merck, germany; benzaldehyde was purchased from national pharmaceutical group chemical reagents ltd; potassium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, glycine, L-threonine, L-histidine, L-tryptophan, and L-cysteine were all obtained from Shanghai Michelin Biochemical technology, Inc.; the D101 type macroporous adsorption resin is purchased from Beijing Solaibao science and technology Limited; both formic acid and methanol were chromatographically pure and purchased from merck, germany; ultrapure water was self-made by a Mili-Q ultrapure water preparation system, and other reagents were analytical grade and purchased from Nanjing chemical reagents, Inc.
2 methods and results
2.1 Gardenia blue Synthesis
2.1.1 preparation of substrates geniposide, 6 beta-hydroxygeniposide
Dried ripe fruits (50kg) of Gardenia jasminoides Ellis (Gardenia jasminoides Ellis) were extracted with 8 times of 75% aqueous ethanol solution under heating and refluxing for three times for two hours each time. Concentrating the extractive solution until no alcohol smell exists, dissolving in 15L water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol with equal volume for three times to obtain petroleum ether solubility, ethyl acetate solubility, n-butanol solubility and water solubility samples. Concentrating n-butanol soluble sample under reduced pressure to obtain soft extract 3.1kg, dissolving in water, performing D101 macroporous resin column chromatography, sequentially eluting with water, 20% ethanol water, 40% ethanol water, and 70% ethanol water, and concentrating under reduced pressure to obtain water part extract (1kg), 20% alcohol part extract (500g), 40% alcohol part extract (1kg), and 70% alcohol part extract (500 g). Collecting 20% ethanol part extract (50g), and separating by liquid chromatography with Hanbang DAC-HB 50 (C)18Semi-preparing a column; isocratic elution with 80% methanol water; flow rate 40ml/min) to obtain 10 fractions (Fr)20-1 to 10). Fr taking20-10, purification by preparative liquid chromatography (C)18Semi-preparing a column; the mobile phase is equal to 70 percent of methanol water; the flow rate is 3ml/min), and the compound (1) geniposide (5g) is obtained; fr taking20-8, obtaining two subfraction Fr by Sephadex LH-20 gel column chromatography (methanol elution)20-8-1 and Fr20-8-2。Fr20Purification of (E) -8-1 by preparative liquid chromatography (C)18Semi-preparing a column; the mobile phase is 85% methanol water isocratic; flow rate 3ml/min) to give compound (2), 6 β -hydroxygeniposide (500 mg).
2.1.2 structural identification of geniposide and 6 beta-hydroxygeniposide
Geniposide (1): white solid, readily soluble in methanol. HRESIMS M/z389.1486[ M + H ]]-(calcd for C17H25O10 +Calculated 389.1448).1H-NMR(500MHz,DMSO-d6):δppm7.47(1H,d, J＝1.2Hz,H-3),5.68(1H,brs,H-7),5.12(1H,d,J＝6.9Hz,H-1'),5.07(1H,d,J＝ 5.3Hz,H-1),4.13(1H,d,J＝14.0Hz,H-10a),3.97(1H,ddd,J＝15.1,5.6,2.7Hz, H-10b),3.67(1H,m,H-6'a),3.64(3H,s,H-OCH3) 3.41(1H, dt, J ═ 11.6,5.7Hz, H-6' b), 3.06-3.15 (4H, m, H-2' to 5'), 2.98(1H, m, H-5),2.69(1H, dd, J ═ 16.0,8.1Hz, H-6a),2.63(1H, t, J ═ 7.5Hz, H-9),2.05(1H, m, H-6 b). Through comparison with literature reports, the data basically accord with each other, and the compound is identified to be geniposide.
