CN116270788A - Preparation method and application of honeysuckle leaf extract and composition thereof - Google Patents
Preparation method and application of honeysuckle leaf extract and composition thereof Download PDFInfo
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- CN116270788A CN116270788A CN202310155911.0A CN202310155911A CN116270788A CN 116270788 A CN116270788 A CN 116270788A CN 202310155911 A CN202310155911 A CN 202310155911A CN 116270788 A CN116270788 A CN 116270788A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/35—Caprifoliaceae (Honeysuckle family)
- A61K36/355—Lonicera (honeysuckle)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
- A61K36/539—Scutellaria (skullcap)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
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- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention discloses a preparation method and application of a honeysuckle leaf extract and a composition thereof. According to the invention, the acid water extraction method is adopted to prepare the honeysuckle leaf extract, and then the honeysuckle leaf extract and the baical skullcap root extract are compounded, so that MIC values of staphylococcus aureus and escherichia coli are obviously reduced, the dosage is further reduced, and the full utilization of the medicinal material resources of the honeysuckle leaf is realized. The extract and the composition have the advantages of good stability and slow degradation of chlorogenic acid serving as an effective substance in a weak alkaline solvent, and have the advantages of stable content and no precipitation of baicalin serving as the effective substance under an acidic condition. The extract can effectively reduce the death rate of chickens and improve the heat stress resistance of organisms after being fed for a long time; effectively reduces feed conversion ratio, improves feed return and reduces feeding cost.
Description
Technical Field
The invention relates to a preparation method and application of a honeysuckle leaf extract with an antibacterial effect and stable and nondegradable active ingredients and a composition thereof, and belongs to the technical field of traditional Chinese medicine extracts.
Background
The honeysuckle (Lonicera japonica thunder.) is a perennial vine of Caprifoliaceae (Caprifoliaceae) of Caprifolia (Lonicera), and its medicinal part is stem and leaf initially, and later is changed into flower. In modern times, honeysuckle is adopted as a medicinal material, and honeysuckle leaves are always used as a non-medicinal part to cause the waste of traditional Chinese medicine resources although the honeysuckle leaves have similar effective components and clinical effects as the honeysuckle, and the clear herbal literature records that the functions of the honeysuckle leaves and flowers are the same. Modern researches have also shown that the active ingredients and pharmacological actions in honeysuckle leaves are no more than those of honeysuckle. The honeysuckle She Jijin is taken as an important traditional Chinese medicine resource in China, and has various medicinal effects such as anti-inflammation, heat clearing, antivirus, bacteriostasis and the like.
The radix Scutellariae (Scutellariae radix) is dry root of radix Scutellariae (Scutellaria baicalensis Georgi) belonging to Labiatae, is one of the traditional bulk medicinal materials in China, and has effects of clearing heat, eliminating dampness, purging pathogenic fire, removing toxic substances, stopping bleeding, and preventing miscarriage. Baicalin is the main active component of radix Scutellariae, is also index component, and has antiviral, anticancer, antioxidant, and antibacterial effects.
Coli (Escherichia coli), also known as Escherichia coli, is one of the most common gram-negative bacteria in clinical infection, and can cause gastrointestinal tract infection or urinary tract infection of various local tissues and organs of people and various animals under certain conditions. Studies have shown that colibacillosis in animals can occur in a variety of domestic animals, poultry, farm economic animals, and other land animals and in certain aquatic animals, where pigs and chickens are the most susceptible and very dangerous. Staphylococcus aureus (Staphylococcus aureus, s.aureus) is also called "staphylococcus aureus", one of the most common gram-positive bacteria in clinical infection, is commonly found on skin surface and upper respiratory mucosa, can produce enterotoxin under certain conditions, causes food poisoning, and is a common pathogenic bacteria causing food poisoning.
