Preparation method of a silphium perfoliatum extract, feed additive and application thereof

Abstract

The present application relates to the technical field of cultivation, in particular to a preparation method of astringent horehound extract, a feed additive and application thereof. The present application takes astringent horehound as raw material, the plant can be planted in large area, and fresh grass yield per mu can reach about 10 tons, and dry grass can reach more than 1 ton. The natural source feed additive active component (i.e. astringent horehound extract) is obtained by ethanol extraction combined with column chromatography. The content of chlorogenic acid in the component can reach more than 12.9%, and the total content of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid can reach more than 35%. The active component as a feed additive shows good disease resistance in the feeding of Tibetan sheep, Ippy rabbits and small crayfish, and the yield of the animals is greatly improved, and the effect is better than that of the same dose of chlorogenic acid adding group. In addition, the astringent horehound extract prepared by the preparation method can significantly improve the meat quality and increase the yield.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology, and in particular to a method for preparing an extract of *Silphium perfoliatum*, a feed additive, and its application. Background Technology

[0002] "Reducing and banning antibiotics" has become a development trend in livestock and poultry farming. However, with the widespread reduction of antibiotics, how to cope with the resulting high incidence of animal diseases and declining yields has become a key issue facing livestock and poultry farming. The development of plant-derived or natural product-derived natural antibiotic feed additives has received increasing attention. Various natural feed additives are constantly being researched and applied in production practice, such as allicin, Bacillus subtilis, honeysuckle, and Eucommia ulmoides leaves, playing an increasingly important role in replacing antibiotics and showing good effects in animal disease prevention and control and improving meat quality. Among them, plant-derived chlorogenic acid feed additives have shown certain effects in disease control and quality improvement in livestock and poultry farming and have been widely used. At present, natural chlorogenic acid additives generally use plants such as Eucommia ulmoides leaves, honeysuckle, and stevia as raw materials. The variety of plant sources is limited, and due to the limitations of existing plant yields and content, the large-scale supply of chlorogenic acid is restricted, and the price remains high. In addition, using chlorogenic acid alone as a feed additive in production practice still cannot completely replace chemical antibiotics, and its disease resistance performance is relatively poor. Summary of the Invention

[0003] To address the aforementioned problems, this invention provides a method for preparing *Silphium perfoliatum* extract, a feed additive, and its applications. The *Silphium perfoliatum* extract prepared using the method of this invention is not only inexpensive but also exhibits superior disease resistance compared to the treatment group treated with the same dose of chlorogenic acid as a feed additive alone.

[0004] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for preparing an extract of *Silphium perfoliatum*, comprising the following steps: The extract was obtained by hot reflux extraction of *Pteris vittata* using ethanol solution; The extract was evaporated under reduced pressure to constant weight to obtain the extract; The extract was eluted using a D101 macroporous resin column to obtain an eluent containing the extract of *Heliotropium indicum*; the eluent used for elution included an aqueous ethanol solution with a mass concentration of 5% to 45%.

[0005] Preferably, the mass concentration of the ethanol solution is 0% to 70%.

[0006] Preferably, the temperature of the hot reflux extraction is 50~80℃; the hot reflux extraction is performed 2~4 times, each time for 2~4 hours.

[0007] Preferably, the volume ratio of the eluent to the D101 macroporous resin column is 2~10:1.

[0008] This invention provides the application of *Heliotropium indicum* extract prepared using the preparation method described above as a feed additive.

[0009] This invention provides a feed additive, including *Heliotropium indicum* extract prepared using the preparation method described above.

[0010] This invention provides an antibiotic-free feed, comprising a purslane extract prepared using the preparation method described above and a basic feed; the mass ratio of the purslane extract to the basic feed is 0.5~4:100.

[0011] This invention provides the application of *Heliotropium indicum* extract prepared by the preparation method described above, or the feed additive or antibiotic-free feed described above, in reducing antibiotic use during aquaculture.

[0012] This invention provides the application of the *Heliotropium indicum* extract prepared by the preparation method described above, or the feed additive or antibiotic-free feed described above, in one or more aspects of improving animal disease resistance, enhancing meat quality, and increasing yield.

