Method for extracting carotenoids from the fleshy roots of yellow ginseng

Abstract

The invention provides a method for extracting carotenoids from the fleshy roots of yellow ginseng. The invention comprises the following steps: S1: drying the fleshy root of Glehnia littoralis to a constant weight, grinding, and pulverizing to obtain an original sample powder; S2: weighing 10 parts by weight of the original sample powder of S1, extracting, and obtaining a mixed solution; S3: filtering the mixed solution obtained in step S2, washing several times, and obtaining a filtrate; S4: standing the filtrate in step S3 for stratification, adding 15-25 mL of distilled water, mixing, and standing for stratification, and repeating the process several times until the lower layer becomes colorless, thereby obtaining a crude carotenoid extract; and S5: selecting silica gel or petroleum ether-neutral alumina suspension as a filler, adding the filler to a chromatography column, adding the crude carotenoid extract of S4 to the chromatography column, adding an eluent, and eluting. The invention has the following beneficial effects: 1. improving the yield of lutein and β-carotene while maintaining the high purity of the extracted lutein and β-carotene; and 2. obtaining β-carotene with high purity and high yield while ensuring the acquisition of high-purity and high-yield lutein.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine, and particularly relates to a method for extracting carotenoids from the fleshy roots of yellow ginseng. Background Art

[0002] Research on the nutritional composition of the Tibetan medicine plant, Miguo Celery, has primarily focused on its roots. Yellow ginseng roots are harvested twice a year: in autumn (approximately October) of the first year, and in spring (approximately April) of the second year. Chi Xiaofeng (2011) found that yellow ginseng has a high nutritional value, with high levels of protein, polysaccharides, and carotenoids. Carotenoids play an important role in antioxidant protection, preventing tumors and cardiovascular disease, and are precursors for the synthesis of vitamin A and the plant hormone ABA, contributing significantly to the quality of yellow ginseng. The accumulation of carotenoids in the fleshy roots of yellow ginseng is a complex process regulated by various genes.

[0003] Carotenoids are colored compounds, mainly including β-carotene and lutein, and are mainly found in yellow, orange and red plant tissues. About 10% of carotenoids are precursors of vitamin A and have anti-cancer activity. They are an indispensable component of human and animal food. The content and form of carotenoids contained in different species vary. During the extraction process, there are problems such as incomplete extraction and easy loss during operation. Therefore, efficient extraction methods are crucial to actual operations. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides a method for extracting carotenoids from the fleshy roots of Rhizoma Corydalis.

[0005] The present invention provides the following technical solutions:

[0006] The method for extracting carotenoids from the fleshy root of yellow ginseng comprises:

[0007] S1: Dry the fleshy roots of Glehnia littoralis to constant weight, grind, and pulverize to obtain the original sample powder;

[0008] S2: extracting the original sample powder of S1 with one or more of anhydrous ethanol and petroleum ether as an extractant to obtain a mixed solution, wherein the material-liquid ratio of the original sample powder to the extractant is 1:10-20;

[0009] S3: filtering the mixed solution obtained in step S2, and washing it several times with one or more of anhydrous ethanol and petroleum ether to obtain a filtrate;

[0010] S4: The filtrate of step S3 was allowed to stand for stratification, 15-25 mL of distilled water was added, mixed and allowed to stand for stratification, and the mixture was repeated several times until the lower layer was colorless to obtain a crude carotenoid extract;

[0011] S5: Silica gel or petroleum ether-neutral alumina suspension is selected as a filler, and the filler is added to a chromatography column. The crude carotenoid extract of S4 is added to the chromatography column. One or more of petroleum ether, ethanol, and acetone are used as eluents. The eluent is added to the chromatography column, and the petroleum ether eluent is released dropwise at a flow rate of 1-4 drops per second. The eluent is collected. When the first colored component is about to drip out, it is collected in another container to obtain a carotenoid extract.

[0012] As an improvement, in step S2, a mixture of petroleum ether and anhydrous ethanol is used as the extraction agent, and the volume ratio of the petroleum ether to anhydrous ethanol is 1:1.

