Screening method of diketopiperazine alkaloid compounds in dendrobium
By using methanol-aqueous solution extraction method in Dendrobium powder and combined with LC-MS technology to detect electrospray ionization and parent ion scanning mode, the difficulty of screening diketopiperazine alkaloids in Dendrobium in the prior art was solved, and a rapid and accurate screening effect was achieved.
Patent Information
- Application Number
- CN202510278872.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-11
- Publication Date
- 2025-06-06
AI Technical Summary
The prior art is difficult to quickly and accurately screen dikepiperazine alkaloids in Dendrobium, mainly due to the lack of standards, low abundance of mass spectrometry fragments and a large number of interfering fragments.
The target compound was extracted from Dendrobium powder by methanol-aqueous solution extraction method and measured by liquid chromatography-mass spectrometry (LC-MS). Combined with electrospray ionization and precursor ion scanning mode, a specific range of the unborn ion scanning ion fragments and precursor ion scanning is set to identify diketopiperazine alkaloids.
This method can effectively reduce the content of impurities and interfering substances, improve the purity of the compounds and the accuracy of detection, and achieve rapid and accurate screening of diketopiperazine alkaloids in Dendrobium.
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Figure CN120102746A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of rapid screening and relates to a method for screening diketopiperazine alkaloid compounds in dendrobium. Background Art
[0002] Dendrobium belongs to the genus Dendrobium of the Orchidaceae family, and mainly includes species such as Dendrobium officinale, Dendrobium purpurogenum, Dendrobium huoshanense, and Dendrobium chrysotoxum. It has the effects of promoting body fluid and nourishing the stomach, nourishing yin and clearing heat, anti-oxidation, and anti-fatigue. At present, most of the attention paid to the active ingredients in Dendrobium is focused on active ingredients such as polysaccharides, flavonoids, and phenolic acids, while less attention is paid to diketopiperazine alkaloid compounds. The content of diketopiperazine alkaloid compounds in Dendrobium is relatively low, but it has strong biological activities such as anti-tumor, antibacterial, free radical scavenging, and insecticidal. It is often determined by liquid chromatography, but due to the small number of standard products for this type of compound, it is impossible to screen and quantify by external standard method. With the development of mass spectrometry technology, the accurate molecular weight combined with primary mass spectrometry and secondary mass spectrometry fragments is currently a more effective screening method. However, the conventional method of first comparing the accurate molecular weight of the primary mass spectrometry and then comparing the secondary mass spectrometry fragments often results in low abundance of secondary mass spectrometry fragments and more interfering fragments. Therefore, there is a need in this field to establish a method that can quickly and effectively screen diketopiperazine alkaloid compounds in Dendrobium. Summary of the invention
[0003] In view of the problems existing in the prior art, the purpose of the present invention is to provide a screening method for diketopiperazine alkaloid compounds in Dendrobium, so as to solve the existing problems of no corresponding standard, low abundance of mass spectrometry fragments, more interfering fragments, and difficulty in rapid and accurate screening.
[0004] The present invention is achieved through the following technical solutions:
[0005] A method for screening diketopiperazine alkaloid compounds in Dendrobium officinale comprises the following steps:
[0006] S1, adding methanol-water solution to the Dendrobium powder and mixing, and then extracting the supernatant by ultrasonic filtration under ice bath condition;
[0007] S2, the supernatant was measured by LC-MS using electrospray ionization and parent ion scanning mode;
[0008] S3, upon detection, if the daughter ion scanning ion fragments contain 69.07m / z, 196.11m / z or 264.17m / z, and the parent ion scanning range is: 350.00-500.00m / z, the screened compound is determined to be a diketopiperazine alkaloid compound.
[0009] Preferably, the addition ratio of the dendrobium powder to the methanol-water solution is 1 g:5.0 mL.
[0010] Preferably, in the methanol-water solution, the volume ratio of methanol to water is 3-5:7-5.