[0013] Beneficial effects: This invention provides a method for preparing *Silphium perfoliatum* extract, comprising the following steps: hot reflux extraction of *Silphium perfoliatum* with ethanol solution to obtain an extract; evaporation of the extract under reduced pressure to constant weight to obtain an extract; elution of the extract using a D101 macroporous resin column to obtain an eluent containing the *Silphium perfoliatum* extract; the eluent comprising an ethanol aqueous solution with a mass concentration of 5%~40%. This invention uses *Silphium perfoliatum* as raw material, a plant that can be cultivated on a large scale, yielding approximately 10 tons of fresh grass and over 1 ton of dried grass per mu (approximately 0.067 hectares). This invention obtains a natural-source feed additive active component (i.e., *Silphium perfoliatum* extract) through ethanol extraction combined with column chromatography. The chlorogenic acid content in this component can reach over 12.9%, and the total content of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid can reach over 35%. This active component, as a feed additive, has shown excellent disease resistance in Tibetan sheep, Iplu rabbits, and crayfish, and significantly improved the yield of these animals, with better effects than the group supplemented with the same dose of chlorogenic acid.

[0014] In addition, the extract of *Heliotropium indicum* prepared using the method of this invention can significantly improve the quality of the flesh and increase the yield. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0016] Figure 1 Chromatogram of *Silphium perfoliatum* extract; Figure 2 The chemical structural formula of neochlorogenic acid; Figure 3 The chemical structural formula of chlorogenic acid; Figure 4 This is the chemical structural formula of cryptochlorogenic acid. Detailed Implementation

[0017] This invention provides a method for preparing an extract of *Silphium perfoliatum*, comprising the following steps: The extract was obtained by hot reflux extraction of *Pteris vittata* using ethanol solution; The extract was evaporated under reduced pressure to constant weight to obtain the extract; The extract was eluted using a D101 macroporous resin column to obtain an eluent containing the extract of *Heliotropium indicum*; the eluent comprised an aqueous ethanol solution with a mass concentration of 5% to 40%.

[0018] In this invention, the aerial parts of *Silphium ferrugineum* are preferably pulverized and used as the raw material for preparing *Silphium ferrugineum* extract. By selecting the pulverized aerial parts of *Silphium ferrugineum* as the raw material, this invention can obtain more chlorogenic acid and its structural analogs. This invention does not have special requirements for the pulverization method; any pulverization method well known to those skilled in the art can be used.

[0019] After obtaining the raw materials, the present invention uses an ethanol solution to perform hot reflux extraction on the raw materials to obtain an extract.

[0020] In this invention, the mass concentration of the ethanol solution is preferably 0%~70%, more preferably 0~50%, and even more preferably 50%. Experiments have shown that when the ethanol concentration exceeds 70%, more small polar compounds are easily extracted, causing unnecessary trouble in subsequent separation and preparation. While ethanol concentration is 0%, i.e., water extraction, the cost is lower, but the extraction rate of chlorogenic acid and its structural analogs is less than 70%, significantly lower than the extraction effect when the ethanol concentration is 50%. Therefore, the 50% ethanol solution extraction scheme is the optimal scheme, which not only saves costs but also improves the extraction rate and increases the utilization rate of the medicinal materials.

[0021] In this invention, the temperature of the hot reflux extraction is preferably 50~80℃, more preferably 50~60℃, and even more preferably 60℃; the number of hot reflux extractions is preferably 2~4 times, and even more preferably 3 times; the time for each hot reflux extraction is preferably 1~4 hours, more preferably 1~2 hours, and even more preferably 1 hour.

[0022] In this invention, during each hot reflux extraction, the ratio of ethanol solution to extract is preferably 3-30 mL:1 g, more preferably 10-20 mL:1 g, and even more preferably 10 mL:1 g. The extract referred to in this invention is the raw material used in the preparation, or the remaining raw material after each extraction.

[0023] After the hot reflux extraction is completed, the present invention preferably combines the hot reflux extracts from each extraction to obtain the extract.

[0024] After obtaining the extract, the present invention evaporates the extract under reduced pressure to constant weight to obtain the extract. In the present invention, the temperature of the reduced pressure evaporation is preferably 50~70℃, more preferably 60℃, and even more preferably 60℃; the pressure of the reduced pressure evaporation is preferably 50~1000mbar, more preferably 50~300mbar, and even more preferably 100mbar.

[0025] After obtaining the extract, the present invention preferably dissolves the extract in water to obtain a solution containing the extract. In the present invention, the mass ratio of the extract to water is preferably 1:2 to 30, more preferably 1:2 to 15, and even more preferably 1:5.

