Study on the composition analysis and function application of flavonoid extract from seeds of Umbelliferae

Abstract

The present invention relates to an applied study on the component analysis and function of a flavonoid extract of an umbelliferae plant seed. The preparation method of the extract is as follows: the umbelliferae plant seeds are crushed into powder, and then the umbelliferae plant seeds are extracted and concentrated using an ethanol solution, and extracted with solvents of different polarities to obtain a corresponding umbelliferae plant seed extract, and then the umbelliferae plant seed extract is subjected to flavonoid component analysis and content testing. It will be verified that the flavonoid extract provided by the present invention can prevent, improve or treat peroxidation pathways and inflammation-related diseases, and can also be used for diseases related to abnormal expression of α-glucosidase and α-amylase, such as cancer of various tissues and organs, hyperuricemia, diabetes, hyperglycemia, hyperlipidemia, cardiovascular disease, vascular sclerosis or premature aging, etc., and can be used for the development of drugs, health foods or skin care products.

Description

Technical Field

[0001] The present invention belongs to the technical field of flavonoid extracts, and specifically relates to an Umbelliferae plant seed flavonoid extract, a preparation method of the extract, a pharmaceutical composition containing the extract, and use of the extract in preparing an antioxidant preparation, and in treating and preventing diseases caused by excessive oxidation, such as inflammation, cancer, hyperuricemia, hyperglycemia, and hyperlipidemia. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Free radicals are generated by normal biochemical reactions in the body, which increase oxidative stress and may damage biomolecules such as lipids, proteins and DNA. Free radicals are associated with cancer, atherosclerosis, aging, inflammation, ischemic heart disease, diabetes and neurodegenerative diseases. In order to reduce the oxidative stress of free radicals, many synthetic antioxidants delay or inhibit cell damage by scavenging free radicals. Natural antioxidants from plant products have attracted attention because they may be safer and more effective in reducing reactive oxygen species (ROS) levels than synthetic single dietary antioxidants, and can achieve the effect of treating and preventing related diseases within a safer range of dosage.

[0004] Most natural polyphenols are free radical scavengers (FRS), especially flavonoids in polyphenols, which can delay or suppress the initiation step of lipid oxidation or hinder its propagation step. The antioxidant mechanism of polyphenols is usually that the aromatic ring donates H to free radicals during the oxidation process. + , and become free radicals themselves. These free radical intermediates are stabilized by the resonance delocalization of the electrons within the aromatic ring. Due to their excellent antioxidant capacity, polyphenols are considered natural antioxidants.

[0005] Oxidative DNA damage is pathogenically linked to a variety of aging-related degenerative diseases, such as cancer, coronary heart disease, and diabetes. Increasing experimental and clinical research evidence shows that there is a close relationship between hyperglycemia, oxidative stress, and diabetic complications. ROS can lead to defects in insulin gene expression and insulin secretion, as well as increased apoptosis, thereby increasing the risk of diabetes.

[0006] Plants in the Apiaceae family are usually aromatic plants with hollow necks, and are all annual or perennial herbs. This family includes plants such as cumin, carrots, parsley, coriander, fennel, and traditional Chinese medicinal materials such as cnidium monnieri, angelica, Chuanxiong, Duhuo, Bupleurum, Ferula, and Peucedanum. The Apiaceae family is rich in chemical components, mainly containing coumarins, terpenes, small molecule organic acids, essential oils, polyphenols, alkaloids, etc. Plants in the Apiaceae family have a variety of application values ​​and can be used as medicinal materials, vegetables, spices, pesticides, etc. This genus of medicinal plants is used as traditional Chinese medicine to treat a variety of diseases such as neurological disorders, inflammation, digestive disorders, diabetes, rheumatism, cancer, and arthritis.

[0007] Celery seeds are seeds of the umbelliferae plant celery (Apium grauens L. var. dulcedc.). Celery seeds are rich in antioxidant polyphenols, indicating that they have good medical uses, such as antioxidant and anti-inflammatory properties. At present, most researchers at home and abroad mainly conduct relevant antioxidant research and reports on extracts from the roots, stems and leaves of celery, and there are almost no reports on the application research of celery seed extracts in terms of antioxidant, anti-inflammatory and hypoglycemic effects. Celery is a common vegetable in daily life, and there are two types of celery: water celery and dry celery. There are many types of celery in China, mainly Chinese celery (ben celery), such as Jinnan Shiqin No. 1, Jinnan Dongqin and Tiegan Celery, etc.; Western celery (yang celery), such as California King (Ventura), American White Celery, Italian Winter Celery and American Celery, etc. Celery seeds contain different types and quantities of active ingredients depending on the celery variety and the geographical area where they are planted, which also results in different pharmacological effects.

[0008] In addition, fennel seeds are also a common medicinal and edible plant, mainly distributed in Europe, the Mediterranean coast and all over my country. Fennel seeds are rich in volatile oil components and are often used in the spices and cosmetics industries. Studies have found that fennel contains flavonoids, such as kaempferol and its derivatives, which have antioxidant, anti-inflammatory and antibacterial activities.

