Method for producing safflower extract with high isoquercitrin content

Abstract

The invention discloses a method for producing a flos carthami extract with high isoquercitrin content, which comprises the following steps: 1) extracting flos carthami in a fading period by using methanol, ethanol and / or water; and 2) drying the extracting solution. Research on differential metabolites and differential genes in petals in different flowering stages shows that the content of effective component flavone in safflower in a fading stage is lower than that in safflower in a full-bloom stage, the petal metabolites and genes in the three stages are obviously separated, and samples in the initial flowering stage and the full-bloom stage are close to each other and far away from samples in the fading stage. The content of isoquercitrin in petals in the fading period of the extract extracted by the method is high and can reach 0.164 mg / g, and the content of isoquercitrin in the full-bloom period can reach 0.067 mg / g. The safflower extract with high isoquercitrin content is produced by adopting the fading period safflower, so that the fading period safflower is turned into wealth, and the economic benefit is increased.

Description

BACKGROUND

[0001] Carthamus tinctorius L., a chrysanthemum annual plant, its dried flowers are traditional Chinese herbal medicine. Safflower has the functions of activating blood circulation to remove blood stasis, removing blood stasis and relieving pain, etc., and is often used for treating dysmenorrhea, chest pain, contusion and injury, etc. Safflower contains various effective chemical components, mainly flavonoids, alkaloids, organic acids, etc., among which safflower yellow pigment and safflower red pigment are the main effective components in safflower and the material basis for exerting pharmacological effects. Safflower has the effects of anti-thrombosis, anti-inflammatory and anti-tumor, and has protective effects on brain tissue, myocardial tissue and osteoblasts. The effective compounds or active parts of safflower have been screened for pharmacological activities in vivo and in vitro, showing good effects on maintaining and promoting human health. Some compounds have been applied to the clinical treatment of coronary heart disease, chronic pulmonary heart disease, cerebrovascular disease, orthopedic disease and diabetes. In recent years, more researches have been conducted on safflower, and safflower has gradually attracted widespread attention due to its medicinal value and edible value. In order to improve the utilization efficiency of safflower.

[0002] Isorhamnetin, also known as Quercetin 3-O-glucoside (Q3G or Isotrifoliin), is one of the main glycoside forms of quercetin, and has good expectorant, antitussive and antiasthmatic effects, and is used for treating chronic bronchitis, and has auxiliary therapeutic effects on patients with coronary heart disease and hypertension.

[0003] The effective component of safflower is flavonoids, and the flowering period of safflower is divided into early flowering period, full flowering period and decline period, and the flavonoid content reaches the highest in the full flowering period and then gradually decreases. The decline period of safflower usually refers to the period after the full flowering period, at which time the flower buds gradually wither and the content of effective components decreases. In the production of safflower, the safflower in the decline period is discarded in the field after the full flowering period safflower is picked. SUMMARY

[0004] The purpose of the present application is to utilize the discarded safflower in the decline period, and a method for producing safflower extract with high isorhamnetin content is provided.

[0005] The safflower extract with high isorhamnetin content is prepared by the following method:

[0006] 1) The safflower in the decline period is extracted with methanol, ethanol and water;

[0007] 2) The extract is dried.

[0008] The extraction temperature is 40-60%, and the extraction times are 1-5 times.

[0009] The ethanol is 80% ethanol, ultrasonic extraction is performed for 2 h, the extraction temperature is 55°C, and the extraction times are 3 times.

[0010] After extraction, the obtained extract is purified by the following method:

[0011] 1) The extract is absorbed by macroporous adsorption resin, and eluted;

[0012] 2) The extract is filtered by filter membrane and purified by liquid chromatography.

[0013] In step 1), the extract is injected into D101 macroporous adsorption resin, eluted with pure water until colorless, and then eluted with 70% ethanol. The eluate is collected, concentrated, and freeze-dried.

[0014] 2) The eluate is fully dissolved in methanol, and insoluble substances are removed by 0.45 μm filter membrane. The eluate is purified by liquid chromatography.

[0015] The chromatographic column is a C18 reverse-phase preparation column.

[0016] The mobile phase is 45% A phase and 55% B phase (methanol:acetonitrile = 12:30).

[0017] The A phase is 0.1% formic acid aqueous solution, and the B phase is methanol:acetonitrile = 12:30.

