A method for simultaneously determining benzyl acetone, adenosine and mangiferin in Aquilaria sinensis leaves

Abstract

The present invention relates to a method for simultaneously determining benzyl acetone, adenosine and mangiferin in white aquilegia leaves, which uses a mixed mode chromatographic column bonded with a hydrophobic chain and an ionic group, and performs detection by a liquid chromatography detection method. The present invention can detect benzyl acetone, adenosine and mangiferin in white aquilegia leaves at one time, thereby reducing time consumption and detection costs.

Description

Technical Field

[0001] The invention relates to the field of detection, and in particular to a method for simultaneously determining benzyl acetone, adenosine and mangiferin in aquilaria sinensis leaves. Background Art

[0002] Aquilaria sinensis is a plant of the genus Aquilaria in the family Thymelaeaceae. The resin formed on and around the wound after it is damaged, worm-eaten or decayed is a famous Chinese medicine aquilaria sinensis, which has the effects of promoting qi and relieving pain, warming the middle and stopping vomiting, and relieving asthma. The older the tree, the longer the balsam is deposited, and the better the quality of the medicinal material. Aquilaria sinensis is used in large quantities as a spice and medicinal material. With people's in-depth understanding of the medicinal value of aquilaria sinensis and the comprehensive development and utilization of its resources, more and more people have begun to artificially plant Aquilaria sinensis, and the scale of planting has continued to expand. However, Aquilaria sinensis grows slowly, takes a long time to form resin, and has a long period of economic benefits. The output of aquilaria sinensis medicinal materials is far from meeting market demand. In addition, only the resin-containing wood of Aquilaria sinensis is used as medicine, and other parts such as leaves, branches, and wood without resin are treated as waste, resulting in a large waste of resources.

[0003] Literature research shows that Aucklandia sinensis contains benzyl acetone, adenosine, mangiferin and other substances. Preliminary pharmacological experiments on the substances contained in Aucklandia sinensis show that benzyl acetone has a significant antitussive effect and is related to the resin formation of Aucklandia sinensis; adenosine can resist arrhythmia, convert paroxysmal supraventricular tachycardia to sinus rhythm, and can treat angina pectoris, myocardial infarction, coronary insufficiency, arteriosclerosis, essential hypertension, cerebrovascular disorders, sequelae of stroke, progressive muscular atrophy, etc.; mangiferin connected with carbon glycoside has the effect of treating metabolic syndrome (obesity, hypertension, hyperlipidemia, hyperglycemia, coronary heart disease, cerebrovascular accident and other diseases).

[0004] At present, the determination methods of benzyl acetone, adenosine and mangiferin in white agarwood are all single component determinations. The detection method of benzyl acetone in white agarwood is gas chromatography, and the detection methods of adenosine and mangiferin are liquid chromatography. Although the contents of the above three substances can be determined by different inspection instruments, the three components must be detected separately, and two analytical equipment and two chromatographic columns must be used, which is time-consuming and has high detection costs. At present, there is no method for simultaneously determining the three substances. Benzyl acetone is a weak polar substance, adenosine is a strong polar substance, and mangiferin is a medium polar substance. The polarities of the three substances are very different. Under the conditions of existing instruments and equipment, using ordinary C18 columns or ordinary ion exchange columns to analyze the above three substances cannot obtain a good retention effect, and the separation effect is not ideal, the interference of miscellaneous peaks is large, and the reference value of the detection and analysis results is small, that is, if a weak polar column is used for analysis, adenosine with strong polarity cannot be retained, and if a strong polar column is used for analysis, the retention effect of benzyl acetone with weak polarity and mangiferin with medium polarity is very poor. Summary of the invention

[0005] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a method for simultaneously determining benzyl acetone, adenosine and mangiferin in Aquilaria sinensis leaves, thereby reducing the time and cost of detecting benzyl acetone, adenosine and mangiferin in Aquilaria sinensis leaves.

[0006] To achieve the purpose of the present invention, the present invention provides a method for simultaneously determining benzyl acetone, adenosine and mangiferin in white aquilaria leaves, which uses a mixed-mode chromatographic column bonded with a hydrophobic chain and an ionic group and is detected by a liquid chromatography detection method.

