Production method and application of marigold flavonoid glycoside

Abstract

The invention belongs to the technical field of biology, and particularly relates to a production method and application of marigold flavone glycoside. According to the production method of the marigold flavonoid glycoside, the marigold flavonoid glycoside with high content and the lutein ester ointment with high content can be simultaneously produced on a large scale, the utilization efficiency of flavonoid substances in marigold is improved, and the use cost is reduced. The main component of the marigold flavonoid glycoside produced by the method is quercetagetin, and compared with common quercetagetin in the market, the water solubility of the quercetagetin is thousand times higher than that of the quercetagetin, and the quercetagetin is more easily absorbed and utilized by animals. Furthermore, the invention finds that the marigold flavonoid glycoside and the vitamin C have a remarkable synergistic effect, so that the physiological effects of the vitamin C, such as oxidation resistance, stress resistance and collagen synthesis promotion, can be greatly improved. The compound product is used for animal breeding and can improve the nutritional effect of the vitamin C or the derivative thereof.

Description

Technical Field

[0001] The present invention belongs to the field of extraction of plant active ingredients, and particularly relates to a production method and application of flavonoid glycosides from marigold flowers. Background Art

[0002] Flavonoid glycosides are the main form of flavonoids in marigold flowers. They have good water solubility and can exert various biological functions in trace amounts and with high efficiency. However, since the first processing step in the current marigold extraction industry is to use microbial fermentation of fresh flowers to break the plant cell wall to improve the lutein yield, as a result, the flavonoid glycosides are degraded by the glycosidase produced during the microbial fermentation into water-insoluble flavonoid aglycones, greatly reducing the biological utilization rate.

[0003] CN110003071, CN110105257, CN110372554, CN110746331, and CN110776449 etc. disclose methods for extracting quercetagetin from marigold flower granules, and the marigold flower granules in these methods are obtained by granulating marigold after microbial fermentation. Because of this, these methods can only obtain the flavonoid aglycone quercetagetin.

[0004] CN113615767 discloses a feed containing quercetagetin and its uses, and it is found that adding quercetagetin has effects such as promoting animal growth, improving blood indexes, immune function, antioxidant capacity, and improving intestinal health. However, according to the current public research, quercetagetin in the feed can only affect the antioxidant capacity and intestinal health of broilers, laying hens, and piglets when added to a relatively high dose. This shows that the application potency of marigold flavonoid aglycone in the feed is very low.

[0005] Therefore, it is necessary to develop an extraction method and application of marigold active ingredients with better biological potency. Summary of the Invention

[0006] The purpose of the first aspect of the present invention is to provide a production method of flavonoid glycosides from marigold flowers.

[0007] The purpose of the second aspect of the present invention is to provide the application of flavonoid glycosides from marigold flowers.

[0008] The purpose of the third aspect of the present invention is to provide the application of the combined use of flavonoid glycosides from marigold flowers and vitamins or their derivatives.

[0009] The purpose of the fourth aspect of the present invention is to provide an animal feed.

[0010] In order to achieve the above purposes of the present invention, the technical solutions adopted by the present invention are as follows:

[0011] In the first aspect of the present invention, a method for producing marigold flavonoid glycosides is provided, comprising the following steps:

[0012] 1) Extract marigold using an alkaline solvent;

[0013] 2) Add n-hexane and water to the extracted alkaline solvent, mix, and let it stand for layering;

[0014] 3) Cool the lower-layer liquid for crystallization, filter, and dry to obtain crude marigold flavonoid glycoside crystals;

[0015] 4) Dissolve the crude marigold flavonoid glycoside crystals in boiling water, filter, cool for recrystallization, filter, and dry to obtain marigold flavonoid glycosides.

[0016] In some embodiments of the present invention, the alkaline solvent consists of an alcohol, acetone, ethyl acetate, and an alkaline reagent.

[0017] In some embodiments of the present invention, the volume fractions of the alcohol, acetone, and ethyl acetate are (20 - 80%), (20 - 80%), and (0 - 10%); preferably, the volume fractions of the alcohol, acetone, and ethyl acetate are (47.5 - 52.5%), (42.5 - 47.5%), and (2.5 - 7.5%).

