CN110279735B

CN110279735B - Effective part of Chinese walnut leaf and preparation method thereof

Info

Publication number: CN110279735B
Application number: CN201910734547.7A
Authority: CN
Inventors: 上官清; 洪艳; 李莎
Original assignee: Jinhong Pharmaceutical Co ltd
Current assignee: Jinhong Pharmaceutical Co ltd
Priority date: 2019-08-09
Filing date: 2019-08-09
Publication date: 2021-07-09
Anticipated expiration: 2039-08-09
Also published as: CN110279735A

Abstract

The invention relates to an effective part of Chinese gooseberry leaves and a preparation method thereof. The effective part of the Chinese gooseberry leaf is prepared by adopting the following method: 1) extracting folium Schefflerae Arboricolae with ethanol, and filtering to obtain ethanol extractive solution and residue; 2) extracting the residue with water, precipitating with ethanol to obtain water extractive solution; 3) mixing the ethanol extractive solution and the water extractive solution, concentrating, and drying to obtain extract powder; 4) dissolving the extract powder in water, loading onto resin column, sequentially performing gradient elution with deionized water and 10%, 20%, 30%, 50% and 75% ethanol water solution, collecting eluates of each gradient, and distilling under reduced pressure to obtain concentrated extract; and removing residual water in the concentrated extract to finally obtain freeze-dried powder of each part, and combining elution parts into three parts, namely water, a 10% + 20% + 30% ethanol elution part and a 50% + 75% ethanol elution part according to the similarity degree of the compounds, namely the effective part of the Chinese gooseberry leaves.

Description

Effective part of Chinese walnut leaf and preparation method thereof

Technical Field

The invention relates to the technical field of extraction, separation and application of plant effective parts, in particular to a Chinese gooseberry leaf effective part and a preparation method thereof.

Background

Pain is a common clinical symptom, and many complications of diseases are accompanied by pain, which brings great pain to patients. Currently, the analgesics commonly used in clinic refer to drugs that can partially or completely relieve pain, and include non-steroidal anti-inflammatory drugs and central analgesics. Common analgesics include aspirin, pain-relieving tablet, paracetamol, phenylbutazone, rofecoxib, etc. However, after long-term use, gastrointestinal tract reaction and nephrotoxicity are increased, adverse reaction is large, treatment and recovery of diseases of patients are not facilitated, and some anesthetics have addiction, so that the long-term use cannot be realized. The traditional Chinese medicine has the advantages of obviously improving clinical symptoms and physical signs when treating pain or complications, such as corydalis tuber, pseudo-ginseng and the like, and having less and light adverse reactions. More and more attention is paid to the special efficacy of the Chinese mahogany leaf in treating intractable pain such as sciatica, trigeminal neuralgia and the like.

Hantao leaves, a Chinese medicinal material with Guangxi national features, is collected in Guangxi Chinese herbal medicine, and is dried stem branches or stem branches with leaves of Wuxi schefflera kwangsiensis Merr. It is slightly bitter, astringent and warm in nature and has the effects of dispelling wind, relieving pain, relaxing muscles and tendons, activating collaterals and the like. It is mainly used for treating rheumatic arthralgia, sciatica, trigeminal neuralgia and renal and biliary tract pain. Hantao leaves are mainly produced in Guangxi province and mainly distributed in Wuming, Fusei and other counties in Guangxi province. The efficacy and common usage of Han Tao Ye are described in Guangxi national drug plains: decocting the root and stem with water, and can be used for treating rheumatic heart disease, premenstrual abdominal pain, and common cold; decocted in water and infused with wine for treating rheumatic lumbago; the decoction can be decocted in water for body washing and edema treatment; pounding and applying on affected part to treat fracture; pounding and applying on the periphery of the wound to treat venomous snake bite. At present, few research reports on the Chinese peach leaves are reported in China, Liu refined and the like in the publication of screening effective parts with anti-inflammatory and analgesic effects of the Chinese peach leaves, the petroleum ether, ethyl acetate and n-butyl alcohol parts of the Chinese peach leaves are preliminarily screened, and the results show that the ethyl acetate parts have relatively remarkable anti-inflammatory and analgesic effects, but no reports about further extraction and purification methods and applications suitable for large-scale production exist. Hushuping et al research on organic acid components of Chinese mahogany leaves and development of ointments, determine the optimal extraction process of total organic acid by adopting ion exchange chromatography and modern separation technology, and perform pharmacological research, but the extraction method is complex, the production cost is high, and the method is not suitable for industrial mass production.

