AU2020101758A4 - Preparation Method and Application of Total Flavonoids of Choerospondias Axillaris Leaf - Google Patents

Abstract

The invention discloses a preparation method and application of total flavonoids extracted from leaves of Choerospondias Axillaris. Extract the leaves of Choerospondias Axillaris with ethanol, and extract and separate the extract with petroleum ether to obtain the total flavonoids of Choerospondias Axillaris Leaf; After ethyl acetate extraction and polyamide column purification, anthocyanins, flavones, flavonols, dihydroflavones, dihydroflavonols, chalcones, isoflavones, flavanols and other flavonoids are separated on silica thin-layer plate. The total flavonoids of Choerospondias Axillaris Leaf have obvious function of anti-arrhythmia, protecting myocardial ischemia, improving immune function, anti-oxidation, etc., and can be used to prepare drugs that can prevent and treat cardiovascular and cerebrovascular diseases, diseases with reduced immune function, and free radical damage diseases. -115 0506 0 0.700 0530 400450.0 6600550. 0IM W L Waveleuth:A94.7 Abs:IJ. 0 1 Figure1I

Description

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AUSTRALIA

PATENTS ACT 1990

PATENT SPECIFICATION FOR THE INVENTION ENTITLED:

Preparation Method and Application of Total Flavonoids of Choerospondias

Axillaris Leaf

The invention is described in the following statement: -

Preparation Method and Application of Total Flavonoids of Choerospondias

Axillaris Leaf

TECHNICAL FIELD

The present invention relates to the extraction of total flavonoids isolated from the

leaves of Choerospondias Axillaris (Roxb.) Burttet Hill and the process for its preparation

and use in the field of pharmaceutical health products, in particular to application in that

preparation of product for preventing and treatment of cardiovascular and cerebrovascular

diseases, diseases with reduced immune function and disease related to free radical

damage, which belongs to the field of natural medicine chemistry research.

BACKGROUND

Tibetan and Mongolian medicine, as a component of national medicine, has always

been an important part of China's medical treasure-house. The development of Mongolian

medicine has absorbed the essence of Tibetan and Han ethnic medicine, which has been

organically integrated into a whole in long-term practice. With continuous improvement,

modem Mongolian medicine is improving daily and has formed a complete theoretical

system with distinctive national characteristics and categories of modem Mongolian

Medicine with rich clinical experience. It can be said that it has gone through many

vicissitudes and not declined for a long time, which has shown great vitality and potential

value and function.

Choerospondias Axillaris Fructus (the fructus of Choerospondias Axillaris (Roxb.)

Burttet Hill.) is one of the commonly used medicinal materials in Mongolian medicine,

which has the functions of activating qi and activating blood circulation, nourishing the heart and tranquilizing the mind. It is mainly used for qi stagnation and blood stasis, chest pain, palpitation, shortness of breath, and restlessness of mind. According to the preliminary statistics, there are 101 kinds of oral medicine containing Choerospondias

Axillaris Fructus. As an ancient prescription, Choerospondias Axillaris Fructus has been

recorded for a long time, and in 1990, the Chinese Pharmacopoeia began to record that it

was used as a medicine.

Choerospondias Axillaris (Roxb.) Burttet Hill., also known as South sour jujube, five

eye fruit or snot fruit, is a kind of tall deciduous tree belonging to the family

Anacardiaceae. At present, the Choerospondias Axillaris Fructus and its preparations

contained in whether in the national pharmacopoeia, or in the Inner Mongolia standard

medicine, refers to the dry mature fruit of Choerospondias Axillaris. Choerospondias

Axillaris Fructus, like other plant drugs, contains many chemical components, including

flavonoids, organic acids, sterols, volatile oils, coumarins, sugars, acidic components and

many kinds of amino acids, and it's main active component is flavonoids, that is Total

flavonoids of Choerospondias Axillaris Fructus. In that modem pharmacological

experimental study, Total flavonoids of Choerospondias Axillaris Fructus has obvious

effects of anti-arrhythmia, anti-anoxia and protection of myocardial ischemia, inhibition of

platelet aggregation and improvement of hemorheology, and can enhance the immune

function of mice.

The application of total flavonoids of Choerospondias Axillaris Fructus in medicine

field is very broad, and more and more prescriptions are developed and widely used. As the

medicinal value of the plant is further explored, it is bound to pose a new challenge to the

raw material demand of the medicinal material of Choerospondias Axillaris Fructus.

Therefore, it is very important and necessary to find substitutes and expand new medicinal

parts of it. In the early time, the folk developed the green tea, containing tea pigment and

made from wild leaves of Choerospondias Axillaris, which can reduce weight, reduce

blood pressure, enhance immunity and have the functions of antioxidation, anti-arrhythmia

if drunk often. Inspired by this and drawing lessons from the successful experience of

research and development of ginkgo biloba leaf, we conducted a comparative study on the

effective chemical components of the medicinal material (fruit) and leaves of

Choerospondias Axillaris and observed the pharmacological effects of the flavonoids

extracted from the leaves of Choerospondias Axillaris.

The leaf yield of Choerospondias Axillaris is sufficient, which is not limited by

seasons and years, If Choerospondias Axillaris Leaf can be used as Choerospondias

Axillaris Fructus or made into various pharmaceutical preparations, the medicinal value of

Choerospondias Axillaris will be promoted to a greater extent.

So far, there is no report on the chemical constituents and pharmacological effects of

the leaves of Choerospondias Axillaris.

SUMMARY

The object of the present invention is to develop and utilize the resources of the leaves

of Choerospondias Axillaris, which have been discarded as waste so far, especially the

development and utilization of flavonoid active ingredients therein.