[0013] As a further improvement, the elution in step S6 is performed in three steps:

[0014] Step 1: Use petroleum ether:ethanol (90:10) as eluent to retain lutein on the chromatography column and elute β-carotene.

[0015] Step 2: Use petroleum ether: ethanol: acetone (75:15:10) as the eluent to elute lutein and retain other components on the chromatography column.

[0016] As a further improvement, in step S3, the product is washed three times with petroleum ether to obtain a filtrate.

[0017] As a further improvement, ultrasonic method is selected as an auxiliary method of organic solvent extraction method, including: the extraction solvent is acetone: petroleum ether, the ratio is 3:7, the liquid-to-solid ratio is 1:5, the ultrasonic power is 320.15W, and the ultrasonic treatment is 20 minutes.

[0018] As a further improvement, ultrasonic method is selected as an auxiliary method of organic solvent extraction method, including: ultrasonic temperature 35°C, ultrasonic time 60min, ascorbic acid dosage 6.5%, ultrasonic frequency 85KHz.

[0019] As a further improvement, the material-liquid ratio of the original sample powder to the extractant in step S2 is 1:15.

[0020] As a further improvement, the material-liquid ratio of the original sample powder to the extractant in step S2 is 1:20.

[0021] As a further improvement, in step S4, 20 mL of distilled water is added.

[0022] As a further improvement, in step S4, 25 mL of distilled water was added.

[0023] The beneficial effects of the present invention are: 1. improving the yield of lutein and β-carotene while maintaining the high purity of the extracted lutein and β-carotene; 2. obtaining high-purity and high-yield β-carotene while ensuring the acquisition of high-purity and high-yield lutein. DETAILED DESCRIPTION

[0024] The following examples further illustrate the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention.

[0025] Determination of the β-carotene determination wavelength: Take a certain amount of β-carotene standard and dilute it with petroleum ether. Perform spectral scanning in the wavelength range of 200-1000nm using a UV-visible spectrophotometer to determine the maximum absorption wavelength of β-carotene, which is used as the β-carotene determination wavelength. The β-carotene determination wavelength includes 450nm.

[0026] Draw the regression equation of the β-carotene standard curve: Accurately weigh 10 mg of β-carotene standard, dilute with petroleum ether and make up to the volume in a 50 mL volumetric flask, accurately pipette 1.00, 2.00, 3.00, 4.00, 5.00, 6.00, 7.00, and 8.00 mL respectively and make up to 10 mL, measure the absorbance at the β-carotene determination wavelength, and draw the regression equation of the standard curve. The regression equation of the standard curve is Y=0.0210X+0.011, and the correlation coefficient is 0.996.

[0027] Calculate the content of β-carotene in carotenoid extract:

[0028] Calculated by the following formula:

[0029] β-carotene content

[0030] Where: C——mass concentration obtained by substituting the absorbance value into the β-carotene standard curve, μg / mL;

[0031] V——constant volume, mL;

[0032] N——dilution multiple;

[0033] m——Mass of Psoralea corylifolia root powder, g.

[0034] Determination of the wavelength of lutein: Take a certain amount of lutein standard and dilute it with petroleum ether. Scan the spectrum with a UV-visible spectrophotometer within the wavelength range of 200-1000nm to determine the maximum absorption wavelength of lutein, which is used as the lutein determination wavelength. The lutein determination wavelength includes 474nm.

[0035] Draw the regression equation of the standard curve of lutein: Accurately weigh 10 mg of lutein standard, dilute with petroleum ether and make up to the volume in a 50 mL volumetric flask, accurately pipette 1.00, 2.00, 3.00, 4.00, 5.00, 6.00, 7.00, and 8.00 mL respectively and make up to 10 mL, measure the absorbance at the lutein determination wavelength, and draw the regression equation of the standard curve. The regression equation of the standard curve is Y=1.48*108X-1.36*105, and the correlation coefficient is 0.9979.

[0036] Calculate the lutein content in carotenoid extract:

[0037] Calculated by the following formula:

[0038] Lutein content =

[0039] Where: C——mass concentration obtained by substituting the absorbance value into the lutein standard curve, μg / mL;

[0040] V——constant volume, mL;

[0041] N——dilution multiple;

[0042] m——Mass of Psoralea corylifolia root powder, g.

