GB2597145A - Compound separated from root bark of Ginkgo biloba and use thereof - Google Patents

Abstract

Disclosed is a compound "Ginkgo biloba root acid", which is separated from root bark of Ginkgo biloba, i.e., 1,8-dyhydroxyl-dibenzofuran-2,6-dicarboxylic acid, the molecular formula thereof being C14H8O7. Also disclosed is a method for extracting and separating the compound and the use thereof in identifying an extract of root bark of Ginkgo biloba in a Ginkgo biloba extract.

Description

COMPOUND SEPARATED FROM ROOT BARK OF GINKGO BILOBA AND USE THEREOF

TECHNICAL FIELD

The invention relates to biotechnology and pharmaceutical technology field. The invention further relates to a new compound separated from the root bark of Gingko biloba and use thereof.

BACKGROUND OF THE INVENTION

Gingko biloba extract (GBE) is prepared from dry leaves of Gingko blioba, which is one of most important phytopharmaceutical preparations in the world, and used to cure cardiovascular disease. Havonoids and terpene lactones are main active chemical components of the GBE. These flavonoids mainly include quercetin, kaempferide, isorhamnetin, etc., which have some effects of protecting capillary permeability, dilating coronary arteries, restoring arterial circulation, and alleviating cardiac smooth muscle spasm and so on. These terpene lactones of Gingko blioba mainly include Ginkgolide A (GA), Ginkgolide B (GB), Ginkgolide C (GC), Ginkgolide J (GI) mid bilobalide (BB), etc., which have some effects of inhibiting platelet aggregation, inhibiting thrombosis and so on. Besides, these terpene lactones are effective platelet activating factor antagonists.

Domestic and international GBE quality standards stipulate contents of Ginkgo flavonol glycosides and Ginkgo terpene lactones specifically. The 2015 edition of Chinese Pharmacopoeia (ChP2015) stipulates that Ginkgo flavonol glycosides are detected by "quantitative analysis of multi-components by single-marker. QAMS" method with quercetin as a reference standard. The GBE contains Ginkgo flavonol glycosides (the sum of quercetin, kaempferide and isorhamnetin) content > 24%; Ginkgo terpene lactones (the sum of GA, GB, GC and BB) content > 6%. Both of United State Pharmacopoeia (USP36) and Europe Pharmacopoeia (EP8.0) stipulate that the GBE contains no less than (N LT) 22.0% and no more than (NMT) 27.0% of Ginkgo flavonol glycosides (the sum of quercetin, kaempferide and isorhamnetin), but the content of Ginkgo terpene lactones is different. The GBE contains Ginkgo terpene lactones consisting of between 2.6% and 5.8% of BB, between 2.8% and 6.2% of the sum of GA, GB, and GC and between 5.4% and 12.0% of the sum of BB, GJ, GA and GB in the USP36. However, the GBE contains Ginkgo terpene lactones consisting of between 2.6% and 3.2% of BB, between 2.8% and 3.4% of the sum of GA, GB, and GC in the EP8.0. High performance liquid method (HPLC) is simple to operate, high sensitive and accurate, which makes it to be widely used in detection around the world. It is also employed to detect Ginkgo flavonol glycosides and Ginkgo lemon° lactones in GBE of ChP2015, USP36 and EP8.0. Different structures of Ginkgo flavonol glycosides and Ginkgo terpene lactones have different detect conditions. ChP2015, USP36 and EN.0 employ mobile phase methanol-phosphoric acid solution with different ultraviolet (UV) wavelengths to detect Ginkgo flavonol glycosides For Ginkgo terpene lactones detection, ChP2015 employs n-propanoltetrahydrofuran-water (in a ratio of 1:15:84) as the mobile phase and evaporative light scattering detector as HPLC detector; USP36 employs methanol-water as the mobile phase and the same HPLC detector as ChP2015; EP8.0 employs methanol-tetrahydrofuran-water (in a ratio of 20:10:75) as the mobile phase and refractive index detector as HPLC detector Therefore, the content indexes and detecting methods of Ginkgo flavonoids and Ginkgo terpene lactones in GBE are different in various countries.

Ginkgo terpene lactones are peculiar active ingredients in the Ginkgo biloba leaves, fruits and root bark. It has been reported that content of Ginkgo terpene lactones in Ginkgo biloba root bark, wherein the content of Ginkgolide C is in the majority, is about twice as high as that of Ginkgo biloba leaves, which makes root bark become one of the important sources of the Ginkgo terpene lactones. The root bark of Ginkgo biloba is abundant in Ginkgo terpene lactones, but they are simple in ingredient and free of flavonoids. What's more, Ginkgo biloba root bark extract has not undergone systematic pharmacological efficacy experiments or clinical trial, has not demonstrated therapeutic efficacy for cardiovascular disease, which means root bark cannot be used as a medicinal raw material for GBE. Thus, root bark is subsidiary product of Ginkgo planting and mostly treated as waste. In recent years, there has been a situation, in which root bark is used to extract Ginkgo terpene lactones and added into normal GBE to obtain cheap GBE and meet the content requirement, because the root bark is cheaper than leaves of Ginkgo ()Hobo. The cheap GBE has serious quality and clinical safety risks for person. Therefore, it is necessary to establish a detection method that can distinguish sources of Ginkgo terpene lactones to ensure quality, clinical efficacy and safety of GBE.

