Influence of different extraction processes on yield of macleaya cordata extract and application of macleaya cordata extract in grass carp feed

Abstract

The invention belongs to the technical field of plant extraction, and discloses influence of different extraction processes on yield of macleaya cordata extract and application of the macleaya cordata extract in grass carp feed. The invention discloses a macleaya cordata extract preparation method, which comprises: mixing macleaya cordata and an acidic ethanol solution, carrying out reflux extraction, collecting the extraction liquid, and concentrating to obtain the macleaya cordata extract. According to the method disclosed by the invention, the process conditions mainly involved in the extraction process of the macleaya cordata, such as the concentration of ethanol, the material-liquid ratio of acidic ethanol to macleaya cordata powder, the reflux extraction time and the temperature condition during concentration, are investigated. The extraction process of the macleaya cordata is moderately optimized by detecting the content of sanguinarine in the extract, so that the optimal extraction process of the macleaya cordata extract is obtained, and the extraction efficiency of alkaloid (sanguinarine) in the macleaya cordata can be effectively improved by the process.

Description

Technical Field

[0001] This invention belongs to the field of plant extraction technology, specifically relating to the influence of different extraction processes on the yield of *Botrytis cinerea* extract and its application in grass carp feed. Background Technology

[0002] my country is the world's largest aquaculture producer, ranking first globally in fish farming output. Grass carp is the most produced aquaculture species in my country, reaching 5.941 million tons in 2023. With the development of intensive, high-density aquaculture, environmental stress has negatively impacted the growth performance of grass carp, leading to frequent outbreaks of diseases such as enteritis and gill rot, resulting in economic losses for farmers. Furthermore, the overuse of antibiotics has triggered a chain reaction of problems, including drug residues, increased drug resistance, and aquatic environmental degradation. Developing safe, efficient, and residue-free plant-based feed additives has become an urgent need for promoting the healthy and sustainable development of the industry. To meet the rapid growth needs of grass carp, farmers often overfeed them during the farming process, causing water pollution and making grass carp prone to stress due to water quality changes. Simultaneously, high feed intake can also cause intestinal damage or inflammation in grass carp. Therefore, it is very important to find additives that can promote grass carp growth, improve their stress resistance, and promote intestinal health. In addition, since fat and carbohydrates are both energy sources in feed, improving grass carp's ability to utilize fat and increasing the sugar-to-fat ratio in feed are of great significance for reducing feed costs.

[0003] Plant-derived feed additives and their extracts are considered ideal alternatives to antibiotics due to their natural origin, low residue, and multiple bioactive functional substances. *Polygonum hydropiper*, a medicinal plant of the Papaveraceae family, is rich in sanguisorbin, which has been proven to have multiple functions including anti-inflammatory, antibacterial, and immunomodulatory effects. However, existing technologies have the following problems: 1. The acidic ethanol reflux extraction process of *Polygonum hydropiper* involves many controllable conditions, and changes in the process conditions at each step may affect the extraction efficiency of sanguisorbin. Therefore, optimizing the extraction process of sanguisorbin from *Polygonum hydropiper* is of great significance for reducing extraction costs and improving extraction efficiency. 2. Existing studies have shown that the application of *Polygonum hydropiper* in various animal feeds has good effects, but most of these studies directly evaluate the effects using commercial *Polygonum hydropiper* powder. Different extraction process conditions will affect the sanguisorbin content in the extracted feed, thus causing errors in demand assessment. 3. Existing studies have shown that adding *Polygonum hydropiper* to feed can improve the utilization of feed fat, but research on the effects of *Polygonum hydropiper* on the glucose and lipid metabolism and glucose-lipid ratio requirements of grass carp remains lacking.

[0004] The difficulty in solving the problems of existing technologies lies in the fact that the extraction of sanguisorbin from berberine by acidic ethanol reflux involves many process conditions, such as the concentration of ethanol, the ratio of acidic ethanol to berberine powder, the extraction time of water bath heating, and the temperature conditions during low-temperature vacuum concentration. Changes in each condition may affect the final extraction efficiency of sanguisorbin, so a detailed evaluation is required according to the extraction steps. Summary of the Invention

[0005] This invention aims to at least solve one of the technical problems existing in the prior art. This invention provides an extract of *Polygonum hydropiper* under different extraction process conditions, optimizes the extraction process conditions, and evaluates the application effects and required amounts (preliminarily exploring its suitable addition amount in grass carp feed, and comparing the effects of adding and not adding *Polygonum hydropiper* extract on the suitable sugar-lipid ratio level in grass carp feed), providing a theoretical basis and technical support for the scientific application of *Polygonum hydropiper* in grass carp feed.