6 beta-hydroxy geniposide (2): white solid, readily soluble in methanol. HRESIMS M/z403.1248 [ M-H ]]-(calcd for C17H23O11 -，403.1240)。1H-NMR(500MHz,DMSO-d6):δppm 7.37 (1H,d,J＝1.1Hz,H-3),5.65(1H,p,J＝1.8Hz,H-7),5.28(1H,d,J＝4.7Hz,H-1), 4.46(1H,d,J＝7.9Hz,H-1'),4.35(1H,ddp,J＝5.0,3.3,1.6Hz,H-6),4.10(1H,m, H-10a),3.99(1H,ddd,J＝15.3,5.6,1.6Hz,H-10b),3.68(1H,m,H-6'a),3.65(1H,s, -OCH3),3.43(1H,dt,J＝11.6,5.7Hz H-6'b),3.04～3.17(3H,m,H-3'～5'),3.02(1H, m,H-9),2.95(1H,m,H-2'),2.87(1H,ddd,J＝7.4,3.1,1.2Hz,H-5)；13C-NMR(125 MHz,DMSO-d6):δppm 94.77(C-1),151.56(C-3),109.39(C-4),42.77(C-5),79.30 (C-6),128.88(C-7),145.74(C-8),45.58(C-9),58.89(C-10),167.39(C-11),51.18 (-OCH3) 98.49(C-1'),73.17(C-2'),77.26(C-3'),69.97(C-4'),77.26(C-5'),61.02 (C-6'). Through comparison with literature reports, the data basically accord with each other, and the compound is identified to be 6 beta-hydroxyl geniposide.
2.1.3 Synthesis of gardenia blue A, D, E, G, H (3-7)
Weighing the prepared or commercially available geniposide (600mg) and 6 beta-hydroxy geniposide (200 mg), respectively adding water for dissolving, respectively adding cellulase according to the weight ratio of 2:1 (sample: enzyme), hydrolyzing at 40 ℃ for 48h, ending the reaction, and concentrating under reduced pressure to respectively obtain the geniposide and 6 beta-hydroxy geniposide after enzymolysis.
Dissolving geniposide-reacted extract in phosphate buffer solution with pH of 7.3, dividing into 3 parts, adding appropriate amount of glycine, L-tryptophan and L-cysteine, respectively, heating at 75 deg.C under stirring for 5 hr, taking out reaction solution, performing D101 macroporous resin column chromatography, eluting with pure water for 3 column volumes, eluting with 50% ethanol water solution, collecting blue component, and performing high performance liquid preparative chromatography (C)18Semi-preparing column with mobile phase of methanol and water at flow rate of 3ml/min), and further purifying to obtain gardenia blue pigment A (10mg, 20% methanol water elution), gardenia blue pigment D (12mg, 15% methanol water elution) and gardenia blue pigment E (9mg, 35% methanol water elution).
Taking the extract obtained after the 6 beta-hydroxyl geniposide reaction, dissolving the extract in phosphate buffer with pH 7.3, dividing into two parts, respectively adding appropriate amount of L-threonine and L-tryptophan, heating and stirring at 75 ℃ for 5 h. Taking out the reaction solution, and carrying out D101 macroporous resin column chromatography. Eluting with pure water for 3 column volumes, eluting with 50% ethanol water solution, collecting blue component, and performing high performance liquid chromatography (C)18Semi-preparing a column; mobile phase methanol and water; flow rate 3ml/min) was further purified to give gardenia blue pigment G (10mg, 25% methanol elution) and gardenia blue pigment H (15mg, 30% methanol elution).
