CN114766672A - Old lobular broadleaf holly leaf extract and application thereof in preparing food for relieving alcohol-induced liver injury - Google Patents

Abstract

The invention provides a small-leaf broadleaf holly leaf old leaf extract and application thereof in preparing food for relieving alcohol-induced liver injury, and relates to the technical field of food processing. The preparation process of the old lobular broadleaf holly leaf extract comprises the steps of raw material pretreatment, crushing, extraction, concentration, drying, crushing and the like. The prepared old lobular broadleaf holly leaf extract is rich in 54 effective components such as verbascoside, poliumoside, mannitol, rhoifolin, forsythoside E, D-quinic acid, skimmin and the like, and can be used for preparing functional food for relieving alcohol-induced liver injury.

Description

Old lobular broadleaf holly leaf extract and application thereof in preparing food for relieving alcohol-induced liver injury

Technical Field

The invention relates to the technical field of food processing, in particular to a small-leaf broadleaf holly leaf old leaf extract and application thereof in preparing food for relieving alcohol-induced liver injury.

Background

The lobular broadleaf holly leaf is tea processed from fresh leaves of ligustrum quihoui of oleaceae ligustrum, is commonly called as tea leaves, fuding tea and tea-like, and is a traditional pure natural health care beverage good product in China. The finished tea has the advantages of fresh fragrance, bitter taste and sweet and cool taste, and has the effects of clearing away summer heat, improving eyesight, promoting intelligence, promoting the production of body fluid, quenching thirst, promoting urination, strengthening heart, moistening throat, relieving cough, lowering blood pressure, reducing weight, inhibiting and preventing cancers, resisting aging, invigorating blood circulation and the like. From the perspective of traditional Chinese medicine, the broadleaf holly leaf tea has the effects of dispelling wind heat, clearing head and eyes and removing polydipsia, and has very obvious medicinal effects of treating headache, toothache, conjunctival congestion, fever polydipsia, dysentery and the like.

Disclosure of Invention

The invention discloses a method for extracting an old lobular ilex leaf extract by crushing, extracting, concentrating, drying and crushing, measuring the content of the extract by HPLC, and analyzing the content to obtain the extract containing verbascoside, poliumoside, mannitol, rhoifolin, forsythiaside E, dextroquinic acid, skimmin, apigenin-7-O-beta-D-glucopyranoside, oleuropein, luteolin, rutin, salidroside, adenine, citric acid, 5-hydroxyverbascoside, violaxoside C, lonicerin, p-hydroxycinnamic acid, quercetin-3-glucuronide, coumarin, vitamin II, hyperin, L-leucine, mulberrin, L-phenylalanine, mogroside, akebiaquine B, atractyloide A, p-methoxybenzaldehyde, L-tyrosine, D-beta-D-E, Apigenin, hydroxytyrosol, kaempferol-3-O-rutinoside, plantain, bilobalide, guanine, 2-adamantanone, dencichine, protocatechualdehyde, luteolin, protocatechuic acid, camphor, cryptochlorogenic acid, shikimic acid, caffeic acid, 1-caffeoylquinic acid, verbascoside, phillyrin, loganin, 6-gingerol, danshensu, vanillin, and acetyl harpagin. The preparation process of the old lobular broadleaf holly leaf extract comprises the following steps:

(1) pretreatment of raw materials: carrying out steam de-enzyming and drying treatment on fresh and complete lobular Kuding tea to obtain old lobular Kuding tea leaves;

(2) crushing: crushing the old lobular Kuding tea leaves by a crusher and sieving by a sieve of 10 meshes;

(3) extraction: weighing the crushed raw materials, putting the raw materials into a filter bag, adding water, adding distilled water, uniformly mixing, soaking at normal temperature, heating the raw materials, keeping the temperature, performing ultrasonic treatment on the raw materials after stopping heating, performing suction filtration after treatment, repeatedly extracting filter residues for 2-3 times, and combining filtrates;

(4) concentration: carrying out rotary evaporation and concentration on the filtrate until the filtrate is sticky and is taken out;

(5) and (3) drying: pre-freezing the concentrated solution, and freeze-drying to obtain solid concentrated extract;

(6) crushing: pulverizing the solid concentrated extract, and sieving with 300 mesh sieve to obtain small leaf Folum Ilicis old leaf extract.