CN109010396A discloses an effective part of Chinese gooseberry leaf and a preparation method thereof, the effective part is prepared by the steps of crushing a Chinese gooseberry leaf medicinal material, infiltrating with a 5% sodium bicarbonate solution, heating and refluxing for extraction by 30-40% ethanol, filtering, standing filtrate in a refrigeration house at 0-4 ℃ for 24-48 hours, filtering, recovering ethanol from the filtrate under reduced pressure to obtain an extract, adding water, mixing uniformly, adjusting the pH value of the solution to 3-4 by using acid, extracting by using a water saturated n-butyl alcohol solution, and recovering n-butyl alcohol from the extract under reduced pressure to obtain an extract, namely the effective part of Chinese gooseberry leaf. The effective part of the Chinese mahogany leaf is applied to the preparation of health care products or medicines for preventing or treating pain symptoms, in particular to rheumatic arthralgia, sciatica, trigeminal neuralgia, pains of kidney, biliary tract and the like. The preparation method of the effective parts of the Chinese gooseberry leaves, provided by the invention, has the advantages of simple and easy operation, high yield of the extracted effective parts, strong operability and low production cost, and is suitable for industrial large-scale production.

However, the content of the total flavone and the total saponin in the Chinese mahogany leaf extract obtained by the method in the prior art is low, and how to improve the content of the total flavone and the total saponin in the Chinese mahogany leaf extract is difficult in the field.

The present invention has been made in view of this situation.

Disclosure of Invention

The invention aims to provide an effective part of Chinese gooseberry leaves and a preparation method thereof, which greatly improve the content of total flavonoids and total saponins in the Chinese gooseberry leaf extract.

In order to solve the technical problems, the invention adopts the following technical scheme:

the effective part of the Chinese gooseberry leaf is prepared by adopting the following method:

1) extracting folium Schefflerae Arboricolae with ethanol, and filtering to obtain ethanol extractive solution and residue;

2) carrying out water extraction and alcohol precipitation on the residue obtained in the step 1) to obtain water extract;

3) combining the alcohol extract obtained in the step 1) and the water extract obtained in the step 2), concentrating and drying to obtain extract powder;

4) dissolving the extract powder obtained in the step 3) in water, loading the extract powder on a resin column, then sequentially carrying out gradient elution by using deionized water and 10%, 20%, 30%, 50% and 75% ethanol water solution, collecting eluates of various gradients, and carrying out reduced pressure distillation to obtain a concentrated extract state; and removing residual water in the concentrated extract to finally obtain freeze-dried powder of each part, and combining elution parts into three parts, namely water, a 10% + 20% + 30% ethanol elution part and a 50% + 75% ethanol elution part according to the similarity degree of the compounds, namely the effective part of the Chinese gooseberry leaves.

In the prior art, for example, the extraction of the Chinese gooseberry leaves in pharmacopoeia generally adopts a water extraction and alcohol precipitation method, but most of flavonoids and saponins are difficult to extract by water extraction and alcohol precipitation. How to better extract most of the flavonoids and saponins in the Chinese mahogany leaves is always a difficult problem in the field. The inventor analyzes structurally that the flavonoid substance is fat-soluble, is insoluble in water and is soluble in ethanol. Further, a large number of experiments surprisingly find that firstly the Chinese gooseberry leaf medicinal material is subjected to alcohol extraction to obtain alcohol extract and residue, and then the residue after alcohol extraction is subjected to water extraction and alcohol precipitation to obtain water extract; and then combining the alcohol extract and the water extract, concentrating and drying to obtain extract powder. The content of total flavone and total saponin in the obtained extract powder is obviously improved. Further, pharmacodynamic tests prove that the flavonoids and saponins have obvious analgesic effect, most of the flavonoids and saponins are lost by water extraction and alcohol precipitation in the method in the prior art, and the analgesic effect is reduced; the method of the invention obviously improves the content of the total flavone and the total saponin in the extract powder, thereby improving the analgesic effect.

Furthermore, it was found that the amount of water, 10%, 20%, 30%, 50%, and 75% of the powder obtained by gradient elution of the extract powder obtained by water extraction and alcohol precipitation on the resin column was relatively small, whereas the amount of water, 10%, 20%, 30%, 50%, and 75% of the powder obtained by gradient elution of the extract powder obtained by ethanol precipitation followed by water extraction and alcohol precipitation according to the present invention on the resin column was greatly increased.