[0026] After obtaining the solution containing the extract, the present invention uses a D101 macroporous resin column to elute the solution containing the extract, thereby obtaining an eluent containing the *Silphium perfoliatum* extract. In the present invention, the eluent comprises an aqueous ethanol solution with a mass concentration of 5% to 45%, preferably 10% to 30%, more preferably 30%; the volume ratio of the eluent to the D101 macroporous resin column is preferably 2 to 10:1, more preferably 5:1.

[0027] The process of obtaining the eluent preferably further includes evaporating the eluent to constant weight to obtain the *Silphium perfoliatum* extract. In this invention, the evaporation temperature is preferably 55-65°C, more preferably 60°C, and even more preferably 60°C; the evaporation pressure is preferably 50-1000 mbar, more preferably 200 mbar.

[0028] The *Silphium perfoliatum* extract prepared by the method provided in this invention contains chlorogenic acid content of over 12.9%, and the total content of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid can reach over 35%. The *Silphium perfoliatum* extract prepared by this invention, as a feed additive, has shown excellent disease resistance in Tibetan sheep, Ipulu rabbits, and crayfish, and significantly improved the yield of these animals, with better effects than the group supplemented with the same dose of chlorogenic acid. Furthermore, the *Silphium perfoliatum* extract prepared using the method of this invention can significantly improve meat quality and increase yield.

[0029] This invention also provides the application of *Silphium perfoliatum* extract prepared using the preparation method described above as a feed additive. When used as a feed additive, *Silphium perfoliatum* extract prepared using the method of this invention can improve animal disease resistance, significantly enhance meat quality, and increase yield.

[0030] The present invention also provides a feed additive, comprising clematis extract prepared by the preparation method described above.

[0031] This invention also provides an antibiotic-free feed, comprising *Silphium perfoliatum* extract prepared using the method described above and a basic feed. The mass ratio of *Silphium perfoliatum* extract to the basic feed is 0.5–4:100, more preferably 1–3:100, and even more preferably 2:100. This invention, through the appropriate compounding of *Silphium perfoliatum* extract and basic feed, prepares an antibiotic-free feed that can improve animal disease resistance, significantly enhance meat quality, and increase yield.

[0032] This invention also provides the application of *Silphium perfoliatum* extract prepared using the preparation method described above, or the feed additive or antibiotic-free feed described above, in reducing antibiotic use during animal husbandry. The *Silphium perfoliatum* extract prepared using the method of this invention can improve the disease resistance of animals and reduce antibiotic use during animal husbandry, achieving the effect of "antibiotic reduction".

[0033] This invention also provides the application of *Silphium perfoliatum* extract prepared using the preparation method described above, or the feed additive or antibiotic-free feed described above, in improving animal disease resistance, meat quality, and yield in one or more aspects. The *Silphium perfoliatum* extract prepared using the method of this invention can significantly increase the body weight of pigs, sheep, cattle, and chickens, increase slaughter rate, improve meat quality, and significantly reduce common livestock and poultry diseases.

[0034] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a method for preparing a purslane extract, a feed additive, and its application provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1 A *Cephalotaxus fortunei* extract is prepared by the following method: (1) The above-ground parts of the crushed pine needle grass were crushed and extracted by hot reflux at 50°C for 2 hours with water (i.e., ethanol with a mass concentration of 0%) to obtain the initial extract and the extracted pine needle grass; the mass ratio of water to pine needle grass was 5:1. The extracted *Silphium perfoliatum* was extracted with water under reflux at 50°C for 2 hours to obtain a secondary extract. The primary and secondary extracts were then combined to obtain a mixture. The mass ratio of water to extracted *Silphium perfoliatum* was 5:1. (2) The mixture is evaporated to constant weight under reduced pressure at a temperature of 50°C and a pressure of 50 mbar to obtain the extract; (3) Dissolve the extract in water with a mass of 2 times its weight, and load it onto a D101 macroporous resin column. Elute with 2 times the column bed volume of 5% ethanol aqueous solution to obtain an eluent containing chlorogenic acid-like substances. (4) The eluent containing chlorogenic acid is evaporated to constant weight at a temperature of 50°C and a pressure of 50 mbar to obtain the extract of pine sedge.

