CN114478564B - Monascin C with glycosidase activity inhibiting and immunoregulatory activity and preparation method thereof - Google Patents
Monascin C with glycosidase activity inhibiting and immunoregulatory activity and preparation method thereof Download PDFInfo
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Abstract
The invention relates to monascin C with glycosidase inhibition activity and immunoregulation activity and a preparation method thereof, belonging to the technical field of extraction and preparation of biological products. Monascin C is red powder, and molecular formula is C 19 H 22 O 5 Half Inhibition Concentration (IC) of alpha-glucosidase 50 ) The method comprises the following steps: 200 μg/mL, and has a significant downregulating effect on the IL-1 beta, TNF-alpha, and TGF-beta transcriptional levels of mouse macrophage RAW264.7 cultured in vitro: IL-1 beta (0.01.+ -. 0.01) TNF-Alpha (0.02.+ -. 0.01), and TGF-. Beta.s (0.03.+ -. 0.01) were significantly varied (P < 0.05) relative to the DMSO control. The monascus C is obtained by culturing monascus and then extracting and separating. The monascin C can be used for preparing glycosidase inhibitors and anti-inflammatory immunosuppressants, and has the action potential in the aspects of treating diabetes and immune correlation. The monascus is edible fungi, is safe and reliable, adopts microbial fermentation production, is environment-friendly, is not influenced by natural environment and resources, and is easy to realize industrialized, automatic and continuous production.
Description
Technical Field
The invention belongs to the technical field of extraction and preparation of biological products, and particularly relates to extraction, purification, structure identification and activity determination of monascin C (Monascuskalin C) with glycosidase inhibition activity.
Background
Monascus sp. is a small class of filamentous saprophytic fungi belonging to the phylum Eumycota (Eumycophyta), ascomycotina (Ascomycotina), ascomycetes (Plaectomyces), eurotiales (Eurotiales), red Qu Junke (Monascaceae), monascus (Monascus), including species of Monascus purpureus (Monascus purpureus), monascus anka (Monascus anka) and Monascus ruber (Monascus ruber). Monascus can produce various enzyme substances such as saccharifying enzyme, esterifying enzyme, protease and the like, and under the synergistic effect of the enzymes, the monascus can produce flavor substances such as monosaccharide, amino acid, nucleotide and the like and aroma substances such as volatile acids, alcohols, esters and the like, and the monascus contributes significantly to the flavor of fermented foods.
In recent years, due to the improvement of living standard and the reduction of exercise quantity, the number of diabetics is increased year by year, the number of diabetics is 4.4 hundred million people currently, the number of diabetics is increased in proportion of 5% each year, the types of blood glucose reducing medicines currently are few, the choice is small, and development of new high-efficiency blood glucose reducing medicines is needed urgently. The development of related medicaments has great social and economic benefits.
The current society rapidly develops, the life and work rhythm is fast, the pressure is great, and meanwhile, the sub-health crowd suffering from immune disorder is increased year by year due to the lack of exercise and bad habits such as mobile phone watching at night, so that the development of related medicines for regulating the immunity is necessary. As a food microorganism, monascus has the advantages of safety and reliability, so that the related immune preparation and blood glucose reducing preparation have more application potential and great economic value.
Disclosure of Invention
The invention aims to provide monascin C with glycosidase inhibition activity and immunoregulatory activity, and also provides a preparation method of monascin C.
Monascus C having glycosidase inhibitory and immunomodulatory activities is isolated from Monascus sp culture and has the structural formula:
the monascin C is red powder, and the molecular formula is C 19 H 22 O 5 Has effects in inhibiting glycosidase activity, and has half Inhibition Concentration (IC) on alpha-glycosidase (alpha-glycosidase) 50 ) 200 μg/mL, and has a significant down-regulating effect on the IL-1β, TNF- α, and TGF- β transcript levels of in vitro cultured mouse macrophage RAW 264.7: IL-1β (0.01.+ -. 0.01), TNF-. Alpha. (0.02.+ -. 0.01), and TGF-. Beta.0.03.+ -. 0.01) were significantly altered (P < 0.05) relative to the DMSO control.
The Monascus purpureus is Monascus purpureus (Monascus purpureus), monascus anka (Monascus anka) or Monascus ruber (Monascus ruber).
