AU2021100762A4

AU2021100762A4 - Preparation Method and Application of the Extract from Gastrodia elata Bl.

Info

Publication number: AU2021100762A4
Application number: AU2021100762A
Authority: AU
Inventors: Changliang He; Fanglong LI; Xiaoxia Liang; Qiang Wu; Lizi Yin
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2021-02-08
Filing date: 2021-02-08
Publication date: 2021-04-22
Anticipated expiration: 2029-02-08

Abstract

The invention relates to a preparation method of the extract from fresh Gastrodia elata Bl., and its usage for preparing related drugs and foods. -1/2 GasodinT otal content of Processing 4-Hydroxybenzyl Todin andn 4 Polysaccharide method<- atoi< alcohol<- atodnad4 content Hydroxybenzylalcohol<tt Freshly squeezed 0.060%-' 0.785%& 0.845%< 2.77%-' method& Water decoction 0.290% 0.2450/ 0.5350 3.71%< method-' Steaming 0.250%< 0.1230/ 0.3730/ 2.86%< method<___ Figure 1 CastodinTotal content of Processing 4-Hydroxybenzyl ga and 4- Polysaccharide method& alcohol Hydroyezalcohol contentO Hydroxybenzyl alcohol Freshly squeezed 0.030%& 0.736%& 0.766%& 5.17%< method<-' Water decoction 0.270%& 0.299%-I 0.569%& 5.94%o method& Steaming 0.210%& 0.049%< 0.259%& 3.46%< method& Figure 2

Description

-1/2

GasodinT otal content of

Processing 4-Hydroxybenzyl Todin andn 4 Polysaccharide method<- atoi< alcohol<- atodnad4 content Hydroxybenzylalcohol<tt Freshly squeezed 0.060%-' 0.785%& 0.845%< 2.77%-' method& Water decoction 0.290% 0.2450/ 0.5350 3.71%< method-' Steaming 0.250%< 0.1230/ 0.3730/ 2.86%< method<___

Figure 1

CastodinTotal content of

Processing 4-Hydroxybenzyl ga and 4- Polysaccharide method& alcohol Hydroyezalcohol contentO Hydroxybenzyl alcohol Freshly squeezed 0.030%& 0.736%& 0.766%& 5.17%< method<-' Water decoction 0.270%& 0.299%-I 0.569%& 5.94%o method& Steaming 0.210%& 0.049%< 0.259%& 3.46%< method&

Figure 2

Preparation Method and Application of the Extract from Gastrodia elata Bl.

TECHNICAL FIELD

The invention relates to a preparation method of Gastrodia elata Bl. extract and

application thereof in medicine or food, belonging to the field of pharmacy and food.

BACKGROUND

Gastrodia elata is the dried tuber of orchid Gastrodiaelata BL. Gastrodiaelata Bl. is a

genus of orchid Gastrodia plant, which is a Chinese traditional herbal drug with highly

nutritional and medical values, such as anticonvulsant, neuroprotective and memory

improvement activities. Gastrodiaelata Bl. is mainly planted in the southwest of China,

especially in Sichuan and Yunnan Provinces. Its main bioactive constituents include

polysaccharides, phenols, gastrodin, 4-Hydroxybenzyl alcohol and so on.

Chemical components in Gastrodia elata, such as gastrodin, gastrodigenin and

polysaccharide, are important indexes to measure the quality of Gastrodia elata, which

are not only affected by varieties, harvest time, producing areas, fungus materials and

other factors, but also have differences due to different processing methods, further

affecting product quality. Processing is an important part of traditional Chinese medicine

system in China. Different processing methods will cause changes in the content of

gastrodin, polysaccharide and other effective components in Gastrodia elata, thus

affecting the curative effect of Gastrodiaelata in practical application. There are many

records of Gastrodiaelata in herbal medicine of past dynasties. Its quality and efficacy

are obviously related to the processing method and technology of Gastrodia elata.

Gastrodia elata has a long history of processing, and there are various processing methods, such as stir-frying, simmering, wine-making, etc., besides slicing raw products, which are mainly moistening and steaming. At present, the commonly used processing methods include natural sun drying, water boiling, water steaming, microwave processing, hot air drying, vacuum freeze drying and so on.

SUMMARY

One object of the present invention is to provide Gastrodia elata Bl. extract and a

preparation method thereof. Another object of the present invention is to provide the

application of the extract in preparing related medicines and food.

By comparing the effects of fresh processing method and traditional processing method

on the contents of gastrodin, 4-Hydroxybenzyl alcohol and polysaccharide, the invention

determines the best preparation method of Gastrodiaelata extract.