[0011] Preferably, the conditions for the LC-MS measurement are set as follows: the spray voltage is 3.5-4.5 kV, the capillary temperature is 300-500°C, and the probe heater temperature is 300-500°C.
[0012] Preferably, the determination conditions in S2 are set as follows: mobile phase A is a 0.1% formic acid aqueous solution by mass fraction, and mobile phase B is acetonitrile; the gradient elution program is: the first stage, the time is 0-6.5 minutes, 2%-8% mobile phase A and 98%-92% mobile phase B; the second stage, the time is 6.5-21 minutes, 60%-90% mobile phase A, 40%-10% mobile phase B; the third stage, the time is 21-27 minutes, 2% mobile phase A, 98% mobile phase B;
[0013] The injection volume of the gradient elution was 1 μL, the flow rate was 0.2 ml / min, and the temperature was 40° C.
[0014] Preferably, the mixing in S1 includes first oscillating mixing, then ultrasonic mixing, and finally oscillating mixing; the oscillating mixing time is 5 to 20 minutes, and the oscillating mixing speed is 300 rpm; the ultrasonic mixing time is 30 to 45 minutes, and the ultrasonic mixing power is 80 kHz. The ultrasonic mixing needs to be carried out in an ice bath at a temperature of 15-25°C.
[0015] Compared with the prior art, the present invention has the following beneficial technical effects:
[0016] The present invention provides a method for screening diketopiperazine alkaloid compounds in dendrobium, which can effectively extract target compounds from dendrobium powder through a specific extraction step (ultrasonic filtration extraction under ice bath conditions after mixing methanol-water solution), reduce the content of impurities and interfering substances, and provide a purer sample for subsequent detection. At the same time, LC-MS (liquid chromatography-mass spectrometry) technology is used for determination, combined with electrospray ionization and parent ion scanning mode, the target compound can be detected with high sensitivity and high selectivity, and the accuracy and reliability of screening are improved; the method is particularly targeted at screening diketopiperazine alkaloid compounds, and by setting specific daughter ion scanning ion fragments (69.07m / z, 196.11m / z or 264.17m / z) and parent ion scanning range (350.00-500.00m / z), this type of compound can be accurately identified and screened out, avoiding interference from other non-target compounds, and enhancing the pertinence and effectiveness of screening. The invention does not require complicated pre-treatment steps and standard products of diketopiperazine alkaloid compounds, and can realize rapid screening only through compound separation by liquid chromatography and daughter ion and parent ion scanning by mass spectrometry. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.
[0018] Figure 1 The schematic diagram of the chemical structure of diketopiperazine alkaloid compounds screened in Dendrobium officinale;
[0019] Figure 2 This is a typical secondary fragmentation diagram of diketopiperazine alkaloid compounds of Neoechinulin B in the example;
[0020] Figure 3 This is a typical secondary fragmentation diagram of diketopiperazine alkaloid compounds of Neoechinulin D in the embodiment;
[0021] Figure 4 The typical diketopiperazine alkaloid compound secondary fragmentation diagram of Variecolorin O in the examples;
[0022] Figure 5 The typical diketopiperazine alkaloid compound secondary fragmentation diagram of Variecolorin C in the example;
[0023] Figure 6It is a typical secondary fragmentation diagram of diketopiperazine alkaloid compounds of Arestrictin A in the embodiment;
[0024] Figure 7 The secondary fragmentation diagram of a typical diketopiperazine alkaloid compound of Dehydrovariecolorin L in the embodiment;
[0025] Figure 8 This is a typical diketopiperazine alkaloid compound secondary fragment diagram of Dehydroechinulin in the embodiment;
[0026] Fig. 9 This is a typical secondary fragmentation diagram of diketopiperazine alkaloid compounds of Talathermophilin D in the examples. DETAILED DESCRIPTION
[0027] The present invention is further described in detail below in conjunction with specific embodiments, which are intended to explain the present invention rather than to limit it.
[0028] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.
