CN116172201B - Tagetes extract and preparation method and application thereof - Google Patents

Tagetes extract and preparation method and application thereof Download PDF

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CN116172201B
CN116172201B CN202310273082.6A CN202310273082A CN116172201B CN 116172201 B CN116172201 B CN 116172201B CN 202310273082 A CN202310273082 A CN 202310273082A CN 116172201 B CN116172201 B CN 116172201B
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extract
marigold
acid
solution
marigold extract
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CN116172201A (en
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陆柏益
朱宇航
陈祁
王章铁
汪怿璇
彭渴婕
潘倩囡
朱煜康
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Zhejiang University ZJU
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/40—Feeding-stuffs specially adapted for particular animals for carnivorous animals, e.g. cats or dogs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/52—Adding ingredients
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

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Abstract

The invention discloses a marigold extract and a preparation method and application thereof, and belongs to the technical field of plant extraction. The preparation method comprises the following steps: (1) dry marigold products are prepared according to 1g: adding the solution with the volume ratio of 20-60 mL into an organic solvent aqueous solution with the volume ratio of 40-60%, adjusting the pH value to 7-9, leaching at 20-65 ℃, and separating to obtain an extract; (2) Removing organic solvent from the extractive solution, and drying to obtain flos Tagetis Erectae extract. The technology of the invention realizes the efficient enrichment and extraction of the antioxidant and anti-aging functional components in the marigold, and the main components in the extract provided by the invention are relatively high in ellagic acid and quinic acid content. The extract has strong antioxidant and anti-aging capabilities, and has great application prospects in the fields of functional foods, pet foods, medical products and the like.

Description

Tagetes extract and preparation method and application thereof
Technical Field
The invention relates to the technical field of plant extraction, in particular to a marigold extract and a preparation method and application thereof.
Background
Skin aging refers to aging caused by environmental factors such as light and air pollution and lifestyle factors such as diet, sleep and exercise, and is mainly manifested by wrinkles, loose skin, rough, yellowish or sallowness skin discoloration, telangiectasia, pigmentation, etc. Common skin aging includes both natural aging and photoaging. The latter refers to skin aging due to environmental factors (UV radiation, smoking, drinking, blowing, cold and hot stimulation, contact with harmful chemicals, etc.), which are major factors in accelerating skin aging and accelerating the natural aging process. It is estimated that about 90% of the damage to the exposed skin is caused by UV radiation, and long-term exposure to UV radiation causes skin roughness, redness, swelling, pain, pigmentation, telangiectasia, deepening and thickening of wrinkles, etc., which affect beauty and beauty, thereby causing a series of skin lesions such as solar keratosis, solar lentigo, solar elastosis, gum-like papule, etc., severely affecting human health. The mechanisms responsible for photoaging of skin are diverse and extremely complex, mainly involving oxidative stress, DNA damage, collagen loss, inflammatory reactions, etc.
With increasing importance of health and safety of foods, various natural products with unique physiological activities are increasingly favored. Products with various plant extracts (especially from flower parts) as functional ingredients are becoming hot spots in the market. The natural flowers contain a large amount of antioxidant substances, can effectively remove or inhibit the generation of ROS, or can increase the content of collagen fibers and collagen protein of the skin by up-regulating antioxidant defense system and inhibiting skin inflammatory reaction, so that the natural flowers have great development potential in the fields of aging resistance and the like.
Tagetes (TAGETES ERECTA L.) is an annual herb of Tagetes genus of Compositae family, the head flower is single-born, the tongue flower is yellow or dark orange, and the tubular flower corolla is yellow. Tagetes are widely cultivated as ornamental plants worldwide, and the understanding of the Tagetes is mainly focused on ornamental value at present, but the Tagetes have very high edible and medicinal values in practice. The dried flower bud contains abundant bioactive substances (flavonoids, pigments, phenolic acid compounds, volatile oil, tannins, proteins, glycosides, etc.), and has pharmacological effects of resisting tumor, bacteria, viruses, and oxidation, thus having high edible and medicinal values. The petals of the marigold have the effects of clearing heat and detoxicating, resolving phlegm and relieving cough, and have good effects on respiratory tract infection, bronchitis, keratitis and the like.
In the aspect of development and utilization of actual marigold resources, the main processing technology is still dry, and only a small part of the marigold resources are processed into scented tea. The extraction and utilization of the marigold often is to obtain crude water extract, and the actual functional components of the corresponding activity of the marigold are not clear. Literature (Edible Flowers of Tagetes erecta L.as Functional Ingredients:Phenolic Composition,Antioxidant and Protective Effects on Caenorhabditis elegans) reports that the compound is applied to anti-aging medicines or foods, but the main functional factors and the action mechanisms are unknown.