[0009] Cnidium monnieri is the mature fruit of the Apiaceae family Chinese medicinal material Cnidium monnieri. It is distributed in most parts of my country, but less in the Qinghai-Tibet Plateau, and mostly distributed in South China. It has been reported that about 13 flavonoid components have been identified in Cnidium monnieri, mainly including kaempferol glycosides (such as kaempferol-3-O-β-D-glucoside and kaempferol-3-O-β-D-galactoside, etc.), quercetin glycosides (such as quercetin-3-O-β-D-glucoside, hyperoside, rutin), etc. (Isolation and structural identification of flavonoid chemical components in Cnidium monnieri, Chinese Journal of Medicinal Chemistry, 1005-0108 (2020) 09-0542-07). Many reports have found that Cnidium monnieri extracts have good antioxidant, anti-inflammatory and other pharmacological activities. Summary of the invention

[0010] Based on the above technical background, the present invention provides a flavonoid extract from seeds of Umbelliferae plants, wherein the extract and its main flavonoid monomers or compositions have antioxidant, anti-inflammatory and other effects. In addition, the flavonoid components of Umbelliferae seeds and their main flavonoid compound monomers can effectively inhibit α-glucosidase and α-amylase, and lower blood sugar, and are expected to be used as active ingredients in drugs, foods or skin care products.

[0011] In a first aspect, the present invention provides a flavonoid extract from the seeds of an Umbelliferae plant, wherein the extract contains a high-activity monomer flavonoid composition accounting for more than 50%; the high-activity monomer flavonoid composition is composed of flavonoid glycosides such as apigenin A, apigenin B, apigenin, luteolin, kaempferol-7-O-glucoside, apigenin-7-O-glucoside, rutin, hyperoside and kaempferol.

[0012] The Umbelliferae plant seeds described in the first aspect include but are not limited to one or more of celery seeds, fennel seeds, cnidium monnieri, coriander seeds, and caraway seeds; in a preferred embodiment of the present invention, the plant seeds are celery seeds, fennel seeds, or cnidium monnieri; further preferably, they are celery seeds; in a more preferred embodiment, they are celery seeds produced in Shandong, Guangxi, or Northeast China.

[0013] It has been verified that the above-mentioned flavonoid extracts from the seeds of Umbelliferae plants have antioxidant, anti-inflammatory and hypoglycemic effects. Among them, the antioxidant activity of celery seeds is particularly prominent, and has a significant positive correlation with the combination of the above-mentioned six monomer components; in the celery seed extract with better effects, the content of the above-mentioned six monomer flavonoid composition accounts for more than 50% of the total amount of the extract, and in a further preferred embodiment, the composition accounts for more than 54%.

[0014] The second aspect of the present invention provides a method for preparing the Umbelliferae plant seed flavonoid extract according to the first aspect, comprising the following steps:

[0015] The dried plant seeds are crushed and a low-polarity organic reagent is added for degreasing; the defatted plant seeds are transferred into an alcohol solution for reflux extraction, and the extracts are combined and dried to obtain an extract; the extract is added into dichloromethane, fully stirred, and then filtered, the solid part is obtained and dissolved in water, and then n-butanol reagent is added for extraction, and the organic phase is retained and dried to obtain the extract.

[0016] The present invention also investigated the activity of the dichloromethane extract part and the water extract part after n-butanol extraction obtained by the above preparation method; the results showed that the dichloromethane extract part also showed certain antioxidant and hypoglycemic activities, but the pharmacodynamic activity was not as good as the flavonoid extract described in the first aspect. In addition, the dichloromethane extract has poor water solubility and contains some free phenolic compounds. The present invention also tried to directly extract the extract with n-butanol, and the results showed that its antioxidant and hypoglycemic activities were also significantly lower than those of the n-butanol extract part obtained by the method.

[0017] Preferably, the low-polarity organic reagent is one of petroleum ether, n-hexane, ethyl acetate, and dichloromethane, or a mixed solution of two or three of the reagents; further preferably, the low-polarity organic reagent is ethyl acetate.

[0018] Preferably, the defatting treatment steps are as follows: add low-grade organic reagents to the crushed plant seeds and reflux extract at room temperature for 3-5 times, adding 3-5 times the volume of the plant seed powder each time, the total extraction time is 3-5 hours, and the solid powder retained in the reflux system is the defatted plant seeds.

[0019] Preferably, the alcohol solution is methanol, ethanol or a mixed solution of ethanol and water; in a further preferred embodiment, the alcohol solution is ethanol; further, the specific steps for preparing the extract are as follows: add 5-10 times the volume of ethanol to the defatted plant seeds, reflux extract at 75-85°C for 3-5 times, each reflux extraction time is 1-3 hours, and the combined extracts are dried by reduced pressure concentration.

[0020] Preferably, the n-butanol reagent is n-butanol, n-butanol-ethanol solution or n-butanol-acetone solution; in a further preferred embodiment, it is n-butanol.

[0021] In the above preferred embodiment, after adding dichloromethane to the extract, the dichloromethane portion can be fully dissolved by mechanical stirring, and the solid portion is retained after filtration and dissolved in water. In order to improve the dissolution and extraction effect of flavonoid components, the effect of water dissolution can be assisted by ultrasound or mechanical stirring.