[0029] The present invention provides a method for screening diketopiperazine alkaloid compounds in dendrobium, comprising the following steps:
[0030] 1) Weigh the Dendrobium officinale powder, add 5.0 mL of methanol-water solution thereto, extract with 80 kHz ultrasonic wave for 30 min at 15-25° C. in an ice bath, and filter to obtain the supernatant;
[0031] 2) The supernatant of Dendrobium officinale was measured by LC-MS, and the electrospray ionization (ESI) and precursor scan modes were used. The measurement conditions were set as follows: the daughter ion scanning ion fragments were: 69.07, 196.11 or 264.17 m / z; the precursor ion scanning range was: 350.00-500.00 m / z, the spray voltage was 3.5-4.5 kV, the capillary temperature was 300-500°C, and the probe heater temperature was 300-500°C.
[0032] 3) After detection, the daughter ion scanning ion fragments contain 69.07, 196.11, 264.17m / z or other related fragments, and the parent ion scanning range is: 350.00-500.00m / z or other related ranges, and the screened compounds can be determined to be diketopiperazine alkaloid compounds.
[0033] In the present invention, the methanol solution is preferably an aqueous solution of methanol; in the methanol solution, the volume ratio of methanol to water is preferably 3-5:7-5, and more preferably 4:6.
[0034] In the present invention, the mixing includes first oscillating mixing, then ultrasonic mixing, and finally oscillating mixing; the oscillating mixing time is 5 to 20 minutes, more preferably 15 minutes, and the oscillating mixing speed is 200-300rpm, more preferably 250rpm; the ultrasonic mixing time is 30 to 45 minutes, more preferably 40 minutes, the ultrasonic mixing power is 80kHz, and the ultrasound needs to be performed under ice bath conditions, 15-25°C, more preferably 20°C.
[0035] In the present invention, in the step 2), the determination conditions are preferably set as follows: mobile phase A is 0.1% formic acid aqueous solution, B is acetonitrile, the elution program is 0-6.5 minutes, 2%-8% A, 98%-92% B; 6.5-21 minutes, 60%-90% A, 40%-10% B; 21-27 minutes, 2% A, 98% B, the injection volume is 1 μL, the flow rate is 0.2 ml / min, and the temperature is 40°C.
[0036] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.
[0037] Example 1-Example 18
[0038] Provided is a method for screening diketopiperazine alkaloid compounds in Dendrobium officinale, comprising the following steps:
[0039] 1) Weigh 1 g of Dendrobium powder, add 5.0 mL of methanol-water solution, extract with 80 kHz ultrasonic wave at 20°C (ice bath) for 40 min, and filter to obtain the supernatant. The volume ratio of methanol to water is 4:6.
[0040] The mixing includes first oscillating mixing, then ultrasonic mixing, and finally oscillating mixing; the oscillating mixing time is 15 minutes, the oscillating mixing speed is 250rpm; the ultrasonic mixing time is 40 minutes, the ultrasonic mixing power is 80kHz, and the ultrasonic mixing needs to be performed under ice bath conditions;
[0041] 2) The supernatant of Dendrobium officinale was measured by LC-MS, and the electrospray ionization (ESI) and precursor scan (Precursor Scan) modes were used for the measurement. The measurement conditions were set as follows: the daughter ion scanning ion fragments are shown in Table 1 for details, the precursor ion scanning range is shown in Table 1 for details, the spray voltage is 4kV, the capillary temperature is 350°C, and the probe heating temperature is 450°C; the mobile phase A is 0.1% formic acid aqueous solution, and the mobile phase B is acetonitrile; the gradient elution program is: the first stage, the time is 0-6.5 minutes, 2%-8% mobile phase A and 98%-92% mobile phase B; the second stage, the time is 6.5-21 minutes, 60%-90% mobile phase A, 40%-10% mobile phase B; the second stage, the time is 21-27 minutes, 2% mobile phase A, 98% mobile phase B; the injection volume of the gradient elution is 1 μL, the flow rate is 0.2 ml / min, and the temperature is 40°C;
[0042] 3) After detection, the daughter ion scanning ion fragments contain 69.07, 196.11, 264.17m / z or other related fragments, and the parent ion scanning range is: 350.00-500.00m / z or other related ranges, and the screened compounds can be determined to be diketopiperazine alkaloid compounds. The compounds screened in each Example 1 to Example 18 in Table 1 can be determined to be diketopiperazine alkaloid compounds, such as Figure 2-Figure 9 The following is a typical secondary fragmentation diagram of diketopiperazine alkaloid compounds. Figure 1 Schematic diagram of the chemical structure of diketopiperazine alkaloid compounds screened in Dendrobium.
