Method for purifying and extracting gemurrayine in clausena lansium fruits
By employing steps such as preliminary treatment, extraction, centrifugal concentration, hydrochloric acid dissolution, alkalization extraction, and D101 pore resin purification, the problem of low efficiency in the preparation of alkaloids from wampee fruit was solved, achieving efficient extraction and simple purification of alkaloids, reducing costs and providing a detection method.
Patent Information
- Application Number
- CN202511230909.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-30
- Publication Date
- 2025-12-12
AI Technical Summary
Existing technologies have low efficiency in preparing alkaloids from wampee fruit, making it impossible to extract them in large quantities, and the preparation process is complex and costly.
The process involves preliminary treatment, extraction, centrifugal concentration, hydrochloric acid dissolution, alkalization extraction, and purification with D101-well resin, combined with ultraviolet spectroscopy detection, to achieve efficient purification and extraction of keratoside.
This study achieved efficient extraction of alkaloids from wampee fruit, reduced preparation costs, and provided a method for detecting the concentration of alkaloids, which is both efficient and simple.
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Abstract
Description
TECHNICAL FIELD
[0001] The application relates to the technical field of natural medicines, and particularly relates to a purification and extraction method of giseonylonol in Clausena lansium fruit. BACKGROUND
[0002] Alkaloids are a kind of nitrogen-containing basic organic compounds existing in nature (mainly plants), most of which have complex ring structures and are synthesized from different amino acids or direct derivatives thereof, are one of secondary metabolites, have significant biological activities, and are one of important effective components in Chinese herbal medicines. In a plant body, several or tens of alkaloids coexist, and the chemical structures of the alkaloids are similar.
[0003] Clausena lansium belongs to the Rutaceae Clausena genus and is mainly distributed in southern China. The fruit has the effects of food digestion and qi regulation. The carbazole alkaloid is a characteristic component of the Clausena genus, has the characteristics of stable structure and multiple substitution positions, and thus shows various biological and pharmacological activities and is widely applied to multiple fields such as material chemistry, medicinal chemistry and agricultural chemicals, for example, anticancer, antibacterial, antimalarial and antiviral aspects.
[0004] The existing alkaloid preparation technology has low preparation efficiency, cannot obtain a large amount of Clausena lansium alkaloid from the fruit of Clausena lansium, and has a complex preparation process and high cost. Therefore, optimization of the Clausena lansium fruit alkaloid extraction process to more efficiently extract the effective components in Clausena lansium has good scientific research prospects and practical application value. SUMMARY
[0005] The application aims to overcome the deficiencies of the prior art and provide a purification and extraction method of giseonylonol in Clausena lansium fruit.
[0006] In order to achieve the above-mentioned purpose, the technical scheme specifically adopted by the application is as follows.
[0007] A purification and extraction method of giseonylonol in Clausena lansium fruit, comprising the following steps:
[0008] S1, preliminary treatment: 20g of fresh Clausena lansium fruit is placed in a container, an excessive amount of clean water is added, the dust and impurities on the surface of the Clausena lansium fruit are cleaned with the clean water, and the Clausena lansium fruit is made into fruit pulp through a cell wall breaking machine after being drained;
[0009] S2, extraction: the fruit pulp is poured into a container, 80% ethanol solution is poured and the volume is made to be 100ml, and after 6-18h of extraction, filtration is performed to obtain an ethanol extract of Clausena lansium;
[0010] S3, centrifugal concentration:
[0011] S3.1, the ethanol extract of Clausena lansium is subjected to centrifugal treatment, and the supernatant is taken;
[0012] S3.2, the supernatant is reduced pressure rotary evaporated to evaporate the liquid phase, dissolved in 80% ethanol solution, and concentrated to 10 mL by nitrogen blowing to obtain a concentrated supernatant sample;
[0013] S4, extraction:
[0014] S4.1, 1 mL of the concentrated supernatant sample is taken, hydrochloric acid is added to 10 mL, then ethyl acetate is added, shaken, and shaken, and then placed for 30 min;
[0015] S4.2, after standing, the upper layer of ethyl acetate is removed, the pH is adjusted to 9 with ammonia water, and 20 mL of dichloromethane is added twice for extraction;
[0016] S4.3, the two extraction solutions are combined, and the volume is adjusted to 20 mL with dichloromethane, then 10 mL of 80% ethanol solution is added after rotary evaporation at 45 DEG C, and the total alkali solution is obtained after dissolution;
[0017] S5, purification: the total alkali solution is purified and separated by using 20g of treated D101 hole resin to obtain a jijuriacanine solution;
[0018] S6, quantitative detection: 345nm is selected as the detection wavelength to realize the quality control of the jijuriacanine solution.