Through the in-depth and detailed comparative study on the chemical components of

Choerospondias Axillaris Leaf and Choerospondias Axillaris Fructus (i.e. dry and mature

fruit), the invention firstly finds that the total flavonoids in the Choerospondias Axillaris

Leaf are more than those in Choerospondias Axillaris Fructus in type and content. In the

separated components, there are 13 in Choerospondias Axillaris Fructus and 15 in

Choerospondias Axillaris Leaf. In the Choerospondias Axillaris Fructus, there are

flavonoids such as anthocyanins, flavones, flavonols, dihydroflavones, chalcone,

isoflavones, and flavanols (catechins), etc. The flavonoids compounds in the leaves of

Choerospondias Axillaris include anthocyanins, flavones, flavonol, dihydroflavones,

dihydroflavonol, chalcone, isoflavone, flavanol, etc. On this basis, through a series of

comparison and reference experiments of Choerospondias Axillaris Fructus and

Choerospondias Axillaris Leaf, it is found that there are 6 corresponding common

components in the total flavonoids of both fructus and its Leaf. In term of that types of

flavonoid, they are: anthocyanin, dihydroflavone, flavonol, flavonol, chalcone, aurone.

The invention further discloses an extraction method of the flavonoid compounds and an

ideal silica gel thin layer chromatography separation system. In addition, it was found that

that total flavonoids of Choerospondias Axillaris Leaf had obvious effect of

anti-arrhythmia, protecting myocardial ischemia, improving immune function and

anti-oxidation. It can be used to replace Choerospondias Axillaris Fructus to prepare

products which can prevent and treat cardiovascular and cerebrovascular diseases, diseases

with reduced immune function and diseases related to free radical injury, including health

food and pharmaceutical compositions.

In this invention, the Choerospondias Axillaris Leaf is a leaf of the Choerospondias

Axillaris(Roxb.) Burttet Hill., belonging to Anacardiaceae plant; Total flavonoids refers

to the mixture of flavonoids extracted and separated from the leaves of Choerospondias

Axillaris (Roxb.) Burttet Hill.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is the UV Vis absorption spectra of ethanol extracts from Choerospondias

Axillaris Fructus and Choerospondias Axillaris Leaf. The sample has the maximum

absorption value at 506nm.

Figure 2 is the standard curve of rutin reference substance.

Figure 3 is the TLC of alcohol solution of petroleum ether extraction of Choerospondias

Axillaris sample under the ultraviolet lamp (365 nm).

Figure 4 is the TLC of the sample after purification. The above-mentioned left picture is

the thin-layer developed spot observed under visible light, and the right picture is that

observed under ultraviolet lamp (365m).

a- Ethyl acetate extraction layer of ethanol extract from Choerospondias Axillaris Fructus

A-Extraction of water layer by ethyl acetate from ethanol extracts of Choerospondias

Axillaris Fructus

b- Ethyl acetate extraction layer of ethanol extract from Choerospondias Axillaris Leaf

B- Extraction of water layer by ethyl acetate from ethanol extracts of Choerospondias

Axillaris Leaf

Figure 5 is the TLC after purification of ethyl acetate extract on polyamide column.

a- Ethyl acetate extraction layer of ethanol extract from Choerospondias Axillaris Fructus

a'- Purified compounds of sample a on polyamide column

b- Ethyl acetate extraction layer of ethanol extract from Choerospondias Axillaris Leaf

b'- Purified compounds of sample b on polyamide column

S- quercetin

DESCRIPTION OF THE INVENTION

The present invention is accomplished by the following specific technical proposal,

The main contents include extraction, content determination, extraction identification,

separation and identification of flavonoids in fructus and leaves of Choerospondias

Axillaris; In that invention, the total flavonoids of Choerospondias Axillaris Leaf is used

for studying the effects of anti-arrhythmia, anti-immune function, myocardial ischemia

protection and anti-oxidation effect, and finally the invention is completed.

The main contents of this study are as follows: The extraction and determination of

flavonoids in Choerospondias Axillaris Fructus and Choerospondias Axillaris Leaf, the

identification of the extracts and the separation and identification of flavonoids.

In this experiment, the ethanol extracts of Choerospondias Axillaris Fructus and

Choerospondias Axillaris Leaf were extracted with petroleum ether, then extracted with

ethyl acetate and purified with polyamide column and the samples were developed on the

silica gel thin layer plate. More than 10~15 spots were observed under the ultraviolet lamp.

The color reaction was carried out by TLC, and different color spots appear on the

board, which showed there are many and complex kinds of total flavonoids in the leaves of

Choerospondias Axillaris and Choerospondias Axillaris Fructus. In combination with that

thin lay development of silica gel, the color development reaction on the TLC plate, and the

scraping absorption spectrum of each spot component substance under the

ultraviolet-visible spectrophotometry, 13 total flavonoid components of Choerospondias

Axillaris Fructus are finally identified, from the front to the bottom, they are: anthocyanin,

flavonol, flavone, anthocyanins, chalcone, dihydroflavone, flavonol, catechin, flavanol, chalcone, dihydroflavone, aurone and catechin; In Choerospondias Axillaris Leaf, there are flavonoid components, from the front to the bottom: anthocyanin, anthocyanin, anthocyanin, isoflavone, chalcone, anthocyanin, dihydroflavone, flavonol, flavonol, chalcone, chalcone, charlcone, aurone, aurone.

In that experiment, through many times of comparison, it was found that both of them

contain anthocyanin, flavone, flavonol, dihydroflavone, chalcone and aurone, etc.; by

silica gel TLC expansion method, TLC color development method, and UV Vis

spectrophotometry , The results showed that there were six spots of flavonoids in leaves of

Choerospondias Axillaris and Choerospondias Axillaris Fructus. From the leading edge to

the baseline, the sequence is: anthocyanin, dihydroflavone, flavonol, flavanol, chalcone,

aurone. The Rf values of spots 13'and 15 in the figure are the same, but the color reaction

shows that they are completely different chemical composition types.

