CN113899839A - Extraction method of coumarin compound in morinda officinalis - Google Patents
Extraction method of coumarin compound in morinda officinalis Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/89—Inverse chromatography
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Abstract
The invention belongs to the technical field of compound extraction and analysis, and particularly relates to a method for extracting coumarin compounds from morinda officinalis, which comprises the following steps: adjusting pH of the radix Morindae officinalis extract to 1-7; adding trichloromethane to obtain a mixed liquid; treating the mixed liquid at 10-60 ℃, and performing ultrasonic-assisted extraction; and centrifuging the mixed liquid after ultrasonic treatment to obtain a sediment, and finishing extraction. The method combines high performance liquid chromatography, the sensitivity of measuring 5 coumarin compounds in morinda officinalis is high, and the detection limit is 0.5-0.9 ng/mL; the used extraction solvent is less, the consumed time is short, and the method can be used for measuring the coumarin compounds in a large batch of morinda officinalis samples.
Description
Technical Field
The invention belongs to the technical field of compound extraction and analysis, and particularly relates to a method for extracting coumarin compounds from morinda officinalis, which is a method for extracting and analyzing the coumarin compounds from morinda officinalis based on ultrasonic-assisted dispersion liquid-liquid microextraction.
Background
Morinda officinalis contains coumarin components, adverse reactions are mostly related to oral administration, and researches show that coumarin is contained in mice LD for intragastric administration50Toxicity is strong at 1.39g/kg, although species variability is possible with this toxicity, and whether or not coumarin elicits toxicity is associated with polymorphism of the CYP2a6 enzyme in the human population.
At present, no detection method for coumarin compounds in morinda officinalis exists, and for the detection of the coumarin compounds, the key step is sample pretreatment. Through the pretreatment of the sample, on one hand, the interference elimination effect of the trace component to be detected can be achieved, so that the sensitivity of the method is improved, and the detection limit of the method is reduced; on the other hand, substances harmful to the instrument or the pipeline can be removed, so that the service life of the instrument is prolonged. While liquid-in-dispersion micro-extraction (DLLME) is a novel micro-extraction technique. When the extractant and the dispersing agent are added into the sample solution, the dispersing agent enables the extractant to be uniformly distributed in the sample solution, and the contact area between the extractant and the sample solution is increased, so that the object to be detected is quickly extracted by the extractant, and is left at the bottom of the centrifugal tube after centrifugation to form a precipitation phase with a certain volume. Compared with the traditional liquid-liquid extraction method, the dispersion liquid-liquid microextraction method integrates sampling, extraction and concentration, has the characteristics of small usage amount of organic solvent, high extraction efficiency, large enrichment factor, convenience and simplicity in operation, environmental friendliness and the like, is a green sample pretreatment method, and has wide prospects in the field of trace analysis.
Disclosure of Invention
The invention aims to provide an extraction method of coumarin compounds in morinda officinalis, which is an ultrasonic-assisted dispersion liquid-liquid microextraction method with simple operation, high detection speed and low cost, and can be used for simultaneously detecting 5 coumarin compounds, such as coumarin, dihydrocoumarin, vinegatrocoumarin, 6-methylcoumarin and 7-hydroxy-6-methoxycoumarin, by combining high performance liquid chromatography.
According to the technical scheme of the invention, the extraction method of the coumarin compound in the morinda officinalis comprises the following steps,
s1: adjusting pH of the radix Morindae officinalis extract to 1-7;
s2: adding trichloromethane to obtain a mixed liquid;
s3: treating the mixed liquid at 10-60 ℃, and performing ultrasonic-assisted extraction;
s4: and centrifuging the mixed liquid after ultrasonic treatment to obtain a sediment, and finishing extraction.
Further, the preparation method of the morinda officinalis extract comprises the following steps:
SS 1: grinding a morinda officinalis sample, adding water, boiling, extracting, and filtering to obtain a filtrate I and filter residues;
SS 2: adding water into the filter residue, boiling for extraction, and filtering to obtain a filtrate II;
SS 3: mixing the filtrate I and the filtrate II, and freeze-drying to obtain freeze-dried powder;
SS 4: adding water into the freeze-dried powder, performing ultrasonic extraction, and centrifuging to obtain supernatant, namely morinda officinalis extract.