[0006] The first objective of this invention is to provide a method for preparing an extract of *Eupatorium fortunei*.

[0007] A second aspect of the present invention is to provide a herb extract.

[0008] The third aspect of this invention aims to provide a preparation method according to the first aspect of this invention or the application of the extract of *Polygonum hydropiper* according to the second aspect of this invention in the preparation of feed or fish farming.

[0009] The fourth aspect of the present invention is to provide a feed.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a method for preparing a *Botrytis cinerea* extract, comprising the following steps: mixing *Botrytis cinerea* with an acidic ethanol solution, refluxing and extracting, collecting the extract, and concentrating it to obtain the *Botrytis cinerea* extract.

[0011] In some embodiments of the present invention, the ratio of the purpureus to the acidic ethanol solution is 1:(5~25).

[0012] In some preferred embodiments of the present invention, the ratio of the purpureus to the acidic ethanol solution is 1:(10~25).

[0013] In some more preferred embodiments of the present invention, the ratio of the purpureus to the acidic ethanol solution is 1:(15~25).

[0014] In some embodiments of the present invention, the concentration of ethanol in the acidic ethanol solution is 60 v / v% to 80 v / v%, and / or the pH value of the acidic ethanol solution is 3.

[0015] In some preferred embodiments of the present invention, the concentration of ethanol in the acidic ethanol solution is 65 v / v% to 75 v / v%, and / or the pH value of the acidic ethanol solution is 3.

[0016] In some more preferred embodiments of the present invention, the concentration of ethanol in the acidic ethanol solution is 70 v / v% to 75 v / v%, and / or the pH value of the acidic ethanol solution is 3.

[0017] In some embodiments of the present invention, the reflux extraction conditions are reflux extraction at 50~90℃ for 1~3 hours.

[0018] In some preferred embodiments of the present invention, the reflux extraction conditions are reflux extraction at 60~90℃ for 1~3 hours.

[0019] In some more preferred embodiments of the present invention, the reflux extraction conditions are reflux extraction at 60~70℃ for 1.5~2.5h.

[0020] In some embodiments of the present invention, the reflux extraction is repeated twice.

[0021] In some embodiments of the present invention, the concentration temperature is 50~70°C.

[0022] In some preferred embodiments of the present invention, the concentration temperature is 55~70°C.

[0023] In some more preferred embodiments of the present invention, the concentration temperature is 60~65°C.

[0024] In some embodiments of the present invention, the concentration is vacuum concentration.

[0025] In some embodiments of the present invention, the *Polygonum hydropiper* is pretreated before being mixed with the acidic ethanol solution. The pretreatment includes pulverizing the dried rhizomes of *Polygonum hydropiper* through a 60-mesh sieve, removing fat-soluble pigments, oils, and other impurities using a Soxhlet extractor, and then drying.

[0026] In some embodiments of the present invention, the preparation method further includes drying.

[0027] In some embodiments of the present invention, the active ingredient in the extract of *Gynostemma pentaphyllum* includes sanguisorbin.

[0028] This invention provides optimized process conditions for the extraction of sanguisorbin from *Botrytis cinerea*, which can increase the yield of sanguisorbin in the extract.

[0029] A second aspect of the present invention provides a *Platycladus orientalis* extract prepared by the preparation method of the first aspect of the present invention.

[0030] In some embodiments of the present invention, the active ingredient in the extract of *Gynostemma pentaphyllum* includes sanguisorbin.

[0031] A third aspect of the present invention provides the preparation method of the first aspect of the present invention or the application of the extract of *Polygonum hydropiper* from the second aspect of the present invention in the preparation of feed or fish farming.

[0032] In some embodiments of the present invention, the feed is fish feed, such as grass carp feed.

[0033] Carbohydrates and fats are important energy sources in feed. This invention verifies whether adding *Polygonum hydropiper* extract to feed can moderately increase the feed's sugar-to-lipid ratio without affecting fish growth rate, thereby reducing feed costs. Results show that its application in fish feed (such as grass carp feed) can improve fish growth performance, antioxidant capacity, and intestinal health, and can moderately increase the feed's sugar-to-lipid ratio, reducing feed costs without affecting fish growth rate.

[0034] In some embodiments of the present invention, the amount of *Platycladus orientalis* extract added to the feed is 25-100 mg / kg.