[0022] The present invention screened and studied the monomer active ingredients in the above-mentioned Umbelliferae seed extracts, and screened out the following monomer components from celery seeds, cnidium monnieri and fennel seeds, which have good antioxidant, anti-inflammatory and hypoglycemic activities: apigenin A, apigenin B, apigenin, luteolin, quercetin, kaempferol, naringenin, hyperoside and rutin.

[0023] Furthermore, the present invention also attempts to verify the synergistic effect between the above-mentioned monomer compounds. According to the investigation results, quercetin and luteolin have very ideal pharmacological activity after being used in combination, reflecting a synergistic effect.

[0024] Therefore, in the third aspect of the present invention, there is provided the use of any one of the following flavonoid monomer compounds as an antioxidant, anti-inflammatory active ingredient or an α-glucosidase, α-amylase inhibitor, wherein the flavonoid monomer compound is apigenin A, apigenin B, apigenin, luteolin, quercetin, kaempferol, naringenin, hyperoside or rutin.

[0025] The application of the third aspect as an antioxidant, anti-inflammatory active ingredient or an α-glucosidase, α-amylase inhibitor is specifically as follows:

[0026] (1) Used to prepare preparations that have the effect of preventing, improving or treating diseases;

[0027] (2) Used to prepare a model agent, wherein the model agent is used to prepare a peroxidation and inflammation inhibition model or an α-glucosidase and α-amylase expression inhibition model.

[0028] In a fourth aspect, the present invention provides a pharmaceutical composition, which comprises an active dose of the flavonoid extract from the seeds of the Umbelliferae plant described in the first aspect, or the flavonoid monomer compound described in the third aspect, or the pharmaceutical composition has a combination of quercetin and luteolin as active ingredients.

[0029] In the above composition, the active dose of the flavonoid extract can be routinely determined according to factors such as the subject and the method of use; calculated based on the total mass of the pharmaceutical composition, the dose of the flavonoid extract is adjusted within the range of 0.01 to 99%, and further, it can be 0.1 to 70%, 1 to 30%, 0.01 to 0.05%, 0.05 to 0.1%, 0.1 to 0.3%, 0.3 to 0.5%, 0.5 to 1%, 1 to 3%, 3 to 5%, 5 to 10%, 10 to 20%, 20 to 30%, 30 to 50%, 50-70% or 70-99%; in addition, the pharmaceutical composition can select the corresponding preparation type according to the purpose of administration, metabolic performance, etc., and the pharmaceutical composition also includes a pharmaceutically acceptable carrier in the art, which should be harmless to the subject and even sterile, including but not limited to buffers, antioxidants, preservatives, bactericides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, tension regulators, sugars, surfactants, salt-forming counterions, metal complexes and / or non-ionic surfactants.

[0030] Preferably, in the combination of quercetin and luteolin, the mass ratio of quercetin to luteolin is adjusted in the range of 1-9:1-9.

[0031] The fifth aspect of the present invention provides the use of the Umbelliferae plant seed flavonoid extract described in the first aspect, the flavonoid monomer compound described in the third aspect, and the pharmaceutical composition described in the fourth aspect in the field of preparing pharmaceutical preparations.

[0032] Preferably, the pharmaceutical preparation is one including but not limited to a medicine or a skin care product.

[0033] Furthermore, the drug is mainly used to prevent, improve or treat diseases related to peroxidation pathways, related diseases with inflammatory manifestations and / or related diseases of abnormal expression pathways of α-glucosidase and α-amylase; specific examples of the diseases are inflammation, cancer of various tissues and organs, hyperuricemia, diabetes, hyperglycemia, hyperlipidemia, cardiovascular disease, vascular sclerosis, premature aging, etc.

[0034] Furthermore, the skin care products are functional skin care products with anti-aging, anti-sugar or anti-inflammatory effects, including emulsions, gels, creams, liquids or films for cleansing, skin care or sun protection.

[0035] The beneficial effects of one or more of the above technical solutions are:

[0036] The present invention uses Umbelliferae plants as raw materials, and screens out highly active flavonoid extracts therefrom in a simple and easy manner. The extracts contain a relatively high content of highly active monomer combinations, which can achieve comprehensive antioxidant, anti-inflammatory and hypoglycemic effects through the synergistic effect between the monomer components, and are expected to be applied to the addition of drugs, nutritional products and skin care products. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0038] Figure 1 is the chromatogram of flavonoid glycosides obtained in Example 1;

[0039] Figure 2 The chromatogram of flavonoid glycosides of the n-butanol extract of Shandong celery seeds obtained in Example 1;

[0040] Figure 3 The chromatogram of flavonoid glycosides of the n-butanol extract of Guangxi celery seeds obtained in Example 1;

[0041] Figure 4 The chromatogram of flavonoid glycosides of the n-butanol extract of Northeastern celery seeds obtained in Example 1;

[0042] Figure 5 The chromatogram of flavonoid glycosides of n-butanol extract of Jiangsu celery seeds obtained in Example 1;

[0043] Figure 6 The chromatogram of flavonoid glycosides of Hubei celery seed n-butanol extract obtained in Example 1;

[0044] Above Figure 1-Figure 6 Among them, peak 1 is apigenin A, peak 2 is luteolin-O-glucoside, peak 3 is apigenin, peak 4 is apigenin B, peak 5 is apigenin-O-glucoside, and peak 6 is keuridin-7-O-glucoside. DETAILED DESCRIPTION

[0045] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0046] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0047] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0048] Example 1

[0049] In this embodiment, a flavonoid extract from seeds of an Umbelliferae plant is provided. Taking celery seeds as an example, the preparation method of the extract is as follows:

[0050] Weigh 500g of dried celery seeds and crush them to obtain celery seed powder. Add 3 times the volume of ethyl acetate to the celery seed powder and extract at room temperature. The extraction is repeated 3 times. The reflux extraction time is 5 hours. The solid powder in the separation reflux system is the defatted celery seed powder.