2.1.4 structural identification of Gardenia blue A, D, E, G, H
Gardenia blue pigment A: a blue powder. HRESIMS M/z 266.1027[ M-H ]]-(calcd for C13H16NO5 -Theoretical value 266.1028). Compound (I)1In the H-NMR spectrum, a methoxy proton signal of 3.65(3H, s, 11-OCH) can be observed3) Three pairs of methylene proton signals 3.24(1H, dd, J ═ 12.3,5.1 Hz, H-1a), 2.76(1H, m, H-1b), 2.73(1H, m, H-6a), 1.90(1H, dd, J ═ 16.2,9.3Hz, H-6b) and 4.07(1H, d, J ═ 17.9Hz, H-12a), 4.00(1H, d, J ═ 17.9Hz, H-12 b). The four alkene hydrogen proton signals 7.54(1H, s, H-3), 2.99(1H, q, J ═ 8.2Hz, H-5), 5.73(1H, s, H-7) and 2.62 (1H, q, J ═ 11.0,8.4 Hz). Combine HSQC, DEPT and13C-NMR spectrum can find a methoxy carbon signal 51.08 (11-OCH)3) Four methylene carbon signals 47.58(C-1), 38.65(C-6), 59.44 (C-10) and 56.49(C-12), two alkene carbon signals 148.36(C-3) and 127.30(C-7), and two carbonyl carbon signals 172.01(C-11) and 174.65 (12-COOH).1H--1The H COSY spectra show four sets of correlation signals, H-1/H-9, H-5/H-6, H-6/H-7 and H-5/H-9, combined with the main correlation peaks of the HMBC spectra: h-3 and C-5, C-12, H-10 and C-8, C-9, H-OCH3With C-11 and H-12 with C-1, C-COOH, the planar structure of the compound was deduced. The spatial configuration of the compound is determined by NOESY, and the map shows that H-5 is related to H-9 in the presence of NOE, which is consistent with the spatial configuration of the known compound genipin. In conclusion, compound 3 was identified as gardenia blue a.
Based on one-dimensional and two-dimensional nuclear magnetic data of each compound, the other compounds were identified as gardenia blue D, E, G, H by a similar method, and the structural data of the compounds were assigned as follows:
gardenia blue pigment D: blue powder, HRESIMS: M/z 393.1448[ M-H ]]-(calcd for C22H21N2O5 -Theoretical value 393.1450).1H-NMR(500MHz,D2O):δppm 7.74(1H,s,H-3), 7.55(1H,d,J＝7.8Hz,H-19),7.40(1H,d,J＝8.1Hz,H-22),7.17(1H,t,J＝7.6Hz, H-21),7.11(1H,t,J＝7.4Hz,H-20),6.05(1H,s,H-7),4.53(1H,d,J＝7.9Hz,H-1), 4.37(1H,d,J＝5.4Hz,H-12),4.16(1H,d,J＝13.5Hz,H-10a),3.95(1H,d,J＝13.5 Hz,H-10b),3.65(3H,s,11-OCH3),3.36(1H,d,J＝15.2Hz,H-13a),3.12(1H,q,J＝ 7.2Hz,H-5),3.01(1H,dd,J＝15.2,5.7Hz,H-13b),2.78(1H,dd,J＝16.2,8.5Hz, H-6a),2.56(1H,t,J＝7.4Hz,H-9),2.21(1H,dd,J＝16.4,7.7Hz,H-6b)；13C-NMR (125MHz,D2O):δppm 51.17(C-1),149.65(C-3),99.26(C-4),38.99(C-5),38.74 (C-6),131.78(C-7),143.74(C-8),47.41(C-9),59.93(C-10),171.83(C-11),51.09 (11-OCH3),64.76(C-12),178.41(12-COOH),24.19(C-13),106.84(C-14),132.79 (C-15),131.78(C-17),126.43(C-18),117.94(C-19),119.39(C-20),121.65(C-21), 119.65(C-22)。