[0019] Further, in step S3, the temperature of the rotary evaporation under reduced pressure is 40-50 DEG C.
[0020] Further, in step S3, the centrifugal speed is 4000-6000 rpm, and the time is 15-20 min.
[0021] Further, in step S4.1, the concentration of hydrochloric acid is 2-6%, and the amount of ethyl acetate is 20 mL.
[0022] Further, in step S5, the total alkali solution has a mass concentration of 1.22 mg / mL, a pH of 6.0, a volume of 20 mL, a flow rate of 1.0 mL / min, a resin column diameter-height ratio of 1:3, a standing time of 50 min, an ethanol eluent volume fraction of 85-95%, an elution flow rate of 2 mL / min, and an ethanol eluent volume of 40 mL.
[0023] The present application has the following characteristics and beneficial effects:
[0024] 1) The present application can prepare a large amount of Huangpi fruit alkaloids without a complex process, and effectively reduces the preparation cost while improving the extraction efficiency.
[0025] 2) The present application can effectively separate the alkaloids in Huangpi fruit by hydrochloric acid dissolution, alkaline extraction, and D101 hole resin elution, which is simple and efficient.
[0026] 3) The present application also provides a method for detecting the concentration of gilliarkine. BRIEF DESCRIPTION OF DRAWINGS
[0027] Other features, objects, and advantages of the present application will become more apparent from the following detailed description of non-limiting embodiments thereof as read in conjunction with the accompanying drawings:
[0028] Figure 1 Comparison of the extraction efficiency of G-base in different kinds of Phellodendron amurense.
[0029] Figure 2 Comparison of the cytotoxicity of G-base in different kinds of Phellodendron amurense.
[0030] Figure 3 Comparison of the antioxidant capacity of G-base in different kinds of Phellodendron amurense.
[0031] Figure 4 Standard curve of the relationship between G-base content and absorbance. DETAILED DESCRIPTION
[0032] The present application will be described in detail below with specific embodiments. The following examples will help those skilled in the art to further understand the present application, but do not limit the present application in any form. It should be noted that the examples in the present application and the features in the examples can be combined with each other without conflict.
[0033] Example 1
[0034] A method for purifying and extracting gilliarkine from Phellodendron amurense fruit, comprising the following steps:
[0035] S1, preliminary treatment: 20g of fresh Phellodendron amurense fruit is placed in a container, an excess of clean water is added, and the dust and impurities on the surface are washed clean with clean water. After draining the water, the fruit pulp is prepared by a cell wall breaking machine;
[0036] S2, extraction: the fruit pulp is poured into a container, 80% ethanol is added to make up to 100mL, and extracted for 12h. After filtration with double-layer gauze, Phellodendron amurense ethanol extract is obtained;
[0037] S3, centrifugal concentration:
[0038] S3.1, centrifugal treatment of the Phellodendron amurense ethanol extract at 4000-6000rpm for 15-20min to obtain supernatant;
[0039] S3.2, pour the supernatant into a rotary evaporation flask, and rotary evaporate at 45℃ under reduced pressure until the liquid is almost evaporated. Then, dissolve with 80% ethanol solution, pour out from the rotary evaporation flask to a test tube, control the volume of the supernatant to 10mL by nitrogen blowing, and obtain the concentrated supernatant sample.
