Method for green and efficient extraction of strictosamide from nauclea officinalis

Abstract

The invention provides a method for green and efficient extraction of strictosamide in nauclea officinalis, and relates to the technical field of traditional Chinese medicine component extraction. The method comprises the following specific steps: crushing nauclea officinalis into powder, and sieving for later use; preparing lactic acid and deionized water according to a certain proportion to form a lactic acid-water solution system; adding nauclea officinalis powder into a lactic acid-water solution system, performing ultrasonic extraction and high-speed centrifugation, collecting supernate, and adding ethanol for dilution to obtain a crude strictosamide extracting solution; and adsorbing the crude extract of the strictosamide on a column, eluting by using a methanol solution with the volume fraction of 40% and pure methanol in sequence, collecting 100% methanol eluent, filtering, and drying in vacuum to obtain the strictosamide. The lactic acid-aqueous solution system is safe and non-toxic, simple in preparation process, low in price and biodegradable, and the lactic acid-aqueous solution system serving as an extraction solvent for extracting strictosamide from nauclea officinalis is green, environmentally friendly, high in component extraction rate and suitable for large-scale industrial production.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine component extraction technology, and in particular to a green and efficient method for extracting isocinolone lactone from *Gnaphalium affine*. Background Technology

[0002] Gallwood Nauclea officinalis It belongs to the genus *Erythrina* of the family Rubiaceae. Nauclea L. is a perennial woody plant and a precious traditional Chinese medicine with extremely high medicinal value. It has anti-inflammatory effects and can be used to treat colds, fever, sore throat, conjunctivitis, and other ailments, earning it the reputation of "Chinese medicine antibiotic." It has few side effects and high safety, making it extremely promising for clinical application.

[0003] Indole alkaloids are the main type of alkaloids in *Gnaphalium affine*, with isoclavillarin lactam being its main active ingredient. Isoclavillarin lactam is an indole alkaloid glycoside, its English name being strictosamide; its molecular formula and molecular weight are C1. 26 H 30 N2O8; 498.20, possesses various pharmacological effects including antibacterial, antiviral, anti-inflammatory, analgesic, antitussive and antiasthmatic, antitumor, and antimalarial effects. It is the main active ingredient in traditional Chinese medicine preparations made from *Gentiana scabra*, and is present in high concentrations. It is also an indicator component for evaluating the quality of *Gentiana scabra* injections, extract syrups, and capsules. Currently, medicines prepared from *Gentiana scabra* have shown good efficacy in clinical applications.

[0004] The traditional extraction method of vinka lactam at present is usually organic solvent extraction and water extraction method. For example, patent CN101185692A discloses a Nauclea officinalis extract and its preparation and use, wherein (1) the Nauclea officinalis slice xylem, branches, stems, bark or leaves are dried and crushed, and then extracted with an alcohol-water mixed solution or an organic solvent, the extract is combined, refrigerated overnight, filtered, and the alcohol-water mixed solution or the organic solvent is recovered; then added to water or an alkaline solution, stirred, and then adjusted to a pH of 8-4 with an acidic solution, refrigerated, and then filtered, and the filtrate is discarded, and the combined filtrate is obtained. Patent CN110960590A discloses a method for extracting Nauclea officinalis extract, wherein Nauclea officinalis is sequentially extracted with a water solvent with a pH of 2.00-5.00, a water solvent with a pH of 7.00-7.50, and a water solvent with a pH of 8.00-9.00, the extract is combined, concentrated, and Nauclea officinalis extract is obtained. The extraction method in the above patents uses organic solvent extraction, which has certain toxic effects and is easy to cause environmental pollution, has poor biodegradability, is not conducive to the development of environment-friendly development, and in addition, the organic solvent may be left in the extract, which is harmful to the human body. The water extraction method has low extraction efficiency and is not conducive to the dissolution of vinka lactam, which causes waste of Nauclea officinalis resources.

[0005] In addition, the traditional separation, purification and enrichment method of vinka lactam mainly depends on macroporous resin, liquid-liquid extraction, silica gel column chromatography, ODS column chromatography, preparative HPLC and preparative thin layer chromatography. However, these methods have the disadvantages of complicated steps, high energy consumption, and dependence on toxic volatile organic solvents.