The extract of the invention takes fresh Gastrodia elata as the extraction object and is

obtained by juicing, freezing and freeze-drying. Specifically, the preparation method

comprises following steps. Cleaning and cutting fresh Gastrodia elata into small pieces

and then putting them into a juicer with 1-10 times of distilled water. After pulping for 1

min, they are filtered with gauze. Adding 1-5 times of water into the filter residue and

repeating above steps once to ensure that the juice is fully extracted. The obtained juice is

frozen at -20°C for 12 h, and then freeze-dried in a freeze dryer.

The invention also provides a pharmaceutical combination of above obtained Gastrodia

elata extract consisting of active ingredients and pharmaceutically acceptable carriers,

which further exists in the form of verified pharmaceutical preparations, such as tablets,

sugar-coated tablets, film-coated tablets, enteric-coated tablets, sustained-release tablets, capsules, hard capsules, soft capsules, sustained-release capsules, oral liquids, mixtures, oral preparations, granules, electuary, pills, powders, ointments, sublimed preparation, suspensions, solutions, injections, powder-injection, freeze-dried powder injection, suppository, unguentum, emplastrum, cream, spray, dripping pills, dropping pills, patches, etc.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 Component content of Gastrodia elata treated by different methods in Ya'an

area.

Figure 2 Component content of Gastrodia elata treated by different methods in Bazhong

area.

Figure 3 Effect of different material-liquid ratio on component content of Gastrodia

elata.

Figure 4 Effect of different beating time on component content of Gastrodiaelata.

DESCRIPTION OF THE INVENTION

Embodiment 1

1. Preparation of Gastrodiaelata sample

Cleaning fresh Gastrodia elata from Ya'an, Sichuan province. In order to ensure the

consistency of sampling by the three treatment methods, the samples were classified into

three grades by weight: 75-150 g, 150-300 g and > 300 g, corresponding to group A,

group B and group C. Further, each group was then divided into three groups, totally

three groups of Al BI C1, A2 B2 C2 and A3 B3 C3.

(1) Preparation of fresh Gastrodia elata Juice. Cutting fresh Gastrodia elata of group Al

BI C1 into pieces, putting them into a juicer and adding 2.5 times distilled water for a 3

minute beating. Then filtering them with four layers of gauze. Adding 2 times of water

into the filter residue and repeating above steps once to ensure sufficient juice extraction.

The obtained juice was frozen at -20°C for 12 h, and then freeze-dried in a freeze dryer.

(2) Preparation of fresh Gastrodia elata by water decoction. Cutting fresh Gastrodia

elata of group A2 B2 C2 into thick slices and then decocting them twice with 5 times of

water for 1 h each time, and filtering. The liquid medicine was concentrated to a small

volume, frozen at -20°C for 12 h, and then freeze-dried in a freezer.

(3) Preparation of dried Gastrodiaelata by water decoction. Steaming Gastrodiaelata of

group A3 B3 C3 through firstly and then slicing and drying them at 60. Adding 5 times

of water to the dried Gastrodia elata and decocting them twice for 1 h each time. The

liquid medicine was concentrated to a small volume, frozen at -20°C for 12 h, and then

freeze-dried in a freezer.

2. Determination of gastrodin and 4-Hydroxybenzyl alcohol

According to the Chinese Pharmacopoeia 2015 Edition (Volume I) Gastrodia elata, the

content of three Gastrodia elata extracts was determined by High Performance Liquid

Chromatography (HPLC).

(1) Chromatographic conditions and system adaptability test

Chromatographic column Kromasil 100-5-C18 (4.6 mm x 150 mm, 5 pm);

Filler: octadecyl silane bonded silica gel;

Mobile phase: acetonitrile- 0.05% phosphoric acid solution (3: 97);

Detection wavelength: 220 nm;

Sample injection volume: 5 pL [8].

(2) Preparation of reference substance solution

Accurately weighing a proper amount of gastrodin reference substance and 4

Hydroxybenzyl alcohol reference substance. Then adding acetonitrile-water (3: 97)

mixed solution to prepare a mixed solution containing 50 pg gastrodin and 25 pg 4

Hydroxybenzyl alcohol per 1 mL, named [8].

