CN116036161B - Raspberry extract and preparation method and application thereof - Google Patents

Abstract

The invention relates to a raspberry extract and a preparation method and application thereof, belongs to the field of functional foods, health products or pharmacy, and relates to the raspberry extract which can effectively relieve intestinal structure damage and intestinal flora disturbance caused by high-fat diet, and the raspberry extract or functional foods, health products or medicines containing the raspberry extract can be used for regulating intestinal environment disturbance caused by poor diet and have higher use safety.

Description

Raspberry extract and preparation method and application thereof

Technical Field

The invention relates to the technical field of functional food, health care product or medicine manufacturing, in particular to a raspberry extract and a preparation method and application thereof.

Background

With the improvement of the living standard of people, high-fat diet is more and more common, and long-term high-fat diet can lead to metabolic syndrome, such as obesity, hyperglycemia, hyperlipidemia, insulin resistance and the like, and seriously affect the living quality of people. In addition, high-fat diets also disrupt the homeostasis of the intestinal tract, and in particular the effect on the intestinal flora has been reported as a major cause of the induction of metabolic syndrome. High fat diets can also cause changes in intestinal structure, which in turn affect intestinal function. However, the effect of high fat diets on intestinal health is of relatively low interest and there is still a lack of effective means for their prevention and treatment.

The Chinese herbal medicine is Chinese pure, has the effects of preventing and treating various diseases, and has been used for thousands of years. The gastrointestinal tract disease is a disease characterized by repeated or chronic symptoms, has great control difficulty, and the western medicines for treating the gastrointestinal tract disease are few in varieties at present and mainly relieve the symptoms. The Chinese herbal medicine has better efficacy in preventing and treating gastrointestinal diseases due to the characteristics of multiple components and multiple targets, and is a precious resource for researching and developing related health-care products or medicines. Raspberry (Rubus chingii Hu.) is a woody plant of the genus Rubus of the family Rosaceae, and has been listed in the catalogue of homology of medicine and food. The raspberry has various medical values of antioxidation, anti-tumor, bacteriostasis, anti-inflammatory, pain relieving, cough relieving, lipid reducing, blood glucose reducing and the like, and the main functional components of the raspberry comprise phenolic substances, flavonoids, triterpenes, minerals, vitamins, carotenoid and organic acid. The raspberry polysaccharide can be extracted from the root, stem, leaf and fruit of raspberry plant, is one of active ingredients with larger proportion in the raspberry extract, has the effects of resisting tumor, oxidation, aging, reducing blood sugar and lipid, and the like, can be used in food, health care products and pharmaceutical industry, and is widely concerned. However, the effect of raspberry polysaccharide on improving intestinal microecological imbalance and intestinal structural disturbance caused by poor diet has not been reported yet.

Disclosure of Invention

In order to solve the technical defects in the prior art, the invention provides the raspberry extract as well as the preparation method and the application thereof, which can effectively relieve the intestinal tract structure damage caused by high-fat diet.

The technical scheme adopted by the invention is as follows: a raspberry extract comprising polysaccharides and proteins, arabinose, galactose, glucose and galacturonic acid.

The raspberry extract comprises, by mass, 23.70% of polysaccharide and 4.98% of protein, 10.35% of arabinose, 10.70% of galactose, 48.79% of glucose and 19.36% of galacturonic acid.

A preparation method of raspberry extract is characterized by comprising the following steps:

(1) Drying immature fruit of Rubi fructus, pulverizing into powder, sieving to obtain Rubi fructus powder, soaking Rubi fructus powder with ethanol at room temperature to remove small molecules and colored substances, drying, and packaging into plastic bag for sealing and storing;

(2) Mixing pretreated Rubi fructus powder with 25-30% ethanol solution at room temperature;

(3) Then adding 68-76 ℃ preheated distilled water into the solution;

(4) Treating the mixture with ultrasonic device for 9-14min; adding distilled water, extracting at 80deg.C for 1 hr, and centrifuging at low speed to obtain supernatant;

(5) Adding chloroform-n-butanol into the supernatant according to Sevage method to remove protein;

(6) Concentrating the solution using a rotary evaporator;

(7) Adding absolute ethyl alcohol into the solution, standing for precipitation, centrifuging, and discarding the supernatant to obtain a precipitate;

(8) Re-dissolving the precipitate with distilled water, and lyophilizing to obtain Rubi fructus extract.

In the step (1), 500g of raspberry powder is soaked with 4L of 80% ethanol for 2 hours at room temperature to remove small molecules and colored substances.

5g of pretreated raspberry powder in the step (2) is mixed with 20mL of 28% ethanol solution at room temperature for 30min.