[0035] In some preferred embodiments of the present invention, the amount of *Bletilla striata* extract added to the feed is 25-75 mg / kg.

[0036] In some preferred embodiments of the present invention, the amount of *Platycladus orientalis* extract added to the feed is 50 mg / kg.

[0037] Through its application in grass carp feed, the optimal addition amount of the *Botrytis cinerea* extract according to the first aspect of this invention was found to be 50 mg / kg. This extract significantly improved the growth performance, antioxidant capacity, and intestinal health of grass carp. Simultaneously, it was verified that adding 50 mg / kg of *Botrytis cinerea* extract to the feed increased the sugar-to-lipid ratio from 3.8 to 6.15 without negatively impacting the weight gain of grass carp. This reduces feed costs without affecting grass carp growth, contributing to the green, healthy, and sustainable development of grass carp farming.

[0038] In a fourth aspect, the present invention provides a feed comprising the extract of *Gnaphalium affine* from the second aspect of the present invention and a base feed, said base feed comprising protein, fat, carbohydrates, cellulose, calcium dihydrogen phosphate, choline chloride, sodium chloride, multivitamin premix, multimineral premix, and calcium propionate.

[0039] In some embodiments of the present invention, the feed contains 29% to 32% crude protein, 6% to 8% crude fat and 10% to 13% ash.

[0040] In some embodiments of the present invention, the basic feed, by weight parts, comprises 50-52 parts protein, 4-5 parts fat, 10-35 parts carbohydrates, 10-25 parts cellulose, 2-3 parts calcium dihydrogen phosphate, 0.1-0.2 parts choline chloride, 0.1-0.3 parts sodium chloride, 0.1-0.2 parts multivitamin premix, 0.4-0.6 parts mineral premix, and 0.04-0.07 parts calcium propionate.

[0041] In some embodiments of the present invention, the basic feed comprises, by weight parts, 32-34 parts fishmeal, 15-18 parts soybean meal, 10-35 parts starch, 2-9 parts soybean oil, 0.1-0.2 parts multivitamin premix, 0.4-0.7 parts multimineral premix, 2-3 parts calcium dihydrogen phosphate, 0.1-0.2 parts choline chloride, 0.1-0.3 parts sodium chloride, 11-17 parts microcrystalline cellulose, and 0.04-0.06 parts calcium propionate.

[0042] In some embodiments of the present invention, the multivitamin premix includes vitamin A, vitamin D3, vitamin E, vitamin K3, vitamin B12, biotin, folic acid, vitamin B1, vitamin C, niacin, inositol, calcium pantothenate, riboflavin, vitamin B6, and corn starch.

[0043] In some embodiments of the present invention, the multi-mineral premix includes manganese sulfate, magnesium sulfate, ferrous sulfate, zinc sulfate, copper sulfate, potassium iodide, sodium selenite, and corn starch.

[0044] In some embodiments of the present invention, the sugar-to-fat ratio of the feed is 1 to 12; preferably 3.8 to 6.15.

[0045] In some embodiments of the present invention, the feed is fish feed, such as grass carp feed.

[0046] The beneficial effects of this invention are: This invention investigates key process conditions involved in the extraction of *Gynostemma pentaphyllum*, such as ethanol concentration, the ratio of acidic ethanol to *Gynostemma pentaphyllum* powder, reflux extraction time, and concentration temperature. By detecting the sanguinarine content in the extract, the extraction process of *Gynostemma pentaphyllum* was appropriately optimized, resulting in the optimal extraction process for *Gynostemma pentaphyllum* extract. This process can effectively increase the extraction efficiency of alkaloids (sanguinarine) from *Gynostemma pentaphyllum*.

[0047] Furthermore, this invention verified the effect of adding an appropriate amount of *Clerodendrum trichotomum* extract to grass carp feed. The results showed that adding *Clerodendrum trichotomum* extract improved the growth performance of grass carp, enhanced their stress resistance, and increased the expression of genes related to the intestinal barrier. In addition, this invention also verified the effect of adding an appropriate amount of *Clerodendrum trichotomum* extract to the feed on the sugar-lipid ratio of the feed consumed by grass carp. The results showed that grass carp could still exhibit superior growth performance after consuming feed with a higher sugar-lipid ratio, effectively reducing feed costs and providing a theoretical basis and technical support for promoting the green, healthy, and sustainable development of grass carp farming. Attached Figure Description

[0048] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is a schematic diagram showing the effect of ethanol concentration on the extraction rate of erythromycin.

[0049] Figure 2 This is a schematic diagram showing the effect of the material-to-liquid ratio on the extraction rate of berberine.