Modern researches show that the extract of the honeysuckle leaves contains abundant antibacterial active substances, and the flavone substances in the honeysuckle leaves have stronger antibacterial effect on intestinal pathogenic bacteria; the ethyl acetate and n-butanol extract of the lonicera coreana leaves has a good antibacterial effect on escherichia coli. Zhang Ning et al (Zhang Ning, cao Guangqun, lin Guikun, et al, study of antibacterial and antioxidant Activity of honeysuckle leaves [ J ]]The spice essence cosmetics, 2008, (3), 29-32) find that the ethanol extract of the honeysuckle leaves has a wider antibacterial spectrum and has antibacterial effect on escherichia coli and staphylococcus aureus. Baicalin has broad-spectrum antibacterial activity and has remarkable inhibition effect on escherichia coli and staphylococcus aureus. However, wang Shuangshuang and the like have found that the extract of Scutellaria baicalensis Georgi obtained by extracting Scutellaria baicalensis Georgi with pure water has a general inhibitory effect on Staphylococcus aureus. Li Zhen and the like, the MIC of the water extraction of the baical skullcap root on staphylococcus aureus is 15.6mg.mL -1 MIC for E.coli was 31.3 mg.multidot.mL -1 。
Disclosure of Invention
The invention provides a preparation method and application of a honeysuckle leaf extract and a composition thereof. According to the invention, the acid water extraction method is adopted to prepare the honeysuckle leaf extract, and then the honeysuckle leaf extract and the baical skullcap root extract are compounded, so that MIC values of staphylococcus aureus and escherichia coli are obviously reduced, the dosage is further reduced, and the full utilization of the medicinal material resources of the honeysuckle leaf is realized. The extract and the composition have the advantages of good stability and slow degradation of chlorogenic acid which is an effective substance in a weak alkaline solvent (similar to the pH value in the intestinal environment of organisms), and have the advantages of stable and non-precipitation of baicalin content which is an effective substance under acidic conditions.
The invention firstly provides a preparation method of a honeysuckle leaf extract, which is characterized in that the honeysuckle leaf is extracted by adopting a hydrochloric acid aqueous solution with pH of 2.0-5.0 in a heating and decocting way, so as to obtain the honeysuckle leaf extract, wherein the chlorogenic acid content of the honeysuckle leaf extract is more than or equal to 3 percent.
The preparation method of the honeysuckle leaf extract specifically comprises the following steps: extracting by adopting hydrochloric acid aqueous solution with pH of 2.0-5.0 in a heating and decocting mode, wherein the ratio of the first extraction liquid to the second extraction liquid is 1:6-1:15 (mass/volume, g/mL), and the ratio of the second extraction liquid to the first extraction liquid is 1:5-1:15; mixing the extracting solutions, concentrating until the concentration of the liquid medicine is equivalent to 0.5-2.0 g/mL, adjusting the pH to 3.0-6.5 to obtain the concentrated solution of the honeysuckle leaf extract, and spray drying the obtained concentrated solution to obtain the honeysuckle leaf extract.
The invention also provides a composition containing the honeysuckle leaf extract, which is characterized by at least comprising the honeysuckle leaf extract and the baical skullcap root extract, wherein the chlorogenic acid content in the composition is more than or equal to 10.0mg/g, and the baicalin content is more than or equal to 150.0mg/g.
The preparation method of the composition specifically comprises the following steps: dissolving Scutellariae radix extract in concentrated solution of folium Lonicerae extract, and spray drying to obtain composition of radix Lonicerae She Huangqin.
The chlorogenic acid of the honeysuckle leaf extract prepared by the method has good stability, the extract or the composition is diluted by 10-100 times by adding water, the pH value is regulated to 8.0 by using sodium hydroxide solution, and the degradation rate of the chlorogenic acid in the obtained solution within 5 days is less than or equal to 10.0 percent.
The invention also provides application of the extract or the composition in inhibiting staphylococcus aureus and escherichia coli.
The antibacterial effect is as follows: the honeysuckle leaf extract is diluted by 10-500 times by adding water, and has good antibacterial effect on the standard escherichia coli. The honeysuckle She Huangqin composition is diluted by 50-1000 times by adding water, and has good antibacterial effect on the standard escherichia coli.
The invention also provides application of the honeysuckle leaf extract in improving animal immunity and reducing feed conversion ratio.
The invention has the technical effects that:
1. in the preparation process of the extract, a weak acid extraction solvent is adopted, so that partial impurities in the honeysuckle leaves can be removed, and the stability of chlorogenic acid in the extract is improved;
2. in the preparation process of the composition, a method of mixing the honeysuckle leaf extract concentrated solution and the baicalin extract is adopted, so that precipitation of baicalin due to acidity of the solution is avoided; the chlorogenic acid and baicalin which are medicinal substances are fully reserved, so that the stability and antibacterial effect of the product are improved.