[0043] Example 1: S1: The fleshy roots of Glehnia littoralis were dried in a constant temperature oven at 80°C to a constant weight, ground, and pulverized through a 40-mesh sieve to obtain the original sample powder, which was sealed and stored for later use;

[0044] S2: Weigh 10 g of the original sample powder of S1 and place it in a conical flask. Use a mixture of petroleum ether and anhydrous ethanol as the extractant, with a volume ratio of petroleum ether to anhydrous ethanol of 1:1, to obtain a mixed solution with a solid-liquid ratio of the original sample powder to the extractant of 1:15;

[0045] S3: The mixed solution obtained in step S2 was filtered with a suction filtration bottle, and washed three times with petroleum ether to obtain a filtrate;

[0046] S4: Place the filtrate from step S3 in a 250 mL separatory funnel and allow to stand for stratification. Add 20 mL of distilled water, mix well, and allow to stand for stratification. Repeat this process several times until the lower layer is colorless to obtain a crude carotenoid extract.

[0047] S5: Take a 10mm*160mm chromatography column, put a small amount of cotton in the bottom layer of the chromatography column, select silica gel as the filler, add a certain amount of silica gel into the chromatography column, tap the column while adding to make the silica gel evenly deposited in the column and ensure that the upper surface is flat, when it is added to about 10cm, put a small round filter paper on the top of the column, and fix the chromatography column on the iron stand, use a pipette to add the crude carotenoid extract of S4 from the top of the chromatography column to the chromatography column, so that the crude carotenoid extract contacts the inner wall of the chromatography column and slowly spreads along the column wall of the chromatography column. The crude carotenoid extract slowly descends along the inner wall of the chromatography column, so that the entire upper surface is evenly covered by the crude carotenoid extract. At the same time, the pipette should be kept as close to the upper surface as possible to ensure that the crude carotenoid extract enters the chromatography column completely. During the entire column filling process and after the column is filled, a section of eluent liquid column should be maintained on the filler silica gel. Add eluent to the chromatography column, open the piston, and let the eluent release dropwise at a controlled flow rate of 1-4 drops per second. Collect the eluent in a conical flask. When the first colored component is about to drip out, collect it in another conical flask to obtain the carotenoid extract. The elution is carried out in two steps:

[0048] Step 1: Use petroleum ether:ethanol (90:10) as eluent to retain lutein on the chromatography column and elute β-carotene.

[0049] Step 2: Use petroleum ether: ethanol: acetone (75:15:10) as eluent to elute lutein and retain other components on the chromatography column;

[0050] The method also includes selecting an ultrasonic method as an auxiliary method of the organic solvent extraction method, including: the extraction solvent is acetone: petroleum ether, the ratio is 3:7, the liquid-to-material ratio is 1:5, the ultrasonic power is 320.15W, and the ultrasonic treatment is 20 minutes.

[0051] Example 1-1: The difference from Example 1 is that the material-liquid ratio of the original sample powder to the extractant in step S2 is 1:10.

[0052] Example 1-2: The difference from Example 1 is that the material-liquid ratio of the original sample powder to the extractant in step S2 is 1:20.

[0053] Example 1-3: The difference from Example 1 is that in step S4, 15 mL of distilled water is added.

[0054] Example 1-4: The difference from Example 1 is that in step S4, 25 mL of distilled water is added.

[0055] Example 2: Different from Example 1, anhydrous ethanol is used as the extraction agent in step S2.

[0056] Example 3: Different from Example 1, petroleum ether is used as the extraction agent in step S2.

[0057] Example 4: Different from Example 1, step S5 uses ethanol as the first step eluent and acetone as the second step eluent.

[0058] Example 5: Different from Example 1, petroleum ether-neutral alumina suspension is used as the filler in step S5.

[0059] Example 6: Different from Example 1, the conditions for the ultrasonic method are: ultrasonic temperature 35°C, ultrasonic time 60 min, ascorbic acid dosage 6.5%, and ultrasonic frequency 85 KHz.

