Identification Of Seventeen Components in Cistanches Herba Cultured in Tarim Desert By HPLC-MS
Mar 17, 2023
NAN Ze-dong1 ，ＲEN Hua-zhong1 ，ZHAO Ming-bo2 ，JIANG Yong2 ，TU Peng-fei2*
( 1. Leshan Vocational and Technical College，Leshan 614000，China; 2. State Key Laboratory of Natural and Biomimetic Drugs，School of Pharmaceutical Sciences， Peking University Health Science Center，Beijing 100191，China)
［Abstract］ Objective: To identify the chromatographic components of 60% methanol extract in Cistanches deserticola Herba cultured in Tarim desert by HPLC-MS． Method: HPLC-MS analysis was performed on a Agilent ZOＲBAX Eclipse Plus C18 column ( 4. 6 mm × 250 mm，5 μm) ． The mobile phase was acetonitrile-0. 1% formic acid aqueous solution by gradient elution at the flow rate of 1 mL·min － 1，and the detection wavelength was 254 nm． The column temperature was set at 25 ℃，and the injection volume was 10 μL． Ｒesult: Sixteen chromatographic peaks / seventeen components were accurately identified by comparing the retention time， molecular weights and fragment ions of the compounds we isolated and identified． These chromatographic components were determined as coniferin， demethyl syringin， syringin， echinacoside， cistanoside A， verbascoside ( tubuloside A ) ， cistanoside B， isoverbascoside， 2'-acetylcistanoside A， cistanoside C， isocistanoside C，2'-acetylverbascoside，tubuloside B，epimeridinoside A，cistanoside K and cistanoside J．
Conclusion: The HPLC-MS method is suitable for the identification of the chromatographic components of 60% methanol extract in Cistanches Herba． This study will provide the scientific evidence for comprehensively analyzing the chemical constituents of this medicinal herb．
［Key words］ Cistanches deserticola Herba; Cistanche; HPLC-MS; methanol extract
Cistanche deserticola Y.C.Ma
Cistanche deserticola, also known as "desert ginseng", is a famous tonic Chinese medicine, which is distributed in Inner Mongolia, Ningxia, Gansu, and Xinjiang of China [1]. It has the effect of tonifying kidney-yang, benefiting blood essence, moistening intestines, and defecating. It is commonly used in clinics to treat male impotence, female infertility, cold pain in the waist and knee, blood stasis constipation, etc. [2-3]. Cistanche deserticola ma included in the first part of the Chinese Pharmacopoeia (2015 edition) is the dried fleshy stem of Cistanche deserticola and Cistanche tubulosa (4). Our research team has carried out systematic and in-depth research on Cistanche plants for many years. The chemical components of Cistanche plants are mainly phenylethanoid glycosides, in addition to iridoids and their glycosides, lignans and their glycosides, benzyl alcohol glycosides, etc. [5]. Modern pharmacological studies show that phenyl ethanol glycosides, the main components of the genus, have neuroprotective, antioxidant, liver protection, antibacterial, antiviral, anti-tumor, anti-inflammatory, and other effects [6-11].
Desert ginseng
In China, Cistanche deserticola is distributed in western Inner Mongolia, northern Xinjiang, and Qaidam Basin in Qinghai, but not in Tarim Basin in southern Xinjiang. In the late 1990s, to protect the Tazhong oilfield and control the desert, the oilfield headquarters introduced a large number of Haloxylon ammo dendron around the Tazhong oilfield and on both sides of the desert highway and inoculated the Haloxylon ammo dendron with cistanche, which has been more than 10 years. Due to the warm climate in South Xinjiang and the good drip irrigation conditions in the oil base, the yield of cistanche deserticola is very high, and the yield of fresh cistanche deserticola per mu reaches more than 500kg [8]. Many chemical components in Cistanche deserticola have produced a large number of isomers due to different connection positions of substituents.This brings great difficulties to the identification of the chromatographic peak of this medicinal material. It is difficult to accurately identify the components of the chromatographic peak if only relying on LC-MS without monomer compounds as the control. Therefore, there are few literature reports on this work in the early stage. The author isolated many monomer compounds [9-11] from Cistanche deserticola cultivated in the Tazhong Desert in the early stage. Through these monomer compounds obtained in the early stage, the HPLC partial chromatographic peaks of the 60% methanol extract of Cistanche deserticola were accurately identified. This will not only avoid the repeated identification of some known compounds in the chemical constituents of Cistanche deserticola, but also provide some reference for further research on the quality control of the medicinal material. In this paper, 17 components of 60% methanol extract of Cistanche deserticola were identified by HPLC-MS.
