Process for the preparation of a reference substance for plumericin

Abstract

The present disclosure provides a preparation method of a reference substance of plumericin, comprising a specific macroporous resin chromatographic column purification process and a specific polyamide column chromatography process. Through the combination between the specific column chromatography, the target reference substance with high HPLC purity can be obtained in high throughput. The preparation method of the solid reference substance of plumericin can minimize the use of organic solvents and has very good applicability in industrial production.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to a preparation method of plumieride, and in particular to a preparation method capable of preparing a solid reference substance of plumieride which can be used for industrialization. BACKGROUND

[0002] Plumieride is a natural compound mainly derived from Apocynaceae plants such as Plumeria bicolor, Allamanda cathartica and Plumeria obtusifolia. Modern pharmacological studies have shown that it has antifungal activity, showing strong toxicity to some fungi causing fungal diseases of animals and human skin; it also has antitumor activity, showing antitumor activity in in vitro experiments, and its cytotoxicity can be improved by structural modification of plumieride, such as replacing the methyl ester group with alkyl amide of different carbon units; it also has antioxidant and immunomodulatory activity. Studies have also shown that plumieride also has plant growth inhibition effect. Numerous studies have shown that plumieride has multiple pharmacological activities, indicating that it has broad application prospects in the field of medicine and has gradually become a research hotspot. Therefore, the demand for pure plumieride raw materials or reference substances is gradually increasing.

[0003] Due to the presence of numerous hydroxyl groups, this compound is a hydrophilic component in plants and is easily mixed with various tanning and glycoside components. The existing extraction and preparation methods of plumieride disclosed are very limited, and there is currently no efficient related industrial extraction and preparation method of plumieride. There are reports on the separation and purification of plumieride in the literature, but only for research purposes, the target amount obtained by purification is small, the preparation process is complex, the yield is low, and the purity is not high (see Non-Patent Documents 1-3). It is very urgent to develop a preparation method of plumieride reference substance that provides high-throughput, high-purity plumieride and is suitable for mass production.

[0004]

[0005] In addition, with the increasing global awareness of environmental protection, governments have strengthened environmental regulations for the chemical industry, requiring chemical companies to reduce harmful gas emissions and lower TVOC emissions. TVOC (Total Volatile Organic Compounds) refers to all organic compounds that can evaporate at room temperature. Chemical companies face greater social pressure and need to take measures to reduce TVOC emissions. In order to minimize or eliminate the generation of harmful substances such as TVOC in the design, development and implementation of chemical processes, reduce the use of halogenated alkanes, acetone, ethyl acetate and other organic solvents, and use non-toxic or low-toxic organic solvents for chemical processes, the call for green natural ingredient extraction and separation methods to help protect the environment and meet the sustainable development needs of the chemical industry is growing. Therefore, it is necessary to seek a green extraction process for natural ingredients that can help protect the environment and meet the sustainable development needs of the chemical industry.

[0006] Non-patent document 1: Tiwari, T. N.; Pandey, V. B.; Dubey, N. K., Plumieride from Allamanda cathartica as an antidermatophytic agent. Phytotherapy Research 2002, 16(4), 393-394.

[0007] Non-patent document 2: Yamauchi, T.; Abe, F.; Taki, M., Protoplumericin, an iridoid bis-glucoside in Allamanda neriifolia. Chemical and Pharmaceutical Bulletin 1981, 29(10), 3051-3055.

[0008] Non-patent document 3: Coppen, J. J. W.; Cobb, A. L., The occurrence of iridoids in Plumeria and Allamanda. Phytochemistry 1983, 22(1), 125-128. SUMMARY

[0009] The purpose of the present disclosure is to provide a high-throughput, high-purity purity plumeriin preparation method that can provide high-purity plumeriin and is suitable for mass production. The present disclosure also aims to provide a green extraction process that does not use halogenated alkanes, lipids, and ether solvents, and uses water, alcohol and other solvents as much as possible, with low TVOC emissions.