[0018] The flow rate is 7 mL / min.

[0019] The detection wavelength is 360 nm.

[0020] The injection volume is 2 mL.

[0021] The column temperature is room temperature.

[0022] The present application provides a method for producing high isoquercitrin content safflower extract, and the high isoquercitrin content safflower extract is prepared by the following method: 1) using methanol, ethanol and water to extract the safflower in the fading period; and 2) drying the extract. The differential metabolites and differential genes in petals of different flowering periods are studied, and it is found that the flavonoid content of the effective component in the fading period safflower is lower than that in the full-bloom period safflower. The metabolites and genes of petals in the three periods are significantly separated, and the samples in the early flowering period and the full-bloom period are closer to each other and farther away from the sample in the fading period. The isoquercitrin content in the extract obtained by the method is higher in the petals of the fading period, and the content can reach 0.164 mg / g, and the content in the full-bloom period can reach 0.067 mg / g. The production of high isoquercitrin content safflower extract from the fading period safflower makes the fading period safflower become valuable, and increases the economic benefit. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Principal component analysis of metabolites in three flowering periods;

[0024] Figure 2 Principal component analysis of genes in three flowering periods;

[0025] Figure 3 Three-flower-stage differentially expressed metabolite analysis;

[0026] Figure 4 Three-flower-stage differentially expressed gene analysis;

[0027] Figure 5 Venn diagram of differentially expressed metabolites identified between IN, FU and FA groups;

[0028] Figure 6 Venn diagram of differentially expressed genes identified between IN, FU and FA groups;

[0029] Figure 7 KEGG pathway analysis of FUvsFA differentially expressed metabolites;

[0030] Figure 8 KEGG pathway analysis of INvsFU differentially expressed metabolites;

[0031] Figure 9 KEGG pathway analysis of INvsFU differentially expressed metabolites;

[0032] Figure 10 GO enrichment analysis of FUvsFA differentially expressed metabolites;

[0033] Figure 11 GO enrichment analysis of INvsFA differentially expressed metabolites

[0034] Figure 12 GO enrichment analysis of INvsFU differentially expressed metabolites

[0035] Figure 13 Phenotype and total flavonoid content determination at each stage. A: analysis of the phenotype of Crocus sativus L. at each stage; B: analysis of the total flavonoid content at each stage; C: liquid chromatogram of each stage with rutin as the standard

[0036] Figure 14 Metabolomics determination of isoquercitrin content in the fading stage (FA) and the full bloom stage (FU);

[0037] Figure 15 Optimization of flavonoid extraction conditions in fading stage petals (taking isoquercitrin as an example)

[0038] Comparison of the antioxidant activity of Jihong No. 1 full bloom stage and fading stage petal extracts. Note: t-test analysis method was used for analysis (**, P < 0.01);

[0039] Figure 16 Comparison chart of isoquercitrin content in the full bloom stage and the fading stage (P < 0.01); DETAILED DESCRIPTION

[0040] Example 1 Differential metabolites and differential gene analysis based on metabolomics

[0041] 1. Preparation of plant material

[0042] The flower petals of Jihong No. 1 crocus at initial flowering stage, full flowering stage and fading stage were immediately placed in liquid nitrogen and stored in a -80 °C refrigerator.

[0043] 2. Metabolite extraction

[0044] The sample was ground into powder with a grinder (30 Hz, 90 seconds), 100 mg of powder was weighed and dissolved in 1.0 mL of extraction solution, and rotated three times at 4 °C overnight; centrifuged at 10,000 x g for 10 min, and the supernatant was filtered through a microporous filter membrane (0.22 μm pore size) for LC-MS / MS chromatographic analysis.

[0045] 3. RNA extraction and transcriptome sequencing

[0046] Total RNA was extracted from the flower petals of Jihong No. 1 crocus at initial flowering stage, full flowering stage and fading stage. The purity of the RNA was detected by NanoDrop. The raw data was entrusted to Wuhan Maiwei Metabolic Biotechnology Co., Ltd. for cDNA synthesis and transcriptome sequencing. The library was sequenced using the Illumina HiSeq platform. The sequencing data was filtered using fastp to remove a small amount of reads with sequencing connection or low-quality sequencing, and the high-quality sequencing data after sequencing was spliced to obtain the transcriptome.