Disclosure of Invention
The invention aims to provide a method for efficiently extracting active ingredients of marigold, and further research on the antioxidant and anti-inflammatory effects of the marigold extract so as to fill the research gap of the marigold extract in anti-aging ingredients at present.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention provides a preparation method of a marigold extract, which comprises the following steps:
(1) Dried marigold is prepared according to 1g: adding the solution with the volume ratio of 20-60 mL into an organic solvent aqueous solution with the volume ratio of 40-60%, adjusting the pH value to 7-9, leaching at 20-65 ℃, and separating to obtain an extract;
(2) Removing organic solvent from the extractive solution, and drying to obtain flos Tagetis Erectae extract.
The flos Tagetis Erectae refers to flower buds, petals, pistil, etc. of flos Tagetis Erectae plant which is not opened or is initially opened. The variety of Tagetes useful in the present invention is not particularly limited and may be different varieties of Tagetes.
The raw materials adopted by the invention are marigold dry products, namely fresh marigold or common sun-dried marigold which is dried by hot air until the moisture content is not more than 10 percent. The raw materials can be subjected to crushing pretreatment or not. Preferably, the dried marigold product is crushed, which is beneficial to the dissolution of active substances.
The research of the invention shows that the marigold extract has the effects of resisting oxidation, whitening, delaying body aging and the like. Further identifying skin care functional components in the marigold, and definitely determining main active components in the marigold extract to comprise: ellagic acid, quinic acid, syringic acid, gallic acid, isoquercitrin and luteolin, wherein luteolin was first found in marigold. In order to increase the content of effective active ingredients in the extract, the extraction method is optimized by taking ingredient properties as guidance.
In the step (1), the contents of total flavonoids and total phenols in the marigold extract are comprehensively improved by controlling the feed-liquid ratio, the pH condition, the extraction temperature and the water content of the raw materials, so that higher extraction rate is realized.
Preferably, the organic solvent is ethanol, methanol or n-butanol, and the active ingredients can be effectively extracted under the condition of the organic solvent aqueous solution. More preferably, the volume concentration of the aqueous organic solvent solution is 50%.
Preferably, the water content of the marigold dry product is less than or equal to 10%, and the water content is controlled below 10%, so that the content of active ingredients in the extract is improved.
Preferably, the feed liquid ratio of the marigold dry product to the organic solvent aqueous solution is 1g:40mL.
The research of the invention shows that in the extraction process, the pH value of the feed liquid is regulated to 7-9, so that the solubility of ellagic acid and quinic acid can be increased, and the content of the two components in the extract can be improved. Ellagic acid has antioxidant, antiinflammatory and neuroprotective activities, quinic acid has good antioxidant, anticancer and antiaging activities, and the content of the two components is increased to enhance the antioxidant and antiaging effects of the extract.
Preferably, the pH value is adjusted to 8+/-0.3 by using an alkaline agent; the alkaline agent is sodium hydroxide, sodium carbonate, sodium bicarbonate, trisodium bicarbonate, potassium carbonate or potassium bicarbonate.
The extraction temperature has a large influence on the extraction of the target component, particularly the flavonoid component, and preferably, the extraction temperature is 45 to 60 ℃. More preferably, the temperature of leaching is 50 ℃.
Preferably, the time of leaching is 20 to 30 hours.
Preferably, the leaching process is combined with ultrasonic treatment under the following ultrasonic conditions: ultrasonic frequency is 300-500 kHz, ultrasonic intensity is 5-15W/cm 2, time is 1.5-3.0 h, and standing is carried out after ultrasonic treatment is finished. More preferably, the ultrasonic frequency is 400kHz, and the ultrasonic treatment is carried out for 2 hours under the condition of 10W/cm 2.
Separating the extract from the precipitate, evaporating under reduced pressure to remove organic solvent in the extract to obtain concentrated solution, and lyophilizing to obtain flos Tagetis Erectae extract.
The invention provides a marigold extract prepared by the method. The active ingredients of the marigold extract are mainly ellagic acid, quinic acid, syringic acid, gallic acid, isoquercitrin and luteolin.