[0018] In some embodiments of the present invention, the alcohol includes at least one of methanol, ethanol, propanol, isopropanol, butanol, or other lower-carbon-number alcohols.

[0019] In some embodiments of the present invention, the alkaline reagent includes at least one of potassium hydroxide, sodium hydroxide, and sodium bicarbonate.

[0020] In some embodiments of the present invention, the extraction equipment can be an extraction tank, a rotary extraction equipment, a continuous countercurrent extraction equipment, etc., including auxiliary extraction equipment such as microwave and ultrasonic.

[0021] In some embodiments of the present invention, the pH of the alkaline solvent is 7.5 - 9.5.

[0022] In some embodiments of the present invention, the extraction temperature is 40 - 60 °C.

[0023] In some embodiments of the present invention, the marigold is subjected to dehydration treatment.

[0024] In some embodiments of the present invention, the added volumes of n-hexane and water are 0.5 - 3.0 times and 0.1 - 1.0 times the volume of the extracted alkaline solvent, respectively.

[0025] In some embodiments of the present invention, after standing for layering, the upper and lower layers are separately collected by a liquid separation device. The upper layer enters a solvent removal device to volatilize n-hexane to obtain an extract containing lutein esters. The lower layer enters a crystallization device to cool down to crystallize flavone glycosides, and solid-liquid separation is carried out by equipment such as a plate-and-frame filter or a centrifuge. The obtained solid is dried under normal pressure or vacuum to obtain marigold flavone glycosides.

[0026] In some embodiments of the present invention, the content of quercetin marigold glycoside in the marigold flavone glycosides is greater than 80%.

[0027] The second aspect of the present invention provides the use of marigold flavone glycosides in the preparation of products for promoting animal production performance and improving health level.

[0028] In some embodiments of the present invention, the marigold flavone glycosides include quercetin marigold glycoside (CAS: 548-75-4); preferably, the content of quercetin marigold glycoside is greater than 80%.

[0029] In some embodiments of the present invention, the product includes at least one of feed and drinking water additive.

[0030] In some embodiments of the present invention, marigold flavone glycosides can be used as an additional food factor for animal feed for breeding animals and pets, including poultry, livestock, aquatic animals, insects, domesticated wild economic animals, amphibians, reptiles, cats, dogs, etc., and have the effects of improving animal production performance, improving the quality of animal products, and improving the health level of animal bodies. In addition to being added to feed, marigold flavone glycosides can also be used by dissolving them in the drinking water of animals.

[0031] The third aspect of the present invention provides the use of marigold flavone glycosides and vitamin C or its derivatives in the preparation of products for promoting animal production performance and improving health level.

[0032] In some embodiments of the present invention, the marigold flavone glycosides include quercetin marigold glycoside; preferably, the content of quercetin marigold glycoside is greater than 80%.

[0033] In some embodiments of the present invention, the product includes at least one of feed and drinking water additive.

[0034] In some embodiments of the present invention, the mass ratio of the marigold flavone glycosides, vitamin C or its derivatives is 1:(3 - 5).

[0035] The fourth aspect of the present invention provides an animal feed, and the feed includes marigold flavone glycosides prepared by the production method of the first aspect of the present invention.

[0036] In some embodiments of the present invention, the feed contains 10 - 200 mg / kg of marigold flavonoid glycoside.

[0037] In some embodiments of the present invention, the feed contains 25 - 100 mg / kg of marigold flavonoid glycoside; preferably, it is 40 - 60 mg / kg.

[0038] The beneficial effects of the present invention are as follows:

[0039] The present invention discovers a production method of marigold flavonoid glycoside, which can simultaneously produce high - content marigold flavonoid glycoside and lutein ester ointment on a large scale, improving the utilization efficiency of flavonoids in marigold and reducing the usage cost. The main component of the marigold flavonoid glycoside produced by this method is quercetin marigold glycoside. Compared with the common quercetin marigoldin on the market, the water solubility of quercetin marigold glycoside is a thousand times higher than that of quercetin marigoldin, and it is more easily absorbed and utilized by animals. When playing the same effect in animal breeding, the addition amount of quercetin marigold glycoside is only one - twentieth to one - tenth of that of quercetin marigoldin, eliminating the usage limit that marigold flavonoids cannot be added to drinking water. Further, the present invention discovers that marigold flavonoid glycoside and vitamin C or vitamin C derivatives have a significant synergistic effect, which can greatly improve the physiological functions of vitamin C such as antioxidant, anti - stress, and promoting collagen synthesis. The compound product can improve the nutritional effect of vitamin C or its derivatives when used in animal breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The following further describes the present invention with reference to the drawings and embodiments, where:

[0041] Figure 1 It is the liquid chromatography result of the product prepared in Example 1 of the present invention, where a is the standard product of quercetin marigold glycoside; b is the sample of marigold flavonoid glycoside.