[0006] Therefore, it is necessary to develop a high-efficiency green extraction solvent for vinka lactam in Nauclea officinalis, and to find a suitable method for separation, purification and enrichment of vinka lactam, which is very necessary for high-value utilization of vinka lactam. SUMMARY

[0007] Therefore, the present application provides a method for green and efficient extraction of vinka lactam in Nauclea officinalis.

[0008] The technical scheme of the present application is as follows: A method for green and efficient extraction of vinka lactam in Nauclea officinalis, comprising the following specific steps: S1, pretreating Nauclea officinalis: crushing Nauclea officinalis into powder, sieving, and reserving the Nauclea officinalis powder; S2, preparing a lactic acid-water solution system: mixing lactic acid and deionized water to prepare a lactic acid-water solution system; S3, extracting of the vinka lactam component: adding the Nothapodytes pittosporoides powder of S1 into the lactic acid-water solution system of S2, ultrasonic extraction, centrifugation, collecting the supernatant, diluting with ethanol to obtain the vinka lactam crude extract solution; S4, enriching of the vinka lactam: after the vinka lactam crude extract solution of S3 is adsorbed on the column, using the methanol solution with a volume fraction of 40%, pure methanol is used for elution in sequence, and then the pure methanol eluent is collected and filtered to obtain the vinka lactam.

[0009] Further, in step S1, the sieving is sieving through a 50-mesh sieve.

[0010] Further, in step S2, the volume fraction of lactic acid in the lactic acid-water solution system is 10%-90%.

[0011] Further, in step S3, the solid-liquid ratio of the Nothapodytes pittosporoides powder to the lactic acid-water solution system is 1:20-60 g / mL.

[0012] Further, in step S3, the ultrasonic extraction power is 200-600 W, and the time is 5-60 min.

[0013] Further, in step S3, the high-speed centrifugation speed is 8000-12000 rpm, and the time is 5-15 min.

[0014] Further, in step S3, the ethanol is diluted to 2-5 times.

[0015] Further, in step S4, the column is a Sep-Pak C18 cartridges solid-phase extraction column (50 mg, 6 mL), and the Sep-Pak C18 cartridges column is first cleaned with methanol and then cleaned with deionized water for activation.

[0016] Further, in step S4, the filtration is filtration through a 0.22 mu m membrane filter.

[0017] Compared with the prior art, the beneficial effects of the present application are: 1. The raw material of the lactic acid-water solution system of the present application is safe and non-toxic, the preparation process is simple, the price is low, and it is biodegradable, which is used as the extraction solvent for extracting the vinka lactam from Nothapodytes pittosporoides, green and environmentally friendly, high component extraction rate, suitable for large-scale industrial production; avoiding the use of organic solvents in traditional extraction of vinka lactam, which is toxic and volatile, and avoiding the use of deionized water with low extraction rate.

[0018] 2. The present application helps the dissolution and diffusion of intracellular substances in Nauclea officinalis materials through ultrasonic-assisted extraction, greatly shortens the extraction time compared with the traditional heating reflux extraction method, and is simple to operate, and can be used for large-scale industrialized extraction production.

[0019] 3. The present application uses a solid phase extraction column to enrich and purify the vinca alkaloid lactam, has the advantages of simple operation, high recovery rate, high stability, and less solvent consumption, and can realize enrichment, concentration and purification at the same time.

[0020] 4. The method for extracting the vinca alkaloid lactam in Nauclea officinalis in the present application provides a new idea for the extraction of effective components in traditional Chinese medicine resources such as Nauclea officinalis, and has practical significance. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The chromatograms of the vinca alkaloid lactam of Example 1 before and after SPE purification.

[0022] In the figure, a is the chromatogram of the control, b is the chromatogram of the crude extract after SPE purification, and c is the chromatogram of the crude extract after lactic acid-water solution extraction.

[0023] Figure 2 The figure is the effect of the volume fraction of lactic acid in the lactic acid-water solution system on the extraction content of the vinca alkaloid lactam.