The marigold extract prepared under the conditions has the total flavone content of 16-30% and the total phenol content of 50-72%; wherein the weight content of ellagic acid is 1.2% -6.2%, the weight content of quinic acid is 0.4% -1.2%, the weight content of isoquercitrin is 0.5% -2.7%, the weight content of syringic acid is 0.3% -0.45%, the weight content of gallic acid is 0.06% -0.3%, and the weight content of luteolin is 0.04% -0.1%.
The invention performs efficacy study on the prepared marigold extract, and in-vitro and in-vivo experiments prove that the marigold extract has stronger oxidation resistance and anti-aging capability, and can effectively play the roles of delaying aging, whitening and the like after being eaten, so that the marigold extract can be used for preparing related foods or medical products.
Thus, the invention provides the use of the marigold extract in the preparation of a functional food or pet food.
Further, the functional food is a food such as snack (biscuits and the like) or beverage with the effects of resisting aging and prolonging life. The pet food is pet feed, pet can or pet snack with the effects of resisting aging and prolonging life.
The present invention also provides a composition (including food composition, etc.) containing the marigold extract of the present invention as an active ingredient. These compositions are used for delaying aging, resisting oxidation, and relieving inflammation by eating.
Further, the composition is composed of 0.01 to 99.9wt% of the marigold extract and the balance of the carrier.
The marigold extract is used for preparing health food or beverage with anti-aging, anti-oxidation and other effects, and specific forms include, but are not limited to, fruit and vegetable products, meat, egg and milk products, baked products, frozen foods, grain products, aquatic products, candies, cocoa products, sweeteners, seasonings, beverages, wines, special meal foods and the like.
The invention has the beneficial effects that:
(1) The invention realizes the efficient enrichment and extraction of the anti-aging functional components in the marigold, strictly controls the water content of the raw materials and the leaching conditions to improve the extract yield, and further adjusts the pH value of the feed liquid to 7-9 in the leaching process.
(2) The extract provided by the invention has relatively high content of ellagic acid, quinic acid and isoquercitrin, and the marigold extract is reported to contain luteolin for the first time. The extract has strong oxidation resistance and aging resistance, can effectively play roles in delaying aging, reducing skin pigmentation and the like when eaten, and has great application prospects in the fields of functional foods, pet foods and the like.
Drawings
Fig. 1 is a total ion flow diagram of marigold extract.
FIG. 2 shows the mass spectrum of ellagic acid parent ion in the extract.
FIG. 3 shows the mass spectrum of ellagic acid ions in the extract.
FIG. 4 is a mass spectrum of quinic acid parent ion in the extract.
FIG. 5 is a mass spectrum of quinic acid ions in the extract.
FIG. 6 is a mass spectrum of the mother eugenol in the extract.
FIG. 7 shows the mass spectrum of syringic acid ion in the extract.
FIG. 8 is a mass spectrum of gallic acid parent ion in the extract.
FIG. 9 is a mass spectrum of gallic acid ion in the extract.
FIG. 10 is a mass spectrum of isoquercitrin parent ion in the extract.
FIG. 11 is a chart showing the ion mass spectrum of isoquercitrin Pi Ganzi in the extract.
FIG. 12 is a mass spectrum of luteolin parent ion in the extract.
FIG. 13 is a mass spectrum of luteolin seed ions in the extract.
FIG. 14 shows the structural formulae of the 6 compounds.
Detailed Description
The invention will be further illustrated with reference to specific examples. The following examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
The raw material marigold flower buds are purchased from Qi Xitang flagship shops and baked for more than 24 hours at 40 ℃.
The following is a general method:
1) Determination of total flavone content:
An aluminum nitrate-sodium nitrite colorimetric method is adopted. Taking 2.5mL of marigold extract samples or rutin standard solution (0, 30, 60, 90, 120, 150 mg/L) diluted by a certain multiple, adding 150 mu L of 5% sodium nitrite solution, shaking uniformly, standing for 6min, adding 300 mu L of 10% aluminum nitrate solution, shaking uniformly, standing for 5min, adding 1mL of 1.0mol/L sodium hydroxide solution, fixing the volume to 6mL by water, and measuring the absorbance at 510 nm. The concentration of rutin solution and the absorbance value are respectively used as a standard curve, the total flavone content is calculated, and the result is expressed as the rutin equivalent in 100g marigold extract.