[0051] The defatted celery seed powder (60 g) was added with 600 mL of ethanol and heated to reflux at 80° C. for 1.5 h. The above operation was repeated three times, and the extracts were combined and concentrated under reduced pressure to obtain celery seed extract.

[0052] Dichloromethane (DCM) is added to the extract to make pulp, and filtered to obtain a dichloromethane (DCM) extract and a filter cake; water is added to the filter cake to make it evenly dispersed, and n-butanol is added to extract three times, the filtrate is combined, and concentrated under reduced pressure to obtain the n-butanol extract of celery seeds, that is, the Umbelliferae plant seed flavonoids extract provided by the present invention, and the remainder is the celery seed water extract.

[0053] The extraction methods of fennel seeds and cnidium monnieri are similar to the above-mentioned celery seed extract.

[0054] Activity determination of extracts from seeds of Umbelliferae plants

[0055] 1. Determination of antioxidant capacity

[0056] 1. Determination of polyphenol content

[0057] Determination of polyphenol content in Umbelliferae seed extracts: ddH 2 Dilute 0.25N Folin-Ciocalteu reagent (50 μL) with DMSO (0-100 μg / mL). Add each extract or gallic acid (50 μL) to a 96-well microplate. Incubate for 5 min and add 5% Na 2 CO 3 Solution (100 μL). After 15 min of dark incubation at room temperature, the absorbance of the supernatant was measured at 750 nm. The TPC of the extract was determined using a standard calibration curve of gallic acid. In the range of 0-30 μg / mL, R 2 The concentration of TPC is expressed as mg of gallic acid equivalent (GAE) per gram of dry extract. All measurements were performed in triplicate.

[0058] 2. Determination of flavonoid content

[0059] Determination of flavonoid content in Umbelliferae seed extract: 100 μL of 2% AlCl 3 The aqueous solution was added to 100 μL of the sample solution (200 μg / mL, final concentration). After incubation for 15 minutes, the absorbance of each sample was tested at 420 nm. Rutin was used as a reference standard (calibration curve of different concentrations: 0-50 μg; absorbance y = 0.0135 x (μg rutin) + 0.0093, R 2 =0.9996) in order to express TFC as rutin equivalent (RE) in mg / g dry weight of extract.

[0060] Table 1 TPC, TFC, main flavonoid glycosides content and yield of extracts from different fractions of Umbelliferae plants

[0061]

[0062]

[0063] All values ​​are reported as the mean standard deviation of three independent experiments. A is 0-10 mg GAE / g or mg RE / g; B is 10-50 mg GAE / g or mg RE / g; C is 50-100 mg GAE / g or mg RE / g; D>100 mg GAE / g or mg RE / g. GAE: gallic acid equivalent; RE: rutin equivalent. Yield is calculated as % yield = (extract weight / initial weight of dry sample)*100%; average values ​​with different superscripts in the same column (a-n) The difference is significant (p<0.05).

[0064] From the results in Table 1 above, it can be seen that there are great differences in the antioxidant effects of the Umbelliferae seed extracts. Among the three Umbelliferae seed extracts, the n-butanol fraction (i.e., the flavonoid extract provided by the present invention) has the highest TPC and TFC values. The gallic acid equivalent and rutin equivalent in the extract are greater than those in the DCM extract and greater than those in the water extract. In addition, the gallic acid equivalent in the n-butanol extract can reach an average of more than 50 mg / g, and the rutin equivalent can reach more than 100 mg / g, indicating that the n-butanol extract is rich in polyphenols and flavonoid active ingredients.

[0065] 3. Free radical scavenging effect

[0066] (1) DPPH free radical scavenging experiment: 100 μL of sample solutions with different concentration gradients were mixed with 100 μL of DPPH solution, stored at room temperature and protected from light for 30 min, and the absorbance was measured at 513 nm. Each test was repeated three times to obtain the average scavenging rate.

[0067] DPPHRSA(%)=(A 0 -As) / A 0 *100%

[0068] A 0 = absorbance value of blank solution,

[0069] As = absorbance value of sample solution.

[0070] (2) ABTS free radical scavenging experiment: 10 μL of sample solution with different concentration gradients was mixed with 200 μL of ABTS solution, stored at room temperature in the dark for 10 min, and the absorbance was recorded at 734 nm. Each test was repeated three times to obtain the average scavenging rate.

[0071] ABTS RSA (%) = (A 0 -As) / A 0 *100%

[0072] A 0= absorbance value of blank solution,

[0073] As = absorbance value of sample solution.