3. Compared with the extracts extracted by the traditional water extraction process, the honeysuckle leaf extract prepared by the method has higher antibacterial activity and 3 times of MIC (many times of MIC) reduction. Compared with the independent extracts of the honeysuckle leaves and the baical skullcap root, the honeysuckle leaf composition prepared by the method can achieve lower MIC and achieve remarkable synergistic effect. The composition has the advantages of unique antibacterial effect and low cost, and can be used as a traditional Chinese veterinary medicine.
4. Long-term feeding experiments prove that: the honeysuckle leaf extract can effectively improve the immune organ index of the organism, reduce the death rate of chickens, improve the heat stress resistance of the organism, effectively reduce the feed conversion ratio, improve the feed return and reduce the raising cost.
Drawings
FIG. 1 is a total ion flow diagram of a honeysuckle leaf extract, wherein the positive ion mode of FIG. 1-a; FIG. 1-b anion mode;
fig. 2 is a chlorogenic acid mass spectrum (rt=6.31 min, [ M-H ] =353.08734, ppm=8.5);
fig. 3 is a isochlorogenic acid mass spectrum (rt=8.39 min, [ M-H ] =515.11540, ppm=6.7);
FIG. 4 is a comparison of the stability of honeysuckle leaf extract and aqueous extract in slightly alkaline solvents;
FIG. 5 shows the HPLC chart for detecting the content of baicalin and chlorogenic acid (FIG. 5-a: honeysuckle She Huangqin composition, FIG. 5-b: baicalin reference substance, FIG. 5-c: chlorogenic acid reference substance).
Detailed Description
The following examples are given for better illustration of the technical solution of the present invention, but the present invention is not limited thereto.
Example 1: preparation of honeysuckle leaf extract and LC-Q-Orbitrap-MS detection
Preparing the honeysuckle leaf extract by adopting an acid water extraction method: crushing honeysuckle leaves, adding 10 times of dilute hydrochloric acid solution with pH of 4.0, soaking for half an hour, heating, decocting and extracting for 1 hour; filtering, adding 8 times of diluted hydrochloric acid solution with pH of 4.0 into the filter residue, and heating, decocting and extracting for 1 hr; mixing the extractive solutions, concentrating to relative density of 1.10, and spray drying.
Taking 0.1g of honeysuckle leaf extract, precisely adding 100mL of methanol, carrying out ultrasonic treatment for 30min, cooling, supplementing the weight with methanol, filtering, taking 5mL of subsequent filtrate, placing into a 10mL measuring flask, adding methanol to fix the volume to scale, shaking uniformly, and passing through a 0.22 mu m microporous filter membrane. UPLC-Q-orbitrap-MS was sampled under the following conditions, wherein chlorogenic acid content was not less than 0.12mg/mL.
Instrument: ultra high performance liquid chromatography-quaternary rod-electrostatic field orbitrap mass spectrometry (UPLC-Q-Exactive Orbitrap-MS);
chromatographic column: acclaim TM RSLC 120C 18 column (2.1 mm. Times.100 mm,2.2 μm);
mobile phase conditions: the mobile phase A is 0.1% formic acid aqueous solution, the mobile phase B is 0.1% formic acid acetonitrile solution, and the gradient elution is carried out (0-3 min,2% -5% B, 3-15min,5% -25% B, 15-25min,25% -65% B, 25-28min,65% -100% B,28-30min,100% -5% B); volume flow rate is 0.3mL/min; column temperature is 30 ℃; the sample volume was 5.0. Mu.L.
Mass spectrometry conditions: the temperature of the atomized gas is 300 ℃, the volume flow of the atomized gas is 8.0L/min, and the desolventizing gas (N) 2 ) Volume flow rate 8.0L/min, capillary voltage 3500V (positive/negative ion modeFormula), nozzle voltage is 500V, scanning range m/z is 100.0-1700.0. Acquisition time: 2.00-30.00 minutes.
Total ion flow diagram, chlorogenic acid and isochlorogenic acid of the product are shown in figures 1-3.
Example 2: degradation experiment of honeysuckle leaf extract and traditional water extract
100.0g of honeysuckle leaf is taken and the honeysuckle leaf extract is prepared according to the method of the example 1. The chlorogenic acid content of the extract was found to be 4.08%.
Traditional aqueous extracts: taking 100.0g of honeysuckle leaves, adding 1000ml of purified water, soaking for half an hour, heating and reflux-extracting for 1 hour, filtering, adding 800ml of purified water, heating and reflux-extracting for 1 hour, combining filtrates, concentrating to a relative density of 1.10, and spray-drying to obtain the traditional water extract of the honeysuckle leaves. The chlorogenic acid content of the extract was found to be 3.98%.