[0047] 4. Identification of differential metabolites and differential genes in petals at different flowering stages

[0048] Firstly, PCA analysis was performed on the samples at different flowering stages to determine the differences in overall metabolites and genes between different flowering stages and the variation degree between samples within the group. As shown in the figures (Figures Figure 1 and Figure 2 ), the metabolites and genes of the petals at the three stages were clearly separated, and the samples at the initial flowering stage and the full flowering stage were closer to each other and farther from the samples at the fading stage. Then, pairwise comparison of the samples at different flowering stages was performed, and it was found that in the INvsFU group, 19 metabolites were up-regulated, 22 metabolites were down-regulated, 353 genes were up-regulated, and 59 genes were down-regulated; in the INvsFA group, 162 metabolites were up-regulated, 74 metabolites were down-regulated, 2878 genes were up-regulated, and 226 genes were down-regulated; in the FUvsFA group, 150 metabolites were up-regulated, 66 metabolites were down-regulated, 2818 genes were up-regulated, and 67 genes were down-regulated (Figures Figure 3 and Figure 4). Venn analysis showed that there were 6 common metabolites in the three groups, the number of metabolites specific to INvsFU group was 17, the number of metabolites specific to INvsFA group was 46, and the number of metabolites specific to FUvsFA group was 22 Figure 5 ); secondly, the number of genes specific to INvsFU group was 363, the number of genes specific to INvsFA group was 802, and the number of genes specific to FUvsFA group was 564 Figure 6 . All the above indicated that there were significant differences in metabolites and genes among the three groups of flower petals.

[0049] 5. Enrichment analysis of differential metabolites and differential genes in different flower periods

[0050] The KEGG database was used to perform enrichment analysis and annotation of metabolites and differential genes, and the significantly different metabolic pathways and key metabolites in different flower periods were screened, and the results are shown in Figures 7-9 Most of the differential metabolites in the three groups were enriched in phenylpropanoid biosynthesis pathway, flavonoid compound synthesis pathway, etc. In addition, GO enrichment analysis was performed on the differential genes of different groups, and they were annotated and classified according to biological process, cell component and molecular function. In the FUvsFA group and the INvsFA group, "cell process", "metabolic process" and "stimulation response" were the highest enriched biological processes; "cell", "cell part" and "cell organelle" were the highest enriched "cell components"; "binding", "catalytic activity" and "molecular structure activity" were the highest enriched "molecular functions". In INvsFU, the enrichment degree of "biological regulation" was higher than that of "stimulation response", and the enrichment degree of "molecular function" was higher than that of "stimulation response", and in "molecular function", the enrichment degree of "transporter activity" was higher than that of "structure molecular activity" Figures 10-12 .

[0051] Example 2. Comparative analysis of high content compounds in full bloom period and decline period

[0052] By comparing the flavonoid content of petals in different flower periods, it can be seen that the flavonoid content is quite different Figure 13 . Among them, the total flavonoid content in full bloom period is the highest, followed by the decline period. Combined with the metabolomics analysis, it is found that the compounds in full bloom period and decline period are quite different, and the top 50 compound components with the highest expression in full bloom period and decline period are shown in Tables 1 and 2.

[0053] The analysis of Table 1 and Table 2 shows that there are obvious differences in the expression profiles of flavonoids during the full-bloom stage and the decline stage, reflecting the metabolic needs of plants at different growth stages. During the full-bloom stage, Quercetin derivatives are dominant, which may support flower development and pollination. During the decline stage, Kaempferol derivatives, anthocyanins, and dihydroflavonoids are dominant, which may focus more on antioxidant and aging regulation. When transitioning from the full-bloom stage to the decline stage, the expression of Kaempferol derivatives (such as Kaempferol 3-O-galactoside and Kaempferol 3-O-robinobioside) increases, which may be involved in antioxidant protection during the aging process of plants. The expression of Quercetin derivatives decreases, which may be related to the weakening of their UV protection and pollination attraction during flower opening. The expression of anthocyanins (such as Cyanidin) and dihydroflavonoids (such as Naringenin chalcone) significantly increases, which may be involved in petal color changes and aging-related stress responses. Overall, the expression of flavonoids during the decline stage tends to be more protective compounds to cope with environmental stress and aging. These changes provide a theoretical basis for understanding plant physiology and are more conducive to the efficient use of safflower.