[0050] Figure 3 This is a schematic diagram illustrating the effect of extraction temperature on the extraction rate of erythromycin.

[0051] Figure 4 This is a schematic diagram illustrating the effect of extraction time on the extraction rate of purslane.

[0052] Figure 5 A schematic diagram illustrating the effect of reduced pressure concentration temperature on the extraction rate of sanguisorbin.

[0053] Figure 6 A schematic diagram illustrating the effect of adding Boletus extract to feed on the weight gain rate of grass carp.

[0054] Figure 7 A schematic diagram illustrating the effect of adding Boletus extract to feed on the specific growth rate of grass carp.

[0055] Figure 8 A schematic diagram illustrating the effect of adding *Botrytis cinerea* extract to feed on superoxide dismutase in the liver of grass carp.

[0056] Figure 9 A schematic diagram illustrating the effect of adding *Bletilla striata* extract to feed on glutathione peroxidase in the liver of grass carp.

[0057] Figure 10 A schematic diagram illustrating the effect of adding *Botrytis cinerea* extract to feed on catalase in the liver of grass carp.

[0058] Figure 11 A schematic diagram showing the effect of adding Bo Luo Hui extract to feed on malondialdehyde content in grass carp liver.

[0059] Figure 12Adding *Botrytis cinerea* extract to feed can improve the transmembrane protein C in the intestinal tract of grass carp. Claudin c ), Closure protein ( Occludin ) and mucin-2 ( Mucin-2 A schematic diagram illustrating the impact of intestinal barrier-related gene expression on genes such as ( ).

[0060] Figure 13 A schematic diagram illustrating the effect of adding or not adding *Botrytis cinerea* extract to the feed on the weight gain rate of grass carp at different sugar-lipid ratios. Detailed Implementation

[0061] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0062] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0063] This invention optimizes the extraction process of sanguisorbin from *Botrytis cinerea* using acidic ethanol reflux, which can improve the extraction efficiency of sanguisorbin. The appropriate addition amount of this *Botrytis cinerea* extract in grass carp feed is 50 mg / kg, which can improve the growth performance, antioxidant capacity and intestinal health of grass carp. Adding 50 mg / kg of *Botrytis cinerea* extract to the feed can appropriately increase the feed sugar-lipid ratio and reduce feed costs without negatively affecting the growth of grass carp.

[0064] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0065] Example 1: Effect of different process conditions on the extraction efficiency of sanguisorbin from *Polygonum hydropiper* extract 1. Materials and Methods 1.1 Test Methods The dried rhizomes of *Botrytis cinerea* were crushed and passed through a 60-mesh sieve. A Soxhlet extractor was used to remove fat-soluble pigments, oils, and other impurities. Then, an appropriate amount of *Botrytis cinerea* powder was weighed and placed in a flask. An appropriate amount of acidic ethanol (pH = 3.0) was added, and the mixture was heated in a water bath with a condenser. The extract was collected and extracted twice. The extracts were combined, centrifuged, and filtered to obtain the final extract. The extract was concentrated under low temperature and reduced pressure to a paste, and then dried to obtain the *Botrytis cinerea* extract.

[0066] Following the above-mentioned method for obtaining the extract of *Botrytis cinerea*, the effects of single-factor ethanol concentration (60v / v%, 65v / v%, 70v / v%, 75v / v%, 80v / v%), solid-liquid ratio (1:5g / mL, 1:10g / mL, 1:15g / mL, 1:20g / mL, 1:25g / mL), extraction temperature (50℃, 60℃, 70℃, 80℃, 90℃), single extraction time (1.0h, 1.5h, 2.0h, 2.5h, 3.0h), and concentration temperature (50℃, 55℃, 60℃, 65℃, 70℃) on the extraction efficiency of sanguinarine in the extract of *Botrytis cinerea* were investigated.

[0067] 1.2 Detection methods and calculation formulas for sanguinarine content The crude extract of *Botrytis cinerea* is m1 (mg). An appropriate amount of this extract, m2, is accurately weighed and added to methanol solution. After thorough mixing, the supernatant is diluted to a 10mL volumetric flask. The absorbance is then measured using a spectrophotometer. The absorbance value is substituted into the sanguinarine standard curve to calculate the concentration of sanguinarine in the sample solution, and then the extraction content is calculated. The specific calculation method is as follows: W (%) is the extraction efficiency, m1 (mg) is the mass of the crude *Botrytis cinerea* extract, m2 is the mass of the accurately weighed appropriate amount of extract, c is the concentration of the sample solution, and M is the initial mass of *Botrytis cinerea* powder.