A process for preparing monascin C with glycosidase inhibitory and immunoregulatory activities comprises the following steps:
(1) Preparation of solid cultures
Culturing Monascus sp strain, performing primary strain culture, secondary seed culture and tertiary liquid expansion culture to obtain liquid culture;
(1.1) first-order Strain culture
Inoculating monascus suspension on potato agar (PDA) slant culture medium, inoculating 10-30 mu L of the suspension on each slant, culturing for 7-9 days at the temperature of 32-35 ℃ and 220-250 r/min to obtain first-class strain;
(1.2) two-stage seed culture
Preparing the first-class strain into a concentration of 10 by using sterile water 5 ~10 7 Inoculating 200-250 mu L of spore suspension of each spore/mL into 250mL of PDB culture medium, and culturing for 3-5 days at the temperature of 32-35 ℃ and 220-250 r/min by shaking the bottle to obtain liquid secondary strain;
(1.3) three-stage liquid expanded culture
Inoculating 1-3 mL of liquid secondary strain into 250mL of PDB culture medium, shake-flask culturing for 7-9 days at the temperature of 32-35 ℃ and 220-250 r/min, and collecting the culture to obtain a liquid culture;
(2) Extraction and refinement of active principles in liquid cultures
The specific operation is as follows:
the extractant is methanol, ethyl acetate or methanol and ethyl acetate with the volume ratio of 0-10: 10 to 0;
(2.1) extraction of active ingredient in liquid culture
Concentrating and drying the liquid culture under reduced pressure, performing ultrasonic extraction with extractant, filtering with filter membrane or centrifuging, and vacuum removing solvent under reduced pressure to obtain effective component extract;
(2.2) preliminary purification of the extract
Performing primary purification on the active ingredient extract by adopting preparative reverse-phase high performance liquid chromatography to obtain a red primary purified product;
(2.3) refining of the preliminary purified product
Refining the primary purified product by semi-preparative reverse phase high performance liquid chromatography, and collecting the compound corresponding to the highest peak under the condition of eluting with acetonitrile solution to obtain monascin C eluent.
Further technical requirements are as follows:
in the step (1.1), the PDA culture medium is potato dextrose culture medium, and the formula is as follows: 200g peeled potatoes, 20g glucose and 15g agar, and water was added to 1000mL.
In the steps (1.2) and (1.3), the formula of the PDB culture medium is as follows: 200g peeled potatoes, 20g glucose, and water to 1000mL.
In the step (2.1), the liquid culture is concentrated to 3/10 to 1/10 of the original volume under the conditions of vacuum degree of-0.1 to-0.08 MP and temperature of 10 to 60 ℃, dried and crushed under the conditions of temperature of-50 to 130 ℃ and the weight-volume ratio of 1g: adding extractant into 0.5-5 mL, extracting with 40KHz ultrasonic for 20-200 min, centrifuging at 4000-15000 rpm or filtering with 0.1-2.5 μm membrane to obtain filtrate or centrifuging supernatant; the filtrate or the centrifugal supernatant is decompressed and distilled to remove the extractant under the condition of vacuum degree of-0.1 to-0.08 MP and temperature of 10 to 60 ℃ to obtain the active ingredient extract.
In the step (2.2), refining by adopting preparative reverse-phase high performance liquid chromatography; the chromatographic separation preparation conditions are as follows: mobile phase A is ultrapure water, mobile phase B is acetonitrile; elution conditions: gradient elution is carried out on 50% -70% of mobile phase B within 0 to 30 min; the sample injection volume is 30-300 mu L, the column temperature is 20-40 ℃, and the flow rate is 5-50 mL/min; the detection wavelength is 358nm; chromatographic column: preparing a reversed phase C-18 column; collecting the eluent corresponding to the third highest peak, and evaporating the extractant under reduced pressure at the vacuum degree of-0.1 to-0.08 MP and the temperature of 10-60 ℃; drying at 130 ℃ or lower to obtain the red primary purified product.
In the step (2.3), chromatographic separation preparation conditions are as follows: mobile phase A is ultrapure water, mobile phase B is acetonitrile; elution conditions: eluting with 50-70% of mobile phase A for 0-30 min; the sample injection volume is 20-200 mu L, the column temperature is 20-40 ℃ and the flow rate is 2-20 mL/min; the detection wavelength is 358nm; chromatographic column: semi-preparative reverse phase C-18 column; collecting the compound corresponding to the highest peak value as monascin C eluent; vacuum evaporating to remove extractant at 10-60 deg.C and vacuum degree of-0.1 to-0.08 MP; drying at 130 deg.C or below to obtain red powder monascin C. The analytical study of the present invention is described as follows:
1. the invention adopts the preparation type reversed phase high performance liquid chromatography and the semi-preparation type reversed phase high performance liquid chromatography to separate and purify the extract, and 1 new compound is obtained from Monascus sp and named monascin C.
2. Monascin C has the activity of inhibiting glycosidase, has a half inhibition concentration (IC 50) of 200 μg/mL for alpha-glycosidase (alpha-glycosidase), has anti-inflammatory and immunoregulatory effects, and has a significant down-regulating effect on the transcriptional levels of IL-1 beta, TNF-alpha, and TGF-beta of rat 264.7 in vitro cultured mouse macrophages: IL-1β (0.01.+ -. 0.01), TNF-. Alpha. (0.02.+ -. 0.01), and TGF-. Beta.0.03.+ -. 0.01) were significantly altered (P < 0.05) relative to the DMSO control.
3. Chemical structure identification data of monascin C
High-resolution liquid chromatography-mass spectrometry analysis shows that the ionic mass-charge ratio of monascin C is 331.1565, [ M+H ]] + Corresponding molecular formula is C 19 H 22 O 5 Specific nuclear magnetic data are shown in Table 1 below.