2) Determination of total phenol content:
Total phenol was determined by the Fu Lin Fen method. Taking 500 mu L of marigold extract samples or chlorogenic acid standard solution (0, 20, 40, 60, 80 and 100 mg/L) diluted by a certain multiple, adding 100 mu L of Fu Lin Fen reagent, uniformly mixing, reacting for 6min, adding 1mL of 7% Na 2CO3, fixing the volume to 6mL, reacting at the constant temperature of 37 ℃ for 90min, and measuring the absorbance at 750 nm. The concentration of chlorogenic acid solution and the absorbance value are respectively used as a standard curve, the total phenol content is calculated, and the result is expressed as chlorogenic acid equivalent in 100g of marigold extract.
3) Qualitative and quantitative analysis of the extract-UPLC high resolution mass spectrometry
And carrying out component qualitative analysis on the extracted part by UPLC-QE high-resolution mass spectrum. The assay conditions were ACQUITY BEH C 18 column (2.1 mm. Times.100 mm,Waters) was used for UPLC analysis at column temperature 40 ℃, flow rate 0.4mL/min, mobile phase 0.1% formic acid in water (a) and 0.1% formic acid-acetonitrile (B). The gradient procedure for the mobile phase is as follows: 0min (a: b=95:5), 3min (a: b=75:25), 4min (a: b=35:65) and 10min (a: b=35:65). The sample loading was 10. Mu.L. QE operates in positive and negative ion modes. The operating parameters were set as: cone voltage 30v, capillary voltage 2kv, source temperature 100 ℃. Data were recorded at a mass to charge ratio (m/z) range of 95 to 1400, with a scan time of 0.25s, and a scan interval of 0.02s for 10min. On this basis, matching and literature queries are retrieved in conjunction with mass spectrometry database Compound Discoverer TM to determine possible components. The new and higher content ingredients were quantitatively analyzed using a UPLC-PDA.
Example 1
1. The preparation method comprises the following steps: the marigold petals with the moisture content less than or equal to 10 percent (measured according to the GB 5009.3-2016 direct drying method, and dried at 40 ℃ if the moisture content is not met) are dried according to 1g:40mL of a feed liquid ratio, 50% (v/v) of ethanol aqueous solution was added, the pH of the feed liquid was adjusted to 8 to be alkaline by using 1mol/L Na 2CO3 solution, the temperature was controlled to 50 ℃, leaching was performed for 2 hours at an ultrasonic frequency of 400kHz and an ultrasonic intensity of 10W/cm 2 to sufficiently extract and stand for 24 hours. Separating the extract from the Tagetes Erectae precipitate by centrifugation, recovering ethanol by reduced pressure evaporation to obtain concentrated solution, and lyophilizing to obtain Tagetes Erectae extract.
2. The quality and components of the obtained extract are analyzed, the identification result of the components is shown in fig. 1-13, and it is clear that ellagic acid, quinic acid, syringic acid, gallic acid, isoquercitrin and luteolin are used as main active components, and the structural formula is shown in fig. 14. The specific content data are shown in table 1.
Example 2
The preparation method comprises the following steps: the marigold petals with the moisture content less than or equal to 10 percent (measured according to the GB 5009.3-2016 direct drying method, and dried at 40 ℃ if the moisture content is not met) are dried according to 1g:40mL of feed liquid ratio, 60% (v/v) of methanol aqueous solution was added, the pH of the feed liquid was adjusted to 7 using 1mol/L CH 3 COOH solution, the temperature was controlled to 45 ℃, the ultrasonic frequency was 400kHz, the ultrasonic intensity was 10W/cm 2, leaching was performed for 2 hours to sufficiently extract and standing for 24 hours. Separating the extract from the Tagetes Erectae precipitate by filtration, recovering methanol by reduced pressure evaporation to obtain concentrated solution, and lyophilizing to obtain Tagetes Erectae extract.
The quality and composition of the obtained extract were analyzed, and specific data are shown in Table 1.
Example 3
The preparation method comprises the following steps: the marigold flower buds with the moisture content less than or equal to 10 percent (measured according to the GB 5009.3-2016 direct drying method, and dried at 40 ℃ if the moisture content is not met) are dried according to 1g:60mL of feed liquid ratio, 40% (v/v) of n-butanol aqueous solution is added, the pH of the feed liquid is regulated to 9 by using 2mol/L NaOH solution, the temperature is controlled to be 60 ℃, the leaching is carried out for 2 hours at the ultrasonic frequency of 400kHz and the ultrasonic intensity of 10W/cm 2 so as to fully extract and stand for 24 hours. Separating the extract from the marigold precipitate by filtration, recovering n-butanol by reduced pressure evaporation to obtain concentrated solution, and lyophilizing to obtain marigold extract.
The quality and composition of the obtained extract were analyzed, and specific data are shown in Table 1.