In that experiment, the extraction and separation condition of the total flavonoid

components in Choerospondias Axillaris Leaf and Choerospondias Axillaris Fructus were

discussed, and an ideal silica gel thin layer chromatography separation system was found

out, which provides reliable information for the extraction, separation and identification of

the total flavonoids in Choerospondias Axillaris Leaf. At the same time, it provides

conditions for finding the corresponding components or substitutes in the leaves. The

specific process is as follows.

Pretreatment of Experimental Materials

Check whether the purchased or collected Choerospondias Axillaris Fructus and

Choerospondias Axillaris Leaf are moldy, rotten or moth-eaten under visible light, and

mash them separately after removing the residues, and keep them for future use.

Extraction of total flavonoids

Weigh 140 g of each of the above-treated Choerospondias Axillaris Fructus and

Choerospondias Axillaris Leaf, put them in a 2000 mL three-foot round-bottom flask

respectively, and perform reflux extraction with 8 times of 70% ethanol for 1.5 hours in

water bath heating, three times in parallel respectively. The extracted solutions were

collected, filtered to remove the residue, and then concentrated to a paste in a vacuum

rotary evaporator. Drying in oven at proper temperature to obtain solid paste and weighing

to obtain 14.20g and 40.40g of crude paste respectively, with the crude paste yield of

10.14% and 28.86% respectively.

Degreasing by extraction with petroleum ether

The dried extracts of Choerospondias Axillaris Fructus and Choerospondias Axillaris

Leaf were properly dissolved, and then petroleum ether is added for stirring extraction until

the ether layer of each extract has no color. Collect the solution of lower layer of petroleum

ether, concentrate and dry it, weigh it, and obtain 12.70 g and 33.39 g of dry paste

respectively after extracting fatty components from the leaves of Choerospondias Axillaris

and Choerospondias Axillaris Fructus and the paste yield of the two is 9.07% and 23.85%

respectively.

Quantitative test-standard curve method for quantitative determination of total flavonoids

Selection of the best absorption wavelength of flavonoids

It is necessary to determine the optimum wavelength for the quantitative

determination of flavonoids before the determination of total flavonoids by the standard

curve method. The method is as follows: Take the proper concentration of the above-mentioned extracted fructus solution (0.10 mg / mL), operate according to the standard curve. The sample has the maximum absorption value at 506nm, which is close to the optimum wavelength of 51Onm for rutin reference substance, as shown in Figure 1-1. In this experiment, the content of total flavonoids extracted from the leaves of

Choerospondias Axillaris and Choerospondias Axillaris Fructus was determined by

quantitative analysis method of general flavonoids, and rutin was used as control.

Preparation of standard curve

Accurately weigh 10.0mg of dry constant weight rutin reference substance, dilute

with 70% ethanol, dissolve thoroughly, and then fix the volume in a1OOmL volumetric

flask. Measure 0.OmL, 2.5mL, 5.OmL, 7.5mL, 10.OmL and 12.5mL of the

above-mentioned solutions respectively in a 25mL volumetric flask, and pipette 70%

ethanol solution make the volume to 12.5ml. Then, add 1.OmL of 5% NaNO2 solution

prepared fresh into each volumetric flask, shake it up, and stand it still for 6min; then add

1.0 mL of 10% Al (N03) 3 solution and shake up. After standing for 6min, add10.OmL of

4% NaOH solution, fix the volume with 70% ethanol solution to 25mL, shake up, and

stand for 12min. The reagent was used as a blank, and quantitative determination was

carried out at 51Onm. Regression equation: A = 12.125714 x C + 0.006524 r = 0.999795

(see Table 1-1). The resulting standard curve in shown at Figure 1-2.

Table 1-1 Concentration and absorbance of rutin reference substance

S/N 1 2 3 4 5 6 Concentration (mg/mL) 0.0000 0.0100 0.0200 0.0300 0.0400 0.0500 Absorbance A 0.000 0.134 0.256 0.374 0.489 0.611

Determination of the contents of the extracts of Choerospondias Axillaris Fructus and

Choerospondias Axillaris Leaf

Weigh 26.6mg and 11.2mg solid paste sample of Choerospondias Axillaris Fructus

and Choerospondias Axillaris Leaf accurately, put them into 100mL volumetric flasks

respectively, and dissolve them to a constant volume. Then put 2.5mL of the

above-mentioned sample solution into a 25mL volumetric flask and operate according to

the preparation method of the standard curve, measure the absorbance of the fructus A is

0.027, and the absorbance of the leaf A ' is 0.041, and substitute it into the regression

equation to calculate the total flavonoid content of the fructus and leaf of Choerospondias

Axillaris. (See Table 1-2).

Table 1-2 Content of Choerospondias Axillaris Samples

Concentration Ultraviolet Total flavonoids Total flavonoids Sample (mg / mL) absorbance content ofdry content(%.) cream(%o)

fructus 0.0017 0.027 6.39 0.580 leaf 0.0028 0.041 25.0 5.96 Note: Total flavonoid content of Choerospondias Axillaris Fructus = C * dilution multiple

/ sampling amount * paste yield. The content of total flavonoids in leaves of

Choerospondias Axillaris was calculated by the formula.

Identification of the extract

The conditions under which the total flavonoids are extracted are also suitable for the

extraction of alkaloids, polyphenols, and polysaccharide compounds, so that the identification must be carried out .Identification is carried out one by one according to the identification reaction specific to the above compounds.

Identification of polysaccharides

In that method, after the fatty components are extracted, the methanol solution of the

samples of Choerospondias Axillaris Fructus and Choerospondias Axillaris Leaf are taken

into a test tube respectively, and the freshly prepared Fehling's solution is added

respectively, and slightly heated for a while in a boiling water bath to observe the color

change of the test tube. Yellow to red-brown precipitation, positive reaction, shows that

there is polysaccharide.