[0043] Table 1 is a table of test data of each embodiment 1 to embodiment 18
[0044]
[0045]
[0046] Embodiment 19
[0047] Provided is a method for screening diketopiperazine alkaloid compounds in Dendrobium officinale, comprising the following steps:
[0048] 1) Weigh 1 g of Dendrobium officinale powder, add 5.0 mL of methanol-water solution, extract with 80 kHz ultrasonic wave at 15°C (ice bath) for 30 min, and filter to obtain the supernatant. The volume ratio of methanol to water is 3:7.
[0049] The mixing includes first oscillating mixing, then ultrasonic mixing, and finally oscillating mixing; the oscillating mixing time is 5 minutes, the oscillating mixing speed is 200 rpm, the ultrasonic mixing time is 30 minutes, the ultrasonic mixing power is 80 kHz, and the ultrasonication needs to be carried out under ice bath conditions at 15°C.
[0050] 2) The supernatant of Dendrobium officinale was measured by LC-MS, and the electrospray ionization (ESI) and precursor scan (Precursor Scan) modes were used for the measurement. The measurement conditions were set as follows: the daughter ion scanning ion fragments were: 69.07, 196.11 or 264.17 m / z; the precursor ion scanning range was: 350.00-450.00 m / z, the spray voltage was 3.5 kV, the capillary temperature was 300°C, and the probe heating temperature was 300°C.
[0051] Mobile phase A is 0.1% formic acid aqueous solution, and mobile phase B is acetonitrile; the gradient elution program is: the first stage, the time is 0-6.5 minutes, 2%-8% mobile phase A and 98%-92% mobile phase B; the second stage, the time is 6.5-21 minutes, 60%-90% mobile phase A, 40%-10% mobile phase B; the third stage, the time is 21-27 minutes, 2% mobile phase A, 98% mobile phase B; the injection volume of the gradient elution is 1 μL, the flow rate is 0.2 ml / min, and the temperature is 40°C.
[0052] 3) After detection, the daughter ion scanning ion fragments contain 69.07, 196.11, 264.17m / z or other related fragments, and the parent ion scanning range is: 350.00-450.00m / z or other related ranges, and the screened compounds can be determined to be diketopiperazine alkaloid compounds.
[0053] Embodiment 20
[0054] Provided is a method for screening diketopiperazine alkaloid compounds in Dendrobium officinale, comprising the following steps:
[0055] 1) Weigh 1 g of Dendrobium powder, add 5.0 mL of methanol-water solution, extract with 80 KHz ultrasonic wave at 25°C (ice bath) for 45 min, and filter to obtain the supernatant. The volume ratio of methanol to water is 5:5
[0056] The mixing includes first oscillating mixing, then ultrasonic mixing, and finally oscillating mixing; the oscillating mixing time is 20 minutes, the oscillating mixing speed is 300 rpm, the ultrasonic mixing time is 45 minutes, the ultrasonic mixing power is 80 kHz, and the ultrasonic mixing needs to be carried out under ice bath conditions at 25°C.
[0057] 2) The supernatant of Dendrobium officinale was measured by LC-MS, and the electrospray ionization (ESI) and precursor scan (Precursor Scan) modes were used for the measurement. The measurement conditions were set as follows: the daughter ion scanning ion fragments were: 69.07, 196.11 or 264.17 m / z; the precursor ion scanning range was: 350.00-500.00 m / z, the spray voltage was 4.5 kV, the capillary temperature was 500°C, and the probe heating temperature was 500°C.