The current research on the identification of GBE components mainly focuses on the Ginkgo flavonol glycosides. For example, Ch P2015 employs sophoricosidc as a marker for the detection of adulteration of GB E with,S'ophora japonica Linn. In a document "Identification of Ginkgo biloba supplements adulteration using high performance thin layer chromatography and ultra-high performance liquid chromatography-diode array detector-quadrupole time of flight-mass spectrometry", genistein was revealed by chemical analysis with ultra-high performance liquid chromatography-mass spectrometry in Ginkgo biloba, it was proposed as an analytical marker for the detection in adulteration of GBE with Japanese sophora (Styphnolobitun laponicvni). Because Ginkgo biloba leaves and extracts made therefrom do not contain genistein, and some ingredients such as kaempferol-3-0-sophoroside, sophorabiosideis, etc. also clarify that Ginkgo extract mixed fruit or flower of Japanese sophora. The inventor established a QA MS method to improve ChP2015 GBE detection, and published a paper "HPLC fingerprint of Ginkgo biloba extracts and the structural identification of common peaks by LC-MS", which has employed to improve GBE internal control standards. The method analyzes GBE flavonol glycosides by setting up with 20 common peaks in GBE HPLC fingerprint, which has direct guiding significance for identification of GBE flavonoids. However, there is no report about identifying Ginkgo terpene lactoncs of GBE around the world. It is difficult to search a marker to distinguish between GBE and adulterants. What's more, the current GBE detection methods still have some problems, such as long time in detection, difficulty in determining source of ingredients.

The root bark of Ginkgo biloba is a kind of significant material sources of Ginkgo terpene lactones. It is necessary to find a symbolic compound as a marker which is generally stable in the root bark of Ginkgo biloba but not in the leaves, and establish a method to identify the marker in GBE efficiently to promote the improvement of GBE quality standards and ensure that GBE is clinically safe and effective.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a new compound separated from Gingko biloba root bark, wherein the compound is named 1, 8-dihydroxyl-dibenzofuran-2, 6-dicarboxylic acid, molecular formula thereof being Cli4H807, molecular weight thereof being 288.21. The compound has not been reported before, it is extracted and separated from root bark of Ginkgo biloba by inventor for the first time and also named Ginkgo root bark acid (GRBA). The compound as shown in formula is characterized as follows: (1) formula:

HO

HO 4 14 OH

HO

(2) solubility: freely soluble in methanol and ethanol, soluble in 1% v/v sodium hydroxide solution.

(3) mass spectrometric data: El-MS m/z=287, 243, 215, 187, 159.

(4)114 -Nuclear Magnetic Resonance data: 1HNIVIR (400 MHz, Me0D-d4) 6: 7.16 (d, J=8.5 Hz, HT), 7.56 (d, J=2.0 Hz, 1H), 7.77 (d, J=8.5 Hz, 1H), 9.30 (d, J=2.0 Hz, 1H), 10.27 (s, 1H), 11.36 (s, 1H), 12.76 (s, 2H).

(5) -Nuclear Magnetic Resonance data: 13CNMR (100 MHz, Me0D-d4) 8: 126.3, 116.5, 145.2, 120.9, 119.3, 142,5,152.1, 113.0, 123.0, 116.5, 143.3, 121.3, 167.5 and 160.4.

The present invention further provides a method to extract and separate the compound as shown in formula. The method comprising steps as follows: 1) drying and crushing the root bark of Ginkgo biloba to obtain coarse powders; 2) adding ethanol for extracting these coarse powders, concentrating extract solution, treating with alkali solution, filtering, then adjusting pH with acid solution, concentrating mid drying to obtain crude extract solution of Ginkgo biloba root bark; 3) enabling the crude extract solution to pass through a macroporous resin column, eluting with purified water, collecting and concentrating to obtain concentrated eluate; 4) adjusting the concentrated eluate pH with organic acid, passing through another macroporous resin column, sequentially eliding with purified water and 10% -20% v/v ethanol, discarding eluate, then eluting with 30% -50% v/v ethanol and collecting eluate to obtain crude GRBA; 5) purifying the crude GRBA through a polyamidc column, sequentially eluting with purified water and 25% -35% v/v ethanol, discarding eluate, then eluting with 75% -95% v/v ethanol, collecting and concentrating eluate, thereby obtaining 1, 8-dihydroxyl-dibenzofuran-2, 6-d carboxylic acid (GRBA), as shown in formula.

The purity of GRBA prepared by the method above is not less than 65.3%.

Further, in the step 2) the ethanol has 50% -95% v/v concentration for extracting these coarse powders at a temperature of 50'C -90 'C, with 5 -8 times volumes of these coarse powders.

Further, in the step 2) the alkali solution is NaOH or KOH at a concentration of 0.5 -2 mol/L Further, in the step 2) the acid solution is HC1. H2SO4 or H3 PO4 at a concentration of 4 mol/L.

Further, in the step 2) the pH is adjusted to neutral or weakly basic.

Further, in the step 3) the macroporous resin column is selected from D101, DM130, HPD100, IIP20, AB-8 or DA201 Further, in the step 4) the organic acid is formic acid or acetic acid.