5.0g of each of the honeysuckle leaf extract and the water extract is added with 100ml of water, and the pH value is adjusted to 8.0 by sodium hydroxide solution. The chlorogenic acid content of the samples was measured at 37℃for 0, 2, 4, 8, 12, 24, 48, 96, 168 hours by shaking, and the relative chlorogenic acid content of the honeysuckle leaf extract and the conventional aqueous extract of the honeysuckle leaf was calculated (compared with 0 hour).
The method for measuring chlorogenic acid content in the solution comprises the following steps:
instrument: an Agilent 1260 high performance liquid chromatograph (Agilent company, usa) equipped with an autosampler, a column oven, a diode array detector, a quaternary pump;
chromatographic conditions: agilent Zorbax C18 column (4.6 mm. Times.15 mm,5 μm); acetonitrile-0.4% phosphoric acid solution (10:90) is taken as a mobile phase; the detection wavelength was 327nm.
Preparation of a control solution: taking appropriate amount of chlorogenic acid reference substance, precisely weighing, placing into brown measuring flask, and adding 50% methanol to obtain solution containing 40 μg per 1mL (preserved below 10deg.C).
Preparation of test solution: putting 1ml of the solution into a 50ml brown measuring flask, adding a proper amount of 50% methanol, shaking to dissolve, fixing the volume to the scale with 50% methanol, and shaking uniformly to obtain the final product.
The measuring method comprises the following steps: respectively precisely sucking 10 μl of each of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement.
As shown in FIG. 4 and Table 1, compared with the traditional aqueous extract of honeysuckle leaves, the extract of honeysuckle leaves extracted by the method of the invention has slower degradation of chlorogenic acid and better stability.
TABLE 1 chlorogenic acid relative content (%)
| Time (h) | Honeysuckle leaf extract | Honeysuckle She Chuantong |
| 0 | 100.0 | 100.0 |
| 2 | 98.8 | 95.2 |
| 4 | 98.6 | 90.2 |
| 8 | 96.6 | 83.3 |
| 12 | 96.7 | 69.4 |
| 24 | 95.8 | 55.1 |
| 48 | 95.1 | 32.5 |
| 96 | 93.2 | 19.1 |
| 168 | 90.6 | 11.7 |
Example 3: preparation of honeysuckle She Huangqin composition and experiment for measuring content of baicalin and chlorogenic acid
The preparation method comprises the following steps: soaking 100.0g of honeysuckle leaves in 10 times of hydrochloric acid solution with pH of 4.0 for half an hour, and heating, decocting and extracting for 1 hour; adding 8 times of dilute hydrochloric acid solution with pH of 4.0 for the second time, and heating, decocting and extracting for 1 hour; mixing the extractive solutions, and concentrating to 1.0g crude drug/mL. Adding 8.0g of Scutellariae radix extract (baicalin content 85%), heating for dissolving, and spray drying.
And (3) content measurement: putting 0.5g of the honeysuckle leaf and baical skullcap root composition into a 50mL volumetric flask, adding a proper amount of 50% methanol, carrying out ultrasonic treatment for 30 minutes, cooling, and fixing the volume to the scale. The mixture was filtered, and the subsequent filtrate was measured at a precision of 10. Mu.L by HPLC.
Instrument: an Agilent 1260 high performance liquid chromatograph (Agilent company, usa) equipped with an autosampler, a column oven, a diode array detector, a quaternary pump;
chromatographic column: agilent Zorbax C18 column (4.6 mm. Times.15 mm,5 μm);
mobile phase conditions: the mobile phase A is acetonitrile, the mobile phase B is 0.2% phosphoric acid solution, and the gradient elution is carried out (0-15 min, A:5% -20%, 15-30 min, A:20% -30%, 30-40 min, A:30% -70%); the detection wavelength is 327nm; column temperature is 30 ℃; the flow rate is 1.0mL/min; the sample volume was 10.0. Mu.L.
The HPLC chart of baicalin and chlorogenic acid content detection is shown in figure 5, and the detected baicalin content is 176.13mg/g and chlorogenic acid content is 32.47mg/g.