[0010] In the present disclosure, plumeria rubra linn. is also called as the separation target. The natural material selected in the method of the present application is Allemanda neriifolia Hook., which is a shrub of the genus Allemanda in the family Apocynaceae. Allemanda neriifolia Hook. is not a traditional source of plumeria rubra linn., but the inventors of the present application have determined through long-term research that it can be used as an effective material for plumeria rubra linn. and have made great improvements and enhancements to the separation and purification of plumeria rubra linn. by specifically targeting the extract of the branches and leaves, so that it is suitable for the extraction of plumeria rubra linn. After in-depth exploration, the present application realizes the industrialized purification method of the target compound by using the controllable extraction and separation method, which greatly improves the amount and purity of the compound, thereby completing the present disclosure. The method of the present application is also suitable for using the total extract of the branches and leaves of other plants in the family Apocynaceae as a raw material to obtain high-purity plumeria rubra linn.

[0011] If not specifically stated, the percentage of the purity and content of the target in the present disclosure is the content in terms of mass ratio. If not specifically stated, the percentage of the content ratio of liquid to liquid in the present disclosure, such as the content ratio of formic acid in the acetonitrile aqueous solution, is measured in terms of volume fraction.

[0012] The present disclosure can separate plumeria rubra linn. with high purity through a simple and efficient operation process.

[0013] The preparation and separation method of the present disclosure comprises the following steps in sequence: a macroporous resin enrichment process, in which the total extract solution of Allemanda neriifolia Hook. is loaded on a styrene-type non-polar macroporous adsorption resin column for chromatography, and then water, 5-15% ethanol, 45-55% ethanol, and 55% ethanol elution are used to remove the front impurities, collect the 45-55% ethanol eluate, and concentrate the eluate at a temperature below 55°C under reduced pressure to obtain Allemanda neriifolia Hook. crude extract I,

[0014] A polyamide purification process, in which Allemanda neriifolia Hook. crude extract I is loaded on a 30-70 mesh polyamide column for chromatography, and then water, 15-25% ethanol aqueous solution, and 55-65% ethanol are used to elute 3-5 times the column volume of the front impurities, remove the impurities, and collect the 55-65% ethanol eluate, and then sodium bicarbonate is used to adjust the eluate to neutral pH, and the eluate is concentrated at a temperature below 45°C under reduced pressure to obtain plumeria rubra linn. crude extract I;

[0015] The crude plumeria rubra l. extract is loaded into a reversed-phase chromatography column, and eluted with a mixture of acetonitrile and water or a mixture of methanol and water under a pressure of 0.1-1 MPa, wherein the ratio of acetonitrile to water or the ratio of methanol to water is 0.20-0.65:1 by volume. The eluate is collected according to TLC or HPLC detection, concentrated under reduced pressure at a temperature below 45°C, and the acetonitrile or methanol is removed to obtain a reference solution I of plumeria rubra l. The water is removed at room temperature or the reference solution I of plumeria rubra l. is recrystallized to obtain the reference substance of plumeria rubra l.

[0016] In an alternative embodiment of the present application, the preparation method of the total extract solution of Plumeria rubra l. is as follows: Plumeria rubra l. is crushed, 10-20 times the weight of 20%-60% ethanol aqueous solution is added, and reflux extraction is performed for 3-6 hours. After cooling and filtration, the filtrate is concentrated at 60°C until the alcohol smell is removed to obtain the total extract solution of Plumeria rubra l.

[0017] In an alternative embodiment of the present application, in the polyamide purification process, a polyamide column chromatography with nylon 6 or nylon 66 structure is used. 3-4.5 times the column volume of the pre-fraction is eluted with water, 3-4 times the column volume of the pre-fraction is eluted with 15%-20% ethanol, and elution is performed with 48%-53% ethanol. The eluate is collected, the pH is adjusted to neutral with sodium bicarbonate, and the crude plumeria rubra l. extract I is obtained by concentrating under reduced pressure at a temperature below 55°C.

[0018] In an alternative embodiment of the present application, in the reversed-phase chromatography purification process, the reference solution I of plumeria rubra l. is treated by freeze-drying to obtain the reference substance of plumeria rubra l.

[0019] In an alternative embodiment of the present application, the reference solution I of plumeria rubra l. is concentrated at 50-60°C. After the first solid is precipitated, a poorly soluble solvent is added to precipitate plumeria rubra l.. The target semi-finished solid powder is obtained by suction filtration. The poorly soluble solvent is selected from petroleum ether, dichloromethane, trichloromethane, n-hexane, and cyclohexane.