[0007] In some embodiments of the present invention, the mixed mode chromatographic column comprises fully porous silica gel, the hydrophobic chain and the ionic group, the ionic group is at least one of a cation and anion, the hydrophobic chain is a long-chain alkyl group, and the long-chain alkyl group is connected to the ionic group and bonded to the fully porous silica gel.

[0008] In some embodiments of the present invention, the mixed mode chromatography column is Comixsil RP-CX.

[0009] In some embodiments of the present invention, the mixed mode chromatography column is eluted with a calcium ion solution before use.

[0010] In some embodiments of the present invention, the molar concentration of calcium ions in the calcium ion solution is 0.5 mol / L to 0.8 mol / L.

[0011] In some embodiments of the present invention, the calcium ion solution contains at least one of calcium chloride solution, calcium nitrate, and calcium bromide.

[0012] In some embodiments of the present invention, the elution rate is 1.5 mL / min to 2 mL / min, and the elution time is 40 min to 50 min.

[0013] In some embodiments of the present invention, the mixed mode chromatography column is washed with distilled water after being eluted with a calcium ion solution.

[0014] In some embodiments of the present invention, the conditions for detection by liquid chromatography detection method are: flow rate 0.5 mL / min, column temperature 35° C., organic phase in the mobile phase is a mixture of ethanol and acetonitrile, and the aqueous phase is 50 mmol / L ammonium acetate solution.

[0015] In some embodiments of the present invention, the volume ratio of the mixture of ethanol and acetonitrile is 1:1, and the pH of the ammonium acetate solution is 5.5.

[0016] Compared with the prior art, the present invention can achieve the following beneficial effects:

[0017] (1) The present invention adopts a liquid chromatography method, which can accurately, quickly and conveniently determine the three material components of benzyl acetone, adenosine and mangiferin in the leaves of Aquilaria sinensis on the same analytical instrument.

[0018] (2) The present invention uses a mixed-mode liquid phase column to separate and detect three substances with very different polarities, namely benzyl acetone, adenosine and mangiferin, in the leaves of Aquilaria sinensis at one time, with good separation effect, less interference from miscellaneous peaks, and reliable analysis results.

[0019] (3) The present invention realizes the simultaneous determination of three substance components, benzyl acetone, adenosine and mangiferin, in the leaves of Aquilaria sinensis by the same method, which greatly shortens the detection time and reduces the detection cost.

[0020] (4) The present invention reduces the use of one analytical device, thereby reducing the inspection cost and time cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the detection liquid phase spectrum of Example 1 of the present invention.

[0022] Figure 2 This is the detection liquid phase spectrum of Example 2 of the present invention. DETAILED DESCRIPTION

[0023] The present invention provides a method for simultaneously determining benzyl acetone, adenosine and mangiferin in white agarwood leaves, using a mixed mode chromatographic column bonded with a hydrophobic chain and an ionic group, and detecting by a liquid chromatography detection method. Through the above scheme, the present invention overcomes the shortcomings of the prior art and provides a method for quickly and accurately analyzing the material components in white agarwood leaves. The liquid chromatography detection method adopted by the method of the present invention realizes the same instrument and same method detection of the three substances of benzyl acetone, adenosine and mangiferin that need to be detected and analyzed separately on different analytical instruments under the prior art, thereby reducing the use of one analytical device. By using a mixed mode liquid phase column, the simultaneous detection and analysis of the three material components of benzyl acetone, adenosine and mangiferin with a large polarity difference in white agarwood leaves is realized, and the detection and analysis work is completed at one time, which has the advantages of simple and convenient operation, time saving and labor saving. The detection and analysis method adopted by the present invention has a good material component separation effect and is suitable for the determination of benzyl acetone, adenosine and mangiferin in white agarwood leaves.

