CN111035668A - Preparation method and application of instant isatis tinctoria leaf extract - Google Patents

Abstract

The invention relates to a preparation method and application of an instant isatis leaf extract, wherein fresh isatis leaves are used as raw materials, purified water is used for extracting water-soluble substances in the raw materials, and then the raw materials are prepared into dry powder by utilizing a freeze drying technology. The obtained dry powder is dark green, and can convert fresh Isatis tinctoria leaf into powdery stock material, maximally retain original active ingredients of Isatis tinctoria leaf, and realize instant use of plant with season limited growth. The extract mainly contains 31% polysaccharide and 53% protein, has certain antioxidant and antibacterial activity in vitro activity test, and can be used for preparing medicine for treating psoriasis or psoriasis. The fresh Isatis tinctoria leaf used in the method is extracted and squeezed at present, retains the original bioactive components of Isatis tinctoria leaf, and improves the problem that the use of fresh Isatis tinctoria leaf is limited by seasons.

Description

Preparation method and application of instant isatis tinctoria leaf extract

Technical Field

The invention relates to a preparation method and application of an instant isatis tinctoria leaf extract.

Background

Isatis tinctoria belongs to more than 30 species (more than 50 species are considered by some taxonomists) in the middle asia, mainly in the east of the Mediterranean region, and is distributed in the central region of Islam-Tulan, east to Kupfish island and Japan, north to China and Central Europe and North America. 6 varieties of 3 varieties are produced in China, the isatis tinctoria is cultivated all over the country, the isatis tinctoria belongs to three provinces of Xinjiang, inner Mongolia and northeast China, and the cultivated species of isatis tinctoria of special China almost extends all over the country. The Isatis tinctoria is a group with important medicinal economic value, wherein the dried root of Isatis tinctoria is Chinese medicinal radix Isatidis, and the dried leaf is folium Isatidis. The Xinjiang Isatis tinctoria, a peculiar variety of China, and the Uygur medicine name 'Ousma' are often used for dyeing black hair in folk, treating various spots (white spots, purple spots), white hair and the like. Local people collect mature isatis tinctoria seeds in 5 months, store the seeds after airing, sow the seeds in the next year, place the seeds in warm water at 40 ℃ for soaking for 3-4 hours before sowing, take the seeds out, mix the seeds with plant ash or other medicaments, and sow the seeds. The sowing time is selected in late 4 months, a ditch with the depth of 2 cm is formed on the furrow surface or the ridge, the row spacing of the plants is reasonably controlled, the seeds are uniformly sowed in the ditch, then the soil is covered and compacted, the soil is watered to keep the water content of the soil between 60 and 70 percent, and the seedlings can emerge after one week.

According to the ancient legend of Uygur, the distance between the two eyebrows of girls determines the distance of marriage in the future. Two girls with far-spaced eyebrows can be grafted to a far place. Mother always wants daughter to stay around, so that mother uses woad leaf juice to smear her eyebrows, and a little bit, girls learn to smear her eyebrows with woad leaves, starting in the seventh day after birth. The two bends of the growing daughter are tightly connected with the eyebrows every day and year, and the shout at the place where the daughter is grafted can be heard at a glance. Nowadays, the grain for eyebrow is made into eyebrow pencil or eyebrow cream, but the ladies living in the old city of garage still like to apply eyebrow with leaf juice of fresh isatis leaf. During spring bloom, red and green leaves of the woad are collected by Uygur women, and the squeezed leaf juice is mixed with proper mutton fat to prepare the eyebrow cream which is not rotten and is used in winter. The other storage mode is to store the tobacco leaves in the sun like the tobacco leaves, but the eyebrow coating effect is not the same as the former.