[0020] In an alternative embodiment of the present application, in the reversed-phase chromatography purification process, the elution is performed under a pressure of 0.2-0.4 MPa, and a mixture of acetonitrile and water is used for elution. The packing material is octadecylsilane-bonded silica gel with a particle size of 40-60 μm.

[0021] In an alternative embodiment of the present application, in the macroporous resin enrichment process, the macroporous resin is selected from any of AB-8 macroporous resin, HPD-100 macroporous resin, and D-101 macroporous resin. The pore size of the macroporous resin is 5 nm-90 nm.

[0022] The present disclosure also provides a solid reference substance of plumericin with a purity of 99.0% or more, which is prepared by the preparation method of claim 1. The reference substance prepared by the method of the present disclosure has high HPLC purity and excellent cost.

[0023] The present disclosure has the following characteristics:

[0024] 1. The present disclosure is simple and efficient in operation, can be scaled up, and a large amount of plumericin monomer can be separated;

[0025] 2. The source of the target compound is expanded to the branches and leaves of Plumeria alba L., which can greatly reduce the cost of the source; the present disclosure optimizes the total composition of the Plumeria alba L. extract, which can effectively separate the target compound and impurities;

[0026] 3. The present disclosure can reduce the use of organic solvents such as halogenated alkanes, acetone, ethyl acetate, and use non-toxic or low-toxic ethanol, methanol, water, etc. to realize an environmentally friendly, low-cost three-waste treatment extraction and separation process;

[0027] 4. The specific experimental scheme optimized by the present disclosure can separate plumericin monomer with an HPLC purity of more than 99.0%. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 HPLC spectrum of plumericin obtained by the separation method of Example 1;

[0029] Figure 2 HPLC spectrum of plumericin obtained by the separation method of Example 1; 1 H-NMR spectrum (Methanol-d4, 500MHz);

[0030] Figure 3 HPLC spectrum of plumericin obtained by the separation method of Example 1; 13 C-NMR spectrum (Methanol-d4, 125MHz);

[0031] Figure 4 Mass spectrum of plumericin. DETAILED DESCRIPTION

[0032] The specific embodiments of the present disclosure are described below.

[0033] The existing method for separating the target object of the present disclosure, plumeria alba glycoside, mainly uses a combination of various specific reverse phase chromatography, realizes the reduction and greenization of organic solvents, and separates the target object by using solvent gradient elution and the polarity difference between the target object and impurities. Generally speaking, for example, the extract concentrate of the crushed plumeria alba leaf material can be loaded onto a normal phase chromatography column, eluted with a gradient of petroleum ether and ethyl acetate, and the eluted target object is collected and concentrated to obtain a crude product concentrate containing the target object. Then, a second normal phase chromatography column purification process is performed: the semi-finished solid powder of the target object is loaded onto a normal phase chromatography column, eluted with a mixture of dichloromethane and ethyl acetate, and the eluent containing the target product is obtained by eluting with dichloromethane: ethyl acetate. The eluent is collected, concentrated, and freeze-dried to obtain a solid target object. However, the target object of the present disclosure contains sugar units, which have strong polarity and cause irreversible adsorption loss, resulting in low yield. Moreover, the solvents used in the separation process are expensive and do not meet the TVOC prevention specifications. On the other hand, direct use of reverse phase chromatography for separation is difficult to improve the purity of the final product due to the presence of compounds in the plumeria alba raw material that have similar polarity to the target object. In order to adapt to industrial mass production, the present disclosure needs to find a special combination of separation materials to solve the problems of yield and separation efficiency. After in-depth research, the preparation and separation method of the present disclosure comprises the following steps in sequence:

[0034] The macroporous resin enrichment process is as follows: the total extract solution of plumeria alba is loaded onto a styrene-type non-polar macroporous adsorption resin column for chromatography, and the front impurities are removed by eluting with water and 5-15% ethanol, respectively. Then, 45%-55% ethanol is used for elution, and the flow fraction of the 45%-55% ethanol eluent is collected. The flow fraction is concentrated under reduced pressure at a temperature below 55°C to obtain plumeria alba crude extract I,

[0035] The polyamide purification process is as follows: plumeria alba crude extract I is loaded onto a 30-70 mesh polyamide column for chromatography, and the front impurities are eluted with water for 3-5 times the column volume. Then, 15-25% ethanol aqueous solution is used for elution to remove impurities, wherein the 15-25% ethanol aqueous solution contains 0.05-0.1% formic acid by volume ratio. Then, 55-65% ethanol is used for elution, and the 55-65% ethanol eluent is collected. The eluent is adjusted to neutral pH with sodium bicarbonate, and concentrated under reduced pressure at a temperature below 45°C to obtain plumeria alba glycoside crude extract I.