[0042] Figure 2 It is the result of the intestinal effect of marigold flavonoid glycoside on piglets, where A - D are the results of feeding with 0 mg / kg, 25 mg / kg, 50 mg / kg, and 100 mg / kg of marigold flavonoid glycoside respectively.

[0043] Figure 3 It is the standard curve of FeSO4.

[0044] Figure 4 It is the comparison result of the biological potencies of marigold flavonoid glycoside and marigold flavonoid in broilers. DETAILED DESCRIPTION OF THE INVENTION

[0045] The concept of the present invention and the technical effects achieved will be clearly and completely described below in conjunction with embodiments to fully understand the purpose, features, and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0046] Example 1 Production method of flavonoid glycosides from marigold

[0047] The marigold flowers are made into marigold flower granules after pressing and dewatering. 10 tons of granules are put into a horizontal rotary extraction equipment by a scraper, and a weakly alkaline mixed solvent (ethanol-acetone-ethyl acetate mixed solvent with pH = 8.5, volume ratio 47.5:47.5:5) is sprayed according to a solid-liquid ratio of 1:10. The horizontal rotation speed is 15 h / turn, and the extraction is carried out at a temperature of 50 °C ± 2 °C. After the extraction is completed, a total of 127 m 3 of the solvent is collected and transferred to a 500 m 3 tank. 173 m 3 of n-hexane is added to the tank while stirring, and then 40 m 3 of pure water is added. When the addition of pure water is completed, the stirring is stopped and the mixture is allowed to stand for stratification. The lower layer liquid after stratification is discharged from the bottom valve of the tank body, and the discharge is stopped when the liquid turns dark yellow. 75 m 3 of the lower layer liquid is obtained. The lower layer liquid is transferred to a 100 m 3 tank, stirred for 24 h, and cooled to 26 °C ± 2 °C. A filter cloth is installed on the bottom valve to discharge the liquid. The liquid is transferred into a storage tank for standby. The substance on the filter cloth is dried to a constant weight in a vacuum drying oven to obtain 84.26 kg of yellow solid powder. The upper layer liquid in the tank starts to be decompressed and heated, and n-hexane is recovered by condensation. After the recovery is completed, the extract is discharged from the bottom valve to obtain 1189.52 kg of extract.

[0048] By liquid chromatography analysis, the content of lutein ester in the raw material particles is 1.81%, and the content in the extract is 14.60%. The yield of lutein ester is calculated to be 95.95%.

[0049] By liquid chromatography analysis, the content of quercetin marigold glycoside in the raw material particles is 0.76%, and the content in the yellow solid powder is 82.03%. The recovery rate is 90.95%.

[0050] Liquid chromatography conditions for quercetin marigold glycoside: chromatographic column CORTECS C18 column (4.6 mm × 250 mm, particle size 2.5 μm), detection wavelength 360 nm, column temperature 30 °C, injection volume 20 μL, flow rate 0.8 mL / min, mobile phase acetonitrile: 0.2% phosphoric acid aqueous solution (10:90, v / v). The liquid chromatography results are as Figure 1 shown.

[0051] Application Research of Flavonoid Glycosides from Tagetes erecta L. in the Breeding of Weaned Piglets - Example 2

[0052] 1. Experimental Diet

[0053] The experimental diet was corn - soybean meal type, and its various nutritional indexes met the nutritional requirements of weaned piglets. The diet composition and nutritional levels are shown in Table 1.