Identification of alkaloids

Bismuth potassium iodide, mercuric potassium iodide and phosphotungstic acid were

added respectively to the test tubes containing three samples of methanol solution of

Choerospondias Axillaris Fructus and Choerospondias Axillarisye Leaf after extraction of

fatty components, and the color change of the solution was observed. Yellow to orange red

precipitation, which is added acid and not faded, showed positive reaction, and there are

alkaloids.

Identification of Polyphenols

FeCl3 methanol solution was used to react, after extraction of fatty components, 1.0

mL of methanol solution for Choerospondias Axillaris Fructus and 1.0 mL for

Choerospondias Axillaris Leaf were added with 2% FeCl3 respectively, and the color

change of the solution was observed. Yellow to dark green proved it is a positive reaction,

and there is polyphenol.

Identification of flavonoids

Referring documents (Chen Yegao, Plant Chemical Composition [M], Beijing:

Chemical Industry Publishing House, 2004: 3230), the above-mentioned prepared samples

methanol solutions were added with chromogenic reagents, respectively. Observe the

change phenomenon of each reaction (see Table 1-3), and the color reaction is positive.

Table 1-3 Color reaction of flavonoids

Main Sodium Zirconi Saturat Magnes Reactio Phosp HCl- Reactio Conce color (potassi um salt ed lead ium n with homol Mg n of 2% ntrated reacti um) reaction acetate acetate 2% ybdic powd alumin sulfuri on tetrahyd reaction reaction sodium acid er um c acid ride hydroxi reacti reacti trichlori reactio reaction de on on de n frutus No Yellow Yellow Orange Orange Blue- Red Bright Orange change precipit (bright) green purple yellow red ation leaf Magenta Yellow Yellow Orange Orange Green Red Yellow Dark precipit brown purple -green yellow ation

Separation and identification of flavonoids

Preliminary exploration of thin layer (TLC)

Self-made silica gel thin layer chromatography plate (10 x 20 cm 2); the spot sample is

the alcohol solution of Choerospondias Axillaris Fructus and Choerospondias Axillaris

Leaf after the extraction of fatty components by petroleum ether; developing agent:

Chloroform-ethyl acetate-methanol = 15: 20: 2. After the chromatographic plate was

sufficiently dried, 2% aluminum trichloride developer was sprayed, resulting in spots as shown in Figure 1-3 under the ultraviolet lamp (365 nm): The spots were few and there was tailing phenomenon, which had to be purified by ethyl acetate.

Extraction and Purification of Flavonoids from Choerospondias axillaris by Ethyl Acetate

In that invention, appropriate amount of Choerospondias Axillaris Fructus and

Choerospondias Axillaris Leaf ethyl acetate layer extract dry cream are dissolved in

methanol, and are respectively labeled as a and b to be used. The remaining insoluble

ethanol extract is dissolved in proper amount of water, and the water layer is evaporated to

dryness by extraction with ethyl acetate and dissolved in methanol. the fructus and leaf are

labeled as A and B respectively for standby.

Determination of total alkaloids

The samples a and b of Choerospondias Axillaris purified by ethyl acetate showed no

positive reaction in the alkaloid test; the water-soluble part had a positive reaction of

alkaloid (see Table 1-4).

Table 1-4 Alkaloids Detection

Number Bismuth iodide Mercuric iodidea Phosphotungstic Detection results acid fructus a Orange Red* Orange Orange None Orang RedBrown A OrangeRed Brown Precipitation Yes (Micro) Precipitate Precipitation

leaf b Orange Red Yellow Yellow None

B Orange red Yellow grey Orange Yes (Micro) precipitate precipitate

The purity of total flavonoids after purified by ethyl acetate is improved, and most

sugars, phenols and alkaloids have poor solubility in ethyl acetate and can be removed by

this method.

Identification of flavonoids

Combined with the identification experiment of flavonoids, preliminary identification

of the above-mentioned purified flavonoids of Choerospondias Axillaris Fructus and

Choerospondias Axillaris Leaf was done. The preliminary results of the identification

confirmed that both contain flavones, flavonols, dihydroflavones, anthocyanins, chalcones,

aurones, etc. (see Table 1-5).

Table 1-5 Identification of color reaction of flavonoids

Hydroc Hydroc Sodium Zirco Magnes Alkaline Phosp Sulfuric hloric hloric tetrahydr nium ium solution homol acid acid acid- oborate salt acetate ybdic -magne zinc acid sium powder powder Orange Fresh Yellow Green Orange Red Carmine Orange fructus red yellow brown Brown red result 3 4or5 123 123 3 1 12 2 2 or6 Purplish Purplish Fresh Green Light leaf rd ed Orange Orange Nn red red yellow Brown green result 123 123 3 1 12 12

Notes: 1. Flavone 2. Flavonol 3. Dihydroflavone 4. Anthocyanin 5. Chalcones 6. Aurone

References (Chen Xiaoquan. Botany Chemical Taxonomy [M]. Beijing: Higher

Education Press, 1990, 1:65) Take the above-mentioned purified extracts of

Choerospondias Axillaris Fructus and Choerospondias Axillaris Leaf samples with ethyl

acetate extract and insoluble alcohol extraction of the lower layer substance is made into

methanol solution and concentrated on the same self-made silica gel TLC plate, and then

sprayed with different color reagents for color reaction. Through observation and

comparison under visible light and ultraviolet light (365nm), according to the same sample

on the plate, different types of flavonoids can be identified by their respective color

development phenomena under the action of different reagents.