Comparative example
1. Feed-liquid ratio condition
Comparative example 1-1 the feed ratio in example 1 was changed to 1g:10mL, the remainder being as in example 1.
Comparative examples 1-2 the feed ratio in example 1 was changed to 1g:70mL, the remainder being as in example 1.
2. Temperature conditions
Comparative example 2-1 the temperature in example 1 was changed to 15℃and the rest was the same as in example 1.
Comparative example 2-2 the temperature in example 1 was changed to 70℃and the rest was the same as in example 1.
3. PH conditions
Comparative example 3-1 the pH in example 1 was changed to 6.5, the pH adjusting solution was changed to CH 3 COOH solution, and the rest was the same as in example 1.
Comparative example 3-2 the pH in example 1 was changed to 9.5, the remainder being the same as in example 1.
4. Raw material water content condition
Comparative example 4-1 the moisture content in example 1 was changed to 15%, and the rest was the same as in example 1.
TABLE 1 Tagetes extract yield and content of each component under different preparation parameters
Yield unit: the%; total flavone content unit: rutin equivalent per 100g flower extract; total phenol content unit: chlorogenic acid equivalent per 100g flower extract; each main component unit: percent of the total weight of the composition.
As shown in Table 1, the ratio of feed to liquid, temperature, pH and water content of the raw materials all affect the yield of marigold extract and the content of active ingredients. Wherein the influence of temperature is large, and excessive temperature can destroy active ingredients, especially flavonoid ingredients, and ellagic acid can be degraded into gallic acid due to heating. While too low a temperature may result in low extraction yields. In addition, the pH of the leaching solution can increase the solubility of ellagic acid and quinic acid under alkaline conditions, so that the content of the ellagic acid and quinic acid in the extract is higher, but the content of flavonoid components can be reduced when the pH is too strong. In addition, when the moisture content of the raw material marigold is more than 10%, the extract yield may be lowered.
Activity detection example 1: research on in-vitro antioxidant capacity of marigold extract
The antioxidant capacity of the marigold extract in vitro in examples 1,2 and 3 was measured by DPPH, ABTS, ORAC, FRAP method, and the data are shown in Table 2. The specific method comprises the following steps:
1) DPPH method:
Before the experiment, 20mg of DPPH is accurately weighed and dissolved in a proper amount of methanol, and the volume is fixed to 500mL. Taking Trolox with 25-800 μm as reference substance, diluting 100 μl of extract solution or reference substance solution with a certain multiple, adding 3.9ml DPPH, reacting at 37deg.C in dark place for 60min, and measuring absorbance at 515 nm. The absorbance was plotted on the abscissa and the absorbance was plotted on the ordinate, with the results expressed as Trolox equivalents per gram of extract (mmol Trolox/g DW).
2) ABTS method:
Respectively preparing 7.4mM ABTS and 2.6mM potassium persulfate, mixing the materials in equal volume before use, carrying out light-shielding reaction for 12 hours at 37 ℃, and diluting for about 27 times until the absorbance at 734nm is about 0.7. Taking 25-800 mu M Trolox as a reference substance, taking 100 mu L of an extract solution or a reference substance solution diluted by a certain multiple, adding 3.0mL of ABTS reaction solution, performing light-shielding reaction at 37 ℃ for 60min, and measuring the absorbance at 734 nm. The absorbance was plotted on the abscissa and the absorbance was plotted on the ordinate, with the results expressed as Trolox equivalents per gram of extract (mmol Trolox/g DW).
3) ORAC method:
a phosphate buffer solution (pH 7.4) was prepared at a concentration of 75mM, and a solution of fluorescein and AAPH was prepared using the buffer solution as a solvent. Taking 0-80 mu M Trolox as a reference substance, respectively adding 20 mu L of extract solution (after dilution by a certain multiple) and 120 mu L of fluorescein into a black enzyme-labeled 96-well plate, preserving heat at 37 ℃ for 5min, and then adding 60 mu L of AAPH to make final concentrations of the fluorescein and the AAPH be 70nM and 12mM respectively, and immediately measuring by a fluorescence enzyme-labeled instrument. The measurement conditions were measured every 2min for 3 hours. The excitation wavelength and the emission wavelength are 485nm and 530nm respectively. The absorbance was plotted on the abscissa and the absorbance was plotted on the ordinate, with the results expressed as Trolox equivalents per gram of extract (mmol Trolox/g DW).