Meanwhile, analgesic efficacy tests show that water, 10% + 20% + 30% ethanol elution parts and 50% + 75% ethanol elution parts all show analgesic activity, wherein the analgesic activity of the water elution parts is more stable than that of the other two groups, and the activity of the 50% + 75% ethanol elution parts is better than that of the 10% + 20% + 30% ethanol elution parts. Therefore, the effective parts of the Chinese gooseberry leaves are water, the elution parts of 10% + 20% + 30% ethanol and the elution parts of 50% + 75% ethanol.

The invention also provides a preparation method of the effective part of the Chinese gooseberry leaf, wherein the preparation method comprises the following steps:

2) volatilizing the residue obtained in the step 1) to remove residual alcohol solvent, and then carrying out water extraction and alcohol precipitation to obtain water extract;

Further, in the step 1), the alcohol extraction is performed twice by using 70% ethanol.

Further, in step 4), each gradient elutes 4 column volumes.

Further, in the step 4), the resin column is an HP20 macroporous resin column.

Further, in the step 4), before the HP20 macroporous resin column is arranged, the HP20 macroporous resin column is treated as follows:

soaking 1L of HP20 macroporous resin in ethanol solution for 24 hr, washing out the floating and suspended matter, loading on column, washing with ethanol for 3 volumes, and washing out the residual impurities.

In the invention, the related mass percentages are volume percentages.

In the invention, the water extraction and alcohol precipitation in the step 2) are carried out according to a water extraction and alcohol precipitation method in pharmacopoeia, and the specific method comprises the following steps:

volatilizing the residue obtained in the step 1) to remove residual alcohol solvent, adding water, decocting twice for 1.5 hours each time, filtering, combining the filtrates, concentrating to obtain clear paste with the relative density of about 1.2(50 ℃), adding ethanol until the alcohol content reaches 60%, standing, filtering, recovering ethanol, and obtaining the filtrate, namely the water extract.

The water extraction and alcohol precipitation in the step 2) can also be carried out according to the following method: volatilizing the residual alcohol solvent from the residue obtained in the step 1), adding 6-20 times of water by weight, decocting for two times, decocting for 2 hours for the first time and 1 hour for the second time, mixing the decoctions, filtering, concentrating the filtrate to obtain a clear paste with the relative density of 1.0-1.3(60 ℃), adding ethanol until the alcohol content reaches 60%, standing, filtering, recovering the ethanol, and obtaining the filtrate, namely the water extract.

The invention further provides application of the effective part of the Chinese gooseberry leaf in preparing health-care products or medicines for preventing and/or treating pain symptoms.

After adopting the technical scheme, compared with the prior art, the invention has the following beneficial effects:

1. the preparation method comprises the steps of firstly carrying out alcohol extraction on a Chinese gooseberry leaf medicinal material to obtain alcohol extract and residues, and then carrying out water extraction and alcohol precipitation on the residues after alcohol extraction to obtain water extract; mixing the ethanol extractive solution and the water extractive solution, concentrating, and drying to obtain extract powder; loading the extract powder onto resin column, eluting with water and ethanol solution, and screening by combining pharmacological experiment to obtain effective components of folium Schefflerae Arboricolae with remarkable analgesic effect; hot plate tests show that the traditional Chinese medicine composition has obvious analgesic effect;

2. the effective part of the Chinese mahogany leaf with the analgesic effect can be prepared into medicines or health-care products, and can be conveniently used by patients clinically. Meanwhile, the preparation method of the effective parts of the Chinese gooseberry leaves provided by the invention is simple and easy to operate, high in yield of the extracted effective parts, strong in operability, low in production cost and suitable for industrial large-scale production.

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

Drawings

FIG. 1 is a chart of efficacy of a hot plate test of a total extract of Hantao leaves, wherein Indometacin is a positive drug group, HTY is a Hantao leaf group, and Vehicle is a blank control group;

FIG. 2 is a diagram of the pharmacodynamics of different parts of Han Tao Gao in a hot plate test, in which Indometacin is a positive drug group, HTY (organic acid) is an organic acid group, HTY (water) is a water-eluting group, HTY (30% alcohol) is a 10% + 20% + 30% ethanol-eluting group, HTY (50% alcohol) is a 50% + 75% ethanol-eluting group, and Vehicle is a blank control group.