[0036] The crude extract of plumeria alba l. is loaded into a reverse phase chromatography column, and eluted with a mixture of acetonitrile and water or methanol and water as eluent under a pressure of 0.1-1 MPa, wherein the ratio of acetonitrile to water or methanol to water is 0.20-0.65:1 by volume. The eluate is collected according to TLC or HPLC detection, concentrated under reduced pressure at a temperature below 45°C to remove acetonitrile or methanol, and then the water is removed at room temperature to obtain a reference solution I of plumeria alba l. or recrystallized to obtain a reference of plumeria alba l.

[0037] The above process does not use conventional normal phase separation method or reverse phase separation method to obtain the target compound. By changing the order of using different separation methods, it is found that the above method can greatly improve the separation degree of impurities and target substances and improve the separation efficiency. The above process is difficult to obtain good extraction and separation efficiency. Specifically, for example, the order of separation using polyamide and separation using macroporous resin is changed, and the result is also difficult to obtain good separation effect. The order between each separation step of the present disclosure is also an important technical feature of the present disclosure.

[0038] The starting material of the method of the present disclosure can be a commercially available extract powder of Apocynaceae plants, which can be obtained from the market. In alternative embodiments, the starting material is prepared by extracting the total extract solution of yellow trumpet tree by oneself. For example, the following extraction method is recommended to obtain the starting total material, such as the preparation method of the total extract solution of yellow trumpet tree as follows: The branch and leaf parts of yellow trumpet tree are crushed and added with 10-20 times the weight of 20%-60% ethanol aqueous solution, and refluxed for 3-6 hours. After cooling and filtering, the filtrate is concentrated at 60°C until no alcohol taste is left. Generally speaking, the cold soak extraction efficiency is higher, but if the branch and leaf of the raw material yellow trumpet tree in the present disclosure are cold soaked, too much secondary metabolite is dissolved, which directly leads to increased difficulty in separation and decreased total yield. Therefore, reflux extraction is recommended.

[0039] In the present disclosure, the so-called concentration to no methanol or acetonitrile taste means that the methanol or acetonitrile in the mixed solution has been substantially evaporated and removed, leaving only water that is difficult to evaporate.

[0040] The macroporous resin enrichment process in the present disclosure is very important, which can greatly reduce the difficulty of separation of the target substance and has excellent cost. Through comparative experiments, it is found that non-polar macroporous resin is particularly suitable for distinguishing the target substance of the present disclosure from other substances. Here, the non-polar macroporous resin can be easily obtained from the market, and the cost is considered. The macroporous resin is selected from any of AB-8 macroporous resin, HPD-100 macroporous resin, and D-101 macroporous resin. The pore size of the macroporous resin can be any pore size, but from the perspective of easy availability and good adsorption separation effect, the macroporous resin with a pore size of 5-100 nm is preferred.

[0041] The polyamide has a "dual chromatography" property, i.e. it has both non-polar aliphatic chains and polar amide groups in its molecule, which is particularly suitable for separating iridoid glycosides (the type of compound to which plumericin belongs) from other components in the raw plant. When eluted with a polar mobile phase (such as a water-containing solvent system), the polyamide acts as a non-polar stationary phase, and its chromatographic behavior is similar to that of reversed-phase partition chromatography. For the target compound with one sugar unit, the polyamide forms hydrogen bonds with the target compound. The elution ability of various solvents on the polyamide column, from weak to strong, is as follows: water < methanol or ethanol (from low to high concentration) < acetone < dilute sodium hydroxide aqueous solution or ammonia water < formamide < dimethylformamide (DMF) < urea aqueous solution. In the present application, the use of only water, methanol and ethanol solvents can well enrich the target compound. In the preferred embodiment of the present disclosure, polyamide purification is a crucial process, and the inventors have found that after enrichment by macroporous resin, the target compound can be accurately enriched if combined with polyamide purification. In the present disclosure, polyamide column chromatography with nylon 6 or nylon 66 structure can be used, and more preferably, the polyamide column chromatography is performed by eluting 3-4.5 times the column volume of the pre-fraction with water, then eluting 3-4 times the column volume of the pre-fraction with 15%-20% ethanol containing about 0.1% formic acid, eluting with 48%-53% ethanol, collecting the 48%-53% ethanol eluate, adjusting the eluate to neutral pH with sodium bicarbonate, and then concentrating.