[0060] Comparative Example 1: Different from Example 1, the experimental method for extracting lutein from marigold granules as described in 3.3, 3.4, and 3.5 of "Study on Ultrasonic Extraction of Lutein and β-Carotene" by Zhang Haixia was used to extract carotenoids from the fleshy roots of yellow ginseng as follows:

[0061] Grind the fleshy root of Rhizoma Ginseng (Psoralea corylifolia) finely with a mortar and pestle, pass it through a 120-mesh sieve, and weigh 20g of the powder into a 500ml round-bottom flask. Add 200ml of a 40% ethanol solution of petroleum ether in ethanol at room temperature, along with ascorbic acid (an antioxidant). Slowly add 30ml of a 10% ethanol solution of potassium hydroxide in ethanol while stirring, and sonicate. After the reaction is complete, filter the filtrate, and adjust the pH to 7.4 with a 2.5mol / ml HCl solution. Distill the filtrate under reduced pressure at 40°C. Remove the distillation flask, and a dark yellow solid of lutein crystals will be suspended in water. Cool to room temperature (natural cooling), filter the suspension, and collect the lutein crystals and dry them under vacuum at no more than 60°C for 24 hours.

[0062] Column Packing: Clamp a 1.2cm x 20cm chromatography column vertically onto an iron stand using a wet packing method. First, add petroleum ether to the column until it reaches a quarter of its volume. Soak a small amount of absorbent cotton with petroleum ether, squeeze out the bubbles, and then use a clean glass rod to push it into the bottom of the column. Be careful not to press too hard, otherwise the elution will be slow or not at all. Mix silica gel and magnesium oxide in a 1:1 ratio and stir with petroleum ether for 2 hours to form a slurry. Pour the filler into the column at a constant rate while opening the tube clamp to allow the petroleum ether to slowly flow out. While the filler is packing, gently tap the column with a soft rubber hose to ensure even packing and dislodge bubbles. It's best to add all the filler at once, otherwise uneven packing and discontinuation may occur. Maintain a constant column of liquid above the filler throughout the entire packing process and after the column is loaded. Otherwise, air will enter the filler, forming bubbles and affecting the separation. Maintain a flow rate of 1 to 4 drops per second.

[0063] Sample addition: Use dry method to add sample, dissolve the sample in an appropriate solvent, mix it with a small amount of adsorbent, evaporate the solvent to make it loose, and add it to the prepared chromatographic column.

[0064] Elution: Usually the type and proportion of the eluent are changed in increasing order according to the elution capacity of the eluent, and the effluent is collected separately. When the components contained in the effluent are significantly reduced or no longer contained, the type and proportion of the eluent are changed.

[0065] Elution is performed in three steps:

[0066] Step 1: Use petroleum ether:ethanol (90:10) to elute the lutein on the chromatography column and elute the β-carotene.

[0067] Step 2: Elution with petroleum ether: ethanol: acetone (75:15:10) to elute lutein and retain other components on the chromatography column.

[0068] The method also includes selecting an ultrasonic method as an auxiliary method, including: ultrasonic temperature 40° C., ultrasonic time 60 minutes, ascorbic acid dosage 7.5%, and ultrasonic frequency 100 KHz.

[0069] Comparative Example 2: Different from Example 1, the experimental method for extracting β-carotene from carrot powder as described in 4.3, 4.4, and 4.5 of "Study on Ultrasonic Extraction of Lutein and β-Carotene" by Zhang Haixia was used to extract carotenoids from the fleshy roots of yellow ginseng as follows:

[0070] Weigh 5.000g of Psoralea corylifolia root powder into a 250ml round-bottom flask and ultrasonically extract with 30ml of a 1.5:1 mixture of chloroform and acetone. Filter the extract and concentrate under reduced pressure at below 40°C. Freeze-dry the resulting solid sample in the dark.