[0054] Table 1 Basic Diet and Nutritional Levels (Air - dried Basis)

[0055]

[0056]

[0057] 1) The premix provided per kilogram of diet: VA 12000 IU, VD 6000 IU, VE 800 IU, VB1 35 mg, VB2 20 mg, VB6 35 mg, VB 12 0.1 mg, folic acid 1.5 mg, D - pantothenic acid 50 mg, choline chloride 1 g, Fe 90 mg, Cu 20 mg, Zn 80 mg, Mn 40 mg, Se 0.3 mg, I 0.4 mg.

[0058] 2) The digestible energy was calculated according to NRC (2012), and the other nutritional levels were measured values.

[0059] 2. Experimental Animals

[0060] A total of 144 three - way crossbred weaned piglets with a body weight of about 10 kg were selected and randomly divided into 4 groups, with 6 replicates in each group and 6 pigs in each replicate, half male and half female.

[0061] 3. Experimental Grouping

[0062] The 4 groups were randomly fed diets with the addition of flavonoid glycosides from Tagetes erecta L. (prepared in Example 2) at 0, 25, 50, and 100 mg / kg.

[0063] 4. Feeding and Management

[0064] The experimental period was 4 weeks. During the experiment, the piglets had free access to food and water, and the pigsty was kept ventilated and clean.

[0065] 5. Detection Indexes

[0066] (1) Growth Performance

[0067] On the 1st and 28th days of the formal trial, the fasting body weights of piglets in each group were weighed and recorded, and the average daily gain of piglets in each group was calculated accordingly. The added amount and remaining amount of the basal diet of piglets in each group during the trial were weighed and recorded, and the average daily feed intake of piglets in each group was calculated accordingly. The feed-to-gain ratio of piglets in each group was calculated based on the average daily gain and average daily feed intake of piglets in each group. The calculation formula is as follows:

[0068] Average daily gain = (final weight of piglets - initial weight of piglets) / 28;

[0069] Average daily feed intake = (added amount of basal diet - remaining amount of basal diet) / 28;

[0070] Feed-to-gain ratio = average daily feed intake / average daily gain;

[0071] Diarrhea rate = [number of piglets with diarrhea in each replicate / (number of days of the trial × number of pigs in each replicate)]×100.

[0072] (2) Muscle quality and intestinal health

[0073] After the piglets were fasted and weighed at the end of the trial, 4 piglets with body weights close to the average weight of the group were selected and sacrificed in each group. The longissimus dorsi muscle at the first and second thoracic vertebrae from the bottom was excised to measure muscle moisture, crude protein, crude fat and crude ash to evaluate muscle quality. The intestinal status of piglets was judged by HE (hematoxylin-eosin) staining method: the duodenum 8-10 cm below the pylorus was excised and immersed in formalin, embedded in paraffin, the paraffin was cut into 6-mm thick sections, and the sections were stained with standard hematoxylin and eosin. After dehydration, dewaxing and air-drying, the sections were sealed with neutral balsam, and the tissue morphology was observed using an optical microscope.

[0074] 6. Test results

[0075] (1) Growth performance

[0076] As shown in Table 2, adding marigold flavone glycoside can significantly increase the average daily gain and average daily feed intake of piglets (P < 0.05); it can significantly reduce the feed-to-gain ratio and diarrhea rate of piglets (P < 0.05). Among them, the group adding 50 mg / kg marigold flavone glycoside has the best effect.

[0077] Table 2 Effects of marigold flavone glycoside on the production performance and diarrhea rate of weaned piglets

[0078]

[0079] Note: Values with different lowercase superscripts in the same row are significantly different (P < 0.05).

[0080] (2) Muscle quality

[0081] As can be seen from Table 3, the addition of marigold flavonoid glycosides had no significant effect on the moisture and crude protein content of piglet muscle; it could significantly increase the crude fat content of piglet muscle (P < 0.05); it could significantly reduce the crude ash content of piglet muscle (P < 0.05). Among them, the group with the addition of 50 mg / kg marigold flavonoid glycosides had the best effect.

[0082] Table 3 Effects of Marigold Flavonoid Glycosides on the Muscle Quality of Weaned Piglets

[0083]

[0084] Note: Values with different lowercase superscripts in the same row are significantly different (P < 0.05).

[0085] (3) Intestinal Health

[0086] The results were as Figure 2 shown. The intestinal villi of piglets fed with marigold flavonoid glycosides were denser and more complete in morphology, showing a better health status.