[0042] The purity of the crude plumericin extract I obtained by using polyamide after macroporous resin can be more than 60%. Moreover, the impurity components mixed with the target compound are particularly easy to remove by other methods, and it can be said that the introduction of polyamide separation in a suitable process is the key to the present disclosure

[0043] In the preferred embodiment of the present disclosure, the pressure during elution in the reversed-phase chromatography purification process is 0.2-0.4 MPa, the elution is performed using a mixture of methanol and water, and the packing material is octadecylsilane-bonded silica gel with a particle size of 20-80 μm. In the present disclosure, the reversed-phase chromatography purification process is used to separate other components that differ in polarity from the target compound.

[0044] The reversed-phase packing material of the reversed-phase chromatography column used in the column preparation process in the present disclosure can be non-polar, bonded silica gel with an alkane functional group (for example: C18 (ODS), C8, C4, etc.). Preferably, the packing material is a C18 (ODS) silica gel column, i.e. octadecylsilane-bonded silica gel, and a reasonable particle size is conducive to maintaining appropriate column pressure and resolution. Octadecylsilane-bonded silica gel is readily available on the market. From the perspectives of efficiency and availability of packing material, in the preferred preparation method of the present disclosure, the packing material of the reversed-phase chromatography column is octadecylsilane-bonded silica gel with a particle size of 40-60 μm.

[0045] Further, in order to obtain better separation efficiency, the reverse phase chromatography purification process of the present disclosure can further add an additional reverse phase chromatography purification process II after the reverse phase chromatography purification process, which is not included in the technical solutions, but this is not necessary. In the reverse phase chromatography purification process II, the medium pressure reverse phase chromatography purification process and the high pressure reverse phase chromatography purification process can be performed in sequence. In the present disclosure, the medium pressure reverse phase chromatography purification process is used to separate other components that differ in polarity from the target, and a balance between resolution and speed can be achieved by using an elution pressure of 0.05-0.5 MPa. In the medium pressure column separation process, the pressure of the medium pressure reverse phase chromatography column is preferably 0.1-0.2 MPa, which is particularly preferred because it can balance the separation effect and the separation speed. In the medium pressure reverse phase chromatography purification process of the present disclosure, it is further preferred to use an acetonitrile aqueous solution to elute at a pressure of 0.1-0.3 MPa, and the ratio of acetonitrile to water is preferably 0.1-0.3:1. The so-called high pressure reverse phase chromatography column purification generally refers to a chromatography column separation technology with an elution pressure greater than 1 MPa. Such separation efficiency is higher, but the throughput is smaller each time, and it is suitable for further purification at the back end of the process. Elution is performed at a pressure of 1-15.0 MPa to achieve a balance between resolution and speed. The ratio of acetonitrile to water is preferably 0.08-0.25:1. The high pressure reverse phase chromatography column can be any high pressure reverse phase chromatography column as long as the filler load capacity can meet the capacity of the target reverse phase chromatography column. The elution pressure of 5-15 MPa in this process can achieve a balance between resolution and speed, and the further preferred column pressure is 5-10 MPa. More preferably, it is 5-8 MPa, which is particularly preferred because it can balance the separation effect and the separation speed.

[0046] The solution obtained by the reverse phase chromatography purification process is basically a high-purity target aqueous solution. Since the target has an ether structure, it is not suitable to be heated too much, and it is hoped to remove the solvent at low temperature. The freeze-drying method and the difficultly soluble solvent treatment method are preferred.