TABLE 1 Nuclear magnetic resonance Hydrocarbon belonging Table for monascin C
The beneficial technical effects of the invention are shown in the following aspects:
1. in order to find novel natural and efficient substances with glycosidase activity inhibition and anti-inflammatory immune activity, the inventor firstly cultures a large number of Monascus strains, then extracts and activity measurement are carried out on the effective components of the culture, and a Monascus (Monascus sp.) culture extract is found to have the glycosidase activity inhibition and RAW264.7 cell inflammation inhibition and immune activity inhibition. The monascin C (Monascuskaolin C) is prepared by reverse preparation and semi-preparation chromatography separation and purification on the basis of mass culture and extraction, and the compound is red powder, and the structural formula of monascin C is obtained through one-dimensional and two-dimensional nuclear magnetic resonance and accurate mass spectrometry identification. The monascin C has stronger alpha-glycosidase inhibition activity and RAW264.7 cell immunosuppression activity, and has development potential in aspects of diabetes medicines and anti-inflammatory immunity correlation.
2. The invention discovers that Monascus sp has the function of metabolizing the active substances for the first time. On the basis of the invention, the related genes can be expected to be cloned further, and high-yield strains can be constructed.
3. As a food microorganism, monascus has the advantages of safety and reliability, so that the related immune preparation and the blood glucose reducing preparation have great application potential and economic value.
4. The invention adopts microbial fermentation production, is not influenced by natural environment and resources, is easy to realize industrialized, automatic and continuous production, and does not destroy the natural environment and the natural resources.
5. The monascus element product produced by the process method has the advantages of low cost, simple and convenient process, stable process, easy regulation and control and high success rate.
Detailed Description
The invention is further described below with reference to examples.
Example 1
A process for preparing monascin C with glycosidase inhibitory and immunoregulatory activities comprises the following steps:
(1) Preparation of solid cultures
Culturing strain of Monascus purpureus (Monascus purpureus) by primary strain culture, secondary seed culture and tertiary liquid expansion culture to obtain liquid culture;
(1.1) first-order Strain culture
Will 10 7 Inoculating each spore/mL of Monascus purpureus (Monascus purpureus) spore suspension on potato agar (PDA) slant culture medium, inoculating 10 μl of Monascus purpureus spore suspension on each slant, and culturing at 32deg.C and 220r/min for 7 days to obtain first-class strain.
The PDA culture medium is potato glucose culture medium, and the formula is as follows: 200g peeled potatoes, 20g glucose and 15g agar, and water was added to 1000mL.
(1.2) two-stage seed culture
Preparing the first-class strain into a concentration of 10 by using sterile water 5 200 mu L of spore/mL of spore suspension per bottle is inoculated into 250mL of PDB culture medium prepared in advance,culturing at 32deg.C and 220r/min in shake flask for 3 days to obtain liquid secondary strain.
The formula of the PDB culture medium is as follows: 200g peeled potatoes, 20g glucose were added to 1000mL water.
(1.3) three-stage liquid expanded culture
1mL of liquid secondary strain is inoculated into 250mL of PDB culture medium which is sterilized in advance, and the culture is collected after shaking and culturing for 7 days at the temperature of 32 ℃ and 220r/min, so as to obtain the liquid culture.
(2) Extraction and refinement of active principles in liquid cultures
The specific operation is as follows:
(2.1) extraction of active ingredient in liquid culture
Concentrating under reduced pressure, lyophilizing, and extracting with ethyl acetate to obtain effective component extract.
The specific operation is as follows: evaporating the liquid culture under reduced pressure at-10deg.C and vacuum degree of-0.1 MP to 3/10 of original volume, drying at-50deg.C, and pulverizing; according to the weight-volume ratio of 1g: ethyl acetate was added at 0.5mL and extracted by 40KHz ultrasound for 20 minutes. Centrifuging at 4000 rpm, vacuum evaporating supernatant under vacuum degree-0.1 MP at 10deg.C to remove extractant to obtain effective component extract.
(2.2) preliminary purification of the active ingredient extract
And (3) performing preliminary purification on the active ingredient extract by adopting preparative reverse-phase high performance liquid chromatography to obtain a red preliminary purified product.
The specific operation is as follows: refining by preparative reverse phase high performance liquid chromatography; the chromatographic separation preparation conditions are as follows: mobile phase A is ultrapure water, mobile phase B is acetonitrile; elution conditions: gradient elution is carried out on 50% -70% of mobile phase B within 0 to 30 min; sample injection volume 30 μL, column temperature 20 ℃ and flow rate 5mL/min; the detection wavelength is 358nm; chromatographic column: preparing a reversed phase C-18 column; collecting the eluent corresponding to the third highest peak, evaporating the extractant under reduced pressure at-0.1 MP and 10 deg.C, and drying at-40 deg.C to obtain red primary purified product.