[0024] Specifically, in the prior art, the columns in liquid chromatography analysis are usually divided into normal phase columns and reverse phase columns. Normal phase columns generally use polar bonded stationary phases, and the surface of silica gel is bonded with polar organic groups, which are usually used to separate highly polar compounds. Commonly used normal phase columns include ion exchange columns, HILIC columns, etc. The column filler of the reverse phase column uses silica gel as the matrix, and the surface is bonded with a bonded phase with relatively weakly polar functional groups. The order in which the sample flows out of the chromatographic column is that the components with stronger polarity are washed out first, and the components with weaker polarity will have stronger retention on the chromatographic column. The commonly used reverse phase column is C18 column. The target substance component adenosine in the leaves of Aquilaria sinensis is a highly polar substance, mangiferin is a medium polar substance, and benzyl acetone is a weak polar substance. The three are analyzed using reverse phase columns such as C18 columns. Mangiferin and benzyl acetone can have a good retention effect, while adenosine is not retained. When the three substances are analyzed by a normal phase column such as an ion exchange column or a HILIC column, adenosine can have a good retention effect, while mangiferin and benzyl acetone have unsatisfactory retention effects on the column. Due to the hydrophobic effect and anion exclusion effect, this type of column can have a good retention effect on the three substances of benzyl acetone, mangiferin and adenosine in the leaves of Aquilaria sinensis, achieving the expected experimental effect of the present invention.

[0025] In some examples, the mixed mode chromatographic column includes fully porous silica gel, the hydrophobic chain and the ionic group, the ionic group is at least one of a cation and an anion, the hydrophobic chain is a long-chain alkyl group, and the long-chain alkyl group is connected to the ionic group and bonded to the fully porous silica gel. The present invention adopts a mixed mode liquid phase column, and the hydrophobic chain on the stationary phase can provide a hydrophobic effect, and the hydrophobic chain is embedded with a cation, anion or an anionic and cationic group to provide a stable ionic effect. In some examples, the ionic group is an anion, such as a carboxyl group.

[0026] In some examples, the mixed mode chromatographic column is Comixsil RP-CX. The mixed mode liquid phase column of Comixsil RP-CX used in the present invention has a long alkyl chain, and weak anions are embedded and bonded on the alkyl chain, and has a cation exchange effect, and can well separate the three substances of benzyl acetone, mangiferin and adenosine in the white atractylodes leaf.

[0027] In some examples, the mixed mode chromatographic column is eluted with a calcium ion solution before use. The present invention first elutes the mixed mode chromatographic column with a calcium ion solution, and adjusts the ion exclusion effect of the mixed mode chromatographic column through the bonding of calcium ions and anions, thereby improving the separation effect of the chromatographic column on benzyl acetone, mangiferin and adenosine in white aquilegia leaves, and can further reduce the interference of miscellaneous peaks.

[0028] In some examples, the molar concentration of calcium ions in the calcium ion solution is 0.5 mol / L to 0.8 mol / L. When the calcium ion concentration in the calcium ion solution is within the above range, the calcium ion solution has a good elution effect and a good binding effect between calcium ions and anions.

[0029] In some examples, the calcium ion solution contains at least one of calcium chloride solution, calcium nitrate, and calcium bromide. Calcium chloride solution, calcium nitrate, and calcium bromide have good solubility in aqueous solution and are easy to remove negative ions and excess calcium ions from the mixed-mode chromatographic column to avoid ion residues affecting chromatographic column separation.

[0030] In some examples, the elution rate is 1.5 mL / min to 2 mL / min, and the elution time is 40 min to 50 min. Under the above elution conditions, sufficient calcium ions are combined with anions to obtain the desired ion exclusion effect.

[0031] In some examples, the mixed mode chromatographic column is washed with distilled water after being eluted with a calcium ion solution to remove negative ions and excess calcium ions to prevent residual ions from affecting chromatographic column separation.

[0032] In some examples, the conditions for detection by liquid chromatography detection method are flow rate 0.5mL / min, column temperature 35°C, organic phase in the mobile phase is a mixture of ethanol and acetonitrile, and the aqueous phase is 50mmol / L ammonium acetate solution. In some examples, the volume ratio of the ethanol and acetonitrile mixture is 1:1, and the pH of the ammonium acetate solution is 5.5. The above detection conditions and mobile phase can well separate and detect benzyl acetone, adenosine and mangiferin, three substances with very different polarities in white agarwood leaves.

[0033] The present invention is further described in detail below in conjunction with specific implementation modes.

[0034] Example 1

[0035] In this example, a mixed mode liquid phase column of Comixsil RP-CX was used to detect the Aquilaria sinensis leaf sample No. 1.