[0024] Figure 3 The figure is the effect of the solid-liquid ratio of Nauclea officinalis powder and lactic acid-water solution system on the extraction content of the vinca alkaloid lactam.

[0025] Figure 4 The figure is the effect of ultrasonic extraction power on the extraction content of the vinca alkaloid lactam.

[0026] Figure 5 The figure is the effect of ultrasonic extraction time on the extraction content of the vinca alkaloid lactam.

[0027] Figure 6 The figure is the extraction content of the vinca alkaloid lactam of Example 1, Comparative Examples 1-4. DETAILED DESCRIPTION

[0028] In order to better understand the technical content of the present application, the following specific examples are provided to further illustrate the present application.

[0029] The experimental methods used in the embodiments of the present application are conventional methods unless otherwise specified.

[0030] The materials, reagents, etc. used in the embodiments of the present application can be obtained from commercial channels unless otherwise specified.

[0031] Example 1 A method for efficiently extracting the vinca alkaloid lactam from Nothapodytes pittosporoides, comprising the following steps: S1, pretreating the Nothapodytes pittosporoides: crushing the Nothapodytes pittosporoides into powder, passing through a 50-mesh sieve, and reserving the Nothapodytes pittosporoides powder; S2, preparing a lactic acid-water solution system: mixing lactic acid and deionized water to prepare a lactic acid-water solution system, wherein the volume fraction of lactic acid in the lactic acid-water solution system is 50%; S3, extracting the vinca alkaloid lactam component: adding 0.2 g of the Nothapodytes pittosporoides powder of S1 into 10 mL of the lactic acid-water solution system of S2, performing ultrasonic extraction at 500 W for 30 min, centrifuging at 10,000 rpm for 10 min, collecting the supernatant, and adding ethanol to dilute by 2 times to obtain a vinca alkaloid lactam crude extract; S4, enriching the vinca alkaloid lactam: first washing a Sep-Pak C18 cartridges column (50 mg, 6 mL) with methanol, then washing with deionized water to activate, adsorbing the vinca alkaloid lactam crude extract of S3 on the Sep-Pak C18 cartridges solid-phase extraction column, sequentially eluting with a methanol solution with a volume fraction of 40% and pure methanol, collecting the pure methanol eluate, filtering through a 0.22-μm membrane filter, and obtaining the vinca alkaloid lactam, which is subjected to UPLC-DAD analysis.

[0032] The UPLC-DAD analysis results are shown in Figure 1 .

[0033] The UPLC-DAD analysis results show that the purity of the vinca alkaloid lactam is as high as 97%, and the recovery rate is higher than 90%.

[0034] Example 2 Optimization of experimental conditions 2.1 Optimization of the volume fraction of lactic acid in the lactic acid-water solution system Taking the volume fractions of lactic acid in the lactic acid-water solution system, i.e., 10%, 30%, 50%, 70%, and 90%, as variables, the influence of the volume fraction of lactic acid on the extraction rate of the vinca alkaloid lactam was studied, so as to screen the optimal volume fraction of lactic acid, and the results are shown in Figure 2 .

[0035] From the data in Figure 2 , it can be seen that with the increase of the volume fraction of lactic acid in the lactic acid-water solution system, the content of the vinca alkaloid lactam first increases and then decreases, and the optimal value is 12.37±0.06 mg / g, and the optimal volume fraction of lactic acid in the lactic acid-water solution system is 50%.

[0036] 2.2 Optimization of the solid-liquid ratio of the Nothapodytes pittosporoides powder and the lactic acid-water solution system The effect of the material-liquid ratio (1:20 g / mL, 1:30 g / mL, 1:40 g / mL, 1:50 g / mL, and 1:60 g / mL) on the extraction rate of isovinblastin lactam was studied using *Viburnum macrocephalum* powder and lactic acid-water solution as variables, thereby screening out the optimal material-liquid ratio. The results are as follows: Figure 3 As shown.

[0037] from Figure 3 The data shows that as the ratio of *Eucommia ulmoides* powder to lactic acid-water solution increases, the content of isovincinolactam first increases and then decreases. The optimal ratio of *Eucommia ulmoides* powder to lactic acid-water solution in this invention is 1:50 g / mL.