[0047] The freeze-drying method can use a conventional freeze-drying method. As the difficultly soluble solvent treatment method, the control solution I of the plumeria rubra flower glycoside is concentrated at 50-60°C, and after the first solid is precipitated, a difficultly soluble solvent is added to precipitate the plumeria rubra flower glycoside, and then the target semi-finished solid powder is obtained by suction filtration. The difficultly soluble solvent is selected from petroleum ether, dichloromethane, trichloromethane, n-hexane, and cyclohexane.

[0048] In the preferred embodiment of the present disclosure, the freeze-drying method is preferred, which can avoid the excessive introduction of organic solvents, and is of great significance to the realization of a low- TVOC green extraction process.

[0049] The method for preparing frangipani glycoside disclosed in the present invention is reported for the first time. It can prepare a large amount of frangipani glycoside reference substance from the branches and leaves of Cicadae cicadae, providing a solid material basis for the quality standards of medicinal materials such as frangipani and Cicadae cicadae, and has high economic value. Through the simple steps of extraction, macroporous resin separation, polyamide separation, and reverse phase chromatography medium pressure C18 separation, a high-purity frangipani glycoside reference substance with a purity of >99.0% and a yield of greater than 0.1% is obtained from the branches and leaves of Cicadae cicadae. In a typical extraction and separation process, 23g of pure frangipani glycoside with a purity greater than 99.0% was obtained from 17kg of fresh branches and leaves. The preparation method is simple, the yield is as high as 0.13%, and alcohol solvents and water are used, which is environmentally friendly.

[0050] Example

[0051] The following examples further illustrate typical extraction methods disclosed herein. The following experimental schemes are merely examples and are not intended to limit the present disclosure. Those skilled in the art may make any changes based on their understanding of the principles of the embodiments and the spirit of the invention.

[0052] Example 1

[0053] Extraction and separation: The preparation method of frangipani glycoside reference substance is carried out according to the following process steps:

[0054] Raw material extraction: Take 17 kg of yellow bell branches and leaves, cut them into sections and crush them, add 300 L of 50% ethanol, and reflux extract for 3 hours, extract twice. Combine the extracts, filter, and concentrate under reduced pressure at 60°C until there is no alcohol.

[0055] Resin purification: The total extract of the twigs and leaves of Cicadae cicadae was loaded onto 50 kg of treated D101 macroporous adsorption resin (about 50 mesh) for column chromatography and eluted with water, 20% ethanol, 50% ethanol, and 95% ethanol, respectively. The 50% ethanol eluate was collected and concentrated under reduced pressure at 50°C to obtain the crude extract I of Cicadae cicadae.

[0056] Polyamide purification: The crude extract I of yellow candidiasis was loaded onto 10 kg of treated polyamide column chromatography and eluted with water for 4 column volumes, 20% ethanol aqueous solution containing 0.1% formic acid for 4 column volumes, and 60% ethanol aqueous solution for 4 column volumes. The fractions eluted with 60% ethanol aqueous solution were collected, the pH was adjusted to neutral with sodium bicarbonate, and the fractions were concentrated under reduced pressure at 50° C. to obtain a frangipani glycoside-enriched fraction (a solution of the crude extract I of frangipani glycoside);

[0057] Medium pressure preparation purification: The concentrated solution obtained in the polyamide purification was subjected to medium pressure preparation column, the medium pressure preparation column was 100*460mm, produced by Suzhou Huition Chromatography Co., Ltd., and the filler was Daiso ODS-RPS with a particle size of 40-60μm. Gradient elution was performed using methanol water, and the gradient concentration was eluted in turn from 10%, 20%, 30%, 40%, 50%, 60%, and 70%. The effluent was detected by HPLC, and the eluent with a purity of >99.0% (AUC area ratio) was collected according to the HPLC detection, concentrated to no alcohol taste at 55°C in the dark, and freeze-dried to obtain a powder (crude extract II of Plumeria alba Linn), which was vacuum dried at 60°C for 24 hours to obtain 23.0g of Plumeria alba Linn reference substance with an HPLC purity of >99.0%.

[0058] Analytical detection conditions

[0059] Chromatograph: Waters Alliance 2695-2998 HPLC chromatograph;

[0060] Chromatographic column: Welch XB-C18, 4.6*250mm, 5μm;

[0061] Injection volume: 10μL; column temperature: 25°C; detection wavelength: 230nm;

[0062] Gradient setting of mobile phase (A-0.1% formic acid water, B-acetonitrile):

[0063]

[0064] The MS detection conditions of the target obtained in Example 1 are as follows:

[0065] Instrument: Sciex Triple TOF 4600 LC / MS

[0066] Detection mode: Negative ion mode

[0067] ESI source parameters: see the table below.