(2.3) refining of the Red preliminary purification product
Refining the red primary purified product by semi-preparative reverse phase high performance liquid chromatography, and collecting the compound corresponding to the highest peak under the condition of eluting with acetonitrile solution to obtain monascin C eluent.
The specific operation is as follows: the chromatographic mobile phase A is ultrapure water, and the mobile phase B is acetonitrile; elution conditions: eluting with 50% -70% of mobile phase B for 0-30 min; sample injection volume 20. Mu.L, column temperature 20 ℃, flow rate 2mL/min; the detection wavelength is 358nm; chromatographic column: semi-preparative reverse phase C-18 column; collecting the compound corresponding to the highest peak value as monascin C eluent; evaporating under reduced pressure at 10 deg.C and vacuum degree of-0.1 MP to remove extractant; drying at-40deg.C to obtain red powder monascin C.
The structural formula of the spectrum analyzed monascin C is as follows:
the activity measurement results show that: half-maximal Inhibitory Concentration (IC) of monascin C on alpha-glucosidase 50 ) The method comprises the following steps: 200 μg/mL, and has a significant downregulating effect on the IL-1 beta, TNF-alpha, and TGF-beta transcriptional levels of mouse macrophage RAW264.7 cultured in vitro: IL-1β (0.01.+ -. 0.01), TNF-. Alpha. (0.02.+ -. 0.01), and TGF-. Beta.0.03.+ -. 0.01) were significantly altered (P < 0.05) relative to the DMSO control.
Example 2
The operation steps of monascin C with glycosidase inhibition activity and immunoregulatory activity are as follows:
(1) Preparation of solid cultures
Liquid strain of Monascus anka (Monascus anka) is subjected to primary strain culture, secondary seed culture and tertiary liquid expansion culture to obtain a liquid culture;
(1.1) first-order Strain culture
Will 10 6 Inoculating Aspergillus angustifolius (Monascus anka) spore suspension of each spore/mL on potato agar (PDA) slant culture medium, inoculating 30 μl of Aspergillus angustifolius suspension on each slant, and culturing at 35deg.C and rotation speed of 250r/min for 9 daysTo the first level strain.
The PDA culture medium is potato glucose culture medium, and the formula is as follows: 200g peeled potatoes, 20g glucose and 15g agar, and water was added to 1000mL.
(1.2) two-stage seed culture
Preparing the first-class strain into a concentration of 10 by using sterile water 7 The spore suspension of each spore/mL is inoculated into 250mL of PDB culture medium prepared in advance according to 250 mu L of each spore/mL, and the liquid secondary strain is obtained by shaking and culturing for 5 days under the conditions of the temperature of 35 ℃ and the rotating speed of 250 r/min.
The formula of the PDB culture medium is as follows: 200g peeled potatoes, 20g glucose were added to 1000mL water.
(1.3) three-stage liquid expanded culture
Inoculating 3mL of liquid secondary strain into 250mL of PDB culture medium sterilized in advance, shake-flask culturing at 35 ℃ and rotating speed of 250r/min for 9 days, and collecting culture to obtain liquid culture.
(2) Extraction and refinement of active principles in liquid cultures
The specific operation is as follows:
(2.1) extraction of active ingredient in liquid culture
Concentrating the liquid culture under reduced pressure, lyophilizing, and extracting with methanol to obtain effective component extract.
The method comprises the following specific steps: evaporating the liquid culture under reduced pressure at-0.08 MP and 60 deg.C to 1/10 of original volume, drying at 130 deg.C, and pulverizing; according to the weight-volume ratio of 1g: methanol was added to 5mL and the mixture was sonicated at 40KHz for 200 min. Centrifuging at 15000 rpm, and vacuum evaporating the supernatant at-0.08 MP and 60deg.C to obtain effective component extract.
(2.2) preliminary purification of the active ingredient extract
And (3) performing preliminary purification on the active ingredient extract by adopting preparative reverse-phase high performance liquid chromatography to obtain a red preliminary purified product.
The method comprises the following specific steps: refining by preparative reverse phase high performance liquid chromatography; the chromatographic separation preparation conditions are as follows: mobile phase A is ultrapure water, mobile phase B is acetonitrile; elution conditions: gradient elution is carried out on 50% -70% of mobile phase B within 0 to 30 min; sample injection volume 300 μL, column temperature 40 ℃ and flow rate 50mL/min; the detection wavelength is 358nm; chromatographic column: preparing a reversed phase C-18 column; collecting the eluent corresponding to the third highest peak, evaporating the extractant under reduced pressure at the temperature of 60 ℃ and the vacuum degree of-0.08 MP, and drying at the temperature of 130 ℃ to obtain the red primary purified product.
(2.3) refining of the preliminary purified product
Refining the red primary purified product by semi-preparative reverse phase high performance liquid chromatography, and collecting the compound corresponding to the highest peak under the condition of eluting with acetonitrile solution to obtain monascin C eluent.