[0058] The mobile phase A is a formic acid aqueous solution with a mass fraction of 0.1%, and the mobile phase B is acetonitrile; the gradient elution program is: the first stage, the time is 0-6.5 minutes, 2%-8% mobile phase A and 98%-92% mobile phase B; the second stage, the time is 6.5-21 minutes, 60%-90% mobile phase A, 40%-10% mobile phase B; the third stage, the time is 21-27 minutes, 2% mobile phase A, 98% mobile phase B; the injection volume of the gradient elution is 1 μL, the flow rate is 0.2 ml / min, and the temperature is 40°C.
[0059] 3) After detection, the daughter ion scanning ion fragments contain 69.07, 196.11, 264.17m / z or other related fragments, and the parent ion scanning range is: 350.00-500.00m / z or other related ranges, and the screened compounds can be determined to be diketopiperazine alkaloid compounds.
[0060] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any ordinary technician in the industry can smoothly implement the present invention as shown in the drawings and described above. However, any equivalent changes, modifications and evolutions made by technicians familiar with the profession without departing from the scope of the technical solution of the present invention using the technical content disclosed above are all equivalent embodiments of the present invention. At the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention are still within the protection scope of the technical solution of the present invention.
Claims
1. A method for screening diketopiperazine alkaloid compounds in Dendrobium, characterized in that: The steps include: S1, adding methanol-water solution to the Dendrobium powder and mixing, and then extracting the supernatant by ultrasonic filtration under ice bath condition; S2, the supernatant was measured by LC-MS using electrospray ionization and parent ion scanning mode; S3, upon detection, if the daughter ion scanning ion fragments contain 69.07m / z, 196.11m / z or 264.17m / z, and the parent ion scanning range is: 350.00-500.00m / z, the screened compound is determined to be a diketopiperazine alkaloid compound.
2. The method for screening diketopiperazine alkaloid compounds in Dendrobium according to claim 1, characterized in that: The addition ratio of Dendrobium powder to methanol-water solution was 1 g:5.0 mL.
3. The method for screening diketopiperazine alkaloid compounds in Dendrobium according to claim 1, characterized in that: In the methanol-water solution, the volume ratio of methanol to water is 3-5:7-5.
4. The method for screening diketopiperazine alkaloid compounds in Dendrobium according to claim 1, characterized in that: The conditions for the LC-MS measurement were set as follows: the spray voltage was 3.5-4.5 kV, the capillary temperature was 300-500°C, and the probe heater temperature was 300-500°C.
5. The method for screening diketopiperazine alkaloid compounds in Dendrobium according to claim 1, characterized in that: The determination conditions in S2 are set as follows: mobile phase A is a 0.1% formic acid aqueous solution, and mobile phase B is acetonitrile; the gradient elution program is as follows: the first stage, time is 0-6.5 minutes, 2%-8% mobile phase A and 98%-92% mobile phase B; the second stage, time is 6.5-21 minutes, 60%-90% mobile phase A, 40%-10% mobile phase B; the third stage, time is 21-27 minutes, 2% mobile phase A, 98% mobile phase B; The injection volume of the gradient elution was 1 μL, the flow rate was 0.2 ml / min, and the temperature was 40° C.
6. The method for screening diketopiperazine alkaloid compounds in Dendrobium according to claim 1, characterized in that: The mixing in S1 includes oscillating mixing first, ultrasonic mixing second, and oscillating mixing again last; the oscillating mixing time is 5 to 20 minutes, and the oscillating mixing speed is 300 rpm; the ultrasonic mixing time is 30 to 45 minutes, and the ultrasonic mixing power is 80 kHz. The ultrasonic mixing needs to be carried out under ice bath conditions and the temperature is 15-25°C.