[0040] S4, extraction:
[0041] S4.1, take 1 mL of concentrated supernatant sample, add 4% hydrochloric acid to 10 mL, then add 20 mL of ethyl acetate, shake, shake, stand for 30 min;
[0042] S4.2, after standing, remove the upper layer of ethyl acetate, adjust the pH to 9 with ammonia water, then add 20 mL of dichloromethane twice;
[0043] S4.3, combine the two times of extraction solution, dilute to 20 mL with dichloromethane, obtain the crude alkaloid extract, after rotary evaporation of the crude alkaloid extract at 45℃, add 10 mL of 80% ethanol solution, dissolve and pour out to obtain the total alkaline solution;
[0044] S5, purification: the treated D101 hole resin 20g is used to purify and separate the total alkaline solution, wherein the mass concentration of the total alkaline solution is 1.22mg / mL, the pH is 6.0, the volume is 20mL, the flow rate is 1.0mL / min, the ratio of column diameter to height is 1:3, the standing time is 50min, the volume fraction of ethanol eluent is 90%, the elution flow rate is 2mL / min, and the volume of ethanol eluent is 40mL, to obtain the alkaloid extract solution;
[0045] S6, quantitative detection: take 10mL of pH 5.0 disodium hydrogen phosphate-citric acid buffer, respectively add 1mL of gijuijiajian alkaline solution, alkaloid extract solution and blank solvent, and 5.0mL of bromophenol blue solution in a separatory funnel, shake and extract for 2min, stand for layering, and take the upper organic phase in a 1cm cuvette. By ultraviolet spectrum scanning, it is found that the gijuijiajian alkaline solution (control) and the alkaloid extract solution both appear characteristic absorption peaks at 345nm (absorbance is 0.852 and 0.786 respectively), and the blank solvent has no interference at this wavelength, so 345nm is selected as the detection wavelength. With 345nm as the detection wavelength, the quantitative detection of gijuijiajian alkaline solution is realized, and the detection result shows that the content of gijuijiajian alkaloid in the alkaloid extract solution is 374mg / g.
[0046] Control group: traditional method;
[0047] After drying, the fruit peel (0.5kg) is processed into powder, and 95% ethanol is used for extraction for 3 times, 2L each time, and 24h each time at room temperature. The obtained filtrate is concentrated by vacuum reduction pressure to obtain a crude extract, which is dispersed in 2% hydrochloric acid to form a suspension, and extracted with chloroform to obtain an extract (alkaloid).
[0048] Experimental group: the method described in the application;
[0049] Pulping: 20g of fresh fruit of Phellodendri chinensis was placed in a container, and an excess of clean water was added to wash off the dust and impurities on the surface of the fruit. The fruit was then left to stand in a room temperature environment, and the water was drained off. The fruit was then crushed into a pulp using a fruit crusher.
[0050] Extraction: 20g of the prepared fruit pulp was poured into a container, and 80% ethanol was added to make up to 100ml. After 12 hours, the mixture was filtered using double-layered gauze to obtain an ethanol extract of Phellodendri chinensis.
[0051] Centrifugal concentration: The ethanol extract of Phellodendri chinensis was centrifuged for 10 minutes at 6000rpm to obtain a supernatant. The supernatant was poured into a rotary evaporation flask, and rotary evaporation was performed at 45°C under reduced pressure until the liquid was almost evaporated. The supernatant was then dissolved in 80% ethanol and poured out of the rotary evaporation flask into a test tube. The volume of the supernatant was controlled to 10ml using nitrogen blowing to obtain a concentrated supernatant sample.
[0052] Extraction: 1ml of the concentrated supernatant sample was taken, and 4% hydrochloric acid was added to make up to 10ml. Then, 20ml of ethyl acetate was added, and the mixture was shaken and allowed to stand for 30 minutes. The upper layer of ethyl acetate was removed, and the pH was adjusted to 9.0 using ammonia water. Then, 20ml of dichloromethane was added in two portions, and the mixture was extracted. The extracted liquid was combined and made up to 20ml using dichloromethane to obtain a crude alkaloid extract. The crude extract was then dissolved in 10ml of 80% ethanol, and the solution was poured out to obtain a total alkaloid solution.
[0053] Purification: The total alkaloid solution was purified using 20g of treated D101 hole resin. The mass concentration of the total alkaloid solution was 1.22mg / ml, the pH was 6.0, the volume was 20ml, the flow rate was 1.0ml / min, the ratio of column diameter to height was 1:3, the standing time was 50 minutes, the volume fraction of ethanol eluent was 90%, the elution flow rate was 2ml / min, and the volume of ethanol eluent was 40ml. After purification, a G-alkaloid solution was obtained.
[0054] Content determination: 10ml of pH 5.0 disodium hydrogen phosphate-citric acid buffer was taken, and 1ml of G-alkaloid standard solution, G-alkaloid extract solution, and blank solvent were added, respectively. Then, 5.0ml of bromophenol blue solution was added, and the mixture was shaken and extracted for 2 minutes in a separatory funnel. After standing and separating, the upper organic phase was taken in a 1cm cuvette. Through ultraviolet spectrum scanning, it was found that the G-alkaloid standard and the G-alkaloid extract both had characteristic absorption peaks at 345nm (the absorbance values were 0.852 and 0.786, respectively), and the blank solvent had no interference at this wavelength. Therefore, 345nm was selected as the detection wavelength. The results showed that this method had good accuracy and repeatability, and was suitable for the quality control of G-alkaloid. The content of G-alkaloid (mg / ml) was plotted against the absorbance value to draw a standard curve, and the regression equation was y=1.2535x-0.0475 (R 2 =0.9992) Figure 4This indicates that the standard solution exhibits good linearity within the required range of 0.2–1 mg.