It should be noted that the drawings and the description are not intended to limit the scope of the inventive concept in any way, but to illustrate it by a person skilled in the art with reference to specific embodiments.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and the following embodiments are used for illustrating the present invention and are not intended to limit the scope of the present invention.

Example 1

1) Taking 15kg of Chinese peach leaf medicinal material, firstly extracting twice with 70% ethanol, 120kg each time, and filtering to obtain an ethanol extract and residues;

2) volatilizing the residue obtained in the step 1) to remove residual ethanol solvent, adding 6 times of water, decocting twice for 1.5 hours each time, filtering, combining the filtrates, concentrating to obtain fluid extract with relative density of 1.2(50 ℃), adding ethanol until the ethanol content reaches 60%, standing, filtering, recovering ethanol, and obtaining filtrate which is water extract;

3) mixing the alcohol extract obtained in the step 1) with the water extract obtained in the step 2), concentrating and drying to obtain 1.23kg of extract powder;

4) soaking 1L of HP20 macroporous resin in ethanol solution for 24 hr, washing off floating dust and suspended matter, loading on column, washing with ethanol for 3 volumes, and washing off residual impurities in resin. Rinsing with deionized water until the effluent is free of alcohol odor. Taking 65g of the Chinese mahogany leaf extract powder, completely dissolving the powder in 300ml of deionized water, loading, then sequentially carrying out gradient elution by using the deionized water and 10%, 20%, 30%, 50% and 75% ethanol water solution, wherein each gradient elution is 4 times of the column volume, then collecting the eluates of each gradient, and carrying out reduced pressure distillation to obtain a thick extract state. And then placing the concentrated extract on a freeze dryer, removing residual moisture, and finally obtaining freeze-dried powder of each part: water (20.8g, 32%), 10% (3.2g, 4.9%), 20% (5.5g, 8.4%), 30% (4.2g, 6.4%), 50% (10.3g, 15.8%), 75% (2.3g, 3.5%). According to the similarity degree of the compounds, the elution parts are combined into three parts, namely a water elution part with 10% + 20% + 30% ethanol and an elution part with 50% + 75% ethanol, namely the effective part of the Chinese gooseberry leaf.

Example 2

2) volatilizing the residue obtained in the step 1) to remove residual ethanol solvent, adding 10 times of water, decocting twice for 1.5 hours each time, filtering, combining the filtrates, concentrating to obtain fluid extract with relative density of 1.2(50 ℃), adding ethanol until the ethanol content reaches 60%, standing, filtering, recovering ethanol, and obtaining filtrate which is water extract;

3) mixing the alcohol extract obtained in the step 1) and the water extract obtained in the step 2), concentrating and drying to obtain 1.22kg of extract powder;

4) soaking 1L of HP20 macroporous resin in ethanol solution for 24 hr, washing out the floating precipitate and suspended matter, loading on column, washing with ethanol for 3 volumes, washing out the residual impurities in resin, and washing with deionized water until the effluent liquid has no alcohol smell. Dissolving 65g of the extract powder obtained in the step 3) in 300ml of water, loading the extract powder on a resin column, sequentially carrying out gradient elution by using deionized water and 10%, 20%, 30%, 50% and 75% ethanol water solution, wherein each gradient elution is 4 times of the column volume, collecting the eluates of each gradient, and carrying out reduced pressure distillation to obtain a concentrated extract state; and removing residual water in the concentrated extract to finally obtain freeze-dried powder of each part, and combining elution parts into three parts, namely water, a 10% + 20% + 30% ethanol elution part and a 50% + 75% ethanol elution part according to the similarity degree of the compounds, namely the effective part of the Chinese gooseberry leaves.

Example 3

2) volatilizing the residue obtained in the step 1) to remove residual ethanol solvent, adding 14 times of water, decocting twice for 1.5 hours each time, filtering, combining the filtrates, concentrating to obtain fluid extract with relative density of 1.2(50 ℃), adding ethanol until the ethanol content reaches 60%, standing, filtering, recovering ethanol, and obtaining filtrate, namely water extract;

4) dissolving 65g of the extract powder obtained in the step 3) in 300ml of water, loading the extract powder on an HP20 macroporous resin column, then sequentially carrying out gradient elution by using deionized water and 10%, 20%, 30%, 50% and 75% ethanol aqueous solution, wherein each gradient elution is 4 times of the column volume, collecting the eluates of each gradient, and carrying out reduced pressure distillation to obtain a concentrated extract state; and removing residual water in the concentrated extract to finally obtain freeze-dried powder of each part, and combining elution parts into three parts, namely water, a 10% + 20% + 30% ethanol elution part and a 50% + 75% ethanol elution part according to the similarity degree of the compounds, namely the effective part of the Chinese gooseberry leaves.