Thin layer development

On a self-made silica gel thin-layer chromatography plate (10x20 cm 2), use a

capillary tube to draw 10~20ul of the concentrated ethyl acetate extract for spotting, record

as a, b; Quercetin standard S; the same Spot the lower layer alcohol extracts A and B after

the ethyl acetate treatment on the same silica gel plate. Developing solvent: benzene-ethyl

acetate-methanol-formic acid=11:5:2:1. The separation points of the samples can be

clearly observed under visible light and ultraviolet light. Spray 2% aluminum trichloride

developer, observe the spots under visible light and ultraviolet light (365nm), record the

number of spots, color, size and Rf value as shown in Figure 1-4.

Separation and Purification of Total Flavonoids on Amide Column

In order to completely remove that polysaccharides, polyphenol and other impurities

of the paste extracted with ethyl acetate remaining in the above-mentioned frutus and leaf,

and to eliminate the interference of the late analysis of the impurities, a polyamide column

is applied to the paste, eluate with gradient ethanol, extract 10 ~ 70% ethanol eluate, combine and concentrate, and record as a ', b ' (Ref: Baiyune.Polyamide Separation of

Total Flavonoids of Trollius Chinensis [J] .Chinese Journal of Hospital Pharmacy. 2006;

26 (5): 514). Extract samples a b', a, b and quercetin standard sample S were conducted

thin layer chromatography. The above sample chromatographs are basically the same, and

the spots on the plate are compared with each other as shown in Figure 1-5.

According to the principle analysis of the separation and purification of total

flavonoids and other substances on polyamide column chromatography, the ethyl acetate

extract samples of Choerospondias Axillaris Fructus and Choerospondias Axillaris Leaf

were applied to polyamide column, and 10%~70% gradient concentration of ethanol was

collected and eluted. After evaporating to dryness and spotting on a silica gel thin-layer

plate, the developed spots are almost the same as those of the ethyl acetate extract. After

passing the column, the first-stage water eluent and gradient ethanol eluted, the column is

soaked in methanol, the above solution is concentrated and collected, and then the water

eluent, 10% to 70% gradient concentration ethanol eluent and colum soaking solution

with methanol were developed on the same thin-layer silica gel plate with a developing

agent (benzene-ethyl acetate-methanol-formic acid=11:5:2:1). Observation under

ultraviolet light showed that the spots of the water eluate were mainly concentrated at the

origin and did not expand, indicating that the component is relatively polar, and may

contain glycosides, or a small amount of flavonoid glycosides; 10% to 70% gradient

ethanol wash The deliquoring spots are separated well, which is almost the same as the

spots developed by the ethyl acetate extract; the spots separated by the methanol soaking

solution and the gradient ethanol eluate have corresponding spots, but there is a tongue-like

tailing phenomenon on the front. This comparative experiment shows that the content of impurities extracted by ethyl acetate is less, and it has little effect on the separation of fat-soluble flavonoids on silica thin-layer chromatography. This experiment will qualitatively identify the total flavonoids of fat-soluble organisms.

Color reaction on TLC expansion plate

Place the ethyl acetate extracts a , b, the quercetin standard S , the lower layer

insoluble alcohol extracts A and B on the self-made silica gel chromatography plate,. Use

the above condition of TLC to develop. The layers are unfolded, and then different color

reactions are performed on different plates, and the color development of each color

reaction (without color developer, spray 2% AlCl3, ammonia fumigation) under visible

light and ultraviolet light (365nm) is recorded.

TLC developed the color reaction on the plate, and initially identified the

corresponding points of the total flavonoids of Choerospondias Axillaris Fructus (1~13):

anthocyanins, flavonols, flavones, anthocyanins, chalcones, dihydroflavones, flavonols,

catechins, flavonols, chalcones, dihydroflavones, aurones, catechin; Choerospondias

Axillaris Leaf (1~15): anthocyanins, anthocyanins, anthocyanins, isoflavones, chalcones,

anthocyanins, dihydroflavones, flavonols, flavonols, chalcones, chalcones, chalcones,

aurones,aurones.

Qualitative identification of total flavonoids in Choerospondias Axillaris with thin-layer

scraper ultraviolet-visible spectrophotometer

Reference literature (Chen Yegao. Phytochemical components [M]. Beijing:

Chemical Industry Press, 2004, 3:235 ~ 237; Wu Lijun. Natural Medicine Chemistry [M].

Beijing: People's Medical Publishing House, 2005, 6: 190; Li Yunxia Et al.

Characterization of UV-Vis Absorption Spectroscopy of the Active Components of

Scutellaria baicalensis Georgi [J]. Spectroscopy and Spectral Analysis, 2007, 27(1): 131)

According to the UV spectrum characteristics of flavonoids (max/nm, methanol solution,

UV-Vis spectrophotometry), Scan the qualitative spectrum (190~600nm), and identify

the flavonoids based on the characteristic absorption peak spectrum of the scanned

flavonoids.

In the ultraviolet-visible spectrum, because the flavonoids have a cross-conjugated

system composed of cinnamoyl and benzoyl, there are two main ultraviolet absorption

bands at 200-400nm, band I (300-400nm) and band 11 (220 ~280nm). According to the

ultraviolet-visible spectrum characteristics of adding the diagnostic reagent AlCl3 to the

methanol solution and methanol solution, combined the flavonoids core structure and

substituent positions structural, it can infer the type of flavonoids.

Carefully scrape off the corresponding spots developed on the silica gel thin-layer

chromatography plates of Choerospondias Axillaris Fructus and Choerospondias Axillaris

Leaf, eluting with methanol, centrifugation, and take the clear liquid. In addition, scrape off

the blank part of the plate without sample as a blank component. Establish a baseline and

measure the ultraviolet-visible absorption spectrum.