4) FRAP method:
The FRAP working solution (used up in 2 hours) was prepared by mixing 10mM TPTZ solution, 20mM FeCl 3 aqueous solution, 30mM sodium acetate buffer solution at pH=3.6 with 40mM HCl as a solvent at a ratio of 1:1:10 (v/v/v). Taking FeSO 4 as a reference substance, taking 100 mu L of an extract solution or a reference substance solution diluted by a certain multiple, adding 3.0mL of FRAP working solution, performing light-shielding reaction at 37 ℃ for 60min, and measuring the absorbance at 595 nm. The absorption is plotted against the FeSO 4 concentration on the abscissa and the absorbance is plotted against the absorption on the ordinate, the result being expressed in equivalents of FeSO 4 per gram of extract (mmol FeSO 4/g DW).
TABLE 2 antioxidant capacity of Tagetes extract in vitro with different preparation methods
As shown in table 2, the marigold extract prepared by the extraction method in example 1 has the best oxidation resistance in vitro, i.e., by a feed to liquid ratio of 1g:40mL, extraction temperature 50 ℃, ph=8 of the extract.
Activity detection example 2: whitening efficacy research of marigold extract
Tagetes extract and its main ingredient tyrosinase inhibition activity studies are shown in Table 4. The specific method comprises the following steps:
Dissolving the extract or each component substance with 250 mu L of DMSO, preparing a 50mg/L solution by using a buffer solution, diluting the solution into a 1mg/mL solution, preparing a solution of different samples with equal concentration and a positive control solution according to the composition of a reaction solution A 1,A2,A3,A4 in a table 3, fully and uniformly mixing the buffer solution and an enzyme solution, putting the mixture into a gas bath at 37 ℃ for incubation for 10min, adding a corresponding substrate solution, measuring the absorbance of each reaction solution at 475nm after 40min, and measuring the absorbance of each reaction solution at 475nm after 5 min. The inhibition ratio was calculated according to the following formula:
Tyrosine monophenolase/diphenolase inhibition rate/% = [1- (OD A3-ODA4)/(ODA1-ODA2) ] ×100
TABLE 3 reaction System in vitro tyrosinase Activity determination
Component system of reaction liquid A1/mL A2/mL A3/mL A4/mL
Target solution 0 0 0.3 0.3
Buffer solution 0.4 0.5 0.1 0.2
Substrate (tyrosine/L-DOPA) 0.5 0.5 0.5 0.5
Tyrosinase enzyme 0.1 0 0.1 0
TABLE 4 inhibition of the primary ingredients of Tagetes extract on tyrosine monophenolase and tyrosine diphenolase
Group of Tyrosine monophenolase (%) Tyrosine diphenolase (%)
Example 1 65.71 56.93
Example 2 65.45 55.47
Example 3 66.81 57.73
Ellagic acid 69.45 50.28
Quinic acid 72.32 60.56
Syringic acid 37.39 26.88
Gallic acid 42.93 26.34
Isoquercitrin 9.67 11.44
Luteolin 28.32 20.88
Arbutin (positive control) 67.20 /
Kojic acid (positive control) / 62.03
Tyrosinase activity can be divided into monophenolase (tyrosine as substrate) and diphenolase (dopa as substrate) activities. As shown in table 4, the inhibition rate of tyrosine monophenolase of the marigold extract in examples 1,2, and 3 was more than 65%, and the marigold extract had monophenolase inhibitory activity although slightly weaker than the positive control arbutin, in which ellagic acid inhibitory activity as a main component was close to that of arbutin. The inhibition rate of tyrosinase of marigold extract is more than 55%, wherein the inhibition rate of ellagic acid, quinic acid, syringic acid, gallic acid and luteolin diphenolic acid of main components is more than 20%. Therefore, the marigold extract has a certain whitening effect, and the main components of the marigold extract are ellagic acid, quinic acid, syringic acid, gallic acid and luteolin.