[0055] Figure 1 To compare the extraction efficiency of G alkaloids from different types of wampee fruit, the specific information of nine types of wampee fruit is shown in Table 1.
[0056] Table 1. Detailed information on nine types of wampee fruit
[0057]
[0058] from Figure 1 As can be seen, regardless of which type of wampee fruit is used as raw material, the extraction efficiency of this method is significantly higher than that of traditional extraction methods. This result indicates that the method has good accuracy and repeatability and is suitable for the quality control of keratoside.
[0059] Figure 2 The figure shows that, regardless of the type of wampee fruit used as raw material, the extracted G alkaloids can significantly improve cell survival rate. Furthermore, the cell-promoting effect of G alkaloids in most fruits increases with increasing concentration.
[0060] Figure 3 This study compared the antioxidant capacity of G alkaloids in different varieties of wampee. The antioxidant capacity of alkaloid extracts from different wampee varieties responded differently to concentration. For some varieties, the antioxidant capacity increased significantly as the concentration increased from 1000 μg / mL to 2000 μg / mL; for others, increasing the concentration did not improve their antioxidant performance and even exacerbated the negative effects to some extent; and for still others, although the antioxidant capacity changed with increasing concentration, the change was relatively small.
[0061] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.
Claims
1. A method for purifying and extracting eugenol from wampee fruit, characterized in that, Includes the following steps: S1. Preliminary processing: Take 20g of fresh wampee fruit, wash it with clean water, drain the water, and then process it into pulp using a cell wall breaking machine. S2. Extraction: Pour the fruit pulp into a container, extract it with 80% ethanol solution, filter it, and obtain the yellow peel ethanol extract. S3, Centrifugal Concentration: S3.1 Centrifuge the ethanol extract of the yellow peel and take the supernatant; S3.
2. The supernatant is evaporated under reduced pressure until the liquid fraction is basically evaporated. After being dissolved in 80% ethanol solution, it is concentrated to 10 mL by nitrogen blowing to obtain a concentrated supernatant sample. S4. Extraction: S4.1 Take 1 mL of the concentrated supernatant sample, dilute to 10 mL with hydrochloric acid, add ethyl acetate, shake well, and let stand for 30 min. S4.2 After standing, remove the upper layer of ethyl acetate, adjust the pH to 9 with ammonia, and add 20 mL of dichloromethane for extraction in two portions. S4.3 Combine the two extracts, bring the volume to 20 mL with dichloromethane, and after rotary evaporation, add 10 mL of 80% ethanol solution to dissolve completely, obtaining the total alkali solution. S5. Purification: Use 20g of the treated D101 pore resin to purify and separate the total alkali solution to obtain the alkali solution. S6. Quantitative detection: 345nm was selected as the detection wavelength to achieve quality control of the Jijiu Lixiang alkali solution.
2. The method for purifying and extracting eugenol from wampee fruit as described in claim 1, characterized in that, In step S2, 80% ethanol solution is poured in, and the volume is adjusted to 100 mL before extraction for 6–18 h.
3. The method for purifying and extracting eugenol from wampee fruit as described in claim 1, characterized in that, In step S3, the temperature of the vacuum rotary evaporation is 40–50°C.
4. The method for purifying and extracting eugenol from wampee fruit as described in claim 1, characterized in that, In step S3, the centrifugation speed is 4000-6000 rpm and the time is 15-20 min.
5. The method for purifying and extracting eugenol from wampee fruit as described in claim 1, characterized in that, In step S4.1, the concentration of hydrochloric acid is 2-6%, and the amount of ethyl acetate used is 20 mL.
6. The method for purifying and extracting eugenol from wampee fruit as described in claim 1, characterized in that, In step S4.3, the temperature of rotary evaporation of the crude extract is 45°C.
7. The method for purifying and extracting eugenol from wampee fruit as described in claim 1, characterized in that, In step S5, the total alkali concentration is 1.22 mg / mL, the pH is 6.0, the volume is 20 mL, the flow rate is 1.0 mL / min, the resin column diameter-to-height ratio is 1:3, the column is allowed to stand for 50 min, the ethanol eluent volume is 85-95%, the elution flow rate is 2 mL / min, and the ethanol eluent volume is 40 mL.