In the step (3), 75ml of preheated distilled water at 76 ℃ is added to the solution.

In the step (4), the power of the ultrasonic device is 40kHz, and the ultrasonic treatment is adjusted to be carried out for 14min at 76 ℃.

And (3) in the step (4) and the step (7), the centrifugal rotating speed is 3000g, and the centrifugal time is 10min.

And (3) adding chloroform and n-butanol with a volume ratio of 4:1 in the step (5) to remove protein.

The solution was concentrated to one-fourth of the original volume using a rotary evaporator at 42℃in the step (6).

The final ethanol concentration of the absolute ethanol added in the step (7) is 80 percent.

Application of Rubi fructus extract in preparing functional food, health product or medicine for treating flora and intestinal canal structural disorder caused by high fat diet is provided.

The beneficial effects of the invention are as follows: the invention provides a raspberry extract, a preparation method and application thereof, which can effectively relieve intestinal structure damage and intestinal flora disorder caused by high-fat diet. The Rubi fructus extract or functional food, health product or medicine containing the same can be used for regulating intestinal canal environmental disturbance caused by poor diet, and has high safety.

Drawings

FIG. 1 shows an optimized preparation process flow of Rubi fructus extract.

FIG. 2 effect of Rubi fructus extract on intestinal flora diversity of high fat diet mice.

FIG. 3 effect of Rubi fructus extract on intestinal microecology of high fat diet mice.

FIG. 4 effect of Rubi fructus extract on colon structure and barrier function of high fat diet mice.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1: optimized preparation of raspberry extract and component analysis thereof

1. Preparation of Rubi fructus extract

(1) Raw materials: immature fruits of raspberry palmate;

(2) Pretreatment of raw materials: drying fructus Rubi, pulverizing to powder with high-speed grinder, and sieving with 60 mesh sieve to obtain fructus Rubi powder. The powder (500 g) was soaked with 80% ethanol (4L) at room temperature for 2h to remove small molecules and colored substances. And then drying the powder at 37 ℃, and filling the powder into a plastic package bag for sealing and preserving for standby.

(3) Identification of key influencing parameters of raspberry extract preparation: key parameters affecting the extraction rate of raspberry extract were identified using Plackett-Burman experimental design, and 7 extraction parameters, such as ethanol concentration, desorbent feed ratio, desorption time, extractant feed ratio, extraction temperature, ultrasonic power and ultrasonic time, were examined at both low (-1) and high (+1) levels, as shown in table 1. Table 2 shows 12 different extraction conditions and their corresponding extraction rates based on the Plackett-Burman experimental design. The relation model of the extraction parameters and the extraction rate is established based on Plackett-Burman design, as shown in table 3, and the model has statistical significance as shown in the following specific formula by p=0.0093 < 0.01:

Y＝5.23-0.19X ₁ -0.13X ₂ +0.16X ₃ -8×10 ^-3 X ₄ +0.31X ₅ -0.023X ₆ +0.37X ₇

+0.27X ₁ X ₇ +0.19X ₁ X ₆

then, P value is evaluated according to model fitting, and parameters affecting the extraction rate of the raspberry extract are sequentially X ₅ ＞X ₇ ＞X ₁ ＞X ₁ X ₅ ＞X ₃ ＞X ₂ ＞X ₁ X ₆ ＞X ₁ ＞X ₆ ＞X ₄ The first three key extraction parameters are selected for further optimization, namely extraction temperature, extraction time and ethanol concentration.

Table 1 extraction parameters and ranges of raspberry extract for the Plackett-Burman experimental design.

Table 2Plackett-Burman experimental design Table and extraction yield thereof.

Table 3 raspberry extract extraction parameters ANOVA analysis.

(4) Identification of key influence parameter optimization range of raspberry extract preparation: according to the three key parameters obtained by the screening, the fastest rising experiment is adopted to determine each parameterCenter point for further optimization studies. X is shown according to the above formula ₁ 、X ₅ 、X ₇ The experimental step length and positive and negative effects, the extraction time and the extraction temperature show positive effects, the ethanol concentration shows negative effects, and the rest experimental conditions are set as intermediate levels: the desorbing agent liquid ratio is 4mL/g, the desorbing time is 30min, the extracting agent liquid ratio is 15mL/g, the ultrasonic power is 70%, and the design and the results of the fastest rising experiment are shown in Table 4. As the extraction time and the extraction temperature increase, the ethanol concentration decreases, and the extraction rate of the raspberry polysaccharide increases and then decreases. The extraction rate of raspberry polysaccharide was highest when the ethanol concentration was 27%, the temperature was 75 ℃, and the extraction time was 13min, so the level of three factors in group 4 was determined as the center point for further extraction optimization.