(3) Preparation of test solution

The Gastrodia elata samples obtained by the above three processing methods were

crushed by a high-speed grinder, passed through the No. 3 sieve respectively, then

accurately weighed 2.0 g and put into a conical flask with cover. Then 50 ml of 50%

ethanol was precisely added. Weighing them. After ultrasonically treated for 30 minutes,

cooling and taking out to weigh. Adding with dilute ethanol to make up the original

weight and filtering. Accurately measuring 10 mL of the continued filtrate for

evaporation and concentration until it was nearly dry and had no alcohol taste. Dissolving

the residue with acetonitrile-water (3: 97) mixed solution and transferring it to a 25 mL

volumetric flask. Making up the volume to the scale with acetonitrile-water (3:97) mixed

solution, shaking well, filtering, and taking the continued filtrate to obtain three test

solution.

(4) Investigation of linear relationship

Accurately measuring appropriate amount of gastrodin and 4-Hydroxybenzyl alcohol

reference stock solution and adding acetonitrile-water (3: 97) mixed solution to prepare

mixed solution respectively containing 1.25 [g, 2.5 g, 10 g, 20 g, 30 g, 40 g, 50

pg, 60 pg, 80 pg gastrodin and 0.625 [g, 1.25 g, 5 g, 10 , 15 g, 20 g, 25 g, 30 g,

g 4-Hydroxybenzyl alcohol. 5 L of each above solution was precisely absorbed and

injected into HPLC. The absorption peak of the solution was determined by HPLC under

the same conditions. Further, the standard curve was made with the reference sample

injection volume (g) as the abscissa and the integral value of the peak area as the

ordinate, and then the regression equation was calculated

(5) Determination of the sample

Accurately absorbing 5 pL of the reference solution and 5pL of the test solution and

injecting them into the liquid chromatograph. Measuring and calculating the gastrodin

content and 4-Hydroxybenzyl alcohol content in the sample according to the standard

curve.

3. Determination of polysaccharide in Gastrodiaelata

content of polysaccharides in three kinds of Gastrodia elata extracts was determined by

sulfuric acid-phenol method.

(1) Preparation of glucose standard solution

Weighing 50 mg of anhydrous glucose reference substance and putting it in a 100 mL

volumetric flask. Adding an appropriate amount of water to dissolve, then making up the

volume and shaking it well to obtain 0.5 mg / mL glucose standard solution.

(2) Drawing of standard curve

Accurately taking 0.1 ml, 0.2 mL, 0.3 ml, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9

mL, 1.0 mL, 1.1 mL and 1.2 mL of 0.5 mg / mL standard glucose solution. After

replenishing the volume to 10 mL with distilled water, 0.4 mL of each was put into a tube

with cover. Then 1 mL of concentrated sulfuric acid and 0.2 mL of phenol with

concentration of 5% were added for phenol-concentrated sulfuric acid chromogenic

reaction for 30 min. Further, adding 0.4 mL deionized water into 1 mL concentrated

sulfuric acid and 0.2 mL of phenol with concentration of 5% for phenol-concentrated

sulfuric acid chromogenic reaction for 30 min. At the wavelength of 490 nm, the

absorbance was measured with a microplate reader, each with 3 parallel, taking average

value. With absorbance as ordinate and glucose concentration as abscissa, standard curve

is drawn, and regression equation is calculated.

(3) Extraction and refinement of Gastrodiaelata polysaccharide

g of three kinds of samples were weighed respectively and added with petroleum ether

based on the material-liquid ratio of 1: 5 for 2-hour reflux. The filtrate was discarded to

obtain filter residue, and the dry petroleum ether was evaporated. Adding 80% ethanol

based on the material-liquid ratio of 1: 5 for 2-hour reflux. Discarding filtrate to obtain

filter residue and volatilizing dry ethanol. Adding water according to material-liquid ratio

of 1: 5 for 2-hour reflux. The filtrate was filtered and concentrated to 20 mL by rotary

evaporator, then 4 times of absolute ethanol was added, standing overnight, and the

precipitate was obtained by centrifugation. Dissolving the precipitate with proper amount

of deionized water, adding 1% activated carbon, decolorizing in 60°C water bath, and filtering to remove activated carbon. Then it was deproteinized by sevage method. The decolorized Gastrodia elata polysaccharide was extracted with sevage reagent

(chloroform: n-butanol = 1:4) for many times until there was no obvious floc between the

two phases. Abandoning oil phase to obtain water phase. Adding 4 times of absolute

ethanol into that polysaccharide aqueous solution, staying overnight, and filtering to

obtain the precipitate, Gastrodia elata polysaccharide. Repeatedly washing and filtering

the obtained polysaccharide with ethanol, acetone and ether in turn and drying it at low

temperature to obtain refined Gastrodia elata polysaccharide.