[0087] Example 3 Effects of the Compound of Marigold Flavonoid Glycosides and Vitamin C on Antioxidant Capacity

[0088] In this example, the total antioxidant capacity of marigold flavonoid glycosides, vitamin C and their compound with different weight ratios was measured.

[0089] The total antioxidant capacity was evaluated by the "Ferric Reducing / Antioxidant Power Assay (FRAP)".

[0090] Preparation of 2,4,6-tripyridyltriazine (TPTZ) solution: Prepare a 10 mmol / L TPTZ solution with 40 mmol / L hydrochloric acid and store it in the refrigerator for later use.

[0091] Preparation of FRAP solution: Mix 2.5 mL of TPTZ solution, 2.5 mL of 20 mmol / L ferric chloride hexahydrate and 25 mL of 0.3 mol / L acetic acid buffer solution evenly to obtain the FRAP solution.

[0092] Preparation of sample solution: Accurately weigh 5 mg (accurate to 1 mg) of marigold flavonoid glycosides (prepared in Example 1) or vitamin C (purchased commercially, 99%) and make up to 50 mL with pure water to obtain solutions A and B. Solutions A and B were respectively prepared into mixed samples according to the ratios of 19:1, 9:1, 4:1, 1:4, 1:9, and 1:19 for determination.

[0093] Control group: BHT (using ethanol as the solvent).

[0094] Preparation of FeSO4 standard curve: Prepare ferrous sulfate solutions with concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 mmol / L using pure water. Pipette 0.2 mL of each solution, add 6 mL of FRAP solution preheated to 37 °C, mix well, react in the dark for 15 min, use pure water as the blank control, and measure the absorbance value at 593 nm to prepare the standard curve.

[0095] Determination of FRAP value of samples: Pipette 0.2 mL of the sample solution using a pipette gun, add 3 mL of FRAP solution preheated to 37 °C, mix well, react in the dark for 15 min, measure the absorbance value at 593 nm, and perform parallel measurements 3 times. Calculate the FRAP value according to the standard curve.

[0096] The FeSO4 standard curve is as Figure 2 shown. The FRAP values of each sample are shown in Table 4. The results show that there is a synergistic effect between marigold flavonoid glycosides and vitamin C. The antioxidant capacity after compounding at a weight ratio of 1:4 exceeds that of vitamin C, and the antioxidant capacity of 80% vitamin C + 20% marigold flavonoid glycosides (4:1) is the best.

[0097] Table 4 Effects of different compounding ratios of marigold flavonoid glycosides and vitamin C on antioxidant capacity

[0098]

[0099]

[0100] Note: Different lowercase superscripts in the same row indicate significant differences analyzed by IBM SPSS software (P < 0.05).

[0101] Comparative example 1 Comparison of water solubility between marigold flavonoid glycosides and marigold flavonoids

[0102] The water solubility of marigold flavonoid glycosides and marigold flavonoids was determined by the equilibrium solubility method. Marigold flavonoid glycosides were prepared in Example 1, and the content of the active ingredient quercetin marigold glycoside was 82.03%. Marigold flavonoids were purchased products, and the content of the active ingredient quercetin marigoldin was 80.73%.

[0103] Weigh 1.00 g of marigold flavonoid glycosides and marigold flavonoids into 10 mL test tubes, add 9 mL of ultrapure water, and shake in a water bath at 90 °C for 24 h. After taking out, centrifuge, carefully pipette 5 mL of the top supernatant, filter it through a 0.22 μm filter membrane for determination. Use high performance liquid chromatography to determine the content of the active ingredients in the marigold flavonoid glycoside equilibrium solution and the marigold flavonoid equilibrium solution. The results show that the equilibrium solubility of marigold flavonoid glycosides in water is 1.31%, while the equilibrium solubility of marigold flavonoids in water is 0.0012%.

[0104] It can be seen that the solubility of the marigold flavone glycoside prepared by the present invention in water is more than 1000 times greater than that of marigold flavone. Comparison of the biological utilization rates of marigold flavone glycoside and marigold flavone in broilers in Comparative Example 2

[0105] The slope ratio method was used to compare the biological utilization rates of marigold flavone glycoside and marigold flavone in broilers. The marigold flavone glycoside was prepared in Example 1, and the marigold flavone was a commercially available product in Comparative Example 1.