[0038] 2.3 Power Optimization for Ultrasonic Extraction The effects of ultrasonic extraction power (200W, 300W, 400W, 500W, and 600W) on the extraction rate of isoclavicularyl lactam were studied to screen for the optimal ultrasonic extraction power. The results are as follows: Figure 4 As shown.

[0039] from Figure 4 The data shows that as the ultrasonic extraction power increases, the content of isovinctin lactam first increases and then decreases. The optimal ultrasonic extraction power of this invention is 500W.

[0040] 2.4 Optimization of Ultrasonic Extraction Time The effects of ultrasonic extraction time (5 min, 10 min, 20 min, 30 min, and 60 min) on the extraction rate of isocynoplastin were studied to screen for the optimal ultrasonic extraction time. The results are as follows: Figure 5 As shown.

[0041] from Figure 5 The data shows that as the ultrasonic extraction time increases, the content of isovinctin lactam first increases and then decreases. The optimal ultrasonic extraction time of this invention is 30 min.

[0042] Comparative Example 1 The difference from Example 1 is that a 70% methanol solution was used as the extraction solvent, while the rest is the same as in Example 1.

[0043] The method for extracting isocinolone lactone from *Gnaphalium affine* in this comparative example includes the following specific steps: S1. Pre-treatment of *Gnaphalium affine*: Crush *Gnaphalium affine* into powder, pass through a 50-mesh sieve, and set aside the powder. S2, extraction of the vinca alkaloid lactam component: 0.2 g of the Nothapodytes pittosporoides powder of S1 was added into 10 mL of methanol solution with a volume fraction of 70%, and ultrasonic extraction was performed at 500 W for 30 min. Centrifugation was performed at 10,000 rpm for 10 min, the supernatant was collected, and ethanol was added for dilution by 2 times to obtain a crude vinca alkaloid lactam extract. Filtration was performed through a 0.22 μm membrane filter, and UPLC-DAD analysis was performed.

[0044] Comparative Example 2 The difference from Example 1 is that ethanol solution with a volume fraction of 70% was used as the extraction solvent, and the other conditions were the same as those in Example 1.

[0045] That is, the method for extracting the vinca alkaloid lactam in Nothapodytes pittosporoides in the present comparative example, and the specific steps include: S1, pretreatment of Nothapodytes pittosporoides: the Nothapodytes pittosporoides was crushed into powder, and the powder was sieved through a 50-mesh sieve. The powder was used as needed. S2, extraction of the vinca alkaloid lactam component: 0.2 g of the Nothapodytes pittosporoides powder of S1 was added into 10 mL of methanol solution with a volume fraction of 70%, and ultrasonic extraction was performed at 500 W for 30 min. Centrifugation was performed at 10,000 rpm for 10 min, the supernatant was collected, and ethanol was added for dilution by 2 times to obtain a crude vinca alkaloid lactam extract. Filtration was performed through a 0.22 μm membrane filter, and UPLC-DAD analysis was performed.

[0046] Comparative Example 3 The difference from Example 1 is that deionized water was used as the extraction solvent, and the other conditions were the same as those in Example 1.

[0047] That is, the method for extracting the vinca alkaloid lactam in Nothapodytes pittosporoides in the present comparative example, and the specific steps include: S1, pretreatment of Nothapodytes pittosporoides: the Nothapodytes pittosporoides was crushed into powder, and the powder was sieved through a 50-mesh sieve. The powder was used as needed. S2, extraction of the vinca alkaloid lactam component: 0.2 g of the Nothapodytes pittosporoides powder of S1 was added into 10 mL of methanol solution with a volume fraction of 70%, and ultrasonic extraction was performed at 500 W for 30 min. Centrifugation was performed at 10,000 rpm for 10 min, the supernatant was collected, and ethanol was added for dilution by 2 times to obtain a crude vinca alkaloid lactam extract. Filtration was performed through a 0.22 μm membrane filter, and UPLC-DAD analysis was performed.

[0048] Comparative Example 4 The difference from Example 1 is that lactic acid with a volume fraction of 100% was used as the extraction solvent, and the other conditions were the same as those in Example 1.