(4) Calculation of conversion factor

Accurately weighing 75.3 mg of three kinds of refined Gastrodia elata polysaccharides

respectively, and adding deionized water into a 100 mL volumetric flask to supplement

the volume to 100 mL. 1 mL of each was diluted to 25 mL with deionized water, then 0.4

mL of each was added with 1 mL concentrated sulfuric acid and 0.2 mL 5% phenol for

chromogenic reaction by phenol-sulfuric acid method for 30 min. Then 0.4 mL deionized

water was added with 1 mL concentrated sulfuric acid and 0.2 mL 5% phenol for

chromogenic reaction by phenol-sulfuric acid method for 30 min. Measuring absorbance

with microplate reader at the wavelength of 490 nm, with 3 parallel, and taking the

average value. The regression equation was used to calculate the content, wherein, f =

W/(C x D) (W: the quality of Gastrodia elata polysaccharide; C: concentration of

polysaccharide solution; D: dilution multiple of polysaccharide solution).

(5) Preparation of sample solution

Accurately weighing 1 g of Gastrodia elata powder respectively, adding 20 mL of 80%

ethanol for ultrasonic extraction at 60°C for 25 min. Filtering after ultrasonic extraction

twice, adding 50 mL water to filter residue for ultrasonic extraction at 60°C for 25 min,

and filtering again after ultrasonic extraction twice. Combining the filtrates twice and

supplementing the volume to 250 mL. Absorbing 2 mL into a 10 mL centrifuge tube with

cover, adding 8 mL ethanol, shaking well and centrifuging (3000 r/min, 15 min) until the

precipitate has no alcohol smell. Dissolving with deionized water, and transferring to a 50

mL volumetric flask with supplementing to volume of 50 mL.

(6) Sample determination:

0.4 mL deionized water was added to 1 mL concentrated sulfuric acid and 0.2 mL 5%

phenol for chromogenic reaction by phenol-sulfuric acid method for 30 min, and then 0.4

mL deionized water was added to 1 mL concentrated sulfuric acid and 0.2 mL 5% phenol

for chromogenic reaction by phenol-sulfuric acid method for 30 min again. Measuring

absorbance with microplate reader at the wavelength of 490 nm, with 3 parallel, and

taking the average value. Read the weight of glucose in the test solution from the

standard curve and calculate it, that is: polysaccharide dry powder concentration in

sample g/g= m/M (m: polysaccharide mass; M: quality of Gastrodia elata dry powder)

4. Conclusion

The experimental results are shown in Figure 1. The total content of gastrodin and 4

Hydroxybenzyl alcohol in the extract of Ya'an Gastrodia elata by freshly squeezed

method was significantly better than that by water decoction method and traditional

steaming method. The content of polysaccharide in the extract of Gastrodia elata by freshly squeezed method was equivalent to that by water decoction method, but significantly better than that by steaming method.

Embodiment 2

1. Preparation of Gastrodiaelata sample

Cleaning fresh Gastrodia elata from Bazhong, Sichuan province. In order to ensure the

three groups of Al B1 Cl, A2 B2 C2 and A3 B3 C3.

(1) Preparation of fresh Gastrodiaelata Juice. Cutting fresh Gastrodia elata of group Al

BI C into pieces, putting them into a juicer and adding 2.5 times distilled water for a 3

volume, frozen at -20°C for 12 h, and then freeze-dried in a freezer.

group A3 B3 C3 through firstly and then slicing and drying them at 60°C. Adding 5

times of water to the dried Gastrodia elata and decocting them twice for 1 h each time.

The liquid medicine was concentrated to a small volume, frozen at -20'C for 12 h, and

then freeze-dried in a freezer.

2. Determination of gastrodin and 4-Hydroxybenzyl alcohol

The same as Embodiment 1.

3. Determination of polysaccharide in Gastrodiaelata

The same as Embodiment 1.

4. Conclusion

The experimental results are shown in Figure 2. The total content of gastrodin and 4

Hydroxybenzyl alcohol in the extract of Bazhong Gastrodia elata by freshly squeezed

steaming method. The content of polysaccharide in the extract of Gastrodia elata by

freshly squeezed method was equivalent to that by water decoction method, but

significantly better than that by steaming method.