[0074] 4. Determination of total antioxidant capacity

[0075] FRAP experiment: Working solution was mixed with acetate buffer (pH 3.6), ferric chloride solution (20 mM), and TPTZ solution (10 mM TPTZ in 40 mM HCl) in a ratio of 10:1:1, respectively, and prepared freshly before use. 180 μL of working solution was mixed with 20 μL of diluted sample. Incubate at room temperature for 30 min. Absorbance was measured at 593 nm. The standard curve was linear between 0 and 30 μg Trolox, R 2 The value is 0.9999. The results are expressed as mg TE / g dry weight. If the measured FRAP value exceeds the linear range of the standard curve, additional dilutions are required.

[0076] 5. Determination of copper ion reducing ability

[0077] CUPRAC experiment: 50 μL of sample solution was mixed with 150 μL of CUPRAC solution (composed of 10 mM CuCl2, 7.5 mM neocuproine and 1 M NH4Ac buffer pH 7; ratio ratio (v / v / v)). After incubation at room temperature for 15 minutes, the absorbance of the reaction mixture at 450 nm was measured. The results are in mgTE / g extract (calibration curve of different concentrations: 070 μg / mL; absorbance: y=0.0279x+0.0126, R=0.9997).

[0078] 6. Determination of metal chelating ability

[0079] Fe 2+ Complexation experiment: 50 μL of each sample solution was mixed with 1 mM FeCl 2 50 μL was mixed. Then, 100 μL of 2.5 mM ferrous hydrazine solution was added to start the reaction. The solution was stored at room temperature for 10 min and the absorbance at 562 nm was measured. The results were expressed as ethylenediaminetetraacetic acid equivalent (EDTA) (mgEDTAE / g extract).

[0080] Table 2 Antioxidant activity of different fractions of extracts from seeds of Umbelliferae

[0081]

[0082] All values ​​are reported as the mean ± SD of three independent experiments. A is 0-20 μg / mL or mg TE / g; B is 20-100 μg / mL or mg TE / g; C is 100-200 μg / mL or mg TE / g; D is > 200 μg / mL or mg TE / g.

[0083] W is 0-5 mg EDTAE / g; X is 5-10 mg EDTAE / g; Y is 10-15 mg EDTAE / g; Z is >15 mg EDTAE / g. TE: Trolox equivalent; EDTAE: EDTA equivalent. Different superscripts in the same column (a-o) The mean values ​​were significantly different (p<0.05).

[0084] According to the results in Table 2 above, the flavonoid extract provided by the present invention has comprehensive free radical scavenging activity, antioxidant capacity and metal ion complexing ability, and can exert good antioxidant effects in both aqueous and organic matrices. The above extract is more suitable for adding to antioxidants, diseases caused by excessive oxidation, such as inflammation, cancer, hyperuricemia, hyperglycemia, hyperlipidemia drugs or cosmetics.

[0085] In order to further clarify the specific monomer components in the n-butanol extract that exert effective antioxidant activity, this example analyzes the monomer components of the n-butanol extracts of the three Umbelliferae seeds prepared in Example 1. By identifying and screening the monomer components in the n-butanol extracts of the three plant seeds, this example selects 18 monomer components and purchases corresponding reference substances to investigate the antioxidant properties of the monomer compounds (as shown in 1-18 in Table 3 below). In addition, this example also investigates the synergistic effect between the monomer compounds. After investigation, quercetin and luteolin combined in different proportions (as shown in 19-24 in Table 3 below) all show good antioxidant properties:

[0086] Table 3 Antioxidant effects of main flavonoid glycosides and their aglycones in seeds of Umbelliferae

[0087]

[0088]

[0089] All values ​​are reported as mean ± standard deviation of three independent experiments.

[0090] A is 0-20 μg / mL or mg TE / g; B is 20-200 μg / mL or mg TE / g; C is 200-500 μg / mL or mg TE / g; D>500 μg / mL or mg TE / g; N means that the corresponding test was not performed.

[0091] W is 0-10 mg EDTAE / g; X is 10-20 mg EDTAE / g; Y is 20-50 mg EDTAE / g; Z is >50 mg EDTAE / g. TE: Trolox equivalent; EDTAE: EDTA equivalent. Compared with the control group: *P<0.05; **P<0.01; ***P<0.001.

[0092] The 18 monomers in Table 3 above come from the seeds of the three Umbelliferae plants described in Example 1. By comparing with the positive oxidants BHT and VC, it can be seen that some monomer components such as celery glycoside A, celery glycoside B, apigenin, luteolin glycoside, koiricin-7-O-glucoside, apigenin-7-O-glucoside and other six monomer components in celery seeds, rutin, hyperoside, kaempferol, etc. contained in cnidium seeds, kaempferol, etc. contained in fennel seeds, and myricetin in carrot seeds all have good antioxidant effects. In addition, this example also measures the synergistic antioxidant effect between flavonoid monomers, and determines the combined effect by the combined action index CI value. When the CI value is <1, there is a synergistic effect between the monomers; when CI=1, there is an additive effect between the monomers; when CI>1, there is an antagonistic effect between the monomers. The calculation method is as follows:

[0093]

[0094] Wherein: D1 and D2 are the mass concentrations (μg / mL) when the inhibition rate of the combined action of the two monomers is 50%, and Dx1 and Dx2 are the mass concentrations (μg / mL) when the inhibition rate of the two monomers alone is 50%.