Activity detection example 3: research on aging delaying effect of marigold extract on organisms
Adopts the method of preparing different concentration solutions for lavaging mice to observe the effect of the marigold extract on delaying the aging of organisms. The specific method comprises the following steps:
88 healthy Kunming mice were selected and randomly divided into normal control, blank, example 1, example 2, example 3 marigold extract (500 mg/kg). The modeling method adopts a mouse aging model, 10% D-galactose solution is prepared by normal saline, and the rest 6 groups except the normal control group are respectively injected with 0.14g/kg D-galactose solution subcutaneously at the nape of the neck every day to construct the mouse aging model, and D-galactose is continuously administered for 50 days. Starting from the second week, the mice of the low and high dose groups of the marigold extract of each example were respectively perfused with 250mg/kg and 500mg/kg of the marigold extract each day; normal control group and blank control group mice were perfused with an equal amount of physiological saline. The physical behaviors and the activity of the mice are observed and recorded at regular time every day, and the weight change, the hair luster degree, the feeding condition, the sensitivity to stimulus response, the anger response, the sleepiness degree and the like of the mice are recorded. After the last administration for 2 hours, the brain, spleen, kidney and liver are taken out, blood is sucked, and fat and a system film are cut off. The spleen of the mouse was washed with physiological saline, and then, the surface water of the viscera was removed by sticking with filter paper, and the spleen index was calculated by weighing. The brain, spleen, kidney and liver were prepared into 10% homogenate, and the SOD and MAD contents in the brain, spleen, kidney and liver of mice were measured, respectively, according to the method described in the kit. The experimental data are subjected to analysis of variance by SPSS 15.0 statistical software, the results are expressed as (x+ -s), the t-test is adopted for statistics among groups, and the difference is significant when P is less than 0.05.
The results show that: normal control mice have good mental state, agile action, luster hair and difficult grabbing; mice in the blank control group are not in lassitude, slow in action, withered and non-glossy hair color, are not counteracted during unhairing and grabbing, and are easy to grab. Mice in the dosing group showed benign inversions compared to the blank group; the appearance of each high dose group of marigold extract is unchanged compared with that of the normal group, and the activity is normal. The spleen factor of the D-galactose blank control mice is obviously reduced, compared with the normal control mice, the spleen index is obviously increased along with the increase of the marigold extract dosage, and the spleen factor is obviously different (P is less than 0.05). The results are shown in Table 5.
The normal mice had higher SOD levels in tissues, while the aged mice had lower SOD levels in tissues. The marigold extract can improve the SOD content of the brain tissue of the mice, compared with a blank group, the difference of a low-dose group has no statistical significance (P is more than 0.05), and the difference of a high-dose group has obvious statistical significance (P is less than 0.01) and is close to the normal level. After the marigold extract is infused into the stomach, the SOD content of the kidney tissues of the mice can be improved, compared with a blank group, the difference of a low-dose group has no statistical significance (P is more than 0.05), and the difference of a high-dose group has obvious statistical significance (P is less than 0.01). The marigold extract can obviously improve the SOD content of the liver tissue of the mice, and compared with a blank group, the difference between the low dose group and the high dose group has obvious statistical significance (P is less than 0.01). The specific results are shown in Table 6.
Normal mouse tissues have lower MDA content, whereas aged mouse tissues have higher MDA content. The marigold extract can reduce MDA content of brain tissue of mice, compared with a blank group, the difference of a low-dose group has no statistical significance (P is more than 0.05), and the difference of a high-dose group has obvious statistical significance (P is less than 0.01) and is close to a normal level. After the marigold extract is infused into the stomach, the MDA content of kidney tissues of the mice can be reduced, compared with a control group, the difference of a low-dose group has no statistical significance (P is more than 0.05), and the difference of a high-dose group has obvious statistical significance (P is less than 0.01) and is close to the normal level. The marigold extract can obviously reduce the MDA content of liver tissues of mice, compared with a blank group, the difference of the low-dose group has obvious statistical significance (P < 0.05), the difference of the high-dose group has obvious statistical significance (P < 0.01), and the MDA content is lower than the normal level. The specific results are shown in Table 7.
TABLE 5 Effect of Tagetes extract on mouse spleen index
Group of Tagetes extract (mg/kg) Spleen index
Blank group (model group) \ 22.61±2.17
Normal group \ 30.77±1.71*
Example 1 Low dose group 250 27.83±1.54
Example 1 high dose group 500 30.67±2.05*
Example 2 Low dose group 250 25.17±1.95
EXAMPLE 2 high dose group 500 28.24±2.16*
Example 3 Low dose group 250 20.63±1.92
EXAMPLE 3 high dose group 500 29.11±2.72*
Note that: compared with blank group, P is less than 0.05
TABLE 6 Effect of Tagetes extract on the SOD content of mouse brain, kidney and liver tissue
Note that: p < 0.05, P < 0.01 compared to blank group
TABLE 7 Effect of Tagetes extract on MDA content of brain, kidney and liver tissue of mice
Note that: p < 0.05, P < 0.01 compared to blank group
Experimental data show that the marigold extract can obviously improve spleen index and SOD and MDA content in liver tissues of aged mice, and the marigold extract can effectively inhibit spleen and liver injury of mice in the aging process, and has the effects of protecting immune organs and improving immune systems. Therefore, the marigold extract can effectively improve the immunity of organisms, and plays a role in delaying the aging of the organisms by eliminating oxygen free radicals in the bodies and inhibiting lipid peroxidation.