Further, the ratio of the tea leaves to the water in the step (3) is 1 g: 10-20 mL.

Further, the soaking time at normal temperature in the step (3) is 3-5 h.

Further, the raw materials in the step (3) are heated at 90-95 ℃, the heat preservation time is 1-2 hours, and in the heat preservation process, stirring is carried out for 5min every 15 min.

Further, in the step (3), the ultrasonic treatment frequency is 30-40 KHZ, and the ultrasonic treatment time is 10-20 min.

Further, the concentration temperature in the step (4) is 50-70 ℃.

Further, the moisture content of the solid concentrated extract in the step (5) is less than 6%.

The invention also provides the old lobular broadleaf holly leaf extract prepared by the method.

The invention also aims to provide application of the old lobular Kuding tea leaf extract in preparing food for relieving alcohol-induced liver injury.

The main components of the old lobular broadleaf holly leaf extract comprise verbascoside, poliumoside, mannitol, rhoifolin and the like, and the specific effects of the components are as follows:

poliumoside: removing blood stasis, stopping bleeding, relieving swelling and pain, and can be used for hematemesis, hemoptysis, epistaxis, and hematochezia; the external use has the functions of treating traumatic hemorrhage, resisting bacteria, diminishing inflammation and resisting viruses; verbascoside: has antiinflammatory, immunity enhancing, anoxia resisting, and anticancer effects, can be used for content determination/identification/pharmacological experiment, and has pharmacological effects of regulating immunity, resisting oxidation, enhancing physical strength, relieving fatigue, etc.; rhoifolin: anti-tumor; anti-hypertension; xanthine oxidase inhibitors; an antioxidant; mannitol: is a good diuretic in medicine, reduces intracranial pressure and intraocular pressure, and is a drug for treating kidney, dehydrate, sugar substitute, excipient for tablet, and diluent for solid and liquid.

Compared with the prior art, the invention has the following beneficial technical effects:

the extract of the old leaves of the ilex latifolia is rich in functional components such as verbascoside, poliumoside, mannitol, isoergosterol, rhoifolin and the like, can be directly drunk after being dissolved in water or prepared into functional beverage with other components for drinking, can relieve alcoholic liver injury caused by long-term drinking after long-term drinking, and has the effects of resisting bacteria, diminishing inflammation, enhancing immunity, resisting oxidation, enhancing physical strength, resisting fatigue, promoting urination and the like. Meanwhile, the material source of the invention is wide, the cost is low, the preparation process is simple, and the invention has good application prospect.

Drawings

The invention is further illustrated in the following description with reference to the drawings.

FIG. 1 is a graph of the effect on liver in mice with alcoholic liver injury at 4 weeks of administration;

FIG. 2 is a graph showing the changes in serum ALT and AST levels after 4 weeks of administration;

FIG. 3 is a graph showing the change in the amount of TG in serum after 4 weeks of administration.

Detailed Description

The technical solution provided by the present invention is further illustrated by the following examples.

Example 1

A preparation process of an old lobular broadleaf holly leaf extract comprises the following steps:

(1) pretreatment of raw materials: carrying out steam de-enzyming and drying treatment on fresh and complete lobular Kuding tea to obtain old lobular Kuding tea leaves;

(2) crushing: crushing the old lobular Kuding tea leaves by a crusher and sieving the crushed old lobular Kuding tea leaves by a sieve with 10 meshes;

(3) extraction: weighing the crushed raw materials, putting the raw materials into a filter bag, and mixing the raw materials according to a material-liquid ratio of 1 g: adding 20mL of water into distilled water, mixing, soaking at room temperature for 5h, heating the raw materials to 95 deg.C, keeping the temperature for 2h, heating for 15min, stirring for 5min, stopping heating, performing 40KHZ ultrasonic treatment for 15min, pouring out the extractive solution, vacuum filtering, repeatedly extracting the residue for 3 times, and mixing the filtrates;

(4) and (3) concentrating: heating the extractive solution with 50 deg.C water bath, concentrating with rotary distiller to viscous state, and pouring out;

(5) and (3) drying: storing the concentrated solution at-5 deg.C, freezing and drying to obtain solid concentrated extract;

(6) crushing: pulverizing the solid concentrated extract, and sieving with 300 mesh sieve to obtain folium Ilicis Purpureae old leaf extract.