Table 4 design table for the fastest rising extraction rate of raspberry polysaccharide.

Numbering device	Ethanol concentration (%)	Extraction temperature (. Degree. C.)	Extraction time (min)	Extraction yield (%)
					1	50	60	4	6.55±0.24
2	42	65	7	6.70±0.18
					3	35	70	10	7.40±0.26
4	27	75	13	7.95±0.21
					5	20	80	16	6.91±0.18

(5) Preparing key parameter response surface optimization analysis of the raspberry extract: in order to evaluate the influence of the three key extraction parameters on the extraction rate of the raspberry extract, an optimal extraction process is determined, a Box-Behnken response surface optimization experiment is designed by adopting a three-factor three-level experiment, the extraction rate is taken as a response value, and the three key extraction parameters are taken as independent variables. From the results of the steepest ascent experiment, the factors and levels of the Box-Behnken design experiment were determined as shown in Table 5. Table 6 shows 17 different extraction conditions and their corresponding extraction rates based on Box-Behnken response surface optimization experiments. Through multiple regression analysis, a regression model of the extraction rate of the raspberry extract and each parameter is established as follows:

the analysis of variance of the regression model is shown in Table 8, the regression model has statistical significance, R ² Value 0.9705, mismatch termP= 0.4891>0.05, the model fitting degree is better. As shown in table 8, by maximizing the extraction yield, the optimal extraction process conditions were ethanol concentration 28.594%, extraction temperature 76 ℃, and ultrasonic time 13.913 min. According to the actual experimental conditions, the experimental value is adjusted to be 28% of ethanol concentration, the extraction temperature is 76 ℃ and the ultrasonic time is 14min, and three repeated experiments are carried out, so that the actual polysaccharide extraction rate is 8.30+/-0.07%, and the experimental result is well matched with the model result.

Table 5Box-Behnken design parameters and levels of experiments.

Table 6Box-Behnken experimental design table and extraction yield.

Table 7 response surface model analysis of variance based on Box-Behnken experimental design.

Note that: "-" indicates no data. * The terms "0.05", "0.01", "0.001" mean significant differences, respectively.

Table 8 is based on the optimum parameter values, predicted and actual extraction rates for the Box-Behnken experimental design.

(6) The preparation method of the raspberry extract comprises the following steps: the optimal extraction process of the raspberry extract is obtained after the optimization, as shown in fig. 1, 5g of the pretreated sample is placed in a beaker and mixed with 20mL of 28% ethanol solution at room temperature for 30min. Subsequently 75ml of distilled water preheated at 76℃were added to the solution and the mixture was sonicated with an ultrasonic device at 70% total power (40 kHz) at 76℃for 14min. 100mL of distilled water was then added and extracted at 80℃for 1 hour, followed by low-speed centrifugation (3000 g,10 min) to give a supernatant.

(7) Deproteinization: deproteinizing according to Sevage method, and adding 4:1 (v/v) chloroform-n-butanol into supernatant to remove protein.

(8) Concentrating: the solution was concentrated to one-fourth of the original volume using a rotary evaporator at 42 ℃.

(9) Precipitation: adding absolute ethanol to obtain final ethanol concentration of 80%, standing at 4deg.C for precipitation for 12 hr, centrifuging (3000 g,10 min), and discarding supernatant to obtain precipitate.

(10) And (3) drying: re-dissolving the precipitate with distilled water, and lyophilizing to obtain Rubi fructus extract.

2. Component analysis of raspberry extract

Component analysis was performed on the raspberry extract obtained in example 1, which contained 23.70% of total polysaccharide and 4.98% of protein, and mainly contains arabinose, galactose, glucose and galacturonic acid, accounting for 10.35%, 10.70%, 48.79% and 19.36% of total components, respectively, as shown in table 9.

Table 9 raspberry extract extraction yield and main ingredients.

Example 2: animal experiments prove that the raspberry extract has the effect of relieving intestinal structure damage and intestinal flora disorder caused by high-fat diet

1. Animal grouping and experimental protocol

Throughout the study, 54 male C57BL/6 mice of 4-5 weeks old were purchased from beijing villous laboratory animal technology limited, weighing 20.0±2.0g, and kept in a laboratory animal center of university of wenzhou medical science without specific pathogen SPF-grade animal house (room temperature=22±2°c; relative humidity=45±3%) with alternating illumination for 12 hours starting from 8:00 a.m.. The mice were fed with water and food freely during the experiment period for one week. All procedures strictly follow the laboratory animal care and use manual of the university of wenzhou medical science.