Gardenia blue pigment E: blue (B)A colored powder. HRESIMS M/z 310.0732[ M-H ]]-(calcd for C14H16NO5S-Theoretical value 310.0749).1H-NMR(500MHz,D2O):δppm 7.70(1H,s,H-3), 5.86(1H,m,H-7),4.56(1H,dd,J＝6.9,4.1Hz,H-12),4.24(2H,m,H-10),4.17(1H, d,J＝9.7Hz,H-1),3.67(1H,d,J＝1.3Hz,11-OCH3),3.33(1H,dd,J＝11.2,6.8Hz, H-13a),3.18(1H,dd,J＝11.1,4.1Hz,H-13b),3.10(1H,q,J＝8.4Hz,H-5),2.75 (1H,m,H-6a),2.36(1H,m,H-9),1.94(1H,m,H-6b)；13C-NMR(125MHz,D2O): δppm 62.14(C-1),143.39(C-3),99.27(C-4),37.80(C-5),38.42(C-6),128.86(C-7), 144.06(C-8),46.97(C-9),59.94(C-10),171.6(C-11),51.20(11-OCH3),68.73 (C-12),177.47(12-COOH),32.65(C-13)。
Gardenia blue pigment G: a blue powder. HRESIMS M/z 326.1256[ M + H ]]-(calcd for C15H20NO7 +Theoretical value 326.1240).1H-NMR(500MHz,D2O):δppm 7.78(1H,s,H-3), 5.90(1H,d,J＝1.7Hz,H-7),4.59(1H,m,H-6),4.45(1H,d,J＝8.3Hz,H-1),4.40 (2H,m,H-10),4.36(1H,m,H-13),4.12(1H,d,J＝7.3Hz,H-12),3.80(3H,s, 11-OCH3),3.23(1H,t,J＝7.7Hz,H-5),2.54(1H,t,J＝8.1Hz,H-9),1.58(3H,d,J ＝6.1Hz,11-CH3)；13C-NMR(125MHz,D2O):δppm 89.99(C-1),141.86(C-3),97.67 (C-4),45.97(C-5),82.18(C-6),128.44(C-7),146.81(C-8),44.64(C-9),59.79 (C-10),171.72(C-11),51.43(11-OCH3),66.84(C-12),174.73(12-COOH),78.22 (C-13),18.29(13-CH3)。
Gardenia blue pigment H: a blue powder. HRESIMS M/z 411.1563[ M + H ]]-(calcd for C22H23N2O6Calculated 411.1556).1H-NMR(500MHz,DMSO-d6):δppm 7.47(1H,d,J ＝1.1Hz,H-3),7.39(1H,m,H-19),7.30(1H,m,H-22),7.05(1H,ddd,J＝8.2,7.0, 1.2Hz,H-21),6.97(1H,m,H-20),5.76(1H,t,J＝1.9Hz,H-7),4.76(1H,d,J＝6.8 Hz,H-12),4.69(1H,m,H-1),4.26(1H,d,J＝15.0Hz,H-10a),4.08(1H,d,J＝15.0 Hz,H-10b),3.58(1H,t,J＝4.8Hz,H-5),3.55(1H,s,11-OCH3),3.24(1H,d,J＝ 15.6Hz,H-13a),2.92(1H,ddd,J＝15.6,6.9,2.2Hz,H-13b),2.73(1H,m,H-9)；13C-NMR(125MHz,DMSO-d6):δppm 50.01(C-1),147.13(C-3),100.90(C-4),44.70 (C-5),79.24(C-6),129.20(C-7),148.17(C-8),44.15(C-9),58.59(C-10),168.06 (C-11),50.49(11-OCH3),61.26(C-12),172.27(12-COOH),23.43(C-13),105.38 (C-14),135.99(C-15),132.63(C-17),126.62(C-18),17.63(C-19),118.68(C-20), 120.99(C-21),111.28(C-22)。
EXAMPLE 2 MAO-B Activity inhibition assay
1. Laboratory animal
SD rats, 220g-250g, male, supplied by shanghai siepal-bikei laboratory animals ltd, license No.: SCXK (Shanghai) 2018 and 0006.