[0106] 1. Test diet

[0107] A corn-soybean meal type diet was used, and the calculated values of the nutritional indexes were as follows: metabolic energy 3100 Kcal / kg, crude protein 20.5%, calcium 1%, available phosphorus 0.5%, available lysine 1.40%, sulfur-containing amino acids 0.95%, threonine 0.90%, and tryptophan 0.25%.

[0108] 2. Test animals

[0109] 198 1-day-old 817 crossbred broilers were selected and randomly divided into 11 groups, with 3 replicates in each group and 6 chickens in each replicate.

[0110] 3. Test grouping

[0111] The test period was 35 days. The 11 groups were fed a basal diet (control group) and diets with marigold flavone glycoside or marigold flavone added at effective ingredient amounts of 25, 50, 100, 200, and 400 mg / kg. The test design is shown in Table 5.

[0112] Table 5 Test design

[0113]

[0114]

[0115] 4. Detection indexes

[0116] The average body weights of the broilers in each group on the 35th day of the test were recorded, and the body weight-dose regression curves of marigold flavone glycoside and marigold flavone were plotted respectively, and the slope k of the straight line was calculated.

[0117] 5. Test results

[0118] The results of the body weight-dose regression curves of marigold flavone glycoside and marigold flavone are shown in Figure 4, the relevant data are shown in Table 6. When the effective ingredient of marigold flavone glycoside in the diet exceeds 50 mg / kg, a plateau effect appears in the weight gain, so three groups of 0, 25, and 50 mg / kg were selected for the marigold flavone glycoside group to establish a weight-dose regression curve. The k value of marigold flavone glycoside is 2.3054, while the k value of marigold flavone is 0.1876. It can be seen that the biological potency of marigold flavone glycoside is 12.29 times that of marigold flavone.

[0119] Table 6 Average body weight of 35-day-old broilers in each group

[0120]

Claims

1. A method for producing marigold flavonoid glycosides, comprising the following steps: 1) Extracting marigold using an alkaline solvent; 2) adding n-hexane and water to the extracted alkaline solvent, mixing, and standing to separate layers; 3) cooling the lower layer of liquid to crystallize, filtering and drying to obtain crude crystals of marigold flavonoid glycosides; 4) Dissolving crude crystals of marigold flavonoid glycoside in boiling water, filtering, cooling and recrystallizing, filtering and drying to obtain marigold flavonoid glycoside.

2. The production method according to claim 1, characterized in that: The alkaline solvent consists of alcohol, acetone, ethyl acetate, and an alkaline agent; Preferably, the volume fractions of the alcohol, acetone, and ethyl acetate are (20-80%), (20-80%), and (0-10%); Preferably, the alcohol includes at least one of methanol, ethanol, propanol, isopropanol, and butanol; Preferably, the alkaline agent includes at least one of potassium hydroxide, sodium hydroxide, and sodium bicarbonate; Preferably, the pH of the alkaline solvent is 7.5 to 9.

5.

3. The production method according to claim 1, characterized in that: The extraction temperature is 40-60°C; and / or The marigold is dehydrated.

4. The production method according to claim 1, characterized in that: The added volumes of n-hexane and water are 0.5 to 3.0 times and 0.1 to 1.0 times the volume of the alkaline solvent after extraction, respectively.

5. The production method according to claims 1 to 4, characterized in that: The content of quercetin in the marigold flavonoid glycoside is greater than 80%.

6. Application of marigold flavonoid glycosides in the preparation of products for promoting animal production performance and improving health levels.

7. Use of marigold flavonoid glycosides and vitamin C or its derivatives in combination in the preparation of products that promote animal production performance and improve health levels.

8. The use according to claim 6 or 7, characterized in that: The product comprises at least one of feed and drinking water additives.

9. The use according to claim 7, characterized in that: The mass ratio of the marigold flavonoid glycoside to vitamin C or its derivatives is 1:(3-5).

10. A feed, characterized in that: The feed comprises marigold flavonoid glycosides prepared by any one of the production methods of claims 1 to 4; The content of the marigold flavonoid glycoside in the feed is 10-200 mg / kg.

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