In the early nineties, pharmacologists working in Xinjiang are inspired by the fact that local women in Uygur nationality often use isatis tinctoria juice to squeeze eyebrows for girls to draw eyebrows, and can make eyebrows black, bright and dense, have great interest in finding out eyebrow growth and eyebrow nourishing factors from plant isatis tinctoria leaves, and develop related new products in cooperation with research teams. After several years of intensive research, the isatis leaf is found to be rich in effective components such as astragalin, erucic glycoside, indole glycoside and the like which promote the activation of hair follicles, and a series of products such as 'Osman' eyebrow growing pencils, eyebrow growing liquid and the like are developed by taking the natural extract of the isatis leaf as a raw material and by means of a modern technology. In addition, Isatis tinctoria leaf sap has also been reported to promote hair growth in hair loss model animals.

The invention utilizes the freeze drying technology to rapidly lock the water-soluble components of the fresh isatis tinctoria leaves, so that the fresh isatis tinctoria leaves are converted into green dry powder to be stored. The dry powder isatis leaf extract has the following uses: the prepared standard decoction pieces are convenient for local national hospitals to use; provides raw materials for the production and processing of cosmetics such as eyebrow drawing liquid, hair conditioner and the like; provides a raw material medicine for the preparation of Uygur medicine compound patent medicine. The above method is intended to prepare a powder of Isatis tinctoria leaf extract by freeze-drying technique instead of fresh Isatis tinctoria leaves, especially in the seasons and places where the plant is not produced.

Disclosure of Invention

The invention aims to provide a preparation method and application of an instant isatis leaf extract, wherein fresh isatis leaves are used as raw materials, purified water is used for extracting water-soluble substances, and then the isatis leaf extract is prepared by utilizing a freeze drying technology. The main components of the extract are 31 percent of polysaccharide and 53 percent of protein, and in vitro activity tests show that: the isatis leaf extract obtained by the method has certain antioxidant and antibacterial activity, has certain effect in preparing medicaments for treating psoriasis and psoriasis, or can be used as a raw material for providing cosmetics.

The preparation method of the instant isatis tinctoria leaf extract comprises the following steps:

a. freezing fresh Isatis tinctoria leaf at-80 deg.C for 5-10 hr;

b. crushing the frozen isatis leaf in the step a by using a crusher with a cooling device;

c. adding purified water into the isatis tinctoria leaves crushed in the step b according to the weight-volume ratio of 1:3 of the material liquid, stirring and extracting for 4 hours, and repeatedly extracting for 3-4 times;

d. and c, combining the extracts in the step c, filtering an extracting solution, centrifuging at the rotation speed of 4000r/min and the temperature of 4 ℃ for 10min, taking supernatant, freeze-drying to obtain instant isatis indigotica leaf extract dry powder, and storing in a refrigerator at the temperature of-20 ℃.

The application of the isatis tinctoria leaf extract obtained by the method in preparing a medicament for treating psoriasis and psoriasis.

Measuring polysaccharide content of the obtained Isatis tinctoria leaf extract by anthrone-sulfuric acid method; protein content was determined using a bicinchoninic acid (BCA) method; DPPH (1, 1-diphenyl-2-trinitrophenylhydrazine) free radical scavenging method is used for antioxidant activity experiments.

Drawings

FIG. 1 is a graph showing a standard work curve for measuring polysaccharide content in Isatis tinctoria leaf extract according to the present invention;

FIG. 2 is a graph of a standard working curve for determining protein content according to the present invention;

FIG. 3 is a photograph showing the results of the extract of Isatis tinctoria leaf of the present invention against Candida Albicans (CA), Escherichia Coli (EC) and Staphylococcus Aureus (SA).

Detailed Description

Example 1

a. Freezing 500g fresh Isatis tinctoria leaf collected from Hetian at-80 deg.C for 5 hr;

b. crushing the frozen isatis leaf in the step a by using a crusher with cooling function;

c. adding purified water into the crushed isatis tinctoria leaves in the step b according to the weight-volume ratio of 1:3 of the material liquid, stirring and extracting for 4 hours, and repeatedly extracting for 4 times;

d. and c, combining the extracts in the step c, filtering, centrifuging at 4 ℃ for 10min at 4000r/min, taking supernate, freeze-drying to obtain 26.3g of blackish green isatis leaf extract freeze-dried powder, and storing in a refrigerator at the temperature of-20 ℃.