Example 4: antibacterial efficacy experiment of honeysuckle leaf extract and composition
The extracts of honeysuckle leaves, aqueous extracts of honeysuckle leaves and compositions of honeysuckle leaves were prepared as in example 1, example 2 and example 3, and the Minimum Inhibitory Concentration (MIC) of the extracts of scutellaria baicalensis purchased (baicalin content 85%) was examined by a microdilution method using staphylococcus aureus ATCC 25923 and escherichia coli as indicator strain ATCC 25922 and ttc as indicators.
The bacteriostasis experimental scheme is as follows: operating in sterile workbench environment with reference to pharmacopoeia method, diluting the bacteria suspension with sterile physiological saline, making blank with sterile physiological saline, and adjusting the concentration of bacteria solution to OD 600nm =0.5. And sucking 1mL of the prepared bacterial suspension, adding the bacterial suspension into 100mL of LB culture medium, and shaking uniformly for later use. Meanwhile, sample solutions of the honeysuckle leaf extract (100 mg/ml), the honeysuckle leaf composition (100 mg/ml) and the control group baicalin extract (100 mg/ml) were prepared by using a volumetric flask as sample standby solutions. 100. Mu.L of LB medium containing TTC was added to each well of the 96-well plate, and 100. Mu.L of treated stock solution was added to each row of the first wells, which was double diluted. That is, after the sample standby liquid is added into the first hole, the mixture in the first hole is sufficiently mixed by a pipetting gun (at least three times of blowing), 100. Mu.L of the mixture is sucked and added into the second hole, and the mixture is repeatedly blown and mixed by the pipetting gun, and the operation is repeated until the 9 th hole is reached. Adding 100 μl of LB medium containing indicator bacteria into each diluted well, adding into 8 th well, and shaking gently (injection: 9 th well is not added, 10 th well is added) to obtain 2 respectively 2 、2 3 、……2 8 、2 9 And (5) gradient diluted sample solution. The last two wells are controls, i.e. the 9 th well is a negative control (without indicator bacteria solution) and the 10 th well is a growth control (without sample solution).
And (3) result judgment: if bacteria grow, TTC will appear red; TTC does not change color if no bacteria grow. The final well on the 96-well plate that did not reddish corresponds to the MIC value of the sample.
Table 2 MIC of extract for Staphylococcus aureus (mg/ml)
TABLE 3 MIC of extract for E.coli (mg/ml)
Remarks: in the table, the honeysuckle leaf composition (calculated by the honeysuckle leaf extract), i.e. the minimum inhibitory concentration is the concentration of the honeysuckle leaf extract; the honeysuckle leaf composition (calculated by baicalin), namely the minimum antibacterial concentration is the concentration of baicalin; MIC values for the honeysuckle leaf composition are the concentrations of the entire composition.
As can be seen from the results in tables 2 and 3, the honeysuckle leaf extract prepared by the method has higher antibacterial activity and 3 times lower MIC than the extract extracted by the traditional water extraction process. Compared with the independent extracts of the honeysuckle leaves and the baical skullcap root, the honeysuckle leaf composition prepared by the invention can achieve lower MIC and achieve remarkable synergistic effect.
Example 5: long-term feeding experiment of honeysuckle leaf extract
1 Material
1.1 test animals
The large meat chickens are raised from 0 days old to 42 days old. Feeding into semi-closed experimental animal house, and feeding special person with daily ration without functional additive, wherein the daily ration mainly comprises semen Maydis, bean cake, flour, minerals, vitamins, amino acids, enzyme preparation, etc., and the main ingredient assurance values are shown in Table 4.
TABLE 4 daily ration principal ingredient assurance value Table
1.2 test drugs
The honeysuckle leaf extract and the honeysuckle She Chuantong aqueous extract.
1.3 vaccine
Newcastle disease and avian influenza (subtype H9) bivalent inactivated vaccine (La Sota strain + F strain), qingdao Yibang bioengineering company; newcastle disease and infectious bronchitis combined live vaccine (La Sota strain+H2120 strain), harrow group biological vaccine Co., ltd; newcastle disease live vaccine (La Sota strain), qingdao Yibang bioengineering Co., ltd.
2 method
2.1 Experimental drugs
2.1.1 preparation Process of honeysuckle leaf extract
Preparing a honeysuckle leaf extract by adopting an acid water extraction method, crushing the honeysuckle leaves, adding 10 times of dilute hydrochloric acid solution with pH of 4.0, soaking for half an hour, heating, decocting and extracting for 1 hour; adding 8 times of dilute hydrochloric acid solution with pH of 4.0 for the second time, and heating, decocting and extracting for 1 hour; mixing the extractive solutions, concentrating to 1.10 g/mL, and spray drying.