[0036] Example 2 A *Cephalotaxus fortunei* extract is prepared by the following method: (1) The above-ground parts of the crushed pine needle herb were crushed and extracted by hot reflux at 60°C for 3 hours with 50% alcohol by mass concentration to obtain the initial extract and the extracted pine needle herb; the mass ratio of the alcohol to the pine needle herb was 10:1. The extracted *Silphium perfoliatum* was extracted by hot reflux at 60°C for 3 hours using 50% alcohol to obtain a secondary extract and *Silphium perfoliatum* after secondary extraction; the mass ratio of alcohol to extracted *Silphium perfoliatum* was 10:1. The extracted *Silphium perfoliatum* was extracted with 50% alcohol by hot reflux at 60°C for 3 hours to obtain three extracts. The first, second, and third extracts were combined to obtain a mixture. The mass ratio of alcohol to *Silphium perfoliatum* after the second extraction was 10:1. (2) The mixture is evaporated to constant weight under reduced pressure at a temperature of 60°C and a pressure of 200 mbar to obtain the extract; (3) Dissolve the extract in 5 times its mass of water and load it onto a D101 macroporous resin column. Elute with 5 times the column bed volume of 10% ethanol aqueous solution to obtain an eluent containing chlorogenic acid-like substances. (4) The eluent containing chlorogenic acid is evaporated to constant weight at a temperature of 60°C and a pressure of 200 mbar to obtain the extract of Ficus microcarpa.

[0037] Example 3 A *Cephalotaxus fortunei* extract is prepared by the following method: (1) The above-ground parts of the crushed pine needle herb were crushed and extracted by hot reflux at 70°C for 4 hours with 70% alcohol to obtain the initial extract and the extracted pine needle herb; the mass ratio of the alcohol to the pine needle herb was 30:1. The extracted *Silphium perfoliatum* was extracted by hot reflux at 70°C for 4 hours using 70% alcohol to obtain a secondary extract and *Silphium perfoliatum* after secondary extraction; the mass ratio of alcohol to extracted *Silphium perfoliatum* was 30:1. The extracted *Silphium perfoliatum* was extracted by hot reflux at 70°C for 4 hours using 70% alcohol to obtain a triple extract and *Silphium perfoliatum* after triple extraction; the mass ratio of the alcohol to the *Silphium perfoliatum* after the second extraction was 30:1. The extracted *Silphium perfoliatum* was extracted with 70% alcohol by hot reflux at 70°C for 4 hours to obtain four extracts. The first, second, third, and fourth extracts were combined to obtain a mixture. The mass ratio of alcohol to *Silphium perfoliatum* after three extractions was 30:1. (2) The mixture is evaporated to constant weight under reduced pressure at a temperature of 70°C and a pressure of 1000 mbar to obtain the extract; (3) Dissolve the extract in 30 times its weight of water and load it onto a D101 macroporous resin column. Elute with 10 times the column bed volume of 40% ethanol aqueous solution to obtain an eluent containing chlorogenic acid-like substances. (4) The eluent containing chlorogenic acid is evaporated to constant weight at a temperature of 65°C and a pressure of 1000 mbar to obtain the extract of pine sedge.

[0038] Test Example 1 The extracts of *Heliotropium indicum* prepared in Examples 1-3 were subjected to chromatographic analysis, and the results are shown below. Figure 1 .

[0039] Assay method: Column: Shiseido CAPCELL PAK C18 MGⅡ (4.6mm×250mm, 5μm); Mobile phase: 0.1% formic acid aqueous solution (A) and acetonitrile (B); Gradient elution: 0~15min (5%~15% B), 15min~50min (15%~30% B); Column temperature: 25℃; Detection wavelength: 254nm; Injection volume: 10μL.

[0040] The standards were purchased from: chlorogenic acid (China National Institutes for Food and Drug Control, 110753-202018); neochlorogenic acid (Chengdu Purifa Technology Development Co., Ltd., CAS 906-33-2); and cryptochlorogenic acid (Chengdu Purifa Technology Development Co., Ltd., CAS 905-99-7), all with a purity ≥98%. The results are as follows: In Example 1, the content of neochlorogenic acid was measured to be 4.58%, chlorogenic acid content was 6.71%, and cryptochlorogenic acid content was 4.49%.

[0041] In Example 2, the content of neochlorogenic acid was measured to be 8.33%, chlorogenic acid content was 12.94%, and cryptochlorogenic acid content was 14.70%.

[0042] In Example 3, the content of neochlorogenic acid was measured to be 3.28%, chlorogenic acid content was 7.13%, and cryptochlorogenic acid content was 8.67%.