The method comprises the following specific steps: the chromatographic mobile phase A is ultrapure water, and the mobile phase B is acetonitrile; elution conditions: eluting with 50-70% of mobile phase A for 0-30 min; sample injection volume 200 μL, column temperature 40 ℃ and flow rate 20mL/min; the detection wavelength is 358nm; chromatographic column: semi-preparative reverse phase C-18 column; collecting the compound corresponding to the highest peak value as monascin C eluent; evaporating under reduced pressure at 60 deg.C and vacuum degree of-0.08 MP to remove extractant; drying at 149 deg.C to obtain red powder of monascin C.
The structural formula of the spectrum analyzed monascin C is as follows:
the activity measurement results show that: half-maximal Inhibitory Concentration (IC) of monascin C on alpha-glucosidase 50 ) The method comprises the following steps: 200 μg/mL, and has a significant downregulating effect on the IL-1 beta, TNF-alpha, and TGF-beta transcriptional levels of mouse macrophage RAW264.7 cultured in vitro: IL-1β (0.01.+ -. 0.01), TNF-. Alpha. (0.02.+ -. 0.01), and TGF-. Beta.0.03.+ -. 0.01) were significantly altered (P < 0.05) relative to the DMSO control.
Example 3
A process for preparing monascin C with glycosidase inhibitory and immunoregulatory activities comprises the following steps:
(1) Preparation of solid cultures
Culturing Monascus ruber strain by primary strain culture, secondary seed culture and tertiary liquid expansion culture to obtain liquid culture;
(1.1) first-order Strain culture
Will 10 6 Inoculating each spore/mL monascus suspension on potato agar (PDA) slant culture medium, inoculating 20 μl monascus suspension on each slant, and culturing at 33.5deg.C and 235r/min for 8 days to obtain first-class strain.
The PDA culture medium is potato glucose culture medium, and the formula is as follows: 200g peeled potatoes, 20g glucose and 15g agar, and water was added to 1000mL.
(1.2) two-stage seed culture
Preparing the first-class strain into a concentration of 10 by using sterile water 6 The spore suspension of each spore/mL is inoculated into 250mL of PDB culture medium prepared in advance according to 225 mu L of each bottle, and the liquid secondary strain is obtained by shaking and culturing for 4 days under the conditions of the temperature of 33.5 ℃ and the rotating speed of 240 r/min.
The formula of the PDB culture medium is as follows: 200g peeled potatoes, 20g glucose, and water to 1000mL.
(1.3) three-stage liquid expanded culture
Inoculating 2mL of liquid secondary strain into 250mL of PDB culture medium sterilized in advance, shake-flask culturing at 33.5 ℃ and 240r/min for 8 days, and collecting culture to obtain liquid culture.
(2) Extraction and refinement of active principles in liquid cultures
The specific operation is as follows:
(2.1) extraction of active ingredient in liquid culture
The liquid culture was concentrated under reduced pressure and freeze-dried with methanol: ethyl acetate = 1:1 (v/v) extraction to obtain an active ingredient extract.
The specific operation is as follows: evaporating the liquid culture under reduced pressure at 40 deg.C and vacuum degree-0.09 MP to 2/10 of original volume, drying at 70 deg.C, and pulverizing; methanol is added according to the weight-volume ratio of 1g to 2.5 mL: ethyl acetate=1:1 (v/v) mixture, 40KHz ultrasound extraction for 100 minutes. Centrifuging at 10000 rpm, vacuum evaporating supernatant under reduced pressure at-0.09 MP and 40 deg.C to obtain effective component extract. (2.2) preliminary purification of the active ingredient extract
And (3) performing preliminary purification on the active ingredient extract by adopting preparative reverse-phase high performance liquid chromatography to obtain a red preliminary purified product.
The specific operation is as follows: refining by preparative reverse phase high performance liquid chromatography; the chromatographic separation preparation conditions are as follows: mobile phase A is ultrapure water, mobile phase B is acetonitrile; elution conditions: gradient elution is carried out on 50% -70% of mobile phase B within 0 to 30 min; sample injection volume 180 μL, column temperature 30 ℃ and flow rate 25mL/min; the detection wavelength is 358nm; chromatographic column: preparing a reversed phase C-18 column; collecting the eluent corresponding to the third highest peak, evaporating the extractant under reduced pressure at the temperature of 40 ℃ and the vacuum degree of-0.09 MP, and drying at the temperature of 70 ℃ to obtain the red primary purified product.
(2.3) refining of the preliminary purified product
Refining the red primary purified product by semi-preparative reverse phase high performance liquid chromatography, and collecting the compound corresponding to the highest peak under the condition of eluting with acetonitrile solution to obtain monascin C eluent, thereby obtaining red powdery monascin C.
The specific operation is as follows: the chromatographic mobile phase A is ultrapure water, and the mobile phase B is acetonitrile; elution conditions: eluting with 50% -70% of mobile phase B for 0-30 min; sample injection volume 100. Mu.L, column temperature 30 ℃ and flow rate 10mL/min; the detection wavelength is 358nm; chromatographic column: semi-preparative reverse phase C-18 column; collecting the compound corresponding to the highest peak value as monascin C eluent; evaporating under reduced pressure at 40 deg.C and vacuum degree of-0.09 MP to remove extractant; drying at 70deg.C to obtain red powder monascin C.