Example 2

a. Freezing 1kg of fresh Isatis tinctoria leaf collected from Hetian at-80 deg.C for 10 hr;

b. crushing the frozen isatis leaf in the step a by using a crusher with cooling function;

c. adding purified water into the crushed isatis tinctoria leaves in the step b according to the weight-volume ratio of 1:3 of the material liquid, stirring and extracting for 4 hours, and repeatedly extracting for 3 times;

d. and c, combining the extracts in the step c, filtering, centrifuging at 4 ℃ for 10min at 4000r/min, taking supernate, freeze-drying to obtain 53g of dark green isatis tinctoria leaf extract freeze-dried powder, and storing in a refrigerator at the temperature of 20 ℃ below zero.

Example 3

a. Freezing 2kg of fresh Isatis tinctoria leaf at-80 deg.C for 8 hr;

b. crushing the frozen isatis leaf in the step a by using a crusher with a cooling device;

c. adding purified water into the crushed isatis tinctoria leaves in the step b according to the feed liquid, stirring and extracting for 4 hours, and repeatedly extracting for 4 times;

d. and c, combining the extracts in the step c, filtering, centrifuging at the rotation speed of 4000r/min and the temperature of 4 ℃ for 10min, taking supernatant, freeze-drying to obtain 104g of instant isatis tinctoria leaf extract freeze-dried powder, and storing in a refrigerator at the temperature of-20 ℃.

Example 4

The polysaccharide content of the obtained isatis leaf extract was measured by anthrone-sulfuric acid method:

and (3) drawing a polysaccharide content standard curve by using an anthrone-sulfuric acid method: accurately weighing 100.00mg of glucose standard substance, fully dissolving the glucose standard substance in distilled water, fixing the volume in a 100mL volumetric flask, and preparing 1mg/mL solution; taking 10mL, adding water to 100mL, and preparing a standard solution with the concentration of 0.1 mg/mL; taking 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 and 1.8mL respectively, adding water to 2mL, then sequentially adding 8mL of 0.2% anthrone-sulfuric acid reagent, shaking up, placing in an ice water bath for cooling, placing in a water bath with the temperature of 90 ℃ for heating for 10min after cooling, taking out, cooling with running water, placing for 10min at room temperature, measuring absorbance at 620nm by using an ultraviolet spectrophotometer, taking the mass concentration of the standard solution as an abscissa, taking the absorbance as an ordinate, taking distilled water as a blank control, making a standard curve as shown in figure 1, and adopting a regression equation: 0.00899X +0.02405, R²＝0.99742；

And (3) measuring results: 1. a sample size of 2.2mg, an ultraviolet absorbance value (UV) of 0.6351, a percentage of 31.23%; 2. the sample size was 2.2mg, ultraviolet absorbance value (UV)0.7321, percentage 31.5%.

Example 5

The obtained isatis leaf extract was measured for protein content using BCA method:

the protein content of the sample was determined by the bicinchoninic acid (BCA) method: accurately measuring a 2mg/mL standard bovine serum albumin solution, preparing a 25-2000 [ mu ] g/mL standard solution, preparing a working solution according to the operation of a BCA protein measurement kit specification, dividing a BCA reagent into a reagent A and a reagent B, adding 1 time volume of the reagent B into 50 times volume of the reagent A, and fully shaking for later use; measuring 25 mu L of standard solutions with different concentrations in a 96-well plate, respectively adding 175 mu L of BCA working solution, slightly oscillating, fully mixing, keeping the temperature in an incubator at 37 ℃ for 30min, measuring the absorbance of the solution at 562nm by using an enzyme-linked immunosorbent assay, taking the absorbance of the standard as a horizontal coordinate, taking the mass concentration of bovine serum albumin as a vertical coordinate, drawing a standard curve as shown in figure 2, and taking the regression equation as follows: 0.0292x +0.1238, R²＝0.99142；