[0043] It is evident that the methods provided by this invention can effectively extract neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid from *Pteris vittata*, with the highest content of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid obtained by the preparation method in Example 2.

[0044] Application Example 1 Effects of feeding sheep: Tibetan sheep raised on grasslands were selected as the research subjects and divided into a control group, a chlorogenic acid control group, a low-dose additive group, a medium-dose additive group, and a high-dose additive group, with 20 sheep in each group and 3 parallel groups in each group.

[0045] The control group was raised normally and supplemented with conventional feed (purchased from Qinghai Yellow River Livestock Development Co., Ltd., 50kg / bag, product number 20220816). The low-dose additive group was raised in the same manner as the control group, with 5g of the *Cephalotaxus fortunei* extract prepared in Example 2 added to each kilogram of conventional feed. The low-dose additive group was raised in the same manner as the control group, with 20g of the *Cephalotaxus fortunei* extract prepared in Example 2 added to each kilogram of conventional feed. The low-dose additive group was raised in the same manner as the control group, with 40g of the *Cephalotaxus fortunei* extract prepared in Example 2 added to each kilogram of conventional feed. The chlorogenic acid control group was raised in the same manner as the control group. In addition to the regular feed, 2.64g of chlorogenic acid (chlorogenic acid: China National Institutes for Food and Drug Control, 110753-202018, purity ≥98%, fed according to the chlorogenic acid content in the dosage group of Silphium perfoliatum extract, 2.64g = 20g × 12.94% ÷ 98%).

[0046] The experiment lasted for 5 months. During the feeding process, the incidence and mortality rates of different diseases in the sheep were recorded. At the end of the experiment, the weight of the sheep in each group was calculated. The statistical results are shown in Tables 1 and 2.

[0047] Table 1. Disease resistance properties of active components of *Silphium perfoliatum* to sheep.

[0048] Table 2. Effects of active components of *Silphium perfoliatum* on sheep growth performance.

[0049] Tables 1 and 2 show that both low- and high-dose groups of *Silphium perfoliatum* significantly reduced the incidence of the three diseases. The incidence of all three diseases was 0 in the high-dose group throughout the entire feeding cycle. The chlorogenic acid group showed some inhibitory effect on the incidence of several diseases, but there was no significant difference compared to the control group. All doses of *Silphium perfoliatum* extract had a good promoting effect on sheep growth, significantly increasing growth and meat yield. Chlorogenic acid increased sheep carcass weight to some extent, but there was no significant difference compared to the control group.

[0050] Application Example 2 Feeding effects on domestic rabbits: The rabbits were selected for cage breeding, and the Iplu breed was selected for the experiment during the high-temperature season from May to August, which is the peak period for rabbit diseases.

[0051] The animals were divided into a control group, a chlorogenic acid control group, a low-dose additive group, a medium-dose additive group, and a high-dose additive group, with 25 animals in each group and 3 parallel groups in each group.

[0052] The control group was fed conventional rabbit feed (purchased from Chongqing Jinbao Feed Technology Co., Ltd., 40kg / bag, product number Q / CJB06-2021). The low-dose additive group was fed in the same way as the control group, and 5g of the Ficus pumila extract prepared in Example 2 was added to each kilogram of conventional rabbit feed. The low-dose additive group was fed in the same way as the control group, and 20g of the Ficus pumila extract prepared in Example 2 was added to each kilogram of conventional rabbit feed. The low-dose additive group was fed in the same way as the control group, and 40g of the Ficus pumila extract prepared in Example 2 was added to each kilogram of conventional rabbit feed. The chlorogenic acid control group was fed the same as the control group, and 2.64g of chlorogenic acid (chlorogenic acid: China National Institutes for Food and Drug Control, 110753-202018, purity ≥98%, fed according to the chlorogenic acid content in the medium dose group of Silphium perfoliatum extract, 2.64g = 20g × 12.94% ÷ 98%) was added per kilogram of conventional rabbit feed.

[0053] The experiment lasted for 3 months. During the feeding period, the morbidity and mortality rates of the rabbits were recorded. After the experiment, the body weight and carcass weight of each group were measured. The statistical results are shown in Tables 3 and 4.

[0054] Table 3. Disease resistance properties of active components of *Silphium perfoliatum* against rabbits.

[0055] Table 4. Effects of active components of *Silphium perfoliatum* on rabbit growth performance.