[0068]

[0069] 2. Experimental Results 2.1 Effect of ethanol concentration on sanguinarine extraction rate The ethanol concentrations for each treatment group were 60%, 65%, 70%, 75%, and 80%, respectively. All other extraction conditions were the same: a solid-liquid ratio of 1:15, an extraction temperature of 70℃, an extraction time of 2 hours, and a concentration temperature of 60℃. The effect of sanguisorbin extraction rate with ethanol concentration was as follows: Figure 1 As shown, with increasing ethanol concentration, the extraction efficiency of sanguisorbin from *Polygonum hydropiper* exhibits a trend of first increasing and then decreasing. According to... Figure 1 The results show that the optimal ethanol concentration in the extraction process of *Botrytis cinerea* is 70%.

[0070] The solid-liquid ratios for each treatment group were 1:5, 1:10, 1:15, 1:20, and 1:25, respectively. All other extraction conditions were the same: ethanol concentration of 70%, extraction temperature of 70℃, extraction time of 2 hours, and concentration temperature of 60℃. The effect of the solid-liquid ratio on the sanguisorbin extraction rate is shown below. Figure 2 As shown, with the increase of ethanol dosage, the extraction rate of sanguisorbin from *Polygonum hydropiper* showed a trend of first increasing and then decreasing. According to... Figure 2 The results show that the optimal material-to-liquid ratio in the extraction process of *Botrytis cinerea* is 1:15.

[0071] The extraction temperatures for each treatment group were 50℃, 60℃, 70℃, 80℃, and 90℃, respectively. All other extraction conditions were the same: ethanol concentration was 70%, solid-liquid ratio was 1:15, extraction time was 2 hours, and concentration temperature was 60℃. The effect of extraction temperature on sanguinarine extraction rate is as follows: Figure 3 As shown, with increasing extraction temperature, the extraction rate of sanguisorbin from *Polygonum hydropiper* exhibits a trend of first increasing and then decreasing. According to... Figure 3 The results show that the optimal extraction temperature during the extraction process of *Botrytis cinerea* is 60℃.

[0072] The extraction times for each treatment group were 1.0 h, 1.5 h, 2.0 h, 2.5 h, and 3.0 h, respectively. All other extraction conditions were the same: ethanol concentration was 70%, solid-liquid ratio was 1:15, extraction temperature was 60℃, and concentration temperature was 60℃. The effect of sanguisorbin extraction rate on extraction time was as follows: Figure 4 As shown, the extraction rate of sanguisorbin in *Botrytis cinerea* gradually increases with the extension of extraction time. However, after 1.5 hours, the extraction rate of sanguisorbin increases very little with the extension of time. Considering the need to save costs in the extraction process, the optimal extraction time is 1.5 hours.

[0073] The vacuum drying and concentration temperatures for each treatment group were 50℃, 55℃, 60℃, 65℃, and 70℃, respectively. All other extraction conditions were the same: ethanol concentration of 70%, solid-liquid ratio of 1:15, extraction temperature of 60℃, and extraction time of 1.5 h. The effect of sanguisorbin extraction rate with concentration temperature was as follows: Figure 5 As shown, with the gradual increase of concentration temperature, the extraction rate of sanguisorbin from *Polygonum hydropiper* showed a trend of first increasing and then decreasing, as... Figure 5 As shown, the optimal vacuum drying and concentration temperature during the extraction process of *Botrytis cinerea* is 60℃.

[0074] In summary, the optimal extraction process for extracting *Botrytis cinerea* extract using the acid-ethanol method is as follows: ethanol concentration of 70%, material-to-liquid ratio of 1:15, extraction temperature of 60℃, extraction time of 1.5h, and vacuum drying and concentration temperature of 60℃.

[0075] The optimal extraction process for Bo Luo Hui extract includes the following steps: Take dried Bo Luo Hui rhizomes, crush them through a 60-mesh sieve, and use a Soxhlet extractor to remove fat-soluble pigments, oils, and other impurities. Then, weigh an appropriate amount of Bo Luo Hui powder and place it in a flask. Add acidic ethanol (70% ethanol concentration, pH = 3.0) at a material-to-liquid ratio of 1:15. Connect a condenser and heat in a water bath to 60°C for 1.5 hours. Collect the extract, repeat the extraction twice, combine the extracts, centrifuge (3500 rpm, 15 min), filter to obtain the final extract, concentrate under reduced pressure at 60°C to obtain an extract, and then microwave vacuum dry at 40°C to obtain Bo Luo Hui extract.