2.1.2 preparation process of honeysuckle She Putong aqueous extract
Soaking folium Lonicerae in 10 times of purified water for half an hour, reflux-extracting under heating for 1 hr, filtering, adding 8 times of purified water, and reflux-extracting under heating for 1 hr. Mixing the filtrates, concentrating to relative density of 1.10, and spray drying to obtain traditional water extract of folium Lonicerae.
2.2 grouping and processing method
2.2.1 feeding management
The method comprises the steps of feeding chickens to be tested in columns, taking free diet and drinking water in the whole period, controlling the temperature and the humidity according to the feeding requirements of a chicken farm, flushing all the chicken columns, water tanks, trough and other appliances for a week before entering chickens, spraying disinfectant, and spraying quicklime water on the ground and a 1-meter high wall after airing. In the feeding process, the trough and the water tank are cleaned regularly every day, so that excrement is prevented from being mixed into the feed, and the management of each stage is performed by referring to the broiler feeding management.
2.2.2 immunization programs
The immunization schedule for the test chickens is shown in Table 5.
Table 5 test chicken immunization program
2.2.3 test packets and methods of processing
The experiment was conducted in 5 treatment groups of 6 replicates per experimental group of 8 chickens, and the specific treatment is shown in table 6. The medicines are mixed with feed in advance according to the addition amount to prepare premix, and the premix is added into normal daily ration according to the addition amount of 10% during feeding.
TABLE 6 grouping and processing modes
2.3 measurement index and method
2.3.1 determination of growth Properties
Test chickens were fed only, water was not cut, and 14:00 empty weight was weighed and weight changes recorded at 8:00 a morning at 7, 21, and 42 days of age, respectively. Counting feed consumption at ages of 7, 21 and 42 days respectively, calculating average daily feed intake (Average daily feed intake, ADFI), average daily weight gain (Average daily gain, ADG) and feed-to-weight ratio (F/G), recording total number of the dead chickens during the test, and observing the health condition of the test chickens every day; the temperature and humidity of the henhouse environment are detected every day, and the temperature and humidity of the henhouse environment are regulated abnormally and timely.
The calculation method comprises the following steps:
average Daily Feed Intake (ADFI) = (total feed-amount of residual feed)/(number of days tested) number of chickens tested per group
Average Daily Gain (ADG) = (average body weight at test end-average body weight at test beginning)/day of test
Feed to weight ratio (F/G) =average daily feed intake (ADFI)/Average Daily Gain (ADG)
2.3.2 determination of immune organ coefficients
After 6 chickens are randomly extracted from each group at the age of 42 days and weighed, immune organs (thymus coefficient, spleen coefficient and bursa coefficient) of the chickens are collected by cutting, and the immune organs are weighed after moisture is absorbed by filter paper, so that the immune organ coefficients are calculated.
Immune organ coefficient = immune organ dry functional weight (mg)/body weight (g)
2.4 data statistics
Data analysis data processing was performed using Excel 2013 and SPSS 22.0 software.
3. Results
3.1 Rate of death and panning
The death rate of the chickens which died due to heat stress was not counted among the death rate due to the uncontrolled overheating of the animal house temperature in the last few days of the test, and the death rate of the chickens died due to the disease in the test process is shown in table 7.
TABLE 7 mortality rate
As can be seen from Table 7, the death rate and the number of heat stress deaths of the honeysuckle leaf extract are obviously lower than those of the common water extract group and the blank control group of the honeysuckle leaves, so that the honeysuckle leaf extract can effectively reduce the death rate of chickens and enhance the heat stress resistance of chickens.
3.2 body weight results
TABLE 8 influence of various feed additives on weight of white-feather broilers
Note that: compared with the blank control group at the same day of age, * is significant in difference (p<0.05)， ** Is extremely remarkable in difference(p<0.01)。
As can be seen from table 8, after 7 days of age, the weight of the honeysuckle leaf extract group was higher than that of the blank group and also higher than that of the water extract group of honeysuckle She Putong; especially after 21 days of age, the weight of the honeysuckle leaf extract group is significantly different from that of the blank control group.
3.4 growth Performance results
Table 9 influence of each feed additive on growth performance of white feather broilers 1 to 42 days old (n=6)
As shown in Table 9, the high-dose of the extract of honeysuckle leaves was lower in the period of 1-42 days than the water extract of honeysuckle She Putong and the blank group, and the high-dose of the extract of honeysuckle leaves was optimal.