Further, in the step 4) the pH is adjusted to 4 6 The present invention further provides a use of the compound as shown in formula as a characteristic component of Ginkgo biloba root bark in identifying the content of Ginkgo biloba root bark extract in the GBE by instrumental analysis method. The instrumental analysis method contains of HPLC and high performance liquid-mass spectrometry (HPLC-MS), thereof the HPLC method comprising steps as follows: 1) preparation of test sample solution: taking the GBE 600 mg in a 10 m L volumetric flask, dissolving with 50% v/v methanol and diluting final volume to 10 mL, sharking up, filtering, removing initially filtrates and collecting the subsequent filtrates to obtain the test sample solution; 2) preparation of reference substance solution: taking the GRBA 20 mg in 1000 mL voltunetric flask, dissolving with 50% v/v methanol and diluting final volume to 1000 mL, sharking up, filtering, removing initially filtrates and collecting the subsequent filtrates to obtain the reference substance solution; 3) detection method: mobile phase A: a mixture of purified water and formic acid, with a purified water formic acid volume ratio of 100: 0.1; mobile phase B: acetonitrile; gradient elution procedure of the mobile phase as shown in table 1: Table 1 Mobile phase of gradient elution procedure Time (mm) Mobile Phase A (% v/v) Mobile Phase B (°/0 v/v) 0-40 90-464 10-436 40-45 64->50 36->50 45-50 50->0 50-400 50-55 0 100 55-56 0->90 100-40 56-65 90 10 wherein, from 0 min to 40 mm, a volume percentage of mobile phase A gradiently decreasing from 90% v/v to 64% v/v; from 40 min to 45 min, the volume percentage of phase A gradiently decreasing from 64% v/v to 50% v/v; from 45 min to 50 min, the volume percentage of phase A gradiently decreasing from 50% v/v to 0; from 50 min to 55 min, the volume percentage of phase A is 0; from 55 min to 56 min, the volume percentage of phase A gradicntly increasing from 0 to 90% v/v; from 56 min to 65 min, the volume percentage of phase A is 90% v/v; detectionwavelength: 254 nm; column temperature: 30t; flow rate of the mobile phase: 1.0 mL/min The Ginkgo terpene lactones are effective ingredients for the treatment of cardiovascular and cerebrovascular diseases in GBE. The content of Ginkgo terpene lactones is a kind of indicators in GBE related products quality inspection. In order to reduce costs of GBE production and meet the Ginkgo terpene lactones requirements in drug quality standards, some manufactories add root bark extract into normal GBE or add root bark into Ginkgo biloha leaves to obtain cheap GBE. In this way, die cheap GBE may contain some substances without pharmacologic experiments or clinical verifications, which may have serious quality and clinical safety risks for person. The object of the present invention is to provide a method for identifying Ginkgo biloba root bark ingredient efficiently in GBE by using GRBA as a marker.

The GRBA is separated from Ginkgo biloba root bark, which has no similar structure compound in current compound database. The GRBA has an unusual molecular skeleton (dibenzofuran structure), which has not been discovered in other plants or other parts of Ginkgo biloba (such as leaves) except Ginkgo biloba root bark. What's more, the compound has a low content in Ginkgo biloha root bark, has similar polarity to BB with high UV response value and sensitive in detection. It is difficult to separate BB from the compound in GBE that is prepared through a rude and low-cost method completely. Ginkgo biloba root bark crude extract always has GRBA inside. Thus, it can be judged that cheap GBE may be added the root bark of Ginkgo biloba or the extract thereof when the compound is detected in the GBE.

As a characteristic component of Ginkgo biloba root bark, the GRBA can be detected by HPLC or HPLC-MS exclusively and high sensitively to distinguish whether GBE contains Ginkgo biloba root bark extract or not The present invention provides a new compound separated from Ginkgo biloba root bark and use thereof, it has benefits as follows: 1. The present invention provides a new compound named GRBA, which can be used to distinguish whether GBE contains Ginkgo biloba root bark extract or not. It can make market behavior normative and ensure the GBE and products thereof safe and effective in clinical use.

2. The current GBE identification methods still have some problems, such as long time in detection, difficulty in determining source of ingredients. The present invention employs GRBA as a marker in HPLC-UV method to detect Ginkgo biloba root bark extract in GBE. The method is easy to operate, rapid and high sensitive to detect. The detection limit concentrate of GRBA is 0.0014 mg/mL and Ginkgo biloba root bark is 25 mg/kg in GBE through the method.

3. The original HPLC-UV method can detect rutin, quercetin, isorhamnetin, sophoraside and other flavonoids simultaneously in GBE, but cannot detect the Ginkgo terpene lactones or figure out the source of them. The present invention employs GRBA as a marker to distinguish Ginkgo hiloba root bark extract in GBE, which has changed detecting flavonoids only into detecting Ginkgo terpene lactones and figuring out source of these terpene lactones simultaneously. It is batter to achieve QAMS method's goal, suitable for popularizing in industry.

4. The present invention adopts a technique about acidification and extraction to separate between GRBA and BB. The GRBA has purity above 65.3%, which meets the identification requirements of GRBA in GBE. What's more, the compound was separated by eco-friendly technology to reduce environment pollution.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG.1 shows the El-MS spectrogram of the GRBA. FIG.2 shows the El-MS/MS spectrogram of the GRBA. FIG.3 shows the H-N4NR spectrogram of the GRBA.

FIG.4 shows the '3C-IYIN R spectrogram of the GRBA.

FIG.5 shows the H-H COSY spectrogram of the GRBA.

FIG.6 shows the HSQC spectrogram of the GRBA.

FIG.7 shows the HNIBC spectrogram of the GRBA.

FIG.8 shows the HPLC chromatograms of the GRBA detection; the general retention time (RT) of GRBA ranges from 25.4 min to 25.9 min, it can be judged according to the peak shape nearby FIG.8A shows HPLC chromatogram of the GRBA in RT 25.526 min; FIG. 8B shows HPLC chromatogram of GBE without any peak in PART A drawing or partial enlarged drawing; FIG. 8C shows HPLC chromatogram of GBE supplemented with GRBA. in which PART B drawing and partial enlarged drawing have GRBA peaks in RT 25.577 m M. As shown in FIG. 8, GRBA is an unnatural component in Ginkgo biloba leaves, which makes it to be a characteristic component in identifying the content of GRBA in GBE.

FIG.9 shows the HPLC chromatograms of the GRBA for chromatographic column usage; the general RT of GRBA ranges from 25.4 min to 25.9 min, it can be judged according to the peak shape nearby. FIG.9A shows HPLC chromatograms of GBE supplied with GRBA and detected through chromatographic column 1, in which PART C drawing and partial enlarged drawing have GRBA peaks in RT 25.626 min; FIG.9B shows HPLC chromatogram of GBE supplied with GRBA and detected through chromatographic column 2, in which PART D drawing and partial enlarged drawing have GRBA peaks in RT 25.916 min; FIG.9C shows HPLC chromatogram of GRBA detected through chromatographic column 3, in which GRBA has peaks in RT 19.325 min; FIG.9D shows HPLC chromatogram of GBE supplied with GRBA and detected through chromatographic column 3, in which PART E drawing or partial enlarged drawing has no GRBA peak in RT 19.3 min or surround. The result indicates that the column 3 cannot separate GRBA from other elements in GBE effectively.