The absorption spectra measured by ultraviolet-visible spectroscopy showed that the

spectra of Choerospondias Axillaris Fructus and Choerospondias Axillaris Leaf were the

same on the whole, except that the absorption peaks were different in strength and

displacement. Analyze the composition spectrum of the reference literature (Tan

Renxiang. Plant composition analysis [M]. Beijing: Science Press, 2002, 2: 496 ~ 500) to

determine the component type (see Table 1-6).

Table 1-6 UV Spectral Characteristics of Flavonoids (Xmax nm)

MeOH+AlCl3 Tpso MeOH solution Band shift value Typesof Control spot solution flavonoids Band I Band II Band I Band II BandI BandII detected Fructus 285 236 285 225 1 0 10 Anthocyanidins (Strong) (weak) (weak) (weak) 291 257 301 257 (weak) (weak) 10 0 Dihydroflavones 6 (Strong) (weak)

375 278 376 279 7 1 1 Flavonols (weak) (Strong) (weak) (Strong) 296 259 301 265 9 5 6 Flavonols (Strong) (Strong) (Strong) (Strong) 273 284 10 No No - 11 Chalcone (Strong) (Strong) 367 254 419 271 12 52 17 Aurones (Strong) (Strong) (Strong) (Strong) 285 236 287 2 No - 2 Anthocyanidins (Strong) (weak) (weak) 320 mid 277 335 277 (Strong) (Strong) 15 0 Dihydroflavones 8 (Strong) (Strong)

374 275 431 275 9 57 0 Flavonols (Strong) (Strong) (Strong) (Strong) Leaf 329 291 334 297 10 5 6 Flavonols (Strong) (Strong) (Strong) (Strong) 273 276 11 No No 0 3 Chalcone (Strong) (Strong) 325 278 375 289 14 50 11 Aurones (weak) (Strong) (weak) (Strong)

Note: The above is the UV spectrum (a-b) of the corresponding speckles of

Choerospondias Axillaris Fructus and Choerospondias Axillaris Leaf: 'and 2; 6' and 8; 7

'and 9; 9'and 10; 10 'and 11; 12' and 14.

Study on the pharmacological activity of the total flavonoids of Choerospondias Axillaris

Leaf

The effect of total flavonoids from leaves of Choerospondias Axillaris on arrhythmia

induced by aconitine in rats

Test Methods

There are 50 healthy Wistar rats, weighing 200g±20g. Randomly divided into 5

groups, each with 10 animals, half male and half female, respectively, the normal saline

control group, the total flavonoids group of Choerospondias Axillaris Fructus (0.2g/kg),

the high-dose group of total flavonoids of Choerospondias Axillaris Leaf (0.4g/kg), and

the middle-dose group of total flavonoids of Choerospondias Axillaris Leaf (0.2g/kg), and

the low-dose group of total flavonoids of Choerospondias Axillaris Leaf (0.1g/kg). Rats in

each group were administered intragastrically with a volume of 2ml/1OOg for 7 consecutive

days, lh after the last administration, intraperitoneal injection of 3% sodium pentobarbital

1ml/kg anesthetized, fixed supine, with BL-420F biological The functional experiment

system observes and records normal ECGII, then intravenously inject 10tg/ml aconitine

ig/kg, after 5 seconds, observe and record premature ventricular contractions

(ventricular premature) and ventricular tachycardia (ventricular tachycardia) within 30

minutes , Ventricular fibrillation (ventricular fibrillation), cardiac arrest (arrest) time.

Test results

Compared with the normal saline control group, the total flavonoids group of

Choerospondias Axillaris Fructus and the dosage group of total flavonoids of

Choerospondias Axillaris Leaf can significantly prolong the appearance of premature

beats, ventricular tachycardia, ventricular fibrillation and cardiac arrest in rats (see Table

2-1). The results suggest that total flavonoids of Choerospondias Axillaris Leaf are the

same as the total flavonoids of Choerospondias Axillaris Fructus, which has obvious

anti-arrhythmic effects.

Table 2-1 Effect of total flavones of Choerospondias Axillaris on occurrence time of

arrhythmia induced by aconitine in rats (z s,n=10)

Premature Ventricular Ventricular Cardiac Dose Group (g/kg) beat tachycardia fibrillation arrest (min) (min) (min) (min) Saline group - 2.20+0.28 19.38+1.97 23.38+1.81 15.92+1.51

Frutus group 0.2 8.18+1.55** 23.60+1.97** 25.60+0.66** 20.78+1.14**

High dose group of Leaf 0.4 9.08+1.84** 24.09+1.58** 27.08+1.89** 23.84+2.68**

Middle dose group of Leaf 0.2 8.46+1.47** 23.38+1.92** 25.70+2.01** 20.02+1.86**

Low dose group of Leaf 0.1 6.70+1.55** 21.40+1.69* 25.40+2.06* 17.26+1.03*

Compared with normal saline group, t test was performed, *P< 0.05, **P<0.01

The effect of total flavonoids of Choerospondias Axillaris Leaf on ischemic arrhythmia

caused by coronary artery ligation and the content of SOD and MDA in serum.