2. Preparation of MAO
3 SD male rats weighing about 220-250g are selected, subjected to neck removal and death, and then quickly taken out of livers in an ultra-clean bench sterilized by ultraviolet, washed 3 times with 0.9% physiological saline, wiped dry and stored at-80 ℃ for later use. Preparation of MAO method: taking 5g of liver tissue, placing the liver tissue in 175mL of 0.3mol/L sucrose solution for homogenizing, placing the liver homogenate in a high-speed low-temperature (0-4 ℃) freezing centrifuge, centrifuging at 1500rpm for 10min, discarding the precipitate, taking the supernatant, centrifuging at 10000rpm for 30min, discarding the supernatant, taking the precipitate to be suspended in 4mL of 0.3mol/L sucrose solution, adding the precipitate into 40mL of 1.2mol/L sucrose solution, centrifuging at 10500rpm for 40min, discarding the supernatant, and taking the precipitate as MAO. The mixture was washed 1 time with 100mmol/L potassium phosphate buffer (pH7.6), and 40mL of 100mmol/L potassium phosphate buffer was added to suspend the precipitate, which was divided into 1mL portions per EP tube and stored at-80 ℃ for further use.
3. Preparation of test article
MAO-B was diluted to 75mg/mL using 100mmol/L potassium phosphate buffer (pH 7.6). Preparing the gardenia blue pigment A, D, E, G, H, the geniposide and the 6 beta-hydroxyl geniposide to be detected into 1mmol/L, respectively sucking 100ul MAO-B and 1mmol/L medicament (the negative control is buffer solution with the same volume), placing the mixture into a 1.5 mL centrifuge tube, uniformly mixing the mixture by vortex, centrifuging the mixture at low speed to ensure that a reaction system is concentrated at the bottom of the centrifuge tube, incubating the reaction system at 37 ℃ for 10min, and simultaneously adding a substrate 2.5mg/mL benzylamine 50uL, vortex mixing, low-speed centrifugation, incubation at 37 ℃ for 60min, ending the reaction, immediately adding 150uL of methanol (the product benzaldehyde of the system is fully miscible), stopping the reaction, vortex mixing, filtering by a 0.22 mu m microporous membrane, performing liquid phase detection, wherein the chromatographic conditions are as follows: a chromatographic column: tnatural C18Columns (4.6 mm. times.250 mm,5 μm); mobile phase: methanol (A) -50mmol/L potassium phosphate buffer solution (B) at pH 3.2; isocratic elution: 0-20 min, 40% A; the column temperature is 35 ℃; flow rate: 1ml/min, the injection volume is 20uL, and the detection wavelength is 245 nm.
4. Comparison of MAO-B inhibitory Activity
The relative residual activity is represented by calculating the ratio of the benzaldehyde absorption peak area of an administration group to the benzaldehyde peak area of a negative control group to judge the strength of the MAO-B inhibitory activity of the drug, the relative residual activity is smaller, the enzyme inhibitory activity is larger, the specific experimental result is shown in figure 1, the experimental result of figure 1 shows that the gardenia blue pigment D, E has obvious MAO-B enzyme inhibitory activity (P is represented by P < 0.05) compared with gardenia, and the gardenia blue pigment G, H has obvious MAO-B enzyme inhibitory activity (# P is represented by P < 0.05 and # is represented by < 0.01) compared with 6 beta-hydroxygeniposide. The gardenia blue pigment D, E, G, H prepared by the invention has better MAO-B inhibition activity and is improved well.
Relative residual Activity ═ MAO-B inhibition drug peak area/no drug positive control peak area × 100%
5. Statistical analysis
The experimental data are analyzed by single factor variance with SPSS 22.0 statistical software, the comparison among groups adopts LSD multiple comparison, and the difference among groups has statistical significance when P is less than 0.05.
The above embodiments are merely illustrative of the technical concept and features of the present invention, and the present invention is not limited thereto, and equivalent changes and modifications made according to the spirit of the present invention should be covered thereby.