[0049] That is, the method for extracting the vinca alkaloid lactam in Nothapodytes pittosporoides in the present comparative example, and the specific steps include: S1, pretreatment of Nothapodytes pittosporoides: the Nothapodytes pittosporoides was crushed into powder, and the powder was sieved through a 50-mesh sieve. The powder was used as needed. S2, extraction of isocinolone lactam: 0.2g of S1 ginseng powder was added to 10mL of 100% lactic acid and ultrasonically extracted at 500W for 30min. After centrifugation at 10000rpm for 10min, the supernatant was collected and diluted twice with ethanol to obtain crude isocinolone lactam extract. The extract was filtered through a 0.22μm membrane filter and analyzed by UPLC-DAD.

[0050] The results are as follows Figure 6 As shown.

[0051] from Figure 6 It can be seen that the isoclavicularin lactam content obtained in Example 1 of this invention using a lactic acid-water solution system as the extraction solvent is significantly higher than that obtained in Comparative Example 3 using deionized water as the extraction solvent. It is also higher than the isoclavicularin lactam content obtained in Comparative Example 4 using pure lactic acid as the extraction solvent. Although the isoclavicularin lactam content obtained in Comparative Example 1 using 70% methanol and Comparative Example 2 using 70% ethanol as the extraction solvent differs slightly from that of this invention, organic solvents are highly toxic and volatile, which does not conform to the concept of green environmental protection.

[0052] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for efficiently extracting the isovincadifficine lactam from Nothapodytes pittosporoides, characterized by, The specific steps include: S1, pretreating Nauclea officinalis: crushing Nauclea officinalis into powder, sieving, and reserving Nauclea officinalis powder; S2, preparing lactic acid-water solution system: mixing lactic acid and deionized water to prepare lactic acid-water solution system; S3, extracting vinka lactam component: adding Nauclea officinalis powder of S1 into lactic acid-water solution system of S2, ultrasonic extraction, centrifugation, collecting supernatant, diluting with ethanol, and obtaining vinka lactam crude extract; S4, enriching vinka lactam: after column adsorption of vinka lactam crude extract of S3, eluting with 40% methanol solution and pure methanol in sequence, collecting pure methanol eluent, filtering, and obtaining vinka lactam.

2. The method for efficiently extracting isovincadifficine lactam from Nothapodytes pittosporoides according to claim 1, wherein, In step S1, the sieving is through 50 mesh sieve.

3. The method of claim 1, wherein the green and efficient extraction of the seco- vinca alkaloid lactam from Nothapodytes pittosporoides is characterized by, In step S2, the volume fraction of lactic acid in lactic acid-water solution system is 10%-90%.

4. The method for efficiently extracting isovincadifficine lactam from Nothapodytes pittosporoides according to claim 1, wherein, In step S3, the solid-liquid ratio of Nauclea officinalis powder to lactic acid-water solution system is 1:20-60 g / mL.

5. The method of claim 1, wherein the green and efficient extraction of the seco-longiflorin from the Nothapodytes pithecellobium is characterized by, In step S3, the ultrasonic extraction power is 200-600 W, and the time is 5-60 min.

6. The method of claim 1, wherein the green and efficient extraction of the seco-longiflorin from the Nothapodytes pithecellobium is characterized by, In step S3, the high-speed centrifugation speed is 8000-12000 rpm, and the time is 5-15 min.

7. The method of claim 1, wherein the green and efficient extraction of the seco- vinca alkaloid lactam from Nothapodytes pittosporoides is characterized by, In step S3, the ethanol dilution is 2-5 times.

8. The method of claim 1, wherein the green and efficient extraction of the seco- vinca alkaloid lactam from Nothapodytes pittosporoides is characterized by, In step S4, the column is Sep-Pak C18 cartridges solid phase extraction column (50 mg, 6 mL), which is first cleaned with methanol and then with deionized water for activation.

9. The method of claim 1, wherein the green and efficient extraction of the seco-loganic acid of the vinca alkaloid from the Nothapodytes pittosporoides is characterized by, In step S4, the filtering is through 0.22 μm membrane filter.

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