[0054]

[0055]

[0056] Example 3 Optimization of Safflower Extract Process

[0057] Isoquercitrin is a highly active natural plant-derived ingredient. It is not only the key glycoside form of Quercetin, but also has better water solubility and bioavailability than Quercetin. It also has multiple important physiological functions such as antioxidant, anti-inflammatory, antiviral, antitumor, cardiovascular protection, and blood glucose lowering. It is highly valued in the fields of medicine, health products, and cosmetics, and is one of the natural products with extremely high development value. By analyzing Table 1 and Table 2, we found that the content of this compound is higher in the decline stage Figure 14 ).

[0058] The present application determines the final extraction process by changing the extraction conditions, which mainly includes the following points:

[0059] 1) Extract: 100% methanol, 80% methanol, 60% methanol, 20% methanol, 100% ethanol, 80% ethanol, 60% ethanol, 40% ethanol, 20% ethanol, 100% water.

[0060] 2) Ultrasonic extraction time: 0.5h, 1h, 1.5h, 2h, 2.5h, 3h.

[0061] 3) Extraction times: 1 time, 2 times, 3 times, 4 times, 5 times.

[0062] 4) Extraction temperature: 40℃, 45℃, 50℃, 55℃, 60℃.

[0063] Results as shown in Figure 15 When the extraction liquid is 80% ethanol, ultrasonic extraction is 2h, the extraction temperature is 55℃, and the extraction times is 3 times, the extraction effect is better.

[0064] The prepared product is analyzed by liquid chromatography-mass spectrometry, and it is found that the content of isoquercitrin in the petals in the decline period is higher, and the content can reach 0.164mg / g, and the content in the full bloom period can reach 0.067mg / g (see Figure 16 ).

[0065] In order to further extract isoquercitrin, the above extraction liquid is purified through the following experiments:

[0066] 1) The above extraction liquid is injected into D101 macroporous adsorption resin, adsorbed overnight, polysaccharide and other impurities are removed by using pure water, and eluted to be colorless, then eluted by using 70% ethanol, the eluate is collected and concentrated, and then freeze-dried for standby.

[0067] 2) The above crude extraction is fully dissolved with an appropriate amount of methanol, and the insoluble substances are removed by 0.45μm filter membrane, and then purified by preparative liquid chromatography. The chromatographic column is C18 reversed-phase preparative column (20mm × 250mm, 5μm), the mobile phase is 45%A phase (0.1% formic acid water) and 55%B phase (methyl alcohol: acetonitrile = 12:30), the flow rate is 7mL / min, the detection wavelength is 360nm, the injection amount is 2mL, and the column temperature is room temperature.

Claims

1. Safflower extract with high isoquercitrin content, prepared by the following method: 1) The safflower in its decaying stage was extracted with methanol, ethanol and / or water to obtain an extract; 2) Dry the extract.

2. The safflower extract with high isoquercitrin content according to claim 1, characterized in that: The extraction temperature is 40-60%, and the number of extractions is 1-5.

3. The safflower extract with high isoquercitrin content according to claim 1, characterized in that: The extraction was performed using 80% ethanol, ultrasonically for 2 hours at a temperature of 55℃, and the extraction was repeated 3 times.

4. The safflower extract with high isoquercitrin content according to claim 1, 2, or 3, characterized in that: The safflower in its decline period refers to the safflower whose buds begin to gradually wither after its peak bloom.

5. The safflower extract with high isoquercitrin content according to claim 4, characterized in that: After extraction in step 1), the resulting extract was purified using the following method: 1) Elute the extract with macroporous adsorption resin; 2) Membrane filtration and liquid chromatography purification.

6. The safflower extract with high isoquercitrin content according to claim 5, characterized in that: 1) Inject the extract into D101 macroporous adsorption resin, leave overnight, elute with pure water until colorless, then elute with 70% ethanol, collect the eluent and concentrate it, and freeze dry. 2) Dissolve completely in methanol, remove insoluble matter by passing through a 0.45μm filter membrane, and purify by liquid chromatography; Chromatographic column: C18 reversed-phase preparative column; Mobile phase: 45% phase A, 55% phase B; methanol:acetonitrile = 12:30 Phase A is a 0.1% formic acid aqueous solution, and phase B is methanol:acetonitrile = 12:30; Flow rate: 7 mL / min; Detection wavelength: 360 nm; Injection volume: 2 mL; Column temperature: room temperature.

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