Test example 1

(1) Experimental materials: 46 SPF male KM mice with the body weight of 20-25 g are 4-6 weeks old. The experimental reagent comprises a red star Erguotou (purchased from Dewangjia supermarket of New city of Baiyun area in Guiyang); bifendate dripping pills; 4% paraformaldehyde.

(2) The experimental method comprises the following steps:

alcohol liver injury model making

The 60 mice were randomly divided into two groups, 10 normal groups and 50 model groups. The normal weight group measured every week is subjected to intragastric administration for 1 time per day; the module building set is filled with sterile water for 3 days to adapt to stimulation, and then filled with 56-degree Hongxing Erguotou for 3 weeks: the acute alcoholic liver injury model is established by gavage 1 time daily with 0.005ml/g in the first week, gavage 1 time daily with 0.008ml/g in the second week, and gavage 1 time daily with 0.010ml/g in the third week.

Animal grouping and handling

45 mice remained after modeling, 10 mice remained in the normal group, and 10 mice remained in the modeling group. 36 mice of the modeling group are randomly divided into 4 groups of 9 groups, namely a model group, a positive drug group (bifendate dripping pills), a broadleaf holly leaf extract low-dose group and a broadleaf holly leaf extract high-dose group. The administration group is administered by intragastric administration for 1 time every day, and the blank control group and the model group are administered with equal volume of sterile water every time for continuous intragastric administration. The mice were subjected to eye blood sampling and tissue isolation at 2 and 4 weeks of administration, respectively. The grouping situation is as follows:

table 1: grouping of experimental animals

Group of	Quantity (only)	Concentration of Folum Ilicis extract
			Normal group	9	—
Model set	9	—
			Positive drug group	9	150mg/kg
Low dose group	9	150mg/kg
			High dose group	9	300mg/kg

Detecting relevant indexes of experimental animals:

1) general observations were: in the experimental process, the mental state, the activity, the fur color, the water intake, the food intake, the urination and defecation condition, the presence or absence of a bite wound, death and other conditions of the experimental mice are observed every day. If mice die, the number of mice die and the possible cause of the death should be recorded.

2) And (3) determination of serum biochemical indexes: in order to avoid the influence of food on the detection result, after the last administration, the blood is taken from the eyeballs of the experimental mice after being fasted overnight (freely drinking water) for 16 hours, and the fur of the mice is prevented from dropping into the blood. Standing at room temperature for natural coagulation, centrifuging at 4000rpm in a centrifuge for 15min, and separating serum. The serum of each mouse is marked respectively and stored in a-80C ultra-low temperature refrigerator for standby. Respectively measuring the content levels of glutamic-oxaloacetic transaminase (AST), glutamic-pyruvic transaminase (ALT) and Triglyceride (TG) in the serum of the experimental mouse.

3) Mouse liver index determination: before the mice were dissected, the body weight of the mice was weighed and recorded. Immediately after blood collection, the abdomen is opened to pick up mouse liver tissues, filter paper is used for lightly wiping the residual blood on the surface, the appearance of the mouse viscera is observed and photographed for storage, immediately after photographing, the mouse viscera is placed in physiological saline at 4 ℃ for rinsing to remove the residual blood, the filter paper is lightly wiped dry, and the wet weight of the mouse liver is weighed and recorded. Mouse liver index was calculated according to the following formula:

the liver index of the mouse is the wet weight of the liver/body weight multiplied by 100%

Collecting mouse organs:

immediately after blood sampling, the abdomen was opened to extract liver tissue from the mice, and the heart, liver, spleen, and lung were additionally taken out, weighed, and recorded.

And (3) data analysis:

statistical software Excel and SPSS23.0 are adopted for analysis, the measured data are expressed by mean plus minus standard deviation (x plus minus s), the numerical value comparison among a plurality of groups is analyzed by one-factor variance, and the difference with P <0.05 has statistical significance.