FIG.10 shows the HPLC chromatograms of the GRBA for formic acid usage; the general RT of GRBA ranges from 25.4 min to 25.9 min, it can be judged according to the peak shape nearby. FIG.10A shows HPLC chromatogram of GBE supplied with GRBA and detected through 0.1% v/v formic acid -water, in which PART F drawing and partial enlarged drawing have GRBA peaks in RT 25.626 min. FIG.10B shows HPLC chromatogram of GBE supplied with GRBA and detected through 0.08% v/v formic acid -water, in which PART G drawing and partial enlarged drawing have GRBA peaks in RT 25.561 min FIG.10C shows HPLC chromatogram of GBE supplied with GRBA mid detected through 0.12% v/v formic acid -water, in which PART H drawing and partial enlarged drawing have GRBA peaks in RT 25.501 min. The result indicates that the present invention detection method has little effect on the RT of GRBA when mobile phase formic acid ranges from 0.08% v/v to 0.12% v/v.

FIG.11 shows the HPLC chromatograms of the GRBA for column temperature durability; the general RT of GRBA ranges from 25.4 min to 25.9 mM, it can be judged according to the peak shape nearby FIG.11A shows FIPLC chromatogram of GBE supplied with GRBA and detected in 30°C, in which PART I drawing and partial enlarged drawing have GRBA peaks in RT 25.626 min. FIG.1113 shows H PLC chromatogram of GBE supplied with GRBA and detected in 28°C, in which PART J drawing and partial enlarged drawing haveGRBA peaks in RT 25.987 mm, FIG.11C shows HPLC chromatogram of GBE supplied with GRBA and detected in 32°C, in which PART K drawing and partial enlarged drawing have GRBA peaks in RT 25.946 min. The result indicates that the present invention detection method has some effects on the RT of GRBA when column temperature ranges from 25°C to 32"C, it is necessary to determine column temperature in HPLC method.

FIG.12 shows the HPLC chromatograms of the GRBA in Ginkgo biloba root bark extract from different counties; the general RT of GRBA ranges from 25.4 min to 25 9 min, it can be judged according to the peak shape nearby. FIG.] 2A shows HPLC chromatogram of GR BA in Ginkgo bithba root bark extract from Tancheng, in which GRBA peak has RT in 25.723 min FIG.12B shows HPLC chromatogram of GRBA in Ginkgo biloba root bark extract from Lanxi, in which GRBA peak has RT in 25. 815 mm. FIG.12C shows HPLC chromatogram of GRBA in Ginkgo biloba root bark extract from Pizhou, in which GRBA peak has RT in 25.442 min. FIG.12D shows HPLC chromatogram of GRBA in Ginkgo biloba root bark extract from Bazhou, in which GRBA peak has RT in 25.629 min. FIG.12E shows HPLC chromatogram of GBE, in which PART L drawing or partial enlarged drawing has no GRBA peak in RT 25.4 min-25.9 min. The result suggests that GRBA, which is not presented in GBE, is discovered in Ginkgo biloba root bark extract generally.

FIG.13 shows the HPLC chromatograms of GRBA in self-made GBE; the general RT of Ginkgo root bark acid ranges from 25.4 min to 25.9 min, it can be judged according to the peak shape nearby. FIG.13A shows HPLC chromatogram of self-made GBE NO.1, in which PART M drawing or partial enlarged drawing has no GRBA peak in RT 25.4 min-25.9 min FIG.13B shows HPLC chromatogram of self-made GBE NO.2, in which PART N drawing or partial enlarged drawing has no GRBA peak in RT 25.4 m min. The result suggests that GBE NO.1 or NO.2 has no GRBA inside.

FIG.14 shows the HPLC chromatograms of GRBA in commercial GBE; the general RT of GRBA ranges from 25.4 min to 25.9 min, it can be judged according to the peak shape nearby FIG.14A shows HPLC chromatogram of GBE that is prepared by manufactory NO.1, in which PART 0 drawing arid partial enlarged drawing have GRBA peaks in RT 25.362 min. Fl G.14B shows mass spectrometry of GBE that is prepared by manufactoni NO.1, in which GRBA has molecular ion peak I M-H I-m/z=287.02 with marker *. The result suggests that the commercial GBE has GRBA inside.

DETAILED DESCRIPTION OF THE INVENTION

The following further describes the present invention with reference to following examples. The examples below further illustrate the invention, rather than limiting the scope thereof Example 1 Method I of extracting and separating the GRBA 1 Preparation and extraction of Ginkgo biloba root bark Applicant extracted successively 10 kg of dry and crushed Ginkgo biloba root bark coarse powders in 8 volumes and 6 volumes of 70% v/v ethanol at a temperature of 85C to obtain extract solution, 1.5 h per time, concentrated extract solution to about 1 voltune of medicinal materials, added 1.5 mol/L NaOH in volume that equal to extract solution, filtered through filter plate with pore size of 25 pm to obtain filtrate, adjusted filtrate pH with 3 mol/L HO to p1-1 7, then concentrated and dried to obtain crude extract of Ginkgo biloba root bark.

2. The first purification by D101 macroporous resin column chromatography Applicant dissolved the crude extract of Ginkgo bi/oba root bark heatedly in 10 volumes of 50% v/v ethanol, filtered to obtain filtrate and filter residue, dissolved and filtered the filter residue to obtain filtrate again, collected both of filtrate above and concentrated to volatilize ethanol completely, passed through D101 macroporous resin column (diameter 10 cm, 2.5 L bed volume (BV)), eluted with 3BV of purified water, collected and concentrated to obtain 5 L concentrated eluate.