Example 4

1) Taking 15kg of Chinese peach leaf medicinal material, extracting twice with 70% ethanol, 120kg each time, and filtering to obtain ethanol extract and residue;

2) volatilizing the residual alcohol solvent from the residue obtained in the step 1), adding 16 times of water by weight, decocting for 2 times, decocting for 2 hours for the first time and 1 hour for the second time, mixing the decoctions, filtering, concentrating the filtrate to obtain a clear paste with the relative density of about 1.3(60 ℃), adding ethanol until the alcohol content reaches 60%, standing, filtering, recovering ethanol, and obtaining the filtrate, namely water extract;

Example 5

2) volatilizing the residual alcohol solvent from the residue obtained in the step 1), adding 20 times of water by weight, decocting for 2 times, decocting for 2 hours for the first time and 1 hour for the second time, mixing the decoctions, filtering, concentrating the filtrate to obtain a clear paste with the relative density of about 1.0(60 ℃), adding ethanol until the alcohol content reaches 60%, standing, filtering, recovering ethanol, and obtaining the filtrate, namely water extract;

Comparative example 1 preparation of Total extract of Hantao leaves

2) volatilizing the residue obtained in the step 1) to remove residual ethanol solvent, adding water, decocting twice for 1.5 hours each time, filtering, mixing the filtrates, concentrating to obtain fluid extract with relative density of 1.2(50 ℃), adding ethanol until the ethanol content reaches 60%, standing, filtering, and recovering ethanol to obtain filtrate, i.e. water extract;

3) mixing the ethanol extract obtained in the step 1) and the water extract obtained in the step 2), concentrating and drying to obtain 1.23kg of extract powder, namely the Chinese gooseberry leaf total extract.

Comparative example 2 preparation of organic acid fraction of Hantao leaf

4) taking 800ml of 201X 7 anion exchange resin, soaking the resin in deionized water to remove suspended matters, and then soaking the resin in water at 70-80 ℃ for 4 hours to swell the resin. The swollen resin was loaded into a 1.5L column and the resin was activated with 1.5L of 5% NaOH, 1.5L of 5% HCl solution, 2L of 5% NaOH, and 2L of 5% HCl solution in that order. Finally washing with water to neutrality.

5) Dissolving 35g of Chinese gooseberry leaf extract powder in 200ml of deionized water, loading the Chinese gooseberry leaf extract powder on anion exchange resin, washing with deionized water with 4 times of column volume, washing with 1% HCl with 4 times of column volume, collecting 1% HCl eluate, and concentrating by reduced pressure distillation to obtain the extract liquid of the initial organic acid enrichment part.

6) 800ml of 001X 7 cation exchange resin was taken, and the resin was swollen and activated. And (3) loading 100ml of the enriched organic acid extract liquid, eluting with deionized water with 4 times of column volume, recovering the eluent, concentrating under reduced pressure to obtain a concentrated extract, and then placing the concentrated extract on a freeze dryer to remove water to obtain 7.2g (20%) of organic acid powder.

Comparative example 3 effective fraction obtained by water extraction and alcohol precipitation alone

1) Taking 15kg of Chinese gooseberry leaf medicinal material, adding water, decocting twice for 1.5 hours each time, filtering, combining the filtrates, concentrating to obtain clear paste with the relative density of about 1.2(50 ℃), adding ethanol until the ethanol content reaches 60%, standing, filtering, recovering ethanol, concentrating the filtrate to obtain thick paste, and drying to obtain 0.85kg of extract powder.

2) Soaking 1L of HP20 macroporous resin in ethanol solution for 24 hr, washing off floating dust and suspended matter, loading on column, washing with ethanol for 3 volumes, and washing off residual impurities in resin. Rinsing with deionized water until the effluent is free of alcohol odor. Taking 65g of the Chinese mahogany leaf extract powder, completely dissolving the powder in 300ml of deionized water, loading, then sequentially carrying out gradient elution by using the deionized water and 10%, 20%, 30%, 50% and 75% ethanol water solution, wherein each gradient elution is 4 times of the column volume, then collecting the eluates of each gradient, and carrying out reduced pressure distillation to obtain a thick extract state. And then placing the concentrated extract on a freeze dryer, removing residual moisture, and finally obtaining freeze-dried powder of each part: water (16.2g, 25.9%), 10% (2.2g, 3.4%), 20% (3.5g, 5.4%), 30% (2.2g, 3.4%), 50% (4.5g, 6.9%), 75% (0.5g, 0.8%). According to the similarity degree of the compounds, the elution parts are combined into three parts, namely a water elution part with 10% + 20% + 30% ethanol and an elution part with 50% + 75% ethanol, namely the effective part of the Chinese gooseberry leaf.