(3) Results of the experiment

1) Influence of Folum Ilicis extract on body shape and mental status of diabetic mice

In the molding process, the normal control group mice had good mental status, body shape, free movement, bright hair color, normal food intake and drinking, normal urine output, and no injury. 17 mice die in the molding process of the molding mice, wherein 6 mice are dissected to find that liver tissues of the mice are diseased, 2 mice die after being perfused with stomach alcohol, the rest 9 dead mice are emaciated, intestinal flatulence is observed after dissection, and some mice have alcoholic liver injury. When the model is molded for 3 weeks, most mice lose weight.

In the experimental process, the mental state of each alcoholic liver model mouse is worse than that of a normal control group mouse, and the reaction is slightly retarded; the control group was most significant. Along with the increase of the medicine intervention time, the spirit of each dose group of the broadleaf holly leaf extract and the positive medicine group of the alcoholic liver model mouse is slightly better than that of the model group mouse, and padding is relatively clean. 3 days after administration, the buttocks swelling phenomenon appeared in each alcoholic liver model mouse; after 5 days of administration, the positive drug group mice had wounds on 4 buttocks, four mice in the low dose group had wounds on the tail, 1 mouse in the high dose group had wounds, and 1 leg had wounds; after 8 days of administration, these wounds were gradually recovered, but one each died in the high dose group, the low dose group, the positive drug and the model group. After 2 weeks of administration, 4 of the high dose groups had another wound on the buttocks but had not died.

2) Influence of Folum Ilicis extract on body weight and organ weight of mice with alcoholic liver injury

After 2 weeks and 4 weeks of administration, each mouse organ was collected and weighed and recorded. As can be seen from the table, there was no statistical difference in the body weight and the weight of each organ among the groups, indicating that the Yuqing ilex latifolia extract had no significant effect on the body weight and the weight of each organ of the mice with alcoholic liver injury.

Table 2: the weight of mice and the weight of each organ were administered for 2 weeks

Table 3: the weight of mice and the weight of each organ were administered for 4 weeks

Group of	Body weight	Heart of heart	Spleen	Lung (lung)	Kidney (Kidney)
						Normal group	51.40±1.41	0.27±0.06	0.15±0.03	0.29±0.04	0.68±0.04
Model set	48.15±8.95	0.23±0.54	0.11±0.04	0.26±0.04	0.54±0.12
						Low dose group	48.96±3.96	0.25±0.03	0.11±0.01	0.26±0.04	0.58±0.09
High dose group	42.88±3.94	0.20±0.01	0.20±0.12	0.25±0.01	0.55±0.04
						Positive drug group	45.22±5.39	0.24±0.05	0.19±0.17	0.25±0.01	0.54±0.03

3) Influence of broadleaf holly leaf extract on appearance of liver of mice with alcoholic liver injury

As shown in FIG. 1, at week 4 of the intragastric administration of ethanol and the concurrent intragastric administration of the positive control drug and the Folum Ilicis extract, the mice were dissected and sampled to observe pathological changes on the liver surface. The observation result is that the surface of the liver of the normal group is dark red and has no white lesion spots; the surface color of the liver of the model group is light pink, and the liver of the model group has more white lesion spots and slight fibrosis; the surface color of liver tissue of the positive medicine group is pink, and no white lesion spots are few; the surface of the liver tissue of the low-dose group is pink, has slight white lesion spots and is not obvious; the surface of the liver tissue of the high-dose group is dark pink, and white lesion spots do not exist.

4) Effect of Folum Ilicis extract on TG, ALT and AST in mice

And (3) performing eyeball blood collection on the mice at 4 weeks of the intragastric alcohol injection and the intragastric positive control drug and the broadleaf holly leaf extract at the same time, and collecting serum to detect the contents of ALT, AST and TG. As can be seen from FIG. 2, after the 4 th week after the gavage, compared with the model group, the ALT and AST contents in the serum all reached a very significant increase (P <0.01) in the model group, bifendate dripping pills, low dose group and high dose group compared with the normal group; compared with the model group, the content of ALT and AST in serum is also extremely lower than that of the model group in the bifendate dripping pills and the high-dose group (P <0.01), and the content of the bifendate dripping pills and the high-dose group does not reach a significant difference (P >0.05) with the model group; and the content of ALT and AST in serum is not significant (P is more than 0.05) between bifendate dripping pills and high-dose groups. The result shows that the high-dose intragastric extract can relieve alcoholic liver injury to a certain extent, and the effect of the extract is not much different from that of the biphenyl dimethylesterate dropping pill which is a common liver-protecting medicine in the market.