3. The second purification by D I ()I macroporous resin column chromatography Applicant adjusted the concentrated cluatc pH with ethanol° acid solution to pH 4, settled 30 min, then passed through another D101 macroporous resin column (diameter 10 cm, 2.5 L BY), eluted with 3BV of purified water, 3BV of 20% v/v ethanol and 3BV of 40°/0v/v ethanol successively, collected 40% v/v ethanol cluatc, concentrated and dried to obtain crude GRBA.

4. Purification by polyamide column chromatography Applicant dissolved about 7 g of the crude GRBA heatedly in 1L 10% v/v ethanol, passed through a polyamide column (particle size 250-550 pm, diameter 5.5 cm, 250 mL BV), eluted with 6BV of purified water, 6 BY of 35% v/v ethanol and 14 BY of 90% v/v ethanol successively, collected 90% v/v ethanol eMate, concentrated and dried to obtain GRBA, named GRBA NO.1.

Example 2 Method II of extracting and separating the GRBA 1. Preparation and extraction of Ginkgo biloba root bark Applicant extracted successively 1 kg of dry and crushed Ginkgo biloba root bark coarse powders in 6 volumes and 5 volumes of 50% v/v ethanol at a temperature of 90t to obtain extract solution, 2 h per time, concentrated extract solution to about 1 volume of medicinal materials, added 0.5 mol/L NaOH in volume that equal to extract solution, filtered through filter plate with pore size of 25 jun to obtain filtrate, adjusted filtrate pH with 1 moll H2SO4 to pH 8, then concentrated and dried to obtain crude extract of Ginkgo biloba root bark.

2. The first purification by HPD100 macroporous resin column chromatography Applicant dissolved the crude extract of Ginkgo biloba root bark heatedly in 10 volumes of 50% v/v ethanol, filtered to obtain filtrate and filter residue, dissolved and filtered the filter residue to obtain filtrate again, collected both of filtrate above and concentrated to volatilize ethanol completely, passed through HPD100 macroporous resin column (diameter 5.5 cm, 300 mL BV), cluted with 3BV of purified water, collected and concentrated to obtain 500 mL concentrated eluate, 3. The second purification by HPD100 macroporous resin column chromatography Applicant adjusted the concentrated eluate pH with formic acid solution to pH 6, settled 30 min, then passed through another HPD100 macroporous resin column (diameter 5.5 cm, 300 mL BV), eluted with 3BV of purified water, 3BV of 10% v/v ethanol and 3BV of 50% v/v ethanol successively, collected 50% v/v ethanol eluate, concentrated and dried to obtain crude GRBA.

4 Purification by polyamide column chromatography Applicant dissolved about 3g of the crude GRBA heatedly in 500 nth 10% v/v ethanol, passed through a polyamide column (particle size 250-550 pun, diameter 3 cm, 150 mL BV), eluted with 6BV of purified water, 6BV of 30% v/v ethanol and 16 BV of 75% v/v ethanol successively, collected 75% v/v ethanol eluate, concentrated and dried to obtain GRBA. named GRBA NO.2, Example 3 Method III of extracting and separating the GRBA 1. Preparation and extraction of Ginkgo root bark Applicant extracted successively 1 kg of dry and crushed Ginkgo biloba root bark coarse powders in 6 volumes and 5 volumes of 95% v/v ethanol at a temperature of 50°C to obtain extract solution, 1.5 h per time concentrated extract solution to about 1 volume of medicinal materials, added 2 moll KOH in volume that equal to extract solution, filtered through filter plate with pore site of 25 jun to obtain Filtrate, adjusted filtrate pH with 4 mol/L FI3PO4 to pld 7, then concentrated and dried to obtain crude extract of Ginkgo biloba root bark.

2. The first purification by DM130 macroporous resin column chromatography Applicant dissolved the crude extract of Ginkgo biloba root bark heatedly in 10 volumes of 50% v/v ethanol, filtered to obtain filtrate and filter residue, dissolved and filtered the filter residue to obtain filtrate again, collected both of filtrate above and concentrated to volatilize ethanol completely, passed through DM130 macroporous resin column (diameter 5.5 cm, 300 mL BV), eluted with 3BV of purified water, collected and concentrated to obtain 400 mL concentrated eluate.

3. The second purification by DM130 macroporous resin column chromatography Applicant adjusted the concentrated eluate pH with ethanoic acid solution to pH 5, settled 30 min., then passed through another DM130 macroporous resin column (diameter 5.5 cm, 300 mL BV), eluted with 3BV of purified water, 3BV of 15%v/v ethanol and 3BV of 30% v/v ethanol successively, collected 30% v/v ethanol eMate, concentrated and dried to obtain crude GRBA.

4 Purification by polyamide column chromatography Applicant dissolved about 2 g of the crude GRBA heatedly in 500 mL 10% v/v ethanol, passed through a polyamide column (particle size 75150 ttm, diameter 1.5 cm, 20 mL BV), eluted with 6BV of purified water, 6 BV of 25% v/v ethanol and IOBV of 95% v/v ethanol successively, collected 95% v/v ethanol eluate, concentrated and dried to obtain GRBA, named GRBA NO.3.

Example 4 Purity test of the GRBA The GRBA samples from examples Ito 3 were checked purity by this test.

1 Preparation of test sample solution Applicant dissolved -5 mg of GRBA with 80% v/v methanol in volumetric flask and diluted final volume to 25 mL. shaken up, filtered and collected the subsequent filtrates to obtain the test sample solution, detected through HPLC method.

2. Experimental methods Chromatographic column: Intertsil ODS-3 (5 jun, 4.6*250mm): mobile phase A: 0.1% v/v formic acid; mobile phase B: acetonitrile; gradient elution procedure of the mobile phase as shown in table 1; column temperature: 301C; flow rate of the mobile phase: 1.0 mL/min. Evaporative light scattering detector (ELSD) has evaporation temperature 30t, atomization temperature 70t. Ultraviolet (UV) detector had detection wavelength at 254 nm.