The structural formula of the spectrum analyzed monascin C is as follows:
the activity measurement results show that: half-inhibition of alpha-glucosidase by monascin CConcentration (IC) 50 ) The method comprises the following steps: 200 μg/mL, and has a significant downregulating effect on the IL-1 beta, TNF-alpha, and TGF-beta transcriptional levels of mouse macrophage RAW264.7 cultured in vitro: IL-1β (0.01.+ -. 0.01), TNF-. Alpha. (0.02.+ -. 0.01), and TGF-. Beta.0.03.+ -. 0.01) were significantly altered (P < 0.05) relative to the DMSO control.
Example 4
A process for preparing monascin C with glycosidase inhibitory and immunoregulatory activities comprises the following steps:
(1) Preparation of solid cultures
Culturing Monascus ruber strain by primary strain culture, secondary seed culture and tertiary liquid expansion culture to obtain liquid culture;
(1.1) first-order Strain culture
Will 10 6 Inoculating each spore/mL monascus suspension on potato agar (PDA) slant culture medium, inoculating 20 μl monascus suspension on each slant, and culturing at 33.5deg.C and 240r/min for 8 days to obtain first-class strain.
The PDA culture medium is potato glucose culture medium, and the formula is as follows: 200g peeled potatoes, 20g glucose and 15g agar, and water was added to 1000mL.
(1.2) two-stage seed culture
Preparing the first-class strain into a concentration of 10 by using sterile water 6 Inoculating 220 mu L of spore/mL spore suspension into 250mL of PDB culture medium prepared in advance, shake-culturing at 33 ℃ at 240r/min for 4 days, and collecting spores to obtain secondary strain.
The formula of the PDB culture medium is as follows: 200g peeled potatoes, 20g glucose, and water to 1000mL.
(1.3) three-stage liquid expanded culture
Inoculating 2mL of liquid secondary strain into 250mL of PDB culture medium sterilized in advance, shake-flask culturing at 33.5 ℃ and 235r/min for 8 days, and collecting culture to obtain liquid culture.
(2) Extraction and refinement of active principles in liquid cultures
The specific operation is as follows:
(2.1) extraction of active ingredient in liquid culture
Concentrating the liquid culture under reduced pressure, homogenizing, and extracting with methanol to obtain effective component extract.
The specific operation is as follows: evaporating the liquid culture under reduced pressure at 10 deg.C and vacuum degree-0.1 MP to 2/10 of original volume, and directly extracting with extractant for 20 min. Filtering with 0.1 μm membrane, and vacuum evaporating the filtrate under vacuum degree-0.1 MP at 10deg.C to obtain effective component extract.
(2.2) preliminary purification of the active ingredient extract
And (3) performing preliminary purification on the active ingredient extract by adopting preparative reverse-phase high performance liquid chromatography to obtain a red preliminary purified product.
The specific operation is as follows: refining by preparative reverse phase high performance liquid chromatography; the chromatographic separation preparation conditions are as follows: mobile phase A is ultrapure water, mobile phase B is acetonitrile; elution conditions: gradient elution is carried out on 50% -70% of mobile phase B within 0 to 30 min; sample injection volume 200 μL, column temperature 30 ℃ and flow rate 20mL/min; the detection wavelength is 358nm; chromatographic column: preparing a reversed phase C-18 column; collecting the eluent corresponding to the third highest peak, evaporating the extractant under reduced pressure at-0.1 MP and 30 deg.C, and drying at-40 deg.C to obtain red primary purified product.
(2.3) refining of the preliminary purified product
Refining the red primary purified product by semi-preparative reverse phase high performance liquid chromatography, and collecting the compound corresponding to the highest peak under the condition of eluting with acetonitrile solution to obtain monascin C eluent.
The specific operation is as follows: the chromatographic separation preparation conditions are as follows: mobile phase A is ultrapure water, mobile phase B is acetonitrile; elution conditions: eluting with 50% -70% of mobile phase B for 0-30 min; sample injection volume 30 μL, column temperature 30 ℃ and flow rate 5mL/min; the detection wavelength is 358nm; chromatographic column: semi-preparative reverse phase C-18 column; collecting the compound corresponding to the highest peak value as monascin C eluent; evaporating under reduced pressure at 30 deg.C and vacuum degree of-0.1 MP to remove extractant; drying at-40deg.C to obtain red powder monascin C.
The structural formula of the spectrum analyzed monascin C is as follows:
the activity measurement results show that: half-maximal Inhibitory Concentration (IC) of monascin C on alpha-glucosidase 50 ) The method comprises the following steps: 200 μg/mL, and has a significant downregulating effect on the IL-1 beta, TNF-alpha, and TGF-beta transcriptional levels of mouse macrophage RAW264.7 cultured in vitro: IL-1β (0.01.+ -. 0.01), TNF-. Alpha. (0.02.+ -. 0.01), and TGF-. Beta.0.03.+ -. 0.01) were significantly altered (P < 0.05) relative to the DMSO control.