And (3) measuring results: 1. the sampling amount is 62.5 mu g, the ultraviolet absorbance value (UV) is 1.109, and the percentage content is calculated to be 53.98%; 2. a sample size of 75. mu.g, ultraviolet absorbance (UV)1.278, calculated as a percentage of 52.7%, was taken.

Example 6

The obtained isatis leaf extract was subjected to an antibacterial activity test:

detecting the bacteriostatic activity of the sample by using an agar plate method: gram-positive bacteria Staphylococcus Aureus (SA), gram-negative bacteria Escherichia Coli (EC) and Candida Albicans (CA) are cultured in an LB liquid culture medium at 175rpm and 37 ℃ overnight until OD600 is about 2.0 (1 × 106CFU/mL), 10 μ L of overnight-cultured bacterial liquid is taken and mixed with 8mL of LB culture medium containing 0.7% of agar, the mixture is laid on 25mL of LB culture medium containing 1.5% of agar, after the top agar is solidified, a sample solution filtered by a 0.22 μm microporous filter membrane is added dropwise, the temperature is 37 ℃ for overnight culture, the diameter of a bacteriostatic ring is measured, ampicillin is used as a positive control, physiological saline is used as a negative control, and the measurement result is shown in the inhibitory action of the graph 3 and the table 1;

TABLE 1 antibacterial results of Isatis tinctoria leaf extract powder

Example 7

The obtained isatis leaf extract was subjected to an antioxidant activity test:

accurately weighing 7.88mg of DPPH solid powder, fully dissolving in methanol, fixing the volume in a volumetric flask of 100mL, ensuring the concentration to be 0.2mmol/L, accurately sucking 1mL of sample solutions with different concentrations in test tubes with plugs, adding 1mL of prepared DPPH-methanol solution respectively, shaking up, keeping the temperature of a constant temperature box at 37 ℃ for 30min, taking distilled water as a reference, measuring the absorbance at 517 nm of an enzyme labeling instrument, taking 1mL of distilled water as a blank group instead of the solution, taking 1mL of methanol as a control group instead of the DPPH-methanol solution, and calculating the removal rate of the DPPH free radicals of the isatis leaf extract powder sample according to the following formula:

in the formula, A₀Is blank absorbance; a. the_iIs the sample set absorbance; a. the_jIs the control absorbance;

and (3) measuring results: the absorbance Ai of the sample group was 0.34, the absorbance Aj of the control group was 0.026, the absorbance of the blank group was 0.922, and the DPPH radical scavenging rate of the sample of the Isatis tinctoria leaf extract powder was calculated to be 66.2%.

Claims (2)

1. A method for preparing an instant isatis leaf extract is characterized by comprising the following steps:

a. freezing fresh Isatis tinctoria leaf at-80 deg.C for 5-10 hr;

b. crushing the frozen isatis leaf in the step a by using a crusher with a cooling device;

c. adding purified water into the isatis tinctoria leaves crushed in the step b according to the weight-volume ratio of 1:3 of the material liquid, stirring and extracting for 4 hours, and repeatedly extracting for 3-4 times;

d. and c, combining the extracts in the step c, filtering an extracting solution, centrifuging at the rotation speed of 4000r/min and the temperature of 4 ℃ for 10min, taking supernatant, freeze-drying to obtain instant isatis indigotica leaf extract dry powder, and storing in a refrigerator at the temperature of-20 ℃.

2. Use of the extract of isatis tinctoria leaves obtained according to the method of claim 1 for the manufacture of a medicament for the treatment of psoriasis or psoriasis.

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