[0076] Example 2: Effects of adding different amounts of *Clerodendrum trichotomum* extract to feed on grass carp growth, antioxidant capacity, and intestinal health. 1. Materials and Methods 1.1 Experimental Feed The basic formula used fishmeal and soybean meal as the main protein sources, soybean oil as the fat source, and wheat starch as the sugar source. The experimental feed formula and actual nutritional composition are shown in Table 1. The control group (CON) was the basic formula. The MCE25 group had 25 mg / kg of *Polygonum hydropiper* extract (i.e., *Polygonum hydropiper* extract obtained by the optimal extraction process in Example 1, the same below) added to the feed. The MCE50 group had 50 mg / kg of *Polygonum hydropiper* extract added to the feed. The MCE75 group had 75 mg / kg of *Polygonum hydropiper* extract added to the feed. The MCE100 group had 100 mg / kg of *Polygonum hydropiper* extract added to the feed.

[0077] Accurately weigh the raw materials in the basic formula according to the ratio, put them into a mixer and mix them thoroughly. Add an appropriate amount of distilled water, mix thoroughly and adjust the mixture. Process the mixture into pellets with a diameter of 3.0 mm using a twin-screw feed pellet mill. After drying, spray the surface of the pellets with precisely weighed soybean oil and mix them thoroughly again.

[0078] Table 1 Feed formulation and nutrient composition

[0079] 1 Each kg of multivitamin premix contains: Vitamin A, 2.10g; Vitamin D3, 0.4g; Vitamin E, 12.58g; Vitamin K3, 0.83g; Vitamin B12, 0.94g; Biotin, 0.75g; Folic acid, 0.42g; Vitamin B1, 0.11g; Vitamin C, 4.31g; Niacin, 2.58g; Inositol, 19.39g; Calcium pantothenate, 2.56g; Riboflavin, 0.63g; Vitamin B6, 0.62g; Corn starch to fill 1 kg.

[0080] 2 Each kg of multi-mineral premix contains: manganese sulfate monohydrate, 1.89 g; magnesium sulfate monohydrate, 200 g; ferrous sulfate monohydrate, 24.57 g; zinc sulfate monohydrate, 8.25 g; copper sulfate pentahydrate, 0.96 g; potassium iodide, 0.07 g; sodium selenite, 0.02 g; and corn starch to fill 1 kg.

[0081] 1.2 Experimental Fish and Feeding Management The experimental fish used in this invention were all 3-year-old grass carp. Before the formal experiment began, the grass carp were temporarily housed in pond cages (5m×3m×2m, water depth 4m) and fed commercial feed four times a day until full. After two weeks, the fish were starved for 24 hours. 360 healthy, uninjured, and uniformly sized grass carp (initial weight 1924.03±0.80g) were randomly divided into 24 cages, with 15 fish per cage. The experiment consisted of 12 treatment groups, with four replicates per group. The rearing experiment lasted for eight weeks. During the rearing period, the fish were artificially fed four times a day at 7:00, 10:00, 13:00, and 16:00. The pond water quality was measured weekly during the experiment: ammonia nitrogen 0.03~0.04mg / L, dissolved oxygen 7.2~9.3mg / L, pH 7.4-7.6, and water temperature 26~32℃.

[0082] 1.3 Sampling After an 8-week rearing experiment, the fish were fasted for 24 hours, anesthetized with MS222 (100 mg / L, Aladdin, Shanghai), and the number and weight of grass carp in each net cage were counted. Three fish were randomly selected from each net cage, and their livers and intestines were dissected, flash-frozen in liquid nitrogen, and then stored at -80°C.

[0083] Real-time fluorescence quantitative detection Total RNA was extracted from the midgut tissue of grass carp. The concentration and purity of the extracted RNA were determined. RNA that passed quality testing was reverse transcribed into cDNA, which was then analyzed by real-time quantitative PCR (RT-qPCR). The specific primer sequences used are shown in Table 2 below. β-actin As an internal reference gene, it is standardized.