[0036] The main instrument used in this example is a high performance liquid chromatograph (brand: Waters, model: 2695).

[0037] Chromatographic conditions:

[0038] Chromatographic column: Pheromone, Comixsil RP-CX (100 mm × 3.0 mm, 3 μm);

[0039] Detector: 2998 PDA detector (254 nm);

[0040] Flow rate: 0.5ml / min;

[0041] Column temperature: 35°C;

[0042] Mobile Phase:

[0043] Organic phase: ethanol-acetonitrile (1:1);

[0044] Aqueous phase: 50 mmol / L ammonium acetate solution (pH = 5.5);

[0045] Mobile phase gradient:

[0046]

[0047]

[0048] The automatic scale chromatogram obtained in this embodiment is as follows Figure 1 As shown, this embodiment can effectively separate and detect benzyl acetone, mangiferin and adenosine in the leaves of Aquilaria sinensis, realize the detection of the three substances with the same instrument and the same method, shorten the detection time and reduce the detection cost.

[0049] Example 2

[0050] This embodiment uses a mixed mode liquid phase column of Comixsil RP-CX. The mixed mode chromatographic column is eluted with a calcium ion solution before use and then rinsed with distilled water. The calcium ion solution is a calcium chloride solution, the molar concentration of calcium ions is 0.5 mol / L, the elution rate of the calcium ion solution is 1.5 mL / min, and the elution time is 40 min. The mixed mode liquid phase column after elution treatment is used to detect the No. 2 white agarwood leaf sample, and the instruments and chromatographic conditions used are the same as those in Example 1.

[0051] The automatic scale chromatogram obtained in this embodiment is as follows Figure 2 As shown, this embodiment has a good separation effect on benzyl acetone, mangiferin and adenosine in the leaves of Aquilaria sinensis, with little interference from other peaks.

[0052] Finally, it should be emphasized that the above description is only a partial embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for simultaneously determining benzyl acetone, adenosine and mangiferin in Aquilaria sinensis leaves, characterized in that Detection is performed by liquid chromatography using a mixed-mode column bonded with hydrophobic chains and ionic groups; Among them, the conditions for detection by liquid chromatography detection method include: The detector was a 2998PDA detector with a detection wavelength of 254 nm; The flow rate was 0.5 mL / min, the column temperature was 35 °C, the organic phase in the mobile phase was a mixture of ethanol and acetonitrile, and the aqueous phase was a 50 mmol / L ammonium acetate solution; The volume ratio of the ethanol and acetonitrile mixture was 1:1, and the pH of the ammonium acetate solution was 5.5; The mobile phase gradient: Time: 0.00min, organic phase: 5.0%, aqueous phase: 95.0%; Time: 6.00min, organic phase: 5.0%, aqueous phase: 95.0%; Time: 20.00min, organic phase: 75.0%, aqueous phase: 25.0%; Time: 21.00min, organic phase: 75.0%, aqueous phase: 25.0%; Time: 21.50min, organic phase: 5.0%, aqueous phase: 95.0%; Time: 25.00 min, organic phase: 5.0%, aqueous phase: 95.0%; The mixed mode chromatographic column is Comixsil RP-CX, which includes fully porous silica gel, the hydrophobic chain and the ionic group, the ionic group is a weak anion, the hydrophobic chain is a long-chain alkyl, and the long-chain alkyl connects the weak anion and bonds to the fully porous silica gel.

2. A method for simultaneously determining benzyl acetone, adenosine and mangiferin in Aquilaria sinensis leaves according to claim 1, characterized in that The mixed mode chromatographic column is eluted with a calcium ion solution before use; The mixed mode chromatographic column is washed with distilled water after being eluted with the calcium ion solution.

3. A method for simultaneously determining benzyl acetone, adenosine and mangiferin in Aquilaria sinensis leaves according to claim 2, characterized in that The molar concentration of calcium ions in the calcium ion solution is 0.5 mol / L; The elution rate was 1.5 mL / min and the elution time was 40 min.

4. A method for simultaneously determining benzyl acetone, adenosine and mangiferin in Aquilaria sinensis leaves according to claim 3, characterized in that The calcium ion solution contains at least one of calcium chloride solution, calcium nitrate and calcium bromide.

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