3.5 immune organ index results
After the end of the 42-day-old test, 6 chickens were selected for each group, and the immune organ index was calculated by performing a split examination, and the results are shown in Table 10.
Table 10 effect of various feed additives on immune organ index of white feather broilers (n=6)
As shown in Table 10, the thymus index, spleen index and bursa index of the high, medium and low dose group of the honeysuckle leaf extract are higher than those of the water extract group and the blank group of the honeysuckle She Putong. The thymus index and spleen index are obviously improved compared with the blank group.
4.1 the death rate and the heat stress death index of the honeysuckle leaf extract group are lower than those of the common water extract group and the blank control group of the honeysuckle leaves, and the honeysuckle leaf extract can effectively reduce the death rate of chickens and improve the heat stress resistance of organisms.
The feed conversion ratio of the 4.2 honeysuckle leaf extract groups is lower than that of the common water extract groups and the blank control groups of the honeysuckle leaves, and the honeysuckle leaf extracts can effectively reduce the feed conversion ratio, improve feed return and reduce the raising cost.
4.3 the immune organ index of the high, medium and low dose group of the honeysuckle leaf extract is higher than that of the water extract group and the blank group of the honeysuckle She Putong, which shows that the honeysuckle leaf extract can effectively improve the immune organ index of organisms, thereby reducing the morbidity and improving the anti-stress capability of the organisms.
4.4 compared with the blank, the common water extract of the honeysuckle leaves is improved in the aspects of reducing death rate, improving heat stress resistance, reducing feed-meat ratio, improving immune organ index and the like, which shows that the water extract of the honeysuckle She Putong has good effect; all indexes of the honeysuckle leaf extract (acid water extraction) are better than those of the honeysuckle She Putong water extract, which shows that the acid water extraction of the honeysuckle leaves has better effect than the common water extraction.
4.5 comparing the high, medium and low dosage groups of the honeysuckle leaf extract, except for the higher dosage group in the spleen index, other indexes are sequentially reduced according to the high, medium and low dosages, but no significant difference exists, so the administration dosage of the honeysuckle leaf extract is recommended to be added with 0.5 per mill of the honeysuckle leaf extract in daily ration.
Claims (9)
1. A preparation method of a honeysuckle leaf extract is characterized in that the honeysuckle leaf is extracted by adopting a hydrochloric acid aqueous solution with pH of 2.0-5.0 in a heating and decocting mode to obtain the honeysuckle leaf extract.
2. The method for preparing the honeysuckle leaf extract according to claim 1, wherein the extraction is carried out for 2-3 times by heating and decocting with hydrochloric acid aqueous solution with pH of 2.0-5.0; mixing the extracting solutions, concentrating until the concentration of the liquid medicine is equivalent to 0.5-2.0 g/mL, adjusting the pH to 3.0-6.5 to obtain the concentrated solution of the honeysuckle leaf extract, and spray drying the obtained concentrated solution to obtain the honeysuckle leaf extract.
3. The preparation method of the honeysuckle leaf extract as claimed in claim 2, wherein the honeysuckle leaf extract is extracted for 2 times by heating and decocting, the mass-volume ratio of the first honeysuckle leaf to the extracting solution is 1:6-1:15, and the mass-volume ratio of the second honeysuckle leaf to the extracting solution is 1:5-1:15.
4. The honeysuckle leaf extract prepared by the method of any one of claims 1 to 3, wherein the chlorogenic acid content is more than or equal to 3%.
5. A composition containing the honeysuckle leaf extract as claimed in claim 4, wherein the composition at least comprises the honeysuckle leaf extract and the baical skullcap root extract, the chlorogenic acid content in the composition is more than or equal to 10.0mg/g, and the baicalin content is more than or equal to 150.0mg/g.
6. The method of preparing the composition of claim 5, wherein the extract of scutellaria baicalensis is dissolved in the concentrated solution of honeysuckle leaf extract, and spray-dried to obtain the composition of lonicera japonica She Huangqin.
7. The use of the honeysuckle leaf extract of claim 4 for inhibiting staphylococcus aureus and escherichia coli.
8. The use of the honeysuckle leaf extract of claim 4 for improving animal immunity and reducing feed conversion ratio.
9. Use of the composition according to claim 5 for inhibiting staphylococcus aureus and escherichia coli.
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