Embodiment 3

Screening of freshly squeezed technology of Gastrodiaelata

1. Determination of material-liquid ratio

1) Cleaning and cutting fresh Gastrodia elata from Ya'an, Sichuan Province, into small

pieces and then putting them into a juicer with 1 time of distilled water. After beating for

3 min, they are filtered with four layers of gauze. Adding 2 times of water into the filter residue and repeating above steps once to ensure that the juice is fully extracted. The obtained juice is frozen at -20°C for 12 h, and then freeze-dried in a freeze dryer.

2) Cleaning and cutting fresh Gastrodia elata from Ya'an, Sichuan Province, into small

pieces and then putting them into a juicer with 2 times of distilled water. After beating for

3 min, they are filtered with four layers of gauze. Adding 2 times of water into the filter

residue and repeating above steps once to ensure that the juice is fully extracted. The

obtained juice is frozen at -20°C for 12 h, and then freeze-dried in a freeze dryer.

3) Cleaning and cutting fresh Gastrodia elata from Ya'an, Sichuan Province, into small

pieces and then putting them into a juicer with 3 times of distilled water. After beating for

4) Cleaning and cutting fresh Gastrodia elata from Ya'an, Sichuan Province, into small

pieces and then putting them into a juicer with 5 times of distilled water. After beating for

) Cleaning and cutting fresh Gastrodia elata from Ya'an, Sichuan Province, into small

pieces and then putting them into a juicer with 8 times of distilled water. After beating for

6) Cleaning and cutting fresh Gastrodia elata from Ya'an, Sichuan Province, into small

pieces and then putting them into a juicer with 10 times of distilled water. After beating

for 3 min, they are filtered with four layers of gauze. Adding 2 times of water into the

filter residue and repeating above steps once to ensure that the juice is fully extracted.

The obtained juice is frozen at -20°C for 12 h, and then freeze-dried in a freeze dryer.

The results are shown in Figure 3.

2. Determination of beating time

1 min, they are filtered with four layers of gauze. Adding 2 times of water into the filter

min, they are filtered with four layers of gauze. Adding 2 times of water into the filter

8 min, they are filtered with four layers of gauze. Adding 2 times of water into the filter

The results are shown in Figure 4.

Conclusion:

Different material-liquid ratio and beating time have effects on the component content.

With the increase of the material-liquid ratio, the content of components first increases

and then decreases. When the material-liquid ratio is three times, the content of

components is the best. After analysis, the reason for this result is that too little water is

not conducive to the dissolution of components, while the more water is added, the lower

the composition concentration is, because of avoiding heating treatment, without

concentration. In addition, the degree of comminution also affects the dissolution of

components. When the sample is completely broken, the dissolution of components

enters a plateau period, so the comminution time of 3-5 minutes is the best. When the

amount of sample increases, the criterion is that the sample is completely crushed.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. The extract from fresh Gastrodia elata Bl., characterized in that the extract is obtained

from fresh Gastrodiaelata Bl. by fresh pressing process.

2. The preparation method of the extract from Gastrodia elata Bl. according to Claim 1,

characterized by comprising following steps. Cleaning and cutting fresh Gastrodia elata

into small pieces and then putting them into a juicer with 1-10 times of distilled water.

After beating for 1-10 min, they are filtered with four layers of gauze. Adding 1-5 times

of water into the filter residue and repeating above steps once to ensure that the juice is

fully extracted. The obtained juice is frozen at -20°C for 12 h, and then freeze-dried in a

freeze dryer.

3. The preparation method of the extract from Gastrodia elata Bl. according to Claim 1,

characterized in that the method preferably comprises the following steps. Cleaning and

cutting fresh Gastrodia elata into small pieces and then putting them into a juicer with 2

3 times of distilled water. After beating for 3-5 min, they are filtered with four layers of

gauze. Adding 2-3 times of water into the filter residue and repeating above steps once to

ensure that the juice is fully extracted. The obtained juice is frozen at -20°C for 12 h, and

then freeze-dried in a freeze dryer.

4. The Gastrodia elata Bl. extract according to Claim 1 is a pharmaceutical combination

consisting of active ingredients and pharmaceutically acceptable carriers.

5. The pharmaceutical composition according to Claim 4 can exist in the form of

preparations suitable for use, which can be tablets, sugar-coated tablets, film-coated

tablets, enteric-coated tablets, sustained-release tablets, capsules, hard capsules, soft capsules, sustained-release capsules, oral liquids, mixtures, oral preparations, granules, electuary, pills, powders, ointments, sublimed preparation, suspensions, solutions, injections, powder-injection, freeze-dried powder injection, suppository, unguentum, emplastrum, cream, spray, dripping pills, dropping pills, patches, etc.

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Figure 1

Figure 2

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Figure 3

Figure 4