[0056] Tables 3 and 4 show that the high-dose addition of *Silphium perfoliatum* significantly reduced the incidence of diarrhea in Ipru rabbits, and significantly increased rabbit body weight and carcass weight, thus significantly increasing meat yield. Furthermore, both the medium-dose and high-dose *Silphium perfoliatum* groups were superior to the chlorogenic acid-added group.

[0057] Application Example 3 Effects of feeding crayfish: The experiment was conducted during the high-temperature season from April to June. Crayfish were cultured in boxes and divided into a control group, a chlorogenic acid control group, a low-dose additive group, a medium-dose additive group, and a high-dose additive group. Each group had three parallel groups, and the crayfish were raised in separate boxes, with 1,000 crayfish larvae stocked in each box.

[0058] The control group was fed conventional crayfish feed (purchased from Yangzhou Hongda Feed Co., Ltd., 20kg / bag, product number 10043193409985, without additives). The low-dose additive group was fed in the same way as the control group, and 5g of the Ficus pumila extract prepared in Example 2 was added per kilogram of conventional crayfish feed. The low-dose additive group was fed in the same way as the control group, and 20g of the Ficus pumila extract prepared in Example 2 was added per kilogram of conventional crayfish feed. The low-dose additive group was fed in the same way as the control group, and 40g of the Ficus pumila extract prepared in Example 2 was added per kilogram of conventional crayfish feed. The chlorogenic acid control group was fed the same as the control group, and 2.64g of chlorogenic acid (chlorogenic acid: China National Institutes for Food and Drug Control, 110753-202018, purity ≥98%, fed according to the chlorogenic acid content in the medium dose group of Silphium perfoliatum extract, its 2.64g=20g×12.94%÷98%) was added per kilogram of conventional crayfish feed.

[0059] The experiment lasted for two months. During the rearing process, the mortality rate of crayfish was recorded. After the experiment, the number of crayfish harvested and the quantity per box were recorded. The statistical results are shown in Table 5.

[0060] Table 5. Effects of active components of *Silphium perfoliatum* on the growth performance of crayfish.

[0061] Table 5 shows that the *Silphium perfoliatum* experimental group reduced crayfish mortality and significantly improved crayfish growth efficiency, increasing harvest yield. The chlorogenic acid group showed some reduction in crayfish mortality and increased the yield per box, but neither effect was statistically significant.

[0062] In summary, the *Silphium perfoliatum* extract prepared using the method of this invention exhibited excellent disease resistance in Tibetan sheep, Iplu rabbits, and crayfish, and significantly improved the yield of these animals. Moreover, its effect was superior to that of the group with the same dose of chlorogenic acid added. In addition, it could also significantly improve meat quality and increase yield.

[0063] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing an extract of *Silphium perfoliatum*, characterized in that, The process consists of the following steps: hot reflux extraction of *Penicillium schreberi* using an ethanol solution to obtain an extract; the ethanol solution has a mass concentration of 50%. The extract was evaporated under reduced pressure to constant weight to obtain the extract; The extract was eluted using a D101 macroporous resin column to obtain an eluent containing the extract of *Heliotropium indicum*. The eluent included an aqueous ethanol solution with a mass concentration of 5% to 45%. The volume ratio of the eluent to the D101 macroporous resin column was 2 to 10:

1. The temperature of the hot reflux extraction is 50~80℃; the hot reflux extraction is performed 2~4 times, each time for 2~4 hours; The extract of *Symplocos lucida* contains chlorogenic acid of 12.9% or more; the total content of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid is 35% or more.

2. The application of the *Heliotropium indicum* extract prepared by the method described in claim 1 as a feed additive.

3. A feed additive, characterized in that, This includes the extract of *Heliotropium indicum* prepared using the preparation method described in claim 1.

4. An antibiotic-free feed, characterized in that, The mixture includes *Heliotropium indicum* extract prepared by the method described in claim 1 and a basic feed; the mass ratio of the *Heliotropium indicum* extract to the basic feed is 0.5~4:

100.

5. The application of the *Heliotropium indicum* extract prepared by the preparation method of claim 1, or the feed additive of claim 3, or the antibiotic-free feed of claim 4 in reducing the use of antibiotics in the breeding process.

6. The use of the extract of *Heliotropium indicum* prepared by the preparation method of claim 1, or the feed additive of claim 3, or the antibiotic-free feed of claim 4, in one or more aspects of improving animal disease resistance, improving meat quality, and increasing yield.

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