Astragalus stem and leaf extract having colonization-promoting effect on Lactobacillus plantarum and preparation method thereof

Abstract

The present application relates to the field of extracts, and specifically to an astragalus stem and leaf extract having a colonization-promoting effect on Lactobacillus plantarum and a preparation method thereof. The present invention investigates the colonization-promoting effect of the astragalus stem and leaf extract on Lactobacillus plantarum, and screens the functional substances in the astragalus stem and leaf extract that have a colonization-promoting effect on Lactobacillus plantarum by preparative chromatography guided by biological activity.

Description

Technical Field

[0001] The present application relates to the field of extracts, and in particular to an astragalus stem and leaf extract having a colonization-promoting effect on Lactobacillus plantarum and a preparation method thereof. Background Art

[0002] The intestine is an important digestive organ of the body and also the largest immune organ. Intestinal health is closely related to the health of livestock and poultry. Lactobacillus plantarum is one of the common probiotics, which can improve the intestinal microbial environment and thus regulate the intestinal flora. Therefore, research and development of new feed additives that can promote the growth and colonization of Lactobacillus plantarum in the intestine is of great significance to livestock and poultry production.

[0003] Studies have shown that astragalus extract has antioxidant, immune-enhancing, anti-fatigue, anti-viral, anti-tumor effects, treatment of diabetes, atherosclerosis and Alzheimer's disease, and promotion of broiler growth. Astragalus (aerial part) stem and leaf extract has the effects of enhancing immunity, anti-inflammatory, liver protection, anti-aging, and pancreatic protection. However, there are few reports on the promotion of probiotic growth by astragalus stem and leaf extracts and astragalus extracts. Astragalus extract (main component astragalus polysaccharide) can be fermented to culture Bacillus subtilis. Low concentrations of astragalus polysaccharide can effectively increase the number of Bacillus, while high concentrations of astragalus polysaccharide inhibit the growth of Bacillus. However, there are no reports on the promotion of probiotic colonization by astragalus stem and leaf extracts. Summary of the invention

[0004] The invention aims to provide an astragalus stem and leaf extract which has the effect of promoting colonization of Lactobacillus plantarum.

[0005] Another object of the present invention is to provide a method for preparing the astragalus stem and leaf extract having the effect of promoting colonization of Lactobacillus plantarum.

[0006] The Astragalus stem and leaf extract having a colonization-promoting effect on Lactobacillus plantarum according to the present invention is prepared by a method comprising the following steps:

[0007] After the stems and leaves of Astragalus are dried, they are crushed and passed through a 60-mesh sieve for later use;

[0008] Deionized water was added to the crushed stems and leaves of Astragalus membranaceus, and ultrasonic extraction was performed to obtain a crude extract of the stems and leaves of Astragalus membranaceus, wherein the solid-liquid ratio was 1:40, the extraction power was 500 W, the extraction time was 25 min, the extraction temperature was 50° C., and the pH was 7;

[0009] The astragalus stem and leaf crude extract is collected through preparative chromatography and has the effect of promoting colonization of lactobacillus plantarum, wherein 0-10min, 20% acetonitrile; 10-20min, 24% acetonitrile; 20-30min, 35% acetonitrile; 30-40min, 50% acetonitrile; 40-45min, 15% acetonitrile; wavelength 260nm, column room temperature, flow rate 50mL / min, sample concentration 100mg / mL, sample volume 19mL, and the fraction having the effect of promoting colonization of lactobacillus plantarum is collected at 32.53min-36min of the preparation.

[0010] According to the technical scheme of the present application, the ultrasonic extraction conditions were investigated using the four major categories of active substances in Astragalus stem and leaf extract (AME), namely polysaccharides, polyphenols, flavonoids and saponins, as well as the growth-promoting activity of the extract on Lactobacillus plantarum (LP) as indicators, and the Astragalus stem and leaf extraction conditions were optimized by single factor to obtain the optimal ultrasonic extraction process of AME.