Furthermore, in the step SS1, the extraction time is 1.5-4 h; in the step SS2, the extraction time is 1-3 h; in the step SS4, the ultrasonic extraction time is 0.4-0.6 h.
Further, in the step SS4, the mass-to-volume ratio of the freeze-dried powder to the water is 1: 30-50 g/mL.
Further, the volume ratio of the morinda officinalis extract to the trichloromethane is 500: 3-8.
Further, in step S2, the chloroform is dissolved in the methanol, and then the pH-adjusted morinda officinalis extract is added.
Further, the volume ratio of the trichloromethane to the methanol is 1: 8-20.
Further, in the step S3, the time for ultrasonic-assisted extraction is 1-12min, preferably 7 min.
Further, in the step S4, the rotation speed of the centrifugation is 3000-5000rpm, and the time is 6-12 min.
The invention also provides the application of the trichloromethane in the extraction of the coumarin compound.
Compared with the prior art, the technical scheme of the invention has the following advantages:
1) based on the invention, high performance liquid chromatography is combined to determine 5 coumarin compounds in morinda officinalis, so that the sensitivity is high, and the detection limit is 0.5-0.9 ng/mL.
2) The extraction solvent used in the invention is less (50 mu L of trichloromethane and 0.8mL of methanol dispersant), and compared with the traditional reflux extraction method, the method saves a large amount of organic solvent harmful to human body.
3) The method disclosed by the invention is short in consumed time (<30min), and compared with the traditional method, the analysis time is saved, so that the method can be used for measuring the coumarin compounds in a large batch of morinda officinalis samples.
Drawings
FIG. 1 is a diagram showing the effect of different organic solvents as extractants on the extraction effect of coumarins.
FIG. 2 is a graph showing the effect of different volumes of chloroform as an extractant on the extraction results.
FIG. 3 is a graph showing the effect of different dispersants on the extraction results.
Detailed Description
The present invention is further described below in conjunction with specific examples to enable those skilled in the art to better understand the present invention and to practice it, but the examples are not intended to limit the present invention.
Example 1 extraction of coumarins from Morinda citrifolia and determination of control parameters
1. The extraction method of coumarin compounds in morinda officinalis comprises the following steps,
s1: adjusting pH of the radix Morindae officinalis extract to 1-7;
s2: adding an extracting agent (dissolved in a dispersing agent) to obtain a mixed liquid;
s3: treating the mixed liquid at 10-60 ℃, and performing ultrasonic-assisted extraction;
s4: and centrifuging the mixed liquid after ultrasonic treatment to obtain a sediment, and finishing extraction.
2. Determination of control parameters
The main factors influencing the efficiency of ultrasonic-assisted extraction and dispersion liquid-liquid microextraction include: extractant type and amount, extraction temperature, dispersant type and amount, and ionic strength and pH.
2.1, determination of the type of the ultrasonic-assisted extraction agent: the organic solvent with density higher than that of water is selected as the extracting agent, and the selection of the extracting agent is very important for the final extraction result because different organic solvents have different influences on the extraction capacity of the target analyte. The invention selects three organic solvents for enriching and extracting the coumarin compounds in the morinda officinalis extract, wherein the three organic solvents are dichloromethane, trichloromethane and carbon tetrachloride respectively, and the recovery rate is shown in figure 1. The result shows that the coumarin compound in the trichloromethane morinda officinalis has good recovery rate which reaches 82 percent, so that the trichloromethane is finally selected as an extracting agent in the DLLME process.
2.2, determination of chloroform volume: the amount of extractant used is a key factor affecting the extraction efficiency, and the amount of extractant used directly affects the level of recovery. In the invention, 30-80 mu L of trichloromethane (5mL of morinda officinalis extract) is selected. The results of the experiment (fig. 2) show that satisfactory enrichment factors and extraction recoveries were obtained in the experiment using 50 μ L of chloroform.