Activity detection example 4: potential application of marigold extract in anti-pet aging medicines or pet foods
One of the age-related conditions of animals (e.g., cats and dogs) is oxidative damage, and oxidative stress mechanisms are important pathogenesis of neurodegenerative diseases. The experiment uses 3 dogs with aging, which are respectively: 7 years old poodle, 8 years old side pasturing, 7 years old Taidi as study subjects, 1% marigold extract was added to the dog food, and fed for 90 days to verify the potential application of the marigold extract in anti-pet aging drugs or pet foods. The oxidation state in the body of the pet is studied by the urine level of 8-hydroxydeoxyguanosine (biomarker of oxidative damage) in the urine of the pet after feeding the pet dog, and the improvement effect on the aging of the pet is measured by two indexes of activity and cognition. The results are shown in Table 8.
TABLE 8 Effect of Tagetes extract on 8-hydroxydeoxyguanosine content, motility and cognition in urine of pet dogs
Note that: cognition: has improvement+, has obvious improvement+ + and
Conclusion:
(1) After the marigold extract is fed, the level of 8-hydroxydeoxyguanosine in urine of the aged pet dogs is obviously reduced, which indicates that the oxidation state in the pet dogs is obviously improved.
(2) After the marigold extract is fed, the activity and the cognition of the aged pet dogs are obviously improved, which indicates that the marigold extract can improve the cognition of the pet dogs and delay the aging process of the pet dogs.
Finally, it should also be noted that the above list is only a few specific examples and comparative examples of the present invention. Obviously, the present invention is not limited to the above examples and comparative examples, but many variations are possible. All modifications directly derived or linked from the disclosure of the present invention by a person of ordinary skill in the art should be considered as the protection scope of the present invention.

Claims (8)

1. A method for preparing a marigold extract, which is characterized by comprising the following steps:
(1) Dried marigold is prepared according to 1g:20 The ratio of the feed liquid of mL-60 mL is added into an aqueous solution of an organic solvent with the volume ratio of 40% -60%, the organic solvent is ethanol, methanol or n-butanol, the pH value is regulated to 7-9, leaching is carried out at the temperature of 45-60 ℃, and the extract is obtained through separation;
(2) Removing organic solvent in the extractive solution, and drying to obtain flos Tagetis Erectae extract;
The marigold extract comprises ellagic acid, quinic acid, syringic acid, gallic acid, isoquercitrin and luteolin, wherein the weight content of total flavonoids in the marigold extract is 16% -30%, and the total phenols content is 50% -72%; wherein the weight content of ellagic acid is 1.2% -6.2%, the weight content of quinic acid is 0.4% -1.2%, the weight content of isoquercitrin is 0.5% -2.7%, the weight content of syringic acid is 0.3% -0.45%, the weight content of gallic acid is 0.06% -0.3%, and the weight content of luteolin is 0.04% -0.1%.
2. The method for preparing marigold extract according to claim 1, wherein in the step (1), the volume concentration of the aqueous organic solvent solution is 50%.
3. The method for preparing a marigold extract according to claim 1, wherein in the step (1), the water content of the marigold dry product is less than or equal to 10%, and the feed liquid ratio is 1g:40 And (3) mL.
4. The method for preparing marigold extract according to claim 1, wherein in the step (1), the leaching time is 20-30 hours.
5. The method of preparing a marigold extract according to claim 1, wherein in step (1), the leaching process is combined with ultrasonic treatment under the following ultrasonic conditions: the ultrasonic frequency is 300-500 kHz, the ultrasonic intensity is 5-15W/cm 2, the time is 1.5-3.0 h, and the ultrasonic treatment is kept stand after the ultrasonic treatment is finished.
6. The method for preparing marigold extract according to claim 1, wherein in the step (1), the pH is adjusted to 8±0.3 with an alkaline agent; the alkaline agent is sodium hydroxide, sodium carbonate, sodium bicarbonate, trisodium bicarbonate, potassium carbonate or potassium bicarbonate.
7. The marigold extract produced by the production process according to any one of claims 1 to 6.
8. The use of the marigold extract according to claim 7 for preparing a functional food or pet food, wherein the functional food is a food having anti-aging, anti-oxidation and whitening effects.
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