[0011] The collected fractions were determined by preparative chromatograms, based on time- and dose-effects, and bioactivity-oriented screening of fractions with pro-colonization effects on LP. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Liquid phase spectra from different origins;

[0013] Figure 2 Display the content of active ingredients and LP proliferation-promoting activity in different origins;

[0014] Figure 3 Display the liquid phase spectrum of different extraction methods;

[0015] Figure 4 Display the content of ingredients in different extraction methods and their activity in promoting LP proliferation;

[0016] Figure 5 Liquid phase spectra of different material-liquid ratios;

[0017] Figure 6 Display the content of active ingredients and LP proliferation-promoting activity at different material-liquid ratios;

[0018] Figure 7 Liquid phase spectra for different extraction powers;

[0019] Figure 8 Display the content of active ingredients and LP proliferation promoting activity at different extraction powers;

[0020] Fig. 9 Liquid phase spectra at different extraction times;

[0021] Fig.10 It shows the content of active ingredients and the promotion of LP proliferation at different extraction times;

[0022] Fig.11 Liquid phase spectra at different extraction temperatures;

[0023] Fig.12 Display the content of active ingredients and LP proliferation-promoting activity at different extraction temperatures;

[0024] Fig.13 Shows the chromatogram of the preparation of crude extract of Astragalus stem and leaves;

[0025] Fig.14 It shows the liquid chromatogram of crude extract of Astragalus stem and leaves and fractions F1-F9;

[0026] Fig.15 The colonization-promoting activity of crude extracts of stems and leaves of Astragalus membranaceus and fractions F2-F8 against Lactobacillus plantarum was shown;

[0027] Fig.16 The crude extract of Astragalus stems and leaves and fraction F7 showed colonization-promoting activity against Bacillus subtilis;

[0028] Fig.17 The reducing sugar content of fraction F7 is shown. DETAILED DESCRIPTION

[0029] Example 1 Astragalus stem and leaf extract

[0030] The stems and leaves of Astragalus were dried and crushed, passed through a 60-mesh sieve for later use, and ultrasonic extraction was performed with a single factor as the variable as shown in Table 1.

[0031] Table 1 Single factor levels

[0032]

[0033] The contents of active ingredients in the extracts of Astragalus stems and leaves were determined by DNS method, Folin phenol method, sodium nitrite-aluminum nitrate method and vanillin-sulfuric acid colorimetric method, and the growth-promoting activity of the extracts of Astragalus stems and leaves in promoting Lactobacillus plantarum was compared.

[0034] The method for determining the activity of promoting the proliferation of Lactobacillus plantarum is as follows:

[0035] The preserved bacterial liquid was streaked on the plate and cultured in a constant temperature incubator at 37°C for 48 hours; a single colony was picked and inoculated into 40 ml of MRS liquid culture medium and cultured on a shaking table for 24 hours; 400 μL of the bacterial liquid after secondary activation was taken and treated with drugs for 24 hours; the proliferation promotion index was determined:

[0036] OD value: measure the absorbance of bacterial solution at a wavelength of 600nm;

[0037] Self-aggregation ability: Determine the OD of the original bacterial solution 600 , denoted as A 0， Take 5 mL of bacterial solution in a 15 mL sterile centrifuge tube, let it stand at 37°C for 2 h, take the supernatant, and measure the OD600 , denoted as A 1， Self-aggregation ability (%) = (1-A1 / A0)*100%;

[0038] Total protein: Prepare crude enzyme solution and determine total protein by BCA method.

[0039] Organic acid determination: high performance liquid chromatography, liquid phase conditions: detection column: Kromasil 100-5-C18; column temperature: 25°C; detection wavelength 210 nm; mobile phase: methanol: 0.2% phosphoric acid = 2.5:97.5; flow rate: 1.0 mL / min; injection volume: 10 μL.

[0040] 1.1 Comparison of stems and leaves of Astragalus from different origins

[0041] Astragalus stems and leaves from eight different origins were collected. By comparing the content of active ingredients in the extracts of Astragalus stems and leaves from different origins and the activity of promoting the growth of Lactobacillus plantarum, it was found that the extraction rate of Astragalus stems and leaves collected in Pengyang, Ningxia was the highest (as shown in Table 2). Figure 1 As shown, the liquid phase spectrum shows that there are more AME components in Pengyang, Ningxia.