Example 5
A process for preparing monascin C with glycosidase inhibitory and immunoregulatory activities comprises the following steps:
(1) Preparation of solid cultures
Culturing Monascus ruber strain by primary strain culture, secondary seed culture and tertiary liquid expansion culture to obtain liquid culture;
(1.1) first-order Strain culture
Will 10 6 Inoculating each spore/mL monascus suspension on potato agar (PDA) slant culture medium, inoculating 20 μl monascus suspension on each slant, and culturing at 33.5deg.C and 2400r/min for 8 days to obtain first-class strain.
The PDA culture medium is potato glucose culture medium, and the formula is as follows: 200g peeled potatoes, 20g glucose and 15g agar, and water was added to 1000mL.
(1.2) two-stage seed culture
Preparing the first-class strain into a concentration of 10 by using sterile water 6 The spore suspension of each spore/mL is inoculated into 250mL of PDB culture medium prepared in advance according to 220 mu L of each bottle, and the liquid secondary strain is obtained by shaking and culturing for 4 days under the conditions of the temperature of 33 ℃ and the rotating speed of 240 r/min.
The formula of the PDB culture medium is as follows: 200g peeled potatoes, 20g glucose, and water to 1000mL.
(1.3) three-stage liquid expanded culture
Inoculating 2mL of liquid secondary strain liquid into 250mL of PDB culture medium sterilized in advance, shake-flask culturing at 33.5 ℃ and 235r/min for 8 days, and collecting culture to obtain liquid culture.
(2) Extraction and refinement of active principles in liquid cultures
The specific operation is as follows:
(2.1) extraction of active ingredient in liquid culture
Concentrating the liquid culture under reduced pressure, homogenizing, and extracting with ethyl acetate to obtain effective component extract.
The specific operation is as follows: evaporating the liquid culture under reduced pressure to 3/10-1/10 of the original volume under the conditions of vacuum degree of-0.1 to-0.08 MP and temperature of 10-60 ℃, and directly extracting with ethyl acetate for 20-200 minutes. Filtering with 2.5 μm membrane, and vacuum evaporating the filtrate under vacuum degree-0.08 MP at 60deg.C to obtain effective component extract.
(2.2) preliminary purification of the active ingredient extract
And (3) performing preliminary purification on the active ingredient extract by adopting preparative reverse-phase high performance liquid chromatography to obtain a red preliminary purified product.
The method comprises the following specific steps: refining by preparative reverse phase high performance liquid chromatography; the chromatographic separation preparation conditions are as follows: mobile phase A is ultrapure water, mobile phase B is acetonitrile; elution conditions: gradient elution is carried out on 50% -70% of mobile phase B within 0 to 30 min; sample injection volume 200 μL, column temperature 30 ℃ and flow rate 20mL/min; the detection wavelength is 358nm; chromatographic column: preparing a reversed phase C-18 column; collecting the eluent corresponding to the third highest peak, evaporating the extractant under reduced pressure at-0.1 MP and 30 deg.C, and drying at-40 deg.C to obtain red primary purified product.
(2.3) refining of the Red preliminary purification product
Refining the red primary purified product by semi-preparative reverse phase high performance liquid chromatography, and collecting the compound corresponding to the highest peak under the condition of eluting with acetonitrile solution to obtain monascin C eluent.
The specific operation is as follows: the chromatographic separation preparation conditions are as follows: mobile phase A is ultrapure water, mobile phase B is acetonitrile; elution conditions: eluting with 50% -70% of mobile phase B for 0-30 min; sample injection volume 30 μL, column temperature 30 ℃ and flow rate 5mL/min; the detection wavelength is 358nm; chromatographic column: semi-preparative reverse phase C-18 column; collecting the compound corresponding to the highest peak value as monascin C eluent; evaporating under reduced pressure at 30 deg.C and vacuum degree of-0.1 MP to remove extractant; drying at-40deg.C to obtain red powder monascin C.
The structural formula of the spectrum analyzed monascin C is as follows:
the activity measurement results show that: half-maximal Inhibitory Concentration (IC) of monascin C on alpha-glucosidase 50 ) The method comprises the following steps: 200 μg/mL, and has a significant downregulating effect on the IL-1 beta, TNF-alpha, and TGF-beta transcriptional levels of mouse macrophage RAW264.7 cultured in vitro: IL-1β (0.01.+ -. 0.01), TNF-. Alpha. (0.02.+ -. 0.01), and TGF-. Beta.0.03.+ -. 0.01) were significantly altered (P < 0.05) relative to the DMSO control.