[0068] Mass parameters

[0069]

[0070] The structure can be confirmed by mass spectrometry and nuclear magnetic resonance spectrum, and the mass spectrum and nuclear magnetic resonance spectrum are referred to Figures 2-4 , and the HPLC spectrum of the final target can be referred to Figure 1 , which can stably provide high-purity target compounds.

[0071] Comparative Example 1 (without introducing polyamide)

[0072] Extraction and isolation: the preparation method of the plumeria alba glycoside reference substance is carried out according to the following process steps:

[0073] Raw material extraction: 10 kg of plumeria alba branches and leaves are cut into sections and crushed, 50% ethanol 300 L is added, and reflux extraction is carried out for 3 h, and the extraction is carried out twice. The extraction liquid is combined, filtered, and concentrated at 60°C under reduced pressure until there is no alcohol.

[0074] Macroporous resin purification: the plumeria alba branch and leaf total extract is loaded onto a 50 kg D101 macroporous adsorption resin (about 50 mesh) column for chromatography, and water, 20% ethanol water, 50% ethanol water, and 95% ethanol water are sequentially eluted; the 50% ethanol water eluate is collected and concentrated at 50°C under reduced pressure to obtain plumeria alba crude extract I;

[0075] Medium pressure preparation purification: the plumeria alba crude extract I obtained in the polyamide purification is loaded onto a medium pressure preparation column, the medium pressure preparation column is a 100x460 mm column produced by Suzhou Huitong Chromatography Co., Ltd., and the filler is Daicel ODS-RPS with a particle size of 40-60 μm. Methanol water gradient elution is used, and elution is sequentially carried out from 10%, 20%, 30%, 40%, 50%, 60%, and 70% gradient concentrations. The effluent is detected by HPLC, and the eluate with a purity of >99.0% (AUC area ratio) is collected according to the HPLC detection, concentrated at 55°C under light protection until there is no alcohol smell, and freeze-dried to obtain a powder (plumeria alba glycoside crude extract II), which is vacuum dried at 60°C for 24 h to obtain 3.5 g of plumeria alba glycoside reference substance with an HPLC purity of >99.0%.

[0076] Without the polyamide separation process, although high-purity plumeria alba glycoside can also be obtained by medium pressure reverse phase chromatography preparation, the yield is reduced. Without the enrichment effect of polyamide, the separation efficiency of medium pressure reverse phase chromatography is low, and there are fewer fractions with a purity of >99%, so it is not efficient to obtain a good recovery rate.

[0077] Comparative Example 2 (without introducing macroporous resin)

[0078] Extraction and isolation: the preparation method of the plumeria alba glycoside reference substance is carried out according to the following process steps:

[0079] Raw material extraction: 10 kg of plumeria alba branches and leaves are cut into sections and crushed, 50% ethanol 300 L is added, and reflux extraction is carried out for 3 h, and the extraction is carried out twice. The extraction liquid is combined, filtered, and concentrated at 60°C under reduced pressure until there is no alcohol.

[0080] Polyamide purification: the total body of yellow vine was loaded on a 10 kg treated polyamide column chromatography, respectively, with water elution 4 times the column volume, using 20% ethanol aqueous solution containing 0.1% formic acid elution 4 times the volume, and using 60% ethanol aqueous solution elution 4 times the column volume, the 60% ethanol aqueous solution elution fraction was collected, concentrated at 50°C under reduced pressure to obtain the enriched component of Plumeria alba L. (solution of crude extract of Plumeria alba L. I);

[0081] Medium pressure preparation purification: the concentrated solution obtained in the polyamide purification was loaded on a medium pressure preparation column, the medium pressure preparation column was 100x460mm, produced by Suzhou Huitong Chromatography Co., Ltd., and the filler was DIAION ODS-RPS with a particle size of 40-60μm. Gradient elution was performed with methanol water, from 10%, 20%, 30%, 40%, 50%, 60%, 70% gradient concentration in turn. The effluent was detected by HPLC, and the eluent was collected according to the HPLC detection, and the high purity eluent was fitted. However, due to the lack of enrichment process of macroporous resin, the fraction obtained by reverse phase preparation purification is not pure enough, and the target product cannot be obtained efficiently.