[0084] Table 2 Primers used for RT-qPCR

[0085] 2. Experimental Results 2.1 Effects of adding *Pomacea canaliculata* to feed on the growth performance of grass carp Depend on Figure 6 and Figure 7 It can be seen that as the amount of *Clerodendrum trichotomum* extract added to the feed gradually increases, the weight gain rate and specific growth rate of grass carp show a trend of first increasing and then decreasing, both reaching their maximum values ​​in the MCE50 group. Adding 50 mg / kg of *Clerodendrum trichotomum* extract to the feed can significantly improve the growth performance of grass carp. P <0.05), and when the content of *Bletilla striata* continued to increase, although the growth performance did not decrease significantly, it showed a certain downward trend. Therefore, based on growth performance as the evaluation index, the appropriate addition level of *Bletilla striata* extract in grass carp feed is 50 mg / kg.

[0086] 2.2 Liver antioxidant capacity Depend on Figure 8 It was found that with the increase of *Clerodendrum trichotomum* extract in the feed, the activity of superoxide dismutase in the liver of grass carp showed a trend of first increasing and then decreasing, reaching the maximum value in the MCE50 group. Adding 50 mg / kg of *Clerodendrum trichotomum* extract to the feed significantly increased the activity of superoxide dismutase in the liver of grass carp. P <0.05%, further increasing to 100 mg / kg, will significantly reduce hepatic superoxide dismutase activity (SOD). P <0.05). By Figure 9 It can be seen that the activity of hepatic glutathione peroxidase also showed a trend of first increasing and then decreasing with the increase of the content of *Clerodendrum trichotomum* extract in the feed. The addition of *Clerodendrum trichotomum* extract to the feed had no significant effect on the activity of hepatic glutathione peroxidase. P >0.05), the glutathione peroxidase activity in the MCE100 group was significantly lower than that in the MCE50 group, indicating that continuously increasing the amount of *Polygonum hydropiper* extract in the feed may also reduce the liver's antioxidant capacity. Figure 10 and Figure 11 It was found that adding *Polygonum hydropiper* extract to feed had no significant effect on the levels of liver catalase and malondialdehyde. Based on the changes in liver superoxide dismutase and glutathione peroxidase activities, the appropriate addition level of *Polygonum hydropiper* extract to feed is 50 mg / kg. Continuous addition may have adverse effects on antioxidant capacity.

[0087] Depend on Figure 12 It is known that adding 50 mg / kg or 75 mg / kg of *Euphorbia lathyris* extract to feed can significantly improve intestinal health. claudin c Relative expression level ( P <0.05), reaching its maximum at MCE75; adding different concentrations of *Polygonum hydropiper* extract to feed can significantly improve intestinal function. Occludin Relative expression level ( P <0.05), with better effects in MCE50 and MCE75, reaching the maximum in the MCE50 group; adding 50 mg / kg or 75 mg / kg of *Clerodendrum trichotomum* extract to the feed can significantly improve intestinal function. Mucin-2 Relative expression level ( P <0.05), and reached its maximum value in the MCE50 group. The results of the combined diet supplementation with different concentrations of *Clerodendrum trichotomum* extract on the expression levels of intestinal barrier-related genes showed that the addition of 50 mg / kg of *Clerodendrum trichotomum* extract to the diet can significantly increase the expression of intestinal barrier genes and promote intestinal health.

[0088] Example 3: Effect of adding *Bletilla striata* extract to feed on the optimal sugar-lipid ratio requirement of grass carp 1. Materials and Methods 1.1 Experimental Feed Five isonitrogenous and isoenergetic experimental diets were designed, using fishmeal and soybean meal as the main protein sources, soybean oil as the fat source, and wheat starch as the sugar source. The sugar-to-lipid ratio in the diets was calculated based on the actual nitrogen-free extract and crude fat content. The corresponding sugar-to-lipid ratios were 11.58, 6.15, 3.80, 2.50, 1.67, and 1.09. The experimental diet formulations and actual nutrient compositions are shown in Table 3.

[0089] Table 3 Feed formulation and nutrient composition (% dry matter)

[0090] Note: The extract of *Botrytis cinerea* is the extract obtained using the optimal extraction process described in Example 1. The multi-dimensional premix and multi-mineral premix are the same as the feed formulations in Table 1. In the table above, "-" indicates that *Botrytis cinerea* extract was not added, and "+" indicates that *Botrytis cinerea* extract was added.

[0091] 1.2 Experimental Fish and Feeding Management The experimental fish used in this embodiment were all 3-year-old grass carp. Before the formal experiment began, the grass carp were temporarily raised in pond cages (5m×3m×2m, water depth 4m) and fed commercial feed four times a day until full. After 2 weeks, the fish were starved for 24 hours. 360 healthy, uninjured, and uniformly sized grass carp (initial weight 2018.02±1.75g) were randomly divided into 24 cages, with 15 fish in each cage. The experiment consisted of 12 treatment groups, with 4 replicates per group. The rearing experiment lasted for 8 weeks. During the rearing period, the fish were artificially fed four times a day at 7:00, 10:00, 13:00, and 16:00. The pond water quality was measured weekly during the experiment: ammonia nitrogen 0.06~0.09mg / L, dissolved oxygen 7.3~10.5mg / L, pH 7.2~7.6, and water temperature 28~32℃.