The result showed that all of peak area ratios were above 80% in ELSD and above 60% in UV 254 am. It means the UV detector method can reflect more impurities of sample in UV 254 nm than the ELSD method. Thus, the content of GRBA was determined by UV detection, as shown in Table 2, Table 2 Purity of GRBA NO. Purity (°/0) 1 84.1 2 65.3 3 90.2 Example 5 Structure Identification of GRBA 1. MS identification of GRBA As shown in FIG.1, ELMS spectrogram of the compound exhibited m/z at 287.0188 [M-H], 575.0465 [2M1-11. It suggests that molecular ion peak of the compound is 287.0188 [M-Hr, the compound molecular Formula is C1411807.

As shown in FIG.2, El-MS/MS spectrogram of the compound exhibited characteristic fragment ions m/z at 269.01, 243.03, 215.03, 215.03, 199.04, 187.04, 143.05 and 115.06.

2. NMR identification of Ginkgo root bark acid As shown in FIG.3, the II-I-NMR spectrogram of the compound showed all of H signals in low-field. It is preliminarily inferred that the structure may have benzene ring fragments inside. Four labile protons appeared at 812.76, 811.36 and 810.37. The appearance of two protons (69.30, 67.56) as doublet with J=2.0 Hz respectively. It is inferred that one of protons may link to meta position of another. The appearance of two protons (67.77, 67.16) as doublet with =8.5 Hz respectively. That is inferred that one of protons linked to ortho position of another.

As shown in FIG.4, the 1-3C-NMR spectrogram of the compound showed all of C signals in low-field. To combine 'H-NMR spectrogram with DC-NMR spectrogram confirms that the compound has benzene ring. The '3CNMR spectrogram showed 13 C protons in the compound, and two of C protons overlapped in 6116.5.

As shown in FIGS, H-H COSY spectrogram showed that H proton 67.77 linked to H proton 67.16.

As shown in FIG.6. HSQC spectrogram showed that H proton 89.30 linked to C proton 8120.9, H proton 67.77 linked to C proton 6123.0; two H protons (87.56, 87.16) linked to C proton 8116.5, respectively.

As shown in FIG.7, LIMBC spectrogram showed that C-2 connected with C-4; C-6 connected with C-13: C-4 and C-6 connected with C-12. It is inferred that C-2 and C-4 are meta-position connecting in benzene ring A. The spectrogram showed that C-9 and C-11 connected with C14; C-10 and C-8 connected with C-12. It is inferred that C-c and C-10 are ortho-position connecting in benzene ring B. According to connection between C-4 and C-12, it is inferred that benzene ring A and ring B are interlinked with each other.

N MR dates of the compound were curtained and showed in Table 3. According to the molecular weight, the compound molecular formula is CHHK07, as shown as follows: 0 Table 3 NW date of GRBA No. "C NMR 11-1 NMR H M BC 'H-'1-1 COSY 1 126.3 2 116.5 7.56 d. J=2.0 Hz C4. CI, C6_ C3, C13 3 145.2 4 120.9 9.30 d. J=2.0 Hz C10, C12, C6, C3(W), C13 7.56 119.3 6 142.5 7 152.1 113.0 9 123.0 7.77 d, J=8.5 Hz C12, C11, C9, C14 7.16 C8, C12. CI 1. C7.

116.5 7.16 d, J8.5 Hz C14(W) 11 143.3 12 121.3 13 167.5 14 160.4 3-0H 10,27s 13-0H 11.36 s 7-0H and 12.76 s 14-0H Example 6 Study on the &term nation methodology of GRBA in GBE 1. Instnunents and reagents 1.1 Instruments High performance liquid thromatograph: Agilentl 260, Agilent1100, column: Intertsil ODS-3 (5 gm, 4.6*250 mm); electronic analytical balance: NASA 225S-ICE-Du; water purification machine: Milli-QA10.

1.2 Reagents

HO

OH

HO

HO

GRBA (determined in example 5); GBE (batch number: 180107; Zhejiang Conba Pharmaceutical Co., Ltd.). 1.3 Preparation of test sample solution with GBE and GRBA Extract of Ginkgo biloba root bark: the thy Ginkgo biloba root bark was crushed to obtain coarse powders, extracted the coarse powders with 8 volumes of 70% v/v ethanol in boiling slightly twice to obtain extract solution, 1.5 h per time; concentrated the extract solution to volatilize ethanol completely, added purified water in 4 volumes of medicinal materials and filtered to obtain filtrate, added the filtrate to D101 macroporous resin column and eluted with 4BV of purified water and 3BV of 60% v/v ethanol, collected 60% v/v ethanol eluate, concentrated and dried the eluate to obtain extract of Ginkgo biloba root bark (with Ginkgo terpene lactones 15% w/w, GRBA 1.2% w/w).

GBE 93.27 g and extract of Ginkgo biloba root bark 4.85 g were dissolved in ethanol separately, mixed, concentrated and dried to obtain test sample with GBE+ Ginkgo root bark acid which had about 0.05% w/w GRBA and increased 0.7% w/w Ginkgo terpene lactones in theory.

2. Testing method 2.1 Chromatographic condition and system suitability test Column: Intertsil ODS-3 (5 um, 4.6*250 mm); mobile phase A: a mixture of purified water and formic acid, with a purified water formic acid volume ratio of 100: 0.1; mobile phase B: acetonitrile, gradient elution procedure of the mobile phase as shown in Table I; detection wavelength at 254 nm and 202 nm; column temperature at 30 CI; flow rate of the mobile phase is 1.0 m Um in.

Table 1 Mobile phase of gradient elution procedure T (min) Mobile Phase A (V0) Mobile Phase B ( Y0) 0-40 90->64 10->36 40-45 64-,50 36->50 45-50 50-.0 50-.100 50-55 0 100 55-56 0-.90 100-40 56-65 90 10 2.2 Preparation of test sample solution 600 mg of GBE sample was taken in volumetric flask, dissolved with 50% v/v methanol and diluted fmal volume to 10 mL, shaken up, filtered, removed initially filtrates and collected the subsequent filtrates to obtain test sample solution.