[0095] According to the above calculation results, the combined action index is less than 1, indicating that they have a synergistic antioxidant effect, among which quercetin / luteolin = 1 / 3 has the smallest combined action index and the strongest synergistic antioxidant ability.

[0096] From the results in Table 3 above, it can be seen that the six components of celery seed n-butanol extract, including celery glycoside A, celery glycoside B, apigenin, luteolin glycoside, keuridin-7-O-glucoside, and apigenin-7-O-glucoside, showed strong antioxidant capacity in various antioxidant capacity tests and can be considered as monomer compounds with high antioxidant activity.

[0097]

[0098] In order to confirm whether the comprehensive antioxidant capacity of the extract of the present invention is derived from the above-mentioned monomer compounds, the present invention conducted a determination on the monomers of six main highly active flavonoids in the extract, and the results are shown in the following Table 4:

[0099] Table 4 Determination of the contents of six highly active flavonoid monomers in the extracts

[0100]

[0101] Combined with the results in Table 1, it can be seen that the antioxidant activity of celery seeds from Shandong, Guangxi, Northeast China and Hubei is relatively high, and the contents of the six highly active monomer components in the extracts from the above-mentioned production areas are also relatively high.

[0102] 2. Characterization of Anti-inflammatory Activity

[0103] 1. Anti-inflammatory damage:

[0104] Cell culture: RAW264.7 cells were cultured in DMEM containing 10% fetal bovine serum at 37°C and 5.0% CO. 2 cultured in a cell culture incubator.

[0105] Drug administration and grouping: RAW264.7 cells in the logarithmic growth phase were divided into a control group (cultured in complete medium containing 10% fetal bovine serum), a lipopolysaccharide model group (cultured in complete medium containing 1 μg / mL lipopolysaccharide for 24 h), and different components of celery seed extract and its main flavonoid components (different concentrations of celery seed extract and its main flavonoid components) pretreated for 2 h, and then continued to be cultured with complete medium containing 1 μg / mL lipopolysaccharide and corresponding concentrations of celery seed extract and its main flavonoid components for 24 h), with at least 3 replicates for each group.

[0106] 2. Determination of NO content

[0107] Cells in the logarithmic growth phase were inoculated into 96-well plates, and RAW264.7 cells were stimulated with different concentrations of lipopolysaccharide alone for 24 hours, and the cells were pretreated with different concentrations of celery seed extract and its main flavonoid components for 2 hours, and then continued to be cultured with 1μg / mL lipopolysaccharide for the experiment. Each group had 6 replicate wells. After the culture was completed, the NO content was determined according to the instructions of the kit.

[0108] 3. ELISA detection of related cytokines

[0109] RAW264.7 cells in the logarithmic growth phase were taken. After the drug culture was completed, the supernatant of each group of cells was aspirated and added to a 96-well plate. According to the operating instructions of the ELISA kit, the contents of cytokines 1L-6, 1L-1β and 1L-10 were determined and statistically analyzed.

[0110] Table 5 Anti-inflammatory effects of different fractions of extracts from seeds of Umbelliferae

[0111]

[0112]

[0113]

[0114] All values ​​are reported as the mean ± standard deviation of three independent experiments. A is 0-5 μmmol / L; B is 5-10 μmmol / L; C is 10-15 μmmol / L; D is >15 μmmol / L.

[0115] W is 0-10ng / L; X is 10-20ng / L; Y is 20-30ng / L; Z>30ng / L. Compared with the control group: *P<0.05; Compared with the LPS-induced group: # P<0.05.

[0116] The results in Table 5 show that the n-butanol fraction of Umbelliferae seeds has a stronger anti-inflammatory effect than other fractions, followed by DCM fraction and water fraction. Moreover, the anti-inflammatory effects of different Umbelliferae seeds or the same Umbelliferae seeds from different origins are significantly different, indicating that factors such as origin and variety can affect their anti-inflammatory effects. In addition, different fractions of Umbelliferae seeds have certain anti-inflammatory effects, and they are concentration-dependent.

[0117] Similarly, in order to investigate whether the monomer compounds in the three Umbelliferae plant seed extracts also have anti-inflammatory activity, this example screened 11 of the 18 monomer compounds with better effects for anti-inflammatory activity determination, and also determined the anti-inflammatory effect of the combination of quercetin and luteolin. The results are shown in Table 6:

[0118] Table 6 Anti-inflammatory effects of major flavonoid glycosides and their aglycones in seeds of Umbelliferae

[0119]

[0120]

[0121]

[0122] All values ​​are reported as the mean ± standard deviation of three independent experiments. A is 0-5μmmol / L; B is 5-10μmmol / L; C is 10-15μmmol / L; D is >15μmmol / L. W is 0-10ng / L; X is 10-20ng / L; Y is 20-30ng / L; Z>30ng / L. Y is 20-30ng / L; Z>30ng / L. Compared with the control group: *P<0.05; compared with the LPS-induced group: # P<0.05.