[0042] like Figure 2 As shown in the data, the reducing sugar content of Astragalus stems and leaves harvested in Ningxia was the highest, while the content of polyphenols, flavonoids and saponins was the highest in the stems and leaves of Astragalus harvested in Yulin. However, the stems and leaves of Astragalus harvested in Yulin did not have a prominent effect on promoting the proliferation of Lactobacillus plantarum. Therefore, after comprehensive consideration, the stems and leaves of Astragalus harvested in Pengyang, Ningxia were selected for optimization of extraction conditions.

[0043] Table 2 Yields from different origins

[0044]

[0045] 1.2 Comparison of different extraction methods

[0046] like Figure 3 and Figure 4 As shown in the figure, the active substances and the activity of promoting LP proliferation in AME obtained by ultrasonic extraction and decoction extraction were compared. The experimental results showed that the content of active ingredients in the extract obtained by ultrasonic extraction was higher than that in the extract obtained by decoction, and the activity of promoting LP proliferation was also more significant. Therefore, ultrasonic extraction was selected for the next experiment, and the ultrasonic extraction conditions were optimized.

[0047] 1.3 Optimization of extraction conditions

[0048] 1.3.1 Optimization of material-liquid ratio

[0049] By examining the extraction rate, active ingredient content and LP proliferation promoting activity at different solid-liquid ratios, as shown in Table 3, Figure 5 and Figure 6As shown, it can be seen that when the solid-liquid ratio is 1:40, the extraction yield, the content of the four major types of active ingredients and the LP growth promoting activity are higher. Therefore, 1:40 is selected as the optimal extraction solid-liquid ratio after comprehensive consideration.

[0050] Table 3 Yields of different material-liquid ratios

[0051]

[0052] 1.3.2 Extraction power optimization

[0053] By examining the extraction rate, active ingredient content and LP proliferation promoting activity under different extraction powers, the extraction power was optimized. As shown in Table 4, the experimental results show that when the extraction power is 500W, the saponin content and extraction rate are the highest; Figure 7 and Figure 8 As shown, the reducing sugar content showed an increasing trend with the increase of extraction power, while the content of polyphenols and flavonoids showed a decreasing trend; the difference in promoting LP proliferation activity among different groups was small, and 500W was selected as the optimal extraction power for energy saving and environmental protection considerations.

[0054] Table 4 Different extraction power yields

[0055]

[0056] 1.3.3 Extraction time optimization

[0057] The extraction time was optimized by examining the extraction yield, active ingredient content and LP proliferation promoting activity at different extraction times. Fig. 9 and Fig.10 As shown in the figure, the experimental results show that with the extension of extraction time, the extraction rate and saponin content tend to increase; the content of polyphenols and flavonoids shows a downward trend; when the extraction time is 25 minutes, there is a better activity to promote LP proliferation, so 25 minutes is selected as the optimal extraction time.

[0058] Table 5 Yields at different extraction times

[0059]

[0060] 1.3.4 Extraction temperature optimization

[0061] The extraction temperature was optimized by examining the extraction yield, active ingredient content and LP proliferation activity at different extraction temperatures. Fig.11 and Fig.12As shown in the figure, the experimental results show that with the increase of extraction temperature, the extraction rate tends to increase; the reducing sugar content shows a trend of first increasing and then decreasing, and there is no significant difference in the content of polyphenols and saponins among the groups; when the extraction temperature is 50℃, there is a better activity of promoting LP proliferation, so 50℃ is selected as the optimal extraction temperature.

[0062] Table 6 Yields at different extraction temperatures

[0063]

[0064] 1.3.5 Extraction pH optimization

[0065] The extraction pH was optimized by examining the extraction yield, active ingredient content and LP proliferation promoting activity at different extraction pH. Fig.13 and Fig.14 As shown in the figure, the experimental results show that the extraction rate is the highest when pH = 6, the reducing sugar content shows a downward trend with the increase of pH, and there is no significant difference in polyphenol content among the groups. When pH = 7, the content of flavonoids and saponins is the highest, and it also shows good growth-promoting activity, so 7 is selected as the optimal extraction pH.

[0066] Table 7 Different extraction pH yield

[0067]

[0068] Example 2: Separation of functional substances

[0069] The primary fractions were collected by preparative chromatography, concentrated by rotary evaporation, and then freeze-dried. The fractions were screened based on biological activity to determine the fractions that promoted the proliferation of LP and the effective substances were determined by LC / MS. The preparative chromatography conditions are shown in Table 8.