As can be seen from fig. 2, after 4 weeks of administration, compared with the normal group, the TG content in the serum of the mice in the model group and the low dose group was significantly higher than that in the normal group (P <0.01), while the TG content in the serum of the mice in the bifendate dripping pill and the high dose group was significantly higher than that in the normal group (P < 0.05); compared with the model group, the content of the bifendate dripping pills and the high-dose group in the serum of the mice is extremely lower than that of the model group (P <0.01), the difference is not significant compared with other groups, and the difference between the bifendate dripping pills and the high-dose group is not significant (P < 0.05). Therefore, the extract can reduce the increase of TG content in blood caused by alcoholic liver injury by intragastric administration, and the effect is not different from bifendate.

The invention is obtained by combining the above steps, and by carrying out intragastric administration on the ilex latifolia thunb extract, a series of results such as detection of relevant indexes of alcoholic liver injury, behavior observation of intragastric administration animals, weight change, anatomical pathology observation and the like prove that the extract can relieve alcoholic liver injury to a certain degree, and provides experimental basis for further developing and protecting liver products.

The principles and embodiments of the present invention have been described herein using specific examples, which are provided only to help understand the method and the core concept of the present invention; meanwhile, for a person skilled in the art, according to the idea of the present invention, the specific embodiments and the application range may be changed. In view of the above, the present disclosure should not be construed as limiting the invention.

Claims (9)

1. A preparation process of an old leaflet extract of lobular broadleaf holly leaf is characterized by comprising the following steps:

(1) pretreatment of raw materials: carrying out steam de-enzyming and drying treatment on fresh and complete lobular Kuding tea to obtain old lobular Kuding tea leaves;

(2) crushing: crushing the old lobular Kuding tea leaves by a crusher and sieving by a sieve of 10 meshes;

(3) extraction: weighing the crushed raw materials, putting the raw materials into a filter bag, adding water, adding distilled water, uniformly mixing, soaking at normal temperature, heating the raw materials, keeping the temperature, performing ultrasonic treatment on the raw materials after stopping heating, performing suction filtration after treatment, repeatedly extracting filter residues for 2-3 times, and combining filtrates;

(4) and (3) concentrating: carrying out rotary evaporation and concentration on the filtrate until the filtrate is sticky and is taken out;

(5) and (3) drying: pre-freezing the concentrated solution, and freeze-drying to obtain solid concentrated extract;

(6) crushing: pulverizing the solid concentrated extract, and sieving with 300 mesh sieve to obtain folium Ilicis Purpureae old leaf extract.

2. The process for preparing the old small-leaf broadleaf holly leaf extract according to claim 1, wherein the ratio of tea leaves to water in the step (3) is 1 g: 10-20 mL.

3. The process for preparing the old lobular Kuding tea leaf extract according to claim 1, wherein the soaking time in step (3) at normal temperature is 3-5 h.

4. The preparation process of the old small-leaf broadleaf holly leaf extract as claimed in claim 1, wherein the raw material in the step (3) is heated at 90-95 ℃ for 1-2 h, and is stirred for 5min every 15min during the heat preservation process.

5. The process for preparing the old leaflet extract of broadleaf holly leaf according to claim 1, wherein the ultrasonic treatment frequency in the step (3) is 30-40 KHZ, and the ultrasonic treatment time is 10-20 min.

6. The process for preparing the old leaflet extract of lobular Kuding tea as claimed in claim 1, wherein the concentration temperature in the step (4) is 50-70 ℃.

7. The process according to claim 1, wherein the water content of the solid concentrated extract in step (5) is less than 6%.

8. An extract from old leaves of Folum Ilicis, prepared by the method of any one of claims 1-7.

9. The old leaflet extract of lobular Kuding tea as claimed in claim 9, which is used for preparing food for alleviating alcohol-induced liver damage.

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