Claims (6)
1. A method for preparing monascus C with glycosidase inhibition activity and immunoregulatory activity is characterized in that: the structural formula is as follows:
the preparation operation steps of the monascus C with the immunoregulatory activity are as follows:
(1) Preparation of solid cultures
Culturing Monascus sp strain, performing primary strain culture, secondary seed culture and tertiary liquid expansion culture to obtain liquid culture; the Monascus is Monascus purpureus (Monascus purpureus), monascus anka (Monascus anka) or Monascus ruber (Monascus ruber);
(1.1) first-order Strain culture
Inoculating monascus suspension on potato agar PDA slant culture medium, inoculating 10-30 mu L of the monascus suspension on each slant, culturing for 7-9 days at the temperature of 32-35 ℃ and 220-250 r/min to obtain first-class strain;
(1.2) two-stage seed culture
Preparing the first-class strain into a concentration of 10 by using sterile water 5 ~10 7 Inoculating 200-250 mu L of spore suspension of each spore/mL into 250mL of PDB culture medium, and culturing for 3-5 days at the temperature of 32-35 ℃ and 220-250 r/min by shaking the bottle to obtain liquid secondary strain;
(1.3) three-stage liquid expanded culture
Inoculating 1-3 mL of liquid secondary strain into 250mL of PDB culture medium, shake-flask culturing for 7-9 days at the temperature of 32-35 ℃ and 220-250 r/min, and collecting the culture to obtain a liquid culture;
(2) Extraction and refinement of active principles in liquid cultures
The specific operation is as follows:
the extractant is methanol, ethyl acetate or methanol and ethyl acetate with the volume ratio of 0-10: 10 to 0;
(2.1) extraction of active ingredient in liquid culture
Concentrating and drying the liquid culture under reduced pressure, performing ultrasonic extraction with extractant, filtering with filter membrane or centrifuging, and vacuum removing solvent under reduced pressure to obtain effective component extract;
(2.2) preliminary purification of the extract
Performing primary purification on the active ingredient extract by adopting preparative reverse-phase high performance liquid chromatography to obtain a red primary purified product;
(2.3) refining of the preliminary purified product
Refining the primary purified product by semi-preparative reverse phase high performance liquid chromatography, and collecting the compound corresponding to the highest peak under the condition of eluting with acetonitrile solution to obtain monascin C eluent.
2. The method of manufacturing according to claim 1, characterized in that: in the step (1.1), the PDA culture medium is potato dextrose culture medium, and the formula is as follows: 200g peeled potatoes, 20g glucose and 15g agar, and water was added to 1000mL.
3. The method of manufacturing according to claim 1, characterized in that: in the steps (1.2) and (1.3), the formula of the PDB culture medium is as follows: 200g peeled potatoes, 20g glucose, and water to 1000mL.
4. The method of manufacturing according to claim 1, characterized in that: in the step (2.1), the liquid culture is concentrated to 3/10 to 1/10 of the original volume under the conditions of vacuum degree of-0.1 to-0.08 MPa and temperature of 10 to 60 ℃, dried and crushed under the conditions of temperature of-50 to 130 ℃ and the weight-volume ratio of 1g: adding extractant into 0.5-5 mL, extracting with 40KHz ultrasonic for 20-200 min, centrifuging at 4000-15000 rpm or filtering with 0.1-2.5 μm membrane to obtain filtrate or centrifuging supernatant; the filtrate or the centrifugal supernatant is decompressed and distilled to remove the extractant under the condition of vacuum degree of minus 0.1 to minus 0.08MPa and temperature of 10 to 60 ℃ to obtain the active ingredient extract.
5. The method of manufacturing according to claim 1, characterized in that: in the step (2.2), refining by adopting preparative reverse-phase high performance liquid chromatography; the chromatographic separation preparation conditions are as follows: mobile phase A is ultrapure water, mobile phase B is acetonitrile; elution conditions: gradient elution is carried out on 50% -70% of mobile phase B within 0 to 30 min; the sample injection volume is 30-300 mu L, the column temperature is 20-40 ℃, and the flow rate is 5-50 mL/min; the detection wavelength is 358nm; chromatographic column: preparing a reversed phase C-18 column; collecting the eluent corresponding to the third highest peak, and evaporating the extractant under reduced pressure at the vacuum degree of-0.1 to-0.08 MPa and the temperature of 10-60 ℃; drying at 130 ℃ or lower to obtain the red primary purified product.
6. The method of manufacturing according to claim 1, characterized in that: in the step (2.3), chromatographic separation preparation conditions are as follows: mobile phase A is ultrapure water, mobile phase B is acetonitrile; elution conditions: eluting with 50-70% of mobile phase A for 0-30 min; the sample injection volume is 20-200 mu L, the column temperature is 20-40 ℃ and the flow rate is 2-20 mL/min; the detection wavelength is 358nm; chromatographic column: semi-preparative reverse phase C-18 column; collecting the compound corresponding to the highest peak value as monascin C eluent; vacuum evaporating to remove extractant under vacuum degree of-0.1 to-0.08 MPa and temperature of 10-60 ℃; drying at 130 deg.C or below to obtain red powder monascin C.
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