[0123] From the results in Table 6, it can be seen that the above six flavonoid monomer components also have good anti-inflammatory activity. The number of hydroxyl groups, hydroxyl position, saturation at positions 3 and 4 of the B ring, and substituents in flavonoids will affect the anti-inflammatory ability of flavonoid compounds. In addition, in the study of Table 6 above, this embodiment also measured the synergistic anti-inflammatory effect between flavonoid monomers, and determined the combined effect by the combined effect index CI value. When the CI value is <1, there is a synergistic effect between the monomers; when CI=1, there is an additive effect between the monomers; when CI>1, there is an antagonistic effect between the monomers. The calculation method is as follows:

[0124]

[0125] Wherein: D1 and D2 are the mass concentrations (μg / mL) when the inhibition rate of the combined action of the two monomers is 50%, and Dx1 and Dx2 are the mass concentrations (μg / mL) when the inhibition rate of the two monomers alone is 50%.

[0126] According to the above test results, it is shown that different ratios of quercetin and luteolin have synergistic or additive effects, and do not show antagonistic effects. In addition, quercetin / luteolin = 1 / 3 still shows the best synergistic anti-inflammatory ability.

[0127] 3. Characterization of hypoglycemic effect

[0128] 1. α-Glucosidase inhibition test

[0129] Prepare PBS (0.1M, pH=6.8), α-glucosidase (5U / mL) and PNPG (5mM) and store at 4℃ for later use. Add different concentrations of sample solution (dissolved in DMSO) or 20μL of DMSO, 60μL of PBS, and 20μL of α-glucosidase (0.5U / mL) to a 96-well plate and incubate at 37℃ for 20min. Add 20μl of PNPG (5mM) solution and incubate at room temperature for another 15min. Add Na 2 CO 3 The reaction was terminated by adding 80 μL of (0.1 M) solution. After the reaction was completed, the absorbance of each well was measured at 405 nm. The inhibition rate (%) was calculated according to the absorbance, and the formula was as follows: Inhibition rate (%) = (A 0 -As) / A 0 *100%, of which A 0 is the absorbance of the blank group, and As is the absorbance of the test compound.

[0130] 2. α-Amylase inhibition test

[0131] Prepare PBS (0.1M, pH=6.8), α-amylase (10U / mL) and 0.2% soluble starch and store at 4°C for later use. Mix different concentrations of extracts or compounds (dissolved DMSO) or DMSO (20μL) with α-amylase solution (60μL, 1U / mL) in a 96-well plate and incubate at 37°C for 20min. Subsequently, add 0.2% soluble starch (20μL) as a substrate and incubate in a 37°C dry bath for another 15min. Add 1M HCl solution (20μL) to terminate the reaction. Finally, add 80μL of iodine reagent solution and absorbance at 620nm. The inhibition rate (%) is calculated based on the absorbance, and the formula is as follows: Inhibition rate (%) = (A 0 -As) / A 0 *100, where A 0 is the absorbance of the blank group, and As is the absorbance of the test compound.

[0132] Table 7 α-glucoside and α-amylase inhibition effects of different fractions of extracts from seeds of Umbelliferae

[0133]

[0134]

[0135] All values ​​are reported as the mean ± standard deviation of three independent experiments. A is 0-50 μg / mL; B is 50-100 μg / mL; C is 100-200 μg / mL; D is >200 μg / mL. (a-h) The mean values ​​were significantly different (p<0.05).

[0136] The results of in vitro enzyme experiments show that the flavonoid extract provided by the present invention has higher α-glucosidase inhibitory activity than other fractions, and some DCM fractions have moderate α-glucosidase inhibitory activity, such as Northeast celery seeds. The results of amylase inhibition experiments show that the DCM fraction has a stronger inhibitory activity, while the inhibitory activity of the n-butanol fraction is relatively weak.

[0137] Table 8 α-glucosides and α-amylase inhibitory effects of main flavonoid glycosides and their aglycones in seeds of Umbelliferae

[0138]

[0139] All values ​​are reported as the mean ± standard deviation of three independent experiments. A is 0-50 μM; B is 50-100 μM; C is 100-150 μM; D is >150 μM. W is 0-1 mM; B is 1-1.5 mM; C is 1.5-2 mM; D is >2 mM.

[0140] The results show that the flavonoids in the seeds of the Umbelliferae family are the main components of the hypoglycemic effect. The number of hydroxyl groups, the position of hydroxyl groups, the saturation of the 3 and 4 positions of the B ring, and the substituents in the flavonoids will affect the enzyme inhibition ability of flavonoids. The a-glucosidase inhibition experiment showed that monosaccharide glycosylation did not affect the enzyme inhibition effect of flavonoids, but disaccharide glycosylation greatly reduced the enzyme inhibition effect. The a-amylase inhibition experiment was just the opposite, and glycosylation greatly reduced the amylase inhibitory activity of flavonoids. In any case, our experiments found that the main flavonoids in the seeds of the Umbelliferae family have certain a-glucosidase and a-amylase inhibitory effects, and are one of the hypoglycemic components in the seeds of the Umbelliferae family.