[0092] 2. Experimental Results like Figure 13 As shown, regardless of whether 50 mg / kg of *Bletilla striata* extract was added to the feed, the weight gain rate of grass carp showed a trend of first increasing and then decreasing with the decrease of the feed's sugar-lipid ratio, reaching its maximum in the group with a sugar-lipid ratio of 3.8. Adding 50 mg / kg of *Bletilla striata* extract to the feed, at a sugar-lipid ratio of 6.15, did not result in a significant difference in weight gain rate compared to the group without *Bletilla striata* and with a sugar-lipid ratio of 3.8. Therefore, it can be concluded that adding 50 mg / kg of *Bletilla striata* extract to the feed can increase the appropriate sugar-lipid ratio from 3.8 to 6.15 without negatively impacting the growth performance of grass carp. This measure, while maintaining the total energy of the grass carp feed, can reduce the amount of fat raw materials and increase the amount of carbohydrates, thereby reducing feed costs.

[0093] In summary, the optimal extraction efficiency of sanguisorbin from *Botrytis cinerea* using the acid-ethanol method is achieved with an ethanol concentration of 70%, a material-to-liquid ratio of 1:15, an extraction temperature of 60℃, an extraction time of 1.5 h, and a vacuum drying and concentration temperature of 60℃. Adding 50 mg / kg of *Botrytis cinerea* extract to feed promotes the growth performance of grass carp. Based on data on the activity and content of enzymes and substances related to antioxidant capacity in the liver, adding 50 mg / kg of *Botrytis cinerea* extract to feed not only meets the needs of rapid growth but also enhances the antioxidant capacity of grass carp. Furthermore, based on data on the relative expression levels of genes related to the intestinal barrier, adding 50 mg / kg of *Botrytis cinerea* extract to feed not only meets the needs of rapid growth and enhances antioxidant capacity but also improves intestinal health. However, it should be noted that, considering growth performance, antioxidant capacity, and the expression levels of genes related to the intestinal barrier, continuously increasing the content of *Botrytis cinerea* extract in the feed may adversely affect growth performance, antioxidant capacity, and the normal function of genes related to the intestinal barrier. Adding 50 mg / kg of *Bletilla striata* extract to the feed can increase the feed sugar-lipid ratio from 3.8 to 6.15 without affecting the normal growth of grass carp. While maintaining the total energy of the feed, the feed cost can be reduced by decreasing the amount of fat, increasing the amount of carbohydrates, and increasing the sugar-lipid ratio.

[0094] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A method for preparing an extract of *Eupatorium fortunei*, comprising the following steps: Mix *Botrytis cinerea* with an acidic ethanol solution, reflux to extract, collect the extract, and concentrate to obtain *Botrytis cinerea* extract.

2. The preparation method according to claim 1, characterized in that, The ratio of the purpureus to the acidic ethanol solution is 1:(5~25).

3. The preparation method according to claim 1, characterized in that, The concentration of ethanol in the acidic ethanol solution is 60 v / v% to 80 v / v%, and / or the pH value of the acidic ethanol solution is 2 to 4.

4. The preparation method according to any one of claims 1 to 3, characterized in that, The reflux extraction conditions are 50~90℃ for 1~3 hours.

5. The preparation method according to claim 4, characterized in that, The concentration temperature is 50~70℃.

6. The preparation method according to any one of claims 1 to 3, characterized in that, The active ingredient in the extract of *Boluhui* includes sanguisorbin.

7. The extract of *Polygonum hydropiper* prepared by any one of claims 1 to 6.

8. The preparation method according to any one of claims 1 to 6 or the use of the extract of *Botrytis cinerea* according to claim 7 in the preparation of feed or fish farming.

9. The application according to claim 8, characterized in that, The amount of *Botrytis cinerea* extract added to the feed is 25-100 mg / kg.

10. A feed comprising the extract of *Botrytis cinerea* as described in claim 7 and a base feed, said base feed comprising fish meal, soybean meal, starch, oil, multivitamin premix, multimineral premix, calcium dihydrogen phosphate, choline chloride, sodium chloride, and microcrystalline cellulose.

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