After one week of adaptive rearing, 54 mice were randomly divided into 3 groups of 18: (1) a normal diet group (Chow); (2) a high fat diet group (HFD); (3) High fat diet and raspberry extract intervention group (RCP). The raspberry extract was subjected to daily gavage at a dose of 100mg/kg body weight, and 487.80mg was taken daily for 14 consecutive weeks, relative to 60kg adult, with the same dose of physiological saline as that of the HFD group gavage to reduce the variability of irritation caused by gavage to mice. After 14 weeks of intervention, fresh fecal samples of mice were collected and placed in sterile tubes and stored at-80 ℃ for later use. Mice were sacrificed after isoflurane anesthesia and serum samples were collected, followed by rapid separation of colon tissue, one for physiological and biochemical analysis and one for paraformaldehyde for pathological characterization.

2. Experimental results

Through 16S rRNA sequencing analysis, the interference of the raspberry extract can significantly increase the intestinal flora observed_patterns and the Chao1 index (figure 2) of the high-fat diet mice, which shows that the raspberry extract has the effect of improving the diversity of the intestinal flora. Subsequently, at the microbial level, we analyzed the change in key intestinal bacteria between normal and high fat diets (fig. 3 a) and high fat diet and raspberry extract intervention group (fig. 3 b) using volcanic patterns, and identified that 6 intestinal bacteria were co-affected by high fat diet and raspberry extract (fig. 3 c), wherein the relative abundance of Colidextribacter (fig. 3 d) and erysiellotris (fig. 3 e) was significantly increased in the high fat diet group, but raspberry extract intervention was able to reduce the abundance of both types of harmful bacteria, indicating that raspberry extract may have efficacy in inhibiting the growth of harmful bacteria caused by high fat diet.

Subsequently, we examined whether the raspberry extract has an ameliorating effect on intestinal structural disorders induced by high fat diets. As shown in fig. 4a, a high fat diet was found by pathological staining to result in a thinning of the colonic myometrium (fig. 4 b) and mucosal thickness (fig. 4 c), but an effective improvement after intervention with raspberry extract. In addition, we have found that high fat diets significantly reduced colon zon-1 and Occludin levels, whereas raspberry extracts were able to increase zon levels, which was demonstrated at both the protein (FIGS. 4d-4 f) and gene (FIG. 4 g) levels, indicating that raspberry extracts reduced the destructive effect of high fat diets on the colon barrier. Enhanced colonic shielding can reduce endotoxin blood intake, as shown in figure 4h, high fat diet resulted in significant increase in serum endotoxin levels, but decreased serum endotoxin levels following intervention with raspberry extract, indicating that raspberry extract also had the effect of alleviating high fat diet-induced endotoxemia.

The skilled person will know: while the invention has been described in terms of the foregoing embodiments, the inventive concepts are not limited to the invention, and any modifications that use the inventive concepts are intended to be within the scope of the appended claims.

The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above examples, and all technical solutions belonging to the concept of the present invention belong to the protection scope of the present invention. It should be noted that modifications and adaptations to the present invention may occur to one skilled in the art without departing from the principles of the present invention and are intended to be within the scope of the present invention.

Claims (2)

1. Use of a raspberry extract for the preparation of a medicament for the treatment of high fat diet induced dysbacteriosis and intestinal structural disorders, characterized in that the raspberry extract is prepared by the steps of:

(1) Drying immature fruits of Rubus chingii, pulverizing into powder, sieving to obtain Rubi powder, soaking 500g Rubi powder with 4L of 80% ethanol at room temperature for 2 hr to remove small molecules and colored substances, drying, and sealing in plastic bag;

(2) Mixing 5g pretreated Rubi fructus powder with 20mL 28% ethanol solution at room temperature for 30 min;

(3) Then 75ml of preheated distilled water at 76 ℃ is added into the solution;

(4) Treating the mixture with ultrasonic device at 76 deg.C for 14min; adding distilled water, extracting at 80deg.C for 1 hr, centrifuging at low speed to obtain supernatant, wherein the power of ultrasonic device is 40kHz, the centrifuging speed is 3000g, and the centrifuging time is 10min;

(5) Adding chloroform-n-butanol with a volume ratio of 4:1 into the supernatant according to a Sevage method to remove proteins;

(6) Concentrating the solution using a rotary evaporator;

(7) Adding absolute ethyl alcohol into the solution, standing for precipitation, centrifuging, discarding supernatant to obtain precipitate, wherein the centrifuging speed is 3000g, and the centrifuging time is 10min;

(8) Re-dissolving the precipitate with distilled water, and lyophilizing to obtain Rubi fructus extract.

2. The use according to claim 1, wherein the raspberry extract includes polysaccharides and proteins, arabinose, galactose, glucose and galacturonic acid.