[0141] 3. Experiment on hypoglycemic cell activity:

[0142] Mouse NIT-L1 pancreatic β cells were inoculated into RP-MI1640 dry powder culture medium and incubated at an ambient temperature of 37°C and an air condition of 5% CO. 2 , saturated humidity, and cultured under the conditions of 100% to 200% humidity. Use an inverted microscope to observe the growth of cells at any time, and change the medium every 2 to 3 days. When the cells adhere to the wall for 80% to 90%, culture them in bottles. At the same time, take the cells in the logarithmic phase and culture them with alloxan and celery seed extract and its main flavonoid component culture medium, respectively. Set up the experimental group, the blank group is neither of them, and the control group is cultured only with alloxan culture medium. The low dose in the experimental group is 200μg / mL; the medium dose is 400μg / mL; the high dose is 800μg / mL, the culture time is 24h, and the culture medium of the same period is retained.

[0143] The survival rate of NIT-L1 pancreatic β cells was indirectly determined by MTT method, wherein the detection wavelength was 590 nm, and the cell survival rate (%) = (OD value experimental group / OD value control group) × 100%; the detection of NO, SOD, and MDA contents was carried out strictly according to the instructions on the kit.

[0144] Table 9 Cellular hypoglycemic ability of extracts from different fractions of Umbelliferae seeds

[0145]

[0146]

[0147]

[0148]

[0149] All values ​​are reported as the mean standard deviation of three independent experiments. A is 0-50μmol / L; B is 50-100μmol / L; C is 100-200μmol / L; D is >200μmol / L. W is 0-5μmol / L; X is 5-10μmol / L; Y is 10-20μmol / L; Z is >20μmol / L. Compared with the control group: *P<0.05; compared with the model group: # P<0.05.

[0150] The results show that at the cellular level, the flavonoid extract provided by the present invention has the strongest hypoglycemic effect and is concentration-dependent. In addition, there are significant differences in the hypoglycemic effects of different Umbelliferae seeds or the same Umbelliferae seeds from different origins, indicating that factors such as origin and variety can affect their hypoglycemic effects. But in any case, Umbelliferae seeds have a certain ability to lower blood sugar.

[0151] Table 10 Hypoglycemic effects of main flavonoid glycosides and their aglycones in seeds of Umbelliferae at cellular level

[0152]

[0153]

[0154] All values ​​are reported as the mean standard deviation of three independent experiments. A is 0-50μmol / L; B is 50-100μmol / L; C is 100-200μmol / L; D is >200μmol / L. W is 0-5μmol / L; X is 5-10μmol / L; Y is 10-20μmol / L; Z is >20μmol / L. Compared with the control group: *P<0.05; compared with the model group: # P<0.05.

[0155] The results show that the flavonoids in the seeds of the Umbelliferae family are the main components of the hypoglycemic effect. The number of hydroxyl groups, the position of hydroxyl groups, the saturation of the 3 and 4 positions of the B ring, and the substituents in the flavonoids will affect the hypoglycemic ability of flavonoids at the cellular level. In addition, the hypoglycemic ability of flavonoids is concentration-dependent. In any case, our experiments found that the main flavonoids in the seeds of the Umbelliferae family have a hypoglycemic effect at the cellular level and are one of the hypoglycemic components in the seeds of the Umbelliferae family.

[0156] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of a composition of flavonoid monomer compounds in Umbelliferae plant seeds in the preparation of antioxidant and anti-inflammatory drugs, It is characterized in that The flavonoid monomer compound composition consists of quercetin and luteolin, and the ratio of quercetin to luteolin is 1-9:1-9.

2. The use as claimed in claim 1, It is characterized in that The plant seeds are celery seeds, fennel seeds or cnidium monnieri.

3. A method for preparing a flavonoid extract from Umbelliferae seeds, It is characterized in that The high-activity monomer flavonoid composition accounts for more than 50% of the extract; the high-activity monomer flavonoid composition consists of apigenin A, apigenin B, apigenin, luteolin, keuridin-7-O-glucoside, and apigenin-7-O-glucoside; The steps include: The dried plant seeds are crushed and ethyl acetate is added for degreasing; the defatted plant seeds are transferred into ethanol for reflux extraction, and the extracts are combined and dried to obtain an extract; the extract is added into dichloromethane, fully stirred and filtered, the solid part is obtained and dissolved in water, and then n-butanol reagent is added for extraction, and the organic phase is retained and dried.

4. The preparation method according to claim 3, It is characterized in that The defatting treatment steps are as follows: add ethyl acetate to the crushed plant seeds and extract them at room temperature for 3-5 times, adding ethyl acetate 3-5 times the volume of the plant seed powder each time, and the total extraction time is 3-5 hours. The solid powder obtained after filtering is the defatted plant seeds.

5. The preparation method according to claim 3, It is characterized in that The specific steps of preparing the extract are as follows: adding 5-10 times the volume of ethanol to the defatted plant seeds, reflux extraction at 75-85° C. for 3-5 times, each reflux extraction time is 1-3 hours, and the combined extracts are concentrated under reduced pressure and dried to obtain the extract.

6. Use of a composition of flavonoid monomer compounds in Umbelliferae seeds in the preparation of antioxidant skin care products, It is characterized in that The flavonoid monomer compound composition consists of quercetin and luteolin, and the ratio of quercetin to luteolin is 1-9:1-9; The dosage form of the skin care product is emulsion, gel, cream or film.

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