2.3, determination of extraction temperature: during extraction, generally, increasing the temperature accelerates the transfer of analyte between the two phases and also increases the solubility of the extractant and target analyte in solution. In the present invention, the effect of temperature on extraction efficiency was investigated at 5-60 ℃. Wherein the best recovery is obtained in the 20 ℃ range.
2.4, determination of ultrasonic time: performing ultrasonic-assisted extraction at 20 deg.C, and evaluating the effect of ultrasonic treatment time within 1-12 min. And finally, selecting ultrasonic-assisted extraction for 7min in the experiment, wherein the extraction effect is optimal.
2.5, selection of dispersant and volume: the invention selects three organic solvents, acetonitrile, methanol and acetone, which have proper miscibility in ionic liquid and water phase. The recovery rates for the four target analytes were similar (as shown in figure 3), but methanol was chosen as the dispersant in view of the use of methanol as the mobile phase in HPLC. The volume of the dispersant can affect the dispersion of the extractant in the water sample. The dosage of the fixed extractant is 50 mu L, and the methanol with different volumes of 0.4-1.0mL is optimized, wherein the extraction effect is the best when the volume of the methanol is 0.8 mL.
2.6, selection of pH: the pH of the sample solution determines the form of analyte present (ionic or neutral form) and may thereafter affect the extraction efficiency. The effect of the pH of the samples was evaluated in the range of 1-7 by adding appropriate phosphoric acid or sodium hydroxide to the samples to adjust the pH. When the pH value of the sample is equal to 5, the recovery rate of coumarin, dihydrocoumarin, vinpocetine and 7 hydroxy-6-methoxycoumarin is the highest. Thus, a pH of 5 is selected in the examples below.
2.7, selection of ionic strength: ionic strength can increase the solubility of the target analyte in the extractant, thereby increasing extraction efficiency. In the present invention, different amounts of NaH are used2PO4(from 0% to 20%) was added to the sample to evaluate the effect of ionic strength. Adding NaH2PO4After that, the extraction recovery rate decreases. Therefore, Na is not added in the present invention+And (3) salt.
EXAMPLE 2 determination of coumarins in Morinda citrifolia samples
1. Preparation of a standard solution: single standard stock solutions: precisely weighing 2.15mg, 1.25mg, 1.30mg, 1.40mg and 1.30mg of each of coumarin, dihydrocoumarin, acerolol, 6-methylcoumarin and 7-hydroxy-6-methoxycoumarin, adding 50% methanol for dissolving, respectively diluting to 10mL, and preparing into single standard stock solutions with concentrations of 215 μ g/mL, 125 μ g/mL, 130 μ g/mL, 140 μ g/mL and 130 μ g/mL. Mixing standard solutions: precisely absorbing a proper amount of each single standard substance stock solution to prepare a mixed standard substance solution with a corresponding concentration gradient.
2. Sample pretreatment: grinding a morinda officinalis sample into fine powder, putting 100g of the morinda officinalis fine powder into a 3000mL stainless steel pot, adding 1200mL distilled water, boiling and extracting for 2h, filtering, adding 1000mL distilled water into filter residue again, performing boiling preservation and extraction for 1.5h, filtering, combining filtrates, freeze-drying 300mL of extracting solution, and storing. Adding 0.5g of the above lyophilized powder into 20mL of distilled water, and performing ultrasonic extraction for 30 min. The mixed solution was centrifuged for 5min to separate the lyophilized powder residue from the aqueous solution.
3. Dispersion liquid microextraction (DLLME): 5mL of the above extract was placed in a 10mL conical bottom centrifuge tube. 50 μ L of chloroform (dissolved in 800 μ L of methanol) was rapidly injected into the sample solution using a syringe. For the turbid sample solution, vortex for 3min, sonicate at 20 ℃ for 7min followed by ice bath for 10min, and finally centrifuge at 3500rmp for 8 min. The upper aqueous phase was discarded, the IL phase was measured at the bottom of the centrifuge tube, typically 26. mu.L, and carefully removed for injection into the HPLC system.