[0070] Table 8

[0071]

[0072] Nine fractions of Astragalus stem and leaf extract (AME) were collected by preparative chromatography. The self-aggregation ability of F2-F8 against Lactobacillus plantarum (LP) was determined ( Fig.15 ), and screened the fractions that could promote colonization of Lactobacillus plantarum (LP) based on biological activity. The experimental results showed that fraction F7 promoted colonization of LP. Then the colonization promoting effect of F7 on Bacillus subtilis (BS) was determined. The experimental results showed that the crude extract of Astragalus stems and leaves AME not only did not promote the colonization of BS, but inhibited its colonization ability, while fraction F7 had a certain promoting effect on BS ( Fig.16The reducing sugar content in fraction F7 was determined to be 20 mg / g, indicating that the main active ingredient that promotes the colonization of Lactobacillus plantarum is not reducing sugar ( Fig.17 The above embodiments are only used to explain the technical solution of the present application and do not limit the protection scope of the present application.

Claims

1. Astragalus stem and leaf extract that promotes colonization of Lactobacillus plantarum. It is characterized in that The astragalus stem and leaf extract is prepared by a method comprising the following steps: After the stems and leaves of Astragalus are dried, they are crushed and passed through a 60-mesh sieve for later use; Deionized water was added to the crushed stems and leaves of Astragalus membranaceus, and the crude extract of stems and leaves of Astragalus membranaceus was obtained by ultrasonic extraction, wherein the solid-liquid ratio was 1:40, the extraction power was 500 W, the extraction time was 25 min, the extraction temperature was 50 °C, and the pH was 7; The eluate of the crude extract of astragalus stems and leaves is collected by preparative chromatography to obtain an astragalus stem and leaf extract having a colonization-promoting effect on Lactobacillus plantarum, wherein, from 0 to 10 min, 20% acetonitrile and 80% formic acid with a concentration of 0.2%; from 10 to 20 min, 24% acetonitrile and 76% formic acid with a concentration of 0.2%; from 20 to 30 min, 35% acetonitrile and 65% formic acid with a concentration of 0.2%; from 30 to 40 min, 50% acetonitrile and 50% formic acid with a concentration of 0.2%; from 40 to 45 min, 15% acetonitrile and 85% formic acid with a concentration of 0.2%; the wavelength is 260 nm, the column temperature is room temperature, the flow rate is 50 mL / min, the sample concentration is 100 mg / mL, the loading volume is 19 mL, and the astragalus stem and leaf extract having a colonization-promoting effect on Lactobacillus plantarum is collected at 32.53 min-36 min of the preparation.

2. A method for preparing an extract of Astragalus stems and leaves having a colonization-promoting effect on Lactobacillus plantarum, It is characterized in that The method comprises the following steps: After the stems and leaves of Astragalus are dried, they are crushed and passed through a 60-mesh sieve for later use; Deionized water was added to the crushed stems and leaves of Astragalus membranaceus, and the crude extract of stems and leaves of Astragalus membranaceus was obtained by ultrasonic extraction, wherein the solid-liquid ratio was 1:40, the extraction power was 500 W, the extraction time was 25 min, the extraction temperature was 50 °C, and the pH was 7; The eluate of the crude extract of astragalus stems and leaves is collected by preparative chromatography to obtain an astragalus stem and leaf extract having a colonization-promoting effect on Lactobacillus plantarum, wherein, from 0 to 10 min, 20% acetonitrile and 80% formic acid with a concentration of 0.2%; from 10 to 20 min, 24% acetonitrile and 76% formic acid with a concentration of 0.2%; from 20 to 30 min, 35% acetonitrile and 65% formic acid with a concentration of 0.2%; from 30 to 40 min, 50% acetonitrile and 50% formic acid with a concentration of 0.2%; from 40 to 45 min, 15% acetonitrile and 85% formic acid with a concentration of 0.2%; the wavelength is 260 nm, the column temperature is room temperature, the flow rate is 50 mL / min, the sample concentration is 100 mg / mL, the loading volume is 19 mL, and the astragalus stem and leaf extract having a colonization-promoting effect on Lactobacillus plantarum is collected at 32.53 min-36 min of the preparation.

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