Claims (5)
1. The gardenia blue pigment with the function of resisting mental diseases is characterized in that the gardenia blue pigment is the following compounds:
2. the method for preparing gardenia blue pigment having an anti-psychotic disorder according to claim 1, which comprises the steps of:
(1) respectively weighing geniposide and 6 beta-hydroxy geniposide, respectively adding water to dissolve, adding cellulase to perform enzymatic hydrolysis reaction, and concentrating under reduced pressure after hydrolysis to obtain the extractum after enzymolysis of the geniposide and 6 beta-hydroxy geniposide;
(2) taking an extract obtained after enzymolysis of geniposide, dissolving the extract by using a phosphate buffer solution, dividing into 3 parts, respectively adding a proper amount of glycine, L-tryptophan and L-cysteine, heating, stirring, reacting, taking out a reaction solution, carrying out D101 macroporous resin column chromatography, eluting by using pure water, eluting by using a 50% ethanol aqueous solution, collecting blue components, and further purifying each component by using a high performance liquid preparative chromatography to respectively obtain a gardenia blue pigment A, a gardenia blue pigment D and a gardenia blue pigment E;
(3) taking the extract of 6 beta-hydroxyl geniposide after enzymolysis, dissolving the extract by using phosphate buffer solution, dividing the extract into two parts, respectively adding a proper amount of L-threonine and L-tryptophan, and heating and stirring the two parts for reaction; and taking out the reaction liquid, carrying out D101 macroporous resin column chromatography, eluting with pure water, eluting with a 50% ethanol water solution, collecting blue components, and further purifying the components by using a high performance liquid preparative chromatography to obtain the gardenia blue G and the gardenia blue H.
3. The method for preparing gardenia blue pigment having an anti-psychotic disorder according to claim 2, which comprises the steps of:
(1) respectively weighing geniposide and 6 beta-hydroxy geniposide, respectively adding water for dissolving, adding cellulase according to a weight ratio of 2:1, hydrolyzing at 40-50 ℃ for 24-48 h, ending the reaction, and concentrating under reduced pressure to obtain an extract obtained after enzymolysis of the geniposide and the 6 beta-hydroxy geniposide;
(2) dissolving geniposide-enzymolyzed extract with phosphate buffer solution with pH of 7.3, dividing into 3 parts, adding appropriate amount of glycine, L-tryptophan and L-cysteine, heating at 75 deg.C under stirring for 5 hr, taking out reaction solution, performing D101 macroporous resin column chromatography, eluting with pure water for 3 column volumes, eluting with 50% ethanol water solution, collecting blue component, performing high performance liquid chromatography, and collecting blue component18Semi-preparing a column; the mobile phase is methanol and water; purifying the components at a flow rate of 3ml/min to obtain gardenia blue pigment A, gardenia blue pigment D and gardenia blue pigment E respectively;
(3) dissolving the extract obtained after 6 beta-hydroxyl geniposide enzymolysis in phosphate buffer solution with pH of 7.3, dividing into two parts, respectively adding appropriate amount of L-threonine and L-tryptophan, heating and stirring at 75 deg.C for 5 hr; taking out the reaction solution, performing D101 macroporous resin column chromatography, eluting with pure water for 3 column volumes, eluting with 50% ethanol water solution, collecting blue component, and performing high performance liquid chromatography, and preparing column with C18 semi-preparative column; the mobile phase is methanol and water, the flow rate is 3ml/min, and the components are further purified to obtain gardenia blue pigment G and gardenia blue pigment H.
4. The use of gardenia blue pigment having an anti-psychotic disorder according to claim 1 for the preparation of an anti-psychotic disorder medicament.
5. The use of the gardenia blue pigment having an anti-psychotic disorder according to claim 1 for the preparation of a medicament for the treatment of anxiety, depression, parkinson's disease and alzheimer's disease.
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| CN110903677A (en) * | 2019-12-13 | 2020-03-24 | 九江学院 | Method for simultaneously preparing gardenia yellow pigment and blue pigment |
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| US20170260394A1 (en) * | 2014-09-17 | 2017-09-14 | Dsm Ip Assets B.V. | A new process for producing gardenia blue pigment |
| WO2018029338A1 (en) * | 2016-08-12 | 2018-02-15 | Dsm Ip Assets B.V. | A process for producing gardenia blue pigment form geniposide |
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