4. And (3) HPLC determination: the chromatographic analysis was performed on a chromamaster type HPLC system equipped with a Diode Array Detection (DAD) system. Reverse phase C18 chromatography column (inner diameter 250 mm. times.4.6 mm, 5 μm) model Agilent Eclipse XDB-C18. The mobile phase consisted of water (A) with 0.05% phosphoric acid and methanol (B) added (ratio 20:80(v/v)) at a flow rate of 1.0mL min-1. The sample injection amount and the detection wavelength were 10. mu.L and 320nm, respectively.
5. Evaluating the performance of the method: in order to study the performance of the proposed method for determining coumarin compounds in a sample, a series of experiments were designed to evaluate the linearity, reproducibility, detection limits and other characteristics of the method under optimized conditions, and the results are shown in table 1. As can be seen from Table 1, the analytes of coumarin, dihydrocoumarin, aceroloumarin, 6-methylcoumarin and 7-hydroxy-6-methoxycoumarin all show good linearity and correlation coefficient (R)2) Ranging from 0.998 to 0.999. The precision of the method was determined by five extractions and analyses of the spiked samples, with a Relative Standard Deviation (RSD) ranging from 1.25% to 5.28%. The limit of detection (LOD) is calculated to be in the range of 0.50-2.02. mu.g.L based on the signal-to-noise ratio (S/N-3)-1. The parameters show that the method has high sensitivity and reliability and can be used for detecting the concentration of the coumarin compound in the sample.
TABLE 1 method Performance evaluation
6. And (3) sample determination: in order to verify the applicability of the method in the analysis of actual samples, the coumarins in morinda officinalis samples from three different areas (I, II, III) of Tianfangjian pharmaceutical Co. The results obtained by the two methods were compared by the F-test (p 0.95, n 3) and no significant difference was observed, indicating that the method of ultrasound-assisted micro-extraction of the functionalized ex vivo dispersion was used to assay such samples.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.
Claims (10)
1. The extraction method of the coumarin compound in the morinda officinalis is characterized by comprising the following steps,
s1: adjusting pH of the radix Morindae officinalis extract to 1-7;
s2: adding trichloromethane to obtain a mixed liquid;
s3: treating the mixed liquid at 10-60 ℃, and performing ultrasonic-assisted extraction;
s4: and centrifuging the mixed liquid after ultrasonic treatment to obtain a sediment, and finishing extraction.
2. The method of claim 1, wherein said Morinda citrifolia extract is prepared by:
SS 1: grinding a morinda officinalis sample, adding water, boiling, extracting, and filtering to obtain a filtrate I and filter residues;
SS 2: adding water into the filter residue, boiling for extraction, and filtering to obtain a filtrate II;
SS 3: mixing the filtrate I and the filtrate II, and freeze-drying to obtain freeze-dried powder;
SS 4: adding water into the freeze-dried powder, performing ultrasonic extraction, and centrifuging to obtain supernatant, namely morinda officinalis extract.
3. The method of claim 2, wherein in step SS1, the extraction time is 1.5-4 hours; in the step SS2, the extraction time is 1-3 h; in the step SS4, the ultrasonic extraction time is 0.4-0.6 h.
4. The method of claim 2, wherein in step SS4, the mass-to-volume ratio of lyophilized powder to water is 1: 30-50 g/mL.
5. The method of claim 1, wherein the ratio of said Morinda citrifolia extract to said chloroform is 500: 3-8.
6. The method of claim 1 or 5, wherein in step S2, said chloroform is dissolved in methanol and added to said pH adjusted Morinda citrifolia extract.
7. The method of claim 6, wherein the volume ratio of chloroform to methanol is 1: 8-20.
8. The method of claim 1, wherein in step S3, the ultrasonic assisted extraction is performed for a period of 1-12 min.
9. The method as claimed in claim 1, wherein the centrifugation step S4 is performed at 3000-5000rpm for 6-12 min.
10. The application of trichloromethane in extraction of coumarin compounds is provided.
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| CN108752497A (en) * | 2018-06-05 | 2018-11-06 | 上海中医药大学附属曙光医院 | Prepared by Morinda officinalis water extract, oligosaccharides and polysaccharide, and its application |
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