[0071] Column Packing: Clamp a 1.2cm x 20cm chromatography column vertically onto an iron stand using a metal clamp. Use the wet packing method. First, add petroleum ether to the column until it reaches a quarter of the column volume. Soak a small amount of absorbent cotton with petroleum ether, squeeze out the bubbles, and then use a clean glass rod to push it into the bottom of the column. Be careful not to press too tightly, otherwise the liquid will flow out too slowly or not at all during elution. Stir the silica gel filler with petroleum ether for 2 hours to form a slurry. Pour the filler slurry into the column at a uniform rate, while opening the tube clamp to allow the petroleum ether to slowly flow out. During the packing process, gently tap the column with a soft rubber hose to ensure even packing and help dislodge bubbles. It is best to add all the filler at once, otherwise uneven packing and faults may occur. Maintain a liquid column on the filler throughout the entire process and after the column is loaded. Otherwise, air will enter the filler, forming bubbles and affecting the separation effect. Maintain a flow rate of 1 to 4 drops per second.

[0072] Sample addition: Use dry method to add sample, dissolve the sample in an appropriate solvent, mix it with a small amount of adsorbent, evaporate the solvent to make it loose, and add it to the prepared chromatographic column.

[0073] Elution: Usually the type and proportion of the eluent are changed in increasing order according to the elution capacity of the eluent, and the effluent is collected separately. When the components contained in the effluent are significantly reduced or no longer contained, the type and proportion of the eluent are changed.

[0074] Elution is performed in three steps:

[0075] Step 1: Use petroleum ether:acetone (95:5) to elute the lutein on the chromatography column and elute the β-carotene.

[0076] Step 2: Elution with petroleum ether: ethanol: acetone (75:15:10) to elute lutein and retain other components on the chromatography column.

[0077] It also includes selecting an ultrasonic method as an auxiliary method, including: ultrasonic temperature 55°C, ultrasonic time 90min, ultrasonic power 50w, and ultrasonic frequency 100KHz.

[0078] Comparative Example 3: Unlike Example 1, microwave-assisted extraction was used instead of ultrasound-assisted extraction to extract carotenoids from the fleshy roots of yellow ginseng. The specific experimental method was "Study on the Extraction and Properties of Lutein from Marigold" by Xu Xia. The specific steps are as follows:

[0079] Weigh 10.0g of Psoralea corylifolia root powder and perform repeated extractions at a fixed power of 240W and a 1:1 ratio of petroleum ether to acetone (v / v). The first extraction was performed for 45 seconds with a solid-liquid ratio of 1:25. The second extraction was performed for 22.5 seconds each with a solid-liquid ratio of 1:12.5. The filtrate was then vacuum filtered, vacuum concentrated, dried, and the pigment yield was determined.

[0080] Take Example 1, Example 2, Example 3, Example 4, Example 5, Example 6, Control Example 1, Control Example 2, and Control Example 3 as the test group, as shown in the following table

[0081] Table 1 Yield and purity of the main components of carotenoids

[0082]

[0083]

[0084] As can be seen from Table 1, the purity and yield of β-carotene and lutein in Examples 1 and 6 of the present invention are higher than those in Examples 2-3. It can be concluded that the type of extraction agent selected in the present invention has a great influence on the purity and yield of β-carotene and lutein, and the use of a mixed solution of petroleum ether and anhydrous ethanol as the extraction agent for extracting carotenoids from the fleshy roots of Glehnia littoralis is the best.

[0085] The purity and yield of β-carotene and lutein in Examples 1 and 6 of the present invention are higher than those in Example 4. It can be concluded that the type of eluent selected in the present invention has a great influence on the purity and yield of β-carotene and lutein. In the first step, petroleum ether: ethanol (90:10) is used as the eluent, and in the second step, petroleum ether: ethanol: acetone (75:15:10) is used as the eluent for extracting carotenoids from the fleshy roots of yellow ginseng. The best effect is achieved.

[0086] The purity and yield of β-carotene and lutein in Examples 1 and 6 of the present invention are higher than those in Example 5. It can be concluded that the type of filler selected in the present invention has a great influence on the purity and yield of β-carotene and lutein, and silica gel is the best filler for extracting carotenoids from the fleshy roots of Glehnia littoralis.