Test example 1 measurement of Total Flavonoids and Total Saponins in the Total extract of Hantao leaves

In the pharmacopoeia of 2015 edition, the quality of the Chinese gooseberry leaves is controlled by measuring the content of total flavonoids by using an ultraviolet photometry and rutin as a reference substance, so that the total flavonoids content of the total extract is measured by using the measuring method of the total flavonoids in the pharmacopoeia in the experiment. The Chinese gooseberry leaves are known to contain a large amount of triterpenoid saponin components through the existing literature reports. The components have better analgesic and anti-inflammatory effects, so that the invention takes oleanolic acid as a reference substance to measure the content of the total saponin components in each part.

1) Preparation of Chinese peach leaf total extract

1.1 Total extract_{(alcohol extraction + Water extraction)}Preparation of

a. Taking 15kg of Chinese peach leaf medicinal material, firstly extracting twice with 70% ethanol, 120kg each time, and filtering to obtain an ethanol extract and residues;

b. volatilizing the residue obtained in the step a) to remove residual ethanol solvent, adding water, decocting twice for 1.5 hours each time, filtering, mixing the filtrates, concentrating to obtain fluid extract with relative density of 1.2(50 ℃), adding ethanol until the ethanol content reaches 60%, standing, filtering, and recovering ethanol to obtain filtrate, i.e. water extract;

c. combining the alcohol extract obtained in the step a) and the water extract obtained in the step b), concentrating and drying to obtain a total extract_{(alcohol extraction + Water extraction)}。

1.2 Total extract_{(Water extraction)}Preparation of

Decocting folium Schefflerae Arboricolae 15kg with water twice, each for 1.5 hr, filtering, mixing filtrates, concentrating to obtain fluid extract with relative density of 1.2(50 deg.C), adding ethanol until ethanol content reaches 60%, standing, filtering, recovering ethanol, concentrating the filtrate to obtain soft extract, and drying to obtain total extract_{(Water extraction)}。

2) Determination of total flavone content

Taking 10.1mg of anhydrous rutin reference substance, accurately weighing, placing in a 10mL measuring flask, adding 5mL of 60% ethanol, slightly heating to dissolve, cooling, diluting with 60% ethanol to scale, and shaking. Precisely measuring 1mL, placing in a 5mL measuring flask, adding water to the scale, and shaking to obtain the final product (each 1mL contains anhydrous rutin 0.2 mg).

Preparation of a standard curve: precisely measuring reference substance solutions 0.4mL, 0.8mL, 1.2mL, 1.6mL, 2.0mL and 2.4mL, respectively placing in a 10mL measuring flask, adding water to 2.4mL, adding 5% sodium nitrite solution 0.4mL, mixing, standing for 6min, adding 10% aluminum nitrate solution 0.4mL, mixing, standing for 6min, adding sodium hydroxide test solution 4mL, adding water to scale, shaking, standing for 15min, blank with corresponding reagent, measuring absorbance at 500nm wavelength by ultraviolet-visible spectrophotometry (general rule 0401), and drawing a standard curve with absorbance as ordinate and concentration as abscissa.

And (3) sample determination: taking about 0.16g of each sample, precisely weighing, placing in a 20mL measuring flask, adding 15mL of 60% ethanol, heating at 80 ℃ for 30 minutes, shaking constantly, cooling, adding 60% ethanol to the scale, shaking uniformly, and filtering. Precisely absorbing 1.0mL of each subsequent filtrate, respectively placing in a 10-mL measuring flask, adding water to 2.4mL, adding 0.4mL of 5% sodium nitrite solution, uniformly mixing, placing for 6min, adding 0.4mL of 10% aluminum nitrate solution, uniformly mixing, placing for 6min, adding 4mL of sodium hydroxide test solution, adding water to the scale, shaking uniformly, placing for 15min, taking corresponding reagents as a blank, measuring absorbance at the wavelength of 500nm by ultraviolet-visible spectrophotometry (general rule 0401), precisely absorbing 1.0mL of the subsequent filtrate, adding water to dilute to 10mL, shaking uniformly, making a blank, measuring the absorbance by the method, reading the amount of anhydrous rutin in a sample from a standard curve, and calculating to obtain the rutin. The results are shown in Table 1.