Test Methods

60 healthy Wistar rats, half male and half female, weighing 220±60g, were randomly

divided into 6 groups, each with 10 rats, namely the sham operation group (given normal

saline), the ischemia model group (given normal saline), Ischemic model+ total flavonoids

group of Choerospondias Axillaris Fructus (0.2g/kg), ischemia model+ total flavonoids of

Choerospondias Axillaris Leaf high-dose group (0.4g/kg), ischemia model + total

flavonoids of Choerospondias Axillaris Leaf middle-dose group (0.2g /kg), low-dose

group of total flavonoids of Choerospondias Axillaris Leaf (0.1g/kg). Rats in each group

were given intragastrically with a volume of 2ml/100g for 7 consecutive days. Ligate the

left anterior descending coronary artery 1 hour after the last administration, observe and

record the normal II lead ECG before thoracotomy and the occurrence and duration of

arrhythmia within 30 minutes of ischemia, the number of occurrences of ventricular

fibrillation; 5 hours after ischemia, blood is taken Prepare serum and measure the activity

of SOD and MDA according to the kit instructions; take out the heart after blood

collection, wash away residual blood with saline, and immediately put it in the refrigerator

to freeze. After the heart is frozen for 20 minutes in the refrigerator, cut the left ventricle

into 5 pieces of equal thickness from the apex of the heart to the direction parallel to the

atrioventricular sulcus. Place them in 1% TTC solution and stain them in a constant

temperature water bath at 37°C for 20 minutes to distinguish the necrotic area from

non-necrotic Another weighing, calculate the scope of myocardial infarction.

Test results

Compared with the ischemic model group, the total flavonoids of Choerospondias

Axillaris Fructus group and the total flavonoids of Choerospondias Axillaris Leaf in each

dose group can significantly prolong the appearance of arrhythmia caused by ligation of the coronary artery in rats, significantly reduce the number of ventricular abnormal beats, and continue ventricular tachycardia The time is significantly shortened, the incidence of ventricular fibrillation is significantly reduced, and the area of myocardial infarction is significantly reduced, the activity of SOD, an important oxygen free radical scavenger in the body, is significantly increased, and the content of MDA, a metabolite of lipid peroxidation, is significantly reduced (see Table 2-2. Table 2-3). The results suggest that the total flavonoids of Choerospondias Axillaris Leaf are the same as the total flavonoids of

Choerospondias Axillaris Fructus. It has obvious protective effect on myocardial ischemia

injury in rats, effectively resists arrhythmia caused by myocardial ischemia, and has

obvious antioxidant effect.

Table 2-2 Effect of Total Flavonoids of Choerospondias Axillaris Leaf on Arrhythmia

Induced by Coronary Artery Ligation(e*s,n=10)

Number of Sustained Occurrenc Dose Arrhythmia ventricular ventricular ventricular Group of appears (g/kg) extrasystole tachycardia fibrillation time(mi) (pieces) Time (min) Rate(%)

Sham operation group - 0.00±0.00 0+0 0.00+0.00 0

Ischemic model group - 5.57+1.35 961+337 2.30+0.59 90

Frutus group 0.2 8.35+3.52** 380+133** 1.55+0.88* 40**

High dose group of Leaf 0.4 10.773.57** 104+40** 1.100.99** 10**

Middle dose group of Leaf 0.2 8.59+4.38* 279+115** 1.40+0.97* 30*

Low dose group of Leaf 0.1 7.07+2.40 531+320** 1.60+0.52* 50*

Compared with ischemia model group, t test or X 2 test*p<0.05, **p<0.01

Table 2-3 Effects of total flavonoids of Choerospondias Axillaris Leaf on myocardial

infarction range and SOD andMDA contents in mice (z s,n=10)

Dose Myocardial MDA Group (g/kg) infarction SOD (U/mL) (nmol/mL) range (%) Sham operation group - 0.00±0.00 142.11+16.12 7.92±2.14

Ischemic model group - 27.089.70^^ 107.02+14.35^^ 15.27+2.28^^

Frutus group 0.2 17.23+6.33** 137.73+17.35** 11.22+2.11**

High dose group of Leaf 0.4 14.65+8.84** 138.62+19.86** 9.62+1.83**

Middle dose group of Leaf 0.2 17.55+5.77** 136.27+16.59** 10.88+2.54**

Low dose group of Leaf 0.1 18.94+5.90* 137.54+18.12** 12.39+2.07*

Compared with sham operation group, t-test ^^p<0.01;

Compared with the ischemia model group, t-test * p<0.05, **p<0.01

Effect of total flavonoids of Choerospondias Axillaris Leaf on immune function in mice

Test Methods

There were 150 healthy mice with 20 2g body weight. They were randomly

divided into 3 groups. Each group was further divided into 5 groups of 10 animals, half

male and half female, namely the normal saline control group, the total flavonoids group

of Choerospondias Axillaris Fructus (0.3g / kg), high-dose (0.6g / Kg) ,middle-dose (0.3g

/ kg) and low-dose groups (0.15g / kg) of total flavonoids of Choerospondias Axillaris

Leaf. In the first group, carbon clearance method was used to determine the effect of total

flavonoids extract of Choerospondias Axillaris Leaf on phagocytosis of monocyte macrophages referring to (Lu Yinjean, Pei Shaojun, Hu Xingju. "Purple Golden Propolis and Aloe Soft Capsule" on Immune Function of Mice, Chinese Journal of Health Testing,

2009. Nineteen (9): 1984-1985). The second group referring to (Xu Shuyun, Bian Rufu,

Chen Xiu, Pharmacological Experimental Methodology, People's Health Press, 2nd

Edition, 1984) adopts DTH method to measure the effects of each group on delayed-type

hypersensitivity (DTH) in mice. The Third Group of References is (Li Zhi, Chen Bifeng,

Huang Junming. the Effect of Ejiao Oral Liquid on Cellular Immunity and Humoral

Immunity in Mice, Chinese Journal of Health Inspection, 2008, 1196-1120). The effect of

each group on the production of hemolysin antibody induced by sheep red blood cells

(SRBC) in mice was measured by serum hemolytic method.