2.3 Preparation of reference substance solution The GRBA was dissolved with 50% v/v methanol to obtain 20 lag MIL reference substance solution.

3. Verification methodology 3.1 Study on specificity of sample GBE+ GRBA test sample solution, test sample solution and reference substance solution were detected separately through the method 2.1 of this example. As shown in FIGS, the result suggested that test sample solution (GBE only) had no interference in this method which was used to detect GRBA. The method had good specificity in GRBA detection 3.2 Study on detection limit The GRBA was dissolved in 50% v/v methanol to obtain concentrated solution. The concentrated GRBA solution was added test sample solution in different volumes, detected separately through the method 2.1 of this example and determined the detection limit based on the amount of reference substance and the signal-to-noise ratio information. The result suggested that the detection limit concentrate of GRBA is 0.0014 mg/mL and Ginkgo biloba root bark is 25 mg/kg in GBE through the method.

3.3 Study on durability a) Column durability study: GBE+ GRBA test sample solution was detected through the method 2.1 of this example to investigate the separation of GRBA and GBE through different columns. The different manufacturers' chromatographic columns were shown in Table 4.

Table 4 Different chromatographic columns NO. Chromatographic columns SN 1 Inertsilt ODS-3 5 imi. 4 6*250 inmSN 1A7172378 2 WondaCract ODS-25 vim 4.6*250 mm 3DG73062 3 ZORBAX XDB-C15 4.6*250 mm 5-Micron U5NH060434 As shown in FIG.9, column NO.1 and column NO.2 separated GRBA from GBE effectively. The column NO.3 could not separate GRBA from GBE. Duo to different column has different filter, the RI of GRBA in column NO.3 was different from the others. The result suggested that this method was suitable for ODS columns and had some requirements in column durability.

b) Mobile phase durability study: GBE + GRBA test sample solution was detected through the method 2.1 of this example to investigate the separation between GRBA and GBE in different concentration of formic acid solution.

As shown in FIG.10, GRBA was well separated from GBE in different concentration of formic acid solution through the method. The result suggested that concentration of formic acid solution in this method had little effect on GRBA detection.

c) Column temperature durability study: GBE + GRBA test sample solution was detected through the method 2.1 of this example to investigate the separation of GRBA and GBE through different column temperature (28t, 30.0 and 320).

As shown in FIG.11, the RT of GRBA was advanced when column temperature rose up, and delayed when column temperature decreased. The GRBA peak was incorporated into other miscellaneous peaks at 28; conversely, the GRBA peak appeared earlier than before and overlapped with die previous peak at 32 le. The result suggested that column temperature had effect on die compound detection through the method. It was necessary to determine column temperature in the method.

d) Stability study: GBE + GRBA test sample solution was detected through die method 2.1 of this example to investigate stability in test sample solution with different store time.

As shown in Table 5, the test sample solution was stable in 24 hours.

Table 5 Stability of the test sample T (h) Peak area (A) 0 969.38 2 1004.17 4 980.29 948.84 16 1007.97 24 1035.23 RSD% 3.12 Example 7 Determination of GRBA in related samples of Ginkgo biloba leaves in die market 1. Detection of GRBA in Ginkgo biloba root bark 1.1 Preparation of GBE and Ginkgo biloba root bark crude extract Crude extract of Ginkgo biloba root bark: 10 g dry Ginkgo biloba root bark were crushed to obtain coarse powders, extracted die coarse powders with 8 volumes and 6 volumes of 70% v/v ethanol at temperature 85,0 sequentially to obtain extract solution, 1.5 h per time. The extract solution was concentrated and dried to obtain crude extract of Ginkgo bilobct root bark. 20 mg of the crude extract was taken in volumetric flask, dissolved with 80% v/v methanol mid diluted final volume to 10 mL, shaken up, filtered, removed initially filtrates and collected the subsequent filtrates to obtain crude extract solution, detected through El PLC method. Crude extract solutions of Ginkgo biloba root bark from 6 localities were prepared through this method.

GBE (batch number: 180107; Zhejiang Conba Pharmaceutical Co., Ltd.).

1.2 Detection of GBE and Ginkgo biloba root bark crude extract The GBE and crude extract solutions were detected through the method 2.1 in example 6.

1.3 Results As shown in FIG.12, all of crude extract solutions had G RBA peaks in El PLC chromatogram but GBE did not. What's more, GRBA was not interfered by other ingredients in GBE according to chromatogram data of VWD (a UV-detector). Thus, the GRBA is common in Ginkgo biloba root bark and can be a characteristic substance to distinguish between GBE and Ginkgo biloba root bark.

2. Detection of GRBA in Ginkgo biloba leaves 2.1 Preparation of GBE sample solution Ginkgo biloba leaves from twelve localities and collection date were prepared through the method 1.1 in this example to obtain GBE sample solution. Then, the GBE sample solutions were detected through the method 2.1 in example 6.

2.2 Results As shown in Table 6, GBE sample solutions from twelve different localities and collection date were not detected GRBA, which was further confirmed by LC-MS. The LC-MS detection results of GRBA in some GBE sample solution were shown in FIG.13 and Table 6.

Table 6 Content of GRBA in different Ginkgo biloba leaves Collect date Locality Collect Locality Content Content* date 2017 Yunnan province 2016 Lanxi, Zhejiang province 2018 Yunnan province 2017 Lanxi, Zhejiang province 2015 Zhanyi, Yunnan province 2018 Lanx Zhej iang province 2017 Zhanyi, Yunnan province 2016 Hubei province 2017 Pizhou, Jiangsu province 2017 Bazhou, Sichuan province 2018 Pizhou, Jiangsu province 2018 Bazhou, Sichuan province *It was marked "+" by discovering the GRBA; otherwise, marked"-".