TABLE 1 Total flavone content of Hantao leaf extract

The standard curve was established by uv spectrophotometry as y 12.766x + 0.0071.

As shown in table 3, the total flavone content in the total extract powder extracted with ethanol and water was 4.48%, and the total flavone content in the total extract powder extracted with water was 3.68%, which indicates that the total extract powder extracted with water alone had a lower flavone content than the total extract powder extracted with ethanol and water.

3) Determination of total saponin content

Preparing oleanolic acid standard solution: accurately weighing 1mg of oleanolic acid standard substance, placing the oleanolic acid standard substance in a 5ml volumetric flask, and performing constant volume with methanol to obtain 0.2mg/ml oleanolic acid standard substance solution stock solution.

Preparation of oleanolic acid standard curve: weighing 25mg of vanillin and 5ml of glacial acetic acid solution, and mixing to obtain a vanillin solution. Respectively taking 0, 40, 80, 120, 160 and 200ul of the oleanolic acid standard solution stock solution, placing the oleanolic acid standard solution stock solution in a 10ml centrifuge tube, and drying the oleanolic acid standard solution in nitrogen at 40 ℃. Then 50ul of vanillin-glacial acetic acid solution and 200ul of perchloric acid solution are added, and 250ul of the obtained solution is subjected to water bath at 70 ℃ for 15min and then cooled for 5 min. Adding 650ul glacial acetic acid after cooling, mixing the obtained 900ul solution uniformly, standing for 10min, diluting to 2.7ml, and measuring A with ultraviolet spectrophotometer_540nmAnd drawing a standard curve by taking the absorbance as the ordinate and the concentration as the abscissa.

And (3) determination of a sample: 10mg of each sample was dissolved in 5ml of methanol to obtain 5ml of a sample solution. And (3) according to the oleanolic acid standard curve preparation process, measuring the sample liquid, substituting the obtained numerical value into a linear regression equation, and calculating the content of the total saponins in the sample. The results are shown in Table 2.

TABLE 2 Total saponins content of Hantao leaf extract

The standard curve was established by uv spectrophotometry as y 35.569x + 0.0478.

As shown in table 2, the total saponin content in the total extract powder extracted with ethanol and water was 16.51%, and the total saponin content in the total extract powder extracted with water was 8.77%, which indicates that the total saponin content in the total extract powder extracted with water alone was lower than that in the total extract powder extracted with ethanol and water.

Test example 2 Hot plate test

1) Test drug

The total extractive of the Chinese gooseberry leaves and different enrichment parts.

The experimental dose of the total extract is 5g/kg/d, the concentration of the administration solution is the maximum value of the total extract of the Chinese gooseberry leaves which can be dissolved in physiological saline, and the administration amount is 18 times of the administration dose of an adult.

In the second different enriched site activity experiment, the dosage of the four sites is converted on the basis of the experimental dosage (9g/kg/d) which is 1.8 times of the total extract according to the content percentage of the sites in the total extract. The conversion mode considers the mass ratio of different compounds in the total extract while observing the respective pharmacodynamic activities of different parts, and is more consistent with the practical situation of medication. The final dose at several sites was 2.8g/kg/d (water eluted site) (prepared in example 1), 1.8g/kg/d (10% + 20% + 30% ethanol eluted site) (prepared in example 1), 1.7g/kg/d (50% + 75% ethanol eluted site) (prepared in example 1), 2.8g/kg/d (organic acid site) (prepared in comparative example 2).

2) Test animal

SPF grade KM mice, 18-22g, female (hot plate) provided by shenyang pharmaceutical university test animals center, purchased from beijing waukukang biotechnology limited, certification No.: laboratory bottom

3) Pharmaceutical agent

4) Testing instrument

5) Test method

Putting the female mice into a hot plate pain-causing instrument, controlling the temperature of the hot plate pain-causing instrument at (55 +/-0.5) DEG C, and taking the time(s) until the female mice lick the hindpaw as the pain threshold of the mice, wherein the time(s) is less than 5s or more than 30s or the female mice are discarded. And screening qualified female mice, repeatedly measuring the normal pain threshold value of the female mice, and taking the average value of the two normal pain threshold values as the pain threshold value before administration of the female mice. And (4) randomly grouping, wherein each group comprises 12.