Test results

In comparison with that normal saline control group, the total flavonoids group of

Choerospondias Axillaris Fructus and the high and middle dosage groups of total

flavonoids of Choerospondias Axillaris Leaf significantly enhanced the phagocytic

function of monocyte macrophages in mice. The total flavonoids of Choerospondias

Axillaris Fructus group and the total flavonoids of Choerospondias Axillaris Leaf in each

dose group of the date palm could significantly enhance the delayed-type hypersensitivity

in mice. The production of hemolysin antibody in mice was significantly increased in the

total flavonoids group of Choerospondias Axillaris Fructus and the high and middle

dosage groups of total flavonoids of Choerospondias Axillaris Leaf, and the thymus index

of mice was increased significantly (Table 2-4, 2-5, and 2-6). The results showed that the

total flavonoids of Choerospondias Axillaris Leaf had the same effect as the total

flavonoids of Choerospondias Axillaris Fructus on enhancing cellular immunity, humoral immunity and nonspecific immunity.

Table 2-4 Effect of Total Flavonoids of Choerospondias Axillaris Leaf on

PhagocytosisofMonocyteMacrophage (Y±s, n=10)

Dose Phagocytic index Phagocytic Group (g/kg) K (10-') coefficient u

Saline group - 31.96±8.32 4.620±0.792

Frutus Group 0.3 41.31+5.88** 5.220+0.616**

High dose group of Leaf 0.6 42.71+7.04* 5.670+0.359**

Middle dose group of Leaf 0.3 43.41+9.52* 5.786+1.012*

Low dose group of Leaf 0.15 30.99±8.55 4.689±1.003

Compared with Saline group, t-test *p<0.05, **p<0.01

Table 2-5 Effect of Total Flavonoids of Choerospondias Axillaris Leaf on Delayed

Skin Hypersensitivity in Mice ( i±s, n=10)

Weight Dseif c Thymus Index Spleen index Dose difference Group (g/g body (g/g body (g/kg) between ears weight x 10) weight x 10) (mg) Saline group - 2.21+0.33 0.221+0.026 0.818+0.122

Frutus Group 0.3 3.48+0.21* 0.265+0.032* 0.922+0.033

High dose group of Leaf 0.6 4.15+1.90* 0.274+0.046* 1.000+0.111*

Middle dose group of Leaf 0.3 3.54+1.54* 0.202+0.141 0.942+0.419

Low dose group of Leaf 0.15 3.16+1.91* 0.206+0.113 0.806+0.668

Compared with Saline group, t-test*p<0.05, **p<0.01

Table 2-6 Effect of total flavonoids of Choerospondias Axillaris Leaf on hemolysin

antibody production in mice (x±s, n=10)

Spleen index Thymus Index Dose Hemolysin Group content (g/g body (g/g body (g/kg) (HC) weight x 10) weight x 10) Saline group - 301.833+31.775 0.861+0.268 0.238+0.052

Frutus Group 0.3 371.333+28.741** 0.847+0.121 0.326+0.031*

High dose group of Leaf 0.6 356.267+45.408* 0.793+0.114 0.511+0.201*

Middle dose group of Leaf 0.3 350.433+39.519* 0.894+0.246 0.390+0.112*

Low dose group of Leaf 0.15 300.767+65.707 1.037+0.274 0.304+0.133

Compared with Saline group, t-test *p<0.05, **p<0.01

Summary on Pharmacological Activities of Total Flavonoids of Choerospondias Axillaris

Leaf

The results of pharmacological activities of Total Flavonoids of Choerospondias

Axillaris Leaf showed that the total flavonoids in the leaves, like those from the fructus,

had obvious anti-arrhythmic effect, protective effect on myocardial ischemia injury,

obvious anti-oxidation effect and immune function enhancement effect. In that invention,

the Choerospondias Axillaris Leaf can replace the Choerospondias Axillaris Fructus to

extract the total flavonoids, and can be used to prepare products that can prevent and treat

cardiovascular and cerebrovascular diseases, diseases with reduced immune function and

diseases related to free radical injury, including health food and pharmaceutical

compositions.

Claims (11)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. The total flavonoids of Choerospondias Axillaris Leaf is characterized by being extracted

from the leaves of Choerospondias Axillaris, it contains one or more anthocyanins, flavones,

flavonol, dihydroflavones, dihydroflavonol, chalcone, isoflavone and flavanol.

2. The total flavonoids of Choerospondias Axillaris Leaf described in claim 1 is characterized in

that the content of total flavonoids is > 5.00%.

3. The preparation method of total flavonoids of Choerospondias Axillaris Leaf is characterized

in that the leaves are extracted with ethanol and separated by petroleum ether.

4. The ethanol described in claim 3 is characterized in that the concentration is 70% and the

dosage is 8 times of the intended extract.

5. The ethanol extraction described in claim 3 is characterized in that the water bath is heated at

85 °C and refluxed for 1.5h, and each extraction is carried out three times in parallel.

6. The petroleum ether extraction described in claim 3 is characterized in that the extract solution

is concentrated into a paste by a vacuum rotary evaporator, the paste extract is properly

dissolved, and then the petroleum ether is added for stirring extraction. The extraction is

repeated until the ether layer of each extract has no colour. The lower layer solution of

petroleum ether is collected and condensed and dried.

7. The preparation method of claim 3 is characterized in that it comprises further purification

steps of polyamide column separation or silica gel chromatography separation, after extraction

with ethyl acetate.

8. Claim 1 includes the use of the total flavonoids in the preparation of a product for the

prevention and treatment of cardiovascular and cerebrovascular diseases, diseases with

reduced immune function and diseases related to free radical injury.

9. The used of claim 8, wherein the diseases include arrhythmia, arteriosclerosis, coronary heart

disease, cerebral insufficiency, immune dysfunction, dementia, insomnia and amnesia, and

tumour.

10. Pharmaceutical compositions and preparations composed of total flavonoids of claim 1 and

other drugs and medically acceptable pharmaceutical excipients.

11. The preparation of claim 9 includes injection, tablet, capsule, film, pill, granule, oral liquid and

dropping pill.

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Figure 5