[0082] The technical features disclosed above are not limited to the combinations disclosed with other features, and those skilled in the art can also make other combinations between technical features according to the purpose of the application to achieve the purpose of the disclosure, and various improvements to the technical solutions of the disclosure made by those skilled in the art without departing from the spirit of the design of the disclosure shall fall within the protection scope determined by the claims of the disclosure.

[0083] Industrial applicability

[0084] The technical solution of the disclosure can be conveniently scaled up, and a large amount of Plumeria alba L. monomer can be separated, which has a broad industrial prospect.

Claims

1. A method for preparing a reference substance of frangipani glycoside, characterized in that: The following steps are included in sequence: In the macroporous resin enrichment step, the total extract solution of the yellow candied ivy is loaded onto a styrene-type non-polar macroporous adsorption resin column for chromatography, and eluted with water and 5-15% ethanol to remove impurities, and then eluted with 45%-55% ethanol. The fractions of the 45%-55% ethanol eluate are collected and concentrated under reduced pressure at a temperature below 55° C. to obtain the yellow candied ivy crude extract I. The polyamide purification step comprises: loading the yellow candied ivy crude extract I onto a 30-70 mesh polyamide column for chromatography, eluting pre-impurities with water for 3-5 column volumes, eluting with a 15-25% ethanol aqueous solution for 3-5 column volumes to remove impurities, wherein the 15-25% ethanol aqueous solution contains 0.05-0.1% formic acid by volume, eluting with 55-65% ethanol, collecting the 55-65% ethanol eluate, adjusting the eluate to a neutral pH with sodium dihydrogen carbonate, and concentrating under reduced pressure at a temperature below 55° C. to obtain the frangipani glycoside crude extract I; In the reverse phase chromatography purification process, the crude extract of frangipani glycoside is loaded onto the reverse phase chromatography, and eluted with a mixed eluent of acetonitrile-water solution or methanol-water solution at a pressure of 0.1 to 1 MPa, wherein the ratio of acetonitrile to water or methanol to water is 0.20 to 0.65:1 by volume. The eluate is collected according to TLC or HPLC detection, and concentrated under reduced pressure at a temperature below 45°C to remove the acetonitrile or methanol therein to obtain a reference substance solution I of frangipani glycoside, and the water is removed at room temperature or recrystallized to obtain a reference substance of frangipani glycoside.

2. The method for preparing a reference substance of frangipani glycoside according to claim 1, characterized in that: The preparation method of the yellow ican total extract solution is as follows: crush the yellow ican branches and leaves, add them into 10 to 20 times the weight of a 20% to 60% ethanol aqueous solution, reflux extract for 3 to 6 hours, cool and filter, and concentrate the filtrate at 60° C. until there is no alcohol taste, thereby obtaining the yellow ican total extract solution.

3. The method for preparing a reference substance of frangipani glycoside according to claim 1, characterized in that: In the reverse phase chromatography purification step, the frangipani glycoside reference solution I is treated by freeze drying to obtain the frangipani glycoside reference solution.

4. The method for preparing a reference substance of frangipani glycoside according to claim 1, characterized in that: The reference solution I of frangipani glycoside is concentrated at 50-60° C., and after the solid is precipitated for the first time, an insoluble solvent is added to precipitate the frangipani glycoside, and the solution is filtered to obtain a semi-finished solid powder of the target product. The insoluble solvent is selected from petroleum ether, dichloromethane, chloroform, n-hexane, and cyclohexane.

5. The method for preparing the reference substance of frangipani glycoside according to claim 1, characterized in that In the reverse phase chromatography purification process, elution is performed at a pressure of 0.2 to 0.4 MPa, using a mixture of acetonitrile and water, and using 40 to 60 μm octadecylsilane bonded silica gel filler.

6. The preparation method of the reference substance of frangipani glycoside according to claim 1, In the macroporous resin enrichment step, the macroporous resin is any one selected from AB-8 macroporous resin, HPD-100 macroporous resin, and D-101 macroporous resin, and the pore size of the macroporous resin is 5 nm to 90 nm.

Images