Dosing regimen (first time): negative control group, normal saline; positive control group, indomethacin, 20 mg/kg/d; the content of the Chinese gooseberry leaf extract in the high-dose group is 5 g/kg/d. Each group was gavaged 1 time daily for 7 days at a dose of 0.1mL/10g per mouse body weight.

Dosing regimen (second): negative control group, normal saline; positive control group, indomethacin, 20 mg/kg/d; the medicine is prepared by performing intragastric administration on the organic acid extract of the Chinese gooseberry leaves, the aqueous extract of the Chinese gooseberry leaves, the 10% + 20% + 30% ethanol elution parts of the Chinese gooseberry leaves, and the 50% + 75% ethanol elution parts of the Chinese gooseberry leaves for 1 time and 7 days continuously according to the weight of a mouse per day and the dosage of 0.1mL/10 g.

The pain threshold was determined at 30, 60, 90, 120min after administration to mice. If the pain threshold exceeds 60s, recording according to 60s, and calculating the pain threshold increase rate. Pain threshold increase rate (%) (post-administration pain threshold-pre-administration pain threshold)/pre-administration pain threshold × 100.

6) Test results

The test results are shown in tables 3 and 4:

TABLE 3 heat plate test record table for Hantao leaf total extract

TABLE 4 Hot plate test record table for different parts of Hantao leaves

7) Conclusion and discussion

(1) In a hot plate analgesic test, compared with the test before administration, the total extract of the Chinese gooseberry leaves shows analgesic activity at four time points of 30min, 60min, 90min and 120min, the pain threshold value is respectively increased by 30%, 81%, 80% and 50% (table 3, figure 1), and the percentage is weaker than that of the positive drug indometacin group.

(2) In the hot plate analgesic test of the four-part extract, the Chinese mahogany leaf (organic acid) shows no analgesic activity, and the rest three groups have analgesic activity. Wherein, the Chinese peach leaves (10% + 20% + 30% ethanol elution part) only show analgesic activity at 90min, and the pain threshold increase percentage is 77.84%; the analgesic activity of the Chinese peach leaves (50% + 75% ethanol elution part) is better than that of the Chinese peach leaves (10% + 20% + 30% ethanol elution part), the analgesic activity is shown in 90min and 120min, the pain threshold increasing percentage is 116.23% and 84.93%, and the analgesic activity is better than that of the positive medicines indometacin 90min (88.27%) and 120min (58.93%) at the time point. Compared with the group before administration at four time points of 30min, 60min, 90min and 120min, the radix Schefflerae Arboricolae (water elution part) group shows analgesic activity, the pain threshold value is respectively increased by 32.95%, 46.82%, 47.98% and 60.12%, the anti-inflammatory analgesic activity is weaker than that of the positive drug group, but the time points of 30min and 60min are better than that of the radix Schefflerae Arboricolae (50% + 75% ethanol elution part) group.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. The effective part of the Chinese gooseberry leaf is characterized by being prepared by adopting the following method:

1) extracting folium Schefflerae Arboricolae with 70% ethanol twice, and filtering to obtain ethanol extractive solution and residue;

2. A method for preparing effective fraction of folium Schefflerae Arboricolae of claim 1, comprising the following steps:

3. The method according to claim 2, wherein in step 4), each gradient elutes 4 column volumes.

4. The preparation method according to claim 2, wherein in the step 4), the resin column is an HP20 macroporous resin column.

5. The preparation method of claim 4, wherein in the step 4), the HP20 macroporous resin column is treated as follows before the HP20 macroporous resin column is arranged:

6. The preparation method according to any one of claims 2 to 5, wherein the water extraction and alcohol precipitation in step 2) is: volatilizing the residue obtained in the step 1) to remove residual alcohol solvent, adding water, decocting twice for 1.5 hours each time, filtering, combining the filtrates, concentrating to obtain clear paste with a relative density of 1.2 at 50 ℃, adding ethanol until the alcohol content reaches 60%, standing, filtering, and recovering ethanol to obtain filtrate, namely water extract.

7. Use of the effective part of Hantao leaf of claim 1 for the preparation of a medicament for the prevention and/or treatment of pain symptoms.