[0087] The purity and yield of β-carotene and lutein in Example 1 of the present invention were both higher than those in Example 6. This indicates that the parameters selected for the ultrasonic-assisted extraction of the present invention have a significant impact on the purity and yield of β-carotene and lutein. The best results were achieved using an extraction solvent of acetone:petroleum ether in a ratio of 3:7, a liquid-to-solid ratio of 1:5, an ultrasonic power of 320.15 W, and an ultrasonic treatment time of 20 minutes.

[0088] The purity and yield of β-carotene and lutein in Examples 1 and 6 of the present invention were higher than those in Control Examples 1 and 2. This indicates that the ultrasonic-assisted extraction method of the present invention has a synergistic effect with other parameters of the present invention, resulting in higher purity and yield of β-carotene and lutein.

[0089] The purity and yield of β-carotene and lutein in Examples 1 and 6 of the present invention were higher than those in Control Example 3. It can be concluded that the selection of the auxiliary extraction method of the present invention has a significant impact on the purity and yield of β-carotene and lutein, and the ultrasonic-assisted extraction method has a better effect.

[0090] The purity and yield of β-carotene and lutein in Example 1 of the present invention are substantially the same as those in Example 1.1, Example 1.2, Example 1.3, and Example 1.4.

[0091] Although the present invention has been described in detail above using general explanations, specific embodiments, and experiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A method for extracting carotenoids from the fleshy roots of Ginseng Root, characterized in that: include: S1: Dry the fleshy roots of Glehnia littoralis in a constant temperature oven at 80°C to constant weight, grind, and pulverize through a 40-mesh sieve to obtain the original sample powder, which is sealed and stored for later use; S2: Weigh 10 g of the original sample powder of S1 and place it in a conical flask. Use a mixture of petroleum ether and anhydrous ethanol as the extractant, with a volume ratio of petroleum ether to anhydrous ethanol of 1:1, to obtain a mixed solution with a solid-liquid ratio of the original sample powder to the extractant of 1:15; S3: The mixed solution obtained in step S2 was filtered with a suction filtration bottle, and washed three times with petroleum ether to obtain a filtrate; S4: Place the filtrate from step S3 in a 250 mL separatory funnel and allow to stand for separation. Add 20 mL of distilled water, mix well, and allow to stand for separation. Repeat this process several times until the lower layer is colorless to obtain a crude carotenoid extract. S5: Take a 10mm*160mm chromatography column, put a small amount of cotton in the bottom layer of the chromatography column, select silica gel as the filler, add a certain amount of silica gel into the chromatography column, tap the column body while adding to make the silica gel evenly deposited in the column and ensure that the upper surface is flat, when it is added to about 10cm, put a small round filter paper on the top of the column, and fix the chromatography column on the iron stand, use a pipette to add the crude carotenoid extract of S4 from the top of the chromatography column to the chromatography column, so that the crude carotenoid extract contacts the inner wall of the chromatography column and slowly spreads along the column wall to make the crude carotenoid extract The liquid is slowly lowered along the inner wall of the chromatography column so that the entire upper surface is evenly covered with the crude carotenoid extract. At the same time, the pipette should be kept as close to the upper surface as possible to ensure that the crude carotenoid extract enters the chromatography column completely. During the entire column filling process and after the column is filled, a section of eluent liquid column should be kept on the filler silica gel; add eluent to the chromatography column, open the piston, and let the eluent release dropwise at a control flow rate of 1-4 drops per second. Collect the eluent in a conical flask. When the first colored component is about to drip out, collect it in another conical flask to obtain the carotenoid extract. The elution is carried out in two steps: Step 1: Use petroleum ether and ethanol as eluents, petroleum ether: ethanol = 90:10, to retain lutein on the chromatography column and elute β-carotene; Step 2: Use petroleum ether, ethanol and acetone as eluents, petroleum ether: ethanol: acetone = 75:15:10, to elute lutein and retain other components on the chromatography column; The method also includes selecting an ultrasonic method as an auxiliary method of the organic solvent extraction method, including: the extraction solvent is acetone and petroleum ether, acetone: petroleum ether = 3:7; the material-liquid ratio is 1:5; the ultrasonic power is 320.15 W, and the ultrasonic treatment is 20 minutes.

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