The result of these trials suggested that GRBA was discovered generally and stably in the root bark of Ginkgo biloba and was not discovered in the leaves of Ginkgo biloba. Thus, Ginkgo root bark acid is suitable to be a marker to distinguish between root bark mid leaves of Ginkgo biloba.

3. Detection of GRBA in commercial GBEs Different commercial GBEs were prepared through the method 2.2 in example 6 to obtain commercial GBE sample solution. Then, the GBE sample solutions were detected through the method 2.1 in example 6. As shown in Table 7, commercial Ginkgo biloba leaves from four manufacturers were detected GRBA in this test. Table 7 Content of GRBA in commercial GBEs Manufacturer Content* Manufacturer Content NO.1 NO.7 NO.2 NO.8 NO.3 NO.9 NO.4 NO.10 NO.5 NO.11 NO.6 *It was marked "+" by discovering the GRBA; otherwise, marked"-".

The GBE sample solution from manufacturer NO.1 was further detected through LC-MS to determine whether GRBA existed in the sample solution or not. As shown in FIG.14, the GBE sample solution from manufacturer NO.1 was detected characteristic peak of GRBA through UV detector. It was further determined that the characteristic peak had a GRBA molecular ion peak [M-F1] -m/z 287.02. The GBE sample solution from manufacturer NO.3, NO.7 and NO.8 had the same detection result as NO.1.

Therefore, it was speculated that the GBE sample solution of manufacturer NO.1, NO.3, NO.7 and NO.8 were probably added Ginkgo biloba root bark extract.

The applicant declares that the examples above-described in present application are only the preferred examples to make skilled in pharmaceutical industry field understand easy, but the present invention is not limited to the above-described examples. These skilled should be aware that any modifications, equivalent replacement, improvement of the invention or the like, based on principle or spirit of the present invention, are within the protection scope and the scope of disclosure of the present invention.

Claims (1)

1. CLAIMS1. A compound separated from root bark of Ginkgo biloba, wherein the compound is named 1, 8-dihydrovldibenzofuran-2, 6-dicarboxylic acid, molecular formula thereof being C14HO7 as shown in formula: 2. A method of extracting and separating the compound as shown in claim 1, wherein the method comprising steps as follows: 1) drying and crushing the root bark of Ginkgo biloha to obtain coarse powders; 2) adding ethanol for extracting these coarse powders, concentrating extract solution, treating with alkali solution, filtering, then adjusting pH with acid solution, concentrating and drying to obtain crude extract solution of Ginkgo biloba root bark; 3) enabling the crude extract solution to pass through a macroporous resin column, eluting with purified water, collecting and concentrating to obtain concentrated eluate; 4) adjusting the concentrated eluate pH with organic acid, passing through another macroporous resin column, sequentially eliding with purified water and 10% -20% v/v ethanol, discarding eluate, then eluting with 30% -50% v/v ethanol and collecting eluate to obtain crude Ginkgo root bark acid; 5) purifying the crude Ginkgo root bark acid through a polyamide column, sequentially eluting with purified water and 25% -35% v/v ethanol, discarding dilate, then eluting with 75% -95% v/v ethanol, collecting and concentrating eluate, thereby obtaining the compound as shown in claim 1 3. The method according to claim 2, wherein the ethanol of the step 2) has 50% -95% v/v concentration for extracting these coarse powders at a temperature of 50t -90, with 5 -8 times volumes of these coarse powders; the alkali solution is NaOH or KOH at a concentration of 0.5 mol/L -2 moUL; the acid solution is HC1, 112SO4 or H3PO4 at a concentration of 1 moUL -4 moUL; the pH is adjusted to neutral or weakly basic.4. The method according to claim 2, wherein the macroporous resin column of the step 3) is selected from D101, DM130, HPD100, HP20, AB-8 or DA201.5. The method according to claim 2, wherein the organic acid of the step 4) is formic acid or acetic acid, the p1-1 is adjusted to 4 -6.6. Use of the compound according to claim 1 in identifying the content of Ginkgo biloba root bark extract in the Ginkgo biloba extract by instrumental analysis method as a characteristic component of Ginkgo biloba root bark.7. The use according to claim 6, wherein the instrumental analysis method is high performance liquid chromatography or high performance liquid-mass spectrometry 8. The use according to claim 6, wherein the high performance liquid chromatography method comprising steps as follows: 1) preparation of test sample solution: taking the Ginkgo biloba extract 600 mg in volumetric flask, dissolving with 50% v/v methanol and diluting final volume to 10 m L, sharking up, filtering, collecting the subsequent filtrates to obtain the test sample solution; 2) preparation of reference substance solution: taking 20 mg of the compound according to claim 1 in 1000 m L volumetric flask, dissolving with 50% v/v methanol and diluting final volume to 1000 m L, sharking up, filtering, collecting the subsequent filtrates to obtain the reference substance solution; 3) detection method: mobile phase A: a mixture of purified water and formic acid, with a purified water formic acid volume ratio of 100: 0.1; mobile phase B: acetonitrile, gradient elution procedure of the mobile phase as the following table: Time (mm) Mobile Phase A (% v/v) Mobile Phase B (/0 v/v) 0-40 90->64 I0-36 40-45 64->50 36->50 45-50 50->0 50-400 50-55 0 100 55-56 0->90 100-40 56-65 90 10 wherein, from 0 mm to 40 mm, a volume percentage of phase A gradiently decreasing from 90% v/v to 64% v/v; from 40 min to 45 min, the volume percentage of phase A gradiently decreasing from 64% v/v to 50% v/v, from 45 min to 50 mM, the volume percentage of phase A gradiently decreasing from 50% v/v to 0; from 50 min to 55 min, the volume percentage of phase A is O; from 55 min to 56 min, the volume percentage of phase A gradiently increasing from 0 to 90% v/v; from 56 mm to 65 min, the volume percentage of phase A is 90% v/v: detection wavelength: 254 nm; column temperature: 30"C, flow rate of the mobile phase: 1.0 m Um in.