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1. Material
Inova-500 nuclear magnetic resonance instrument (Varian, USA); 6320IonTrap LC-MS mass spectrometer, EclipseXDB-C18 column (4.6mm × 250mm，5 μ m) (Agilent, USA); QUINTIX313-1CN type 1/10000 electronic balance (Sedaris Division); KQ-500DV ultrasonic cleaner (Jiangsu Kunshan Ultrasonic Instrument Co., Ltd.); KMUP - Ⅰ Trace Analytical Ultra Pure Water Generation System (Chengdu Haochun Instrument Equipment Co., Ltd.).
The 39 control substances used in this experiment were all isolated from desert-cultivated Cistanche deserticola in Tazhong by the author in the early stage, and their structures were determined by NMR and other spectroscopy techniques. The structures of 39 control samples were identified as Cistanche deserticoside J, Cistanche deserticoside K, Cistanche deserticoside L, Cistanche deserticoside M, Cistanche deserticoside N, epimeridinosideA, Isocistancheside C, angiocyanin B, Cistanche heterophyllin, Isocornicin, Cistanche deserticoside D, Cistanche deserticoside C, Cistanche cymosin, 2 '- acetyl Cistanche deserticoside A, 2' - acetyl Cistanche deserticoside A, Cistanche deserticola B, Cistanche deserticola A, Echinoside A, Cistanche deserticola glycoside E, Salidroside, 6 '- acetyl salidroside, saliside B, syringin, demethyl syringin, juniperin, (2E, 6E) - 3, 7-dimethyl-8-hydroxyctadien-1-O- β- D-glucoside, (+) - syringin, (+) - syringin-4 '- O- β- D-glucopyranoside, (+) - isoeucomminA, eucomminA, (+) - rosin monomethyl ether- β- D-glucoside, larviciding-4 '- O- β- D-glucoside, larviciding-4-O- β- D-glucoside- β- D glucopyranoside, hesperidin A, area aside A, 6-deoxycatapol. The purity of all the above reference substances is more than 98.0%. Acetonitrile is chromatographically pure (DikmaPure, USA), water is ultrapure water, and other reagents used in the test process are analytical pure reagents, produced by Beijing Chemical Plant.
The medicinal material used in this experiment, Cistanche deserticola, was collected from the desert area of Tazhong, Xinjiang, in November 2010. It was identified by Professor Tu Pengfei of the School of Pharmacy of Peking University as the dried fleshy stem of the Leobaceae plant Cistanche deserticola. The specimen was stored in the Modern Research Center of Traditional Chinese Medicine of Peking University (specimen number: CD201011).
Cistanche deserticola slice
2. Methods and results
2.1 Preparation of reference solution
Take about 1mg of each of the 39 control samples obtained from Cistanche deserticola in the early stage, weigh them accurately, put them into a 5mL volumetric flask respectively, dissolve them with methanol, and volume them to the scale, and then get the single control solution.
2.2 Preparation of test solution
Referring to the Chinese Pharmacopoeia, 2015 edition, take about 1g of the product powder (through the fourth sieve), accurately weigh it, put it into a 100mL brown bottle, precisely add 50mL of 60% methanol, tamp it, shake it well, weigh the mass, soak it for 30min, ultrasonic treatment for 40min, cool it, weigh the mass again, add 50% methanol to make up the reduced mass, shake it well, stand it, take the supernatant, and use 0.45 μ M microporous membrane filtration, take the continuous filtrate to get the test solution.
2.3 Chromatographic conditions
AgilentEclipse XDB-C18 chromatographic column (4.6mm × 250mm，5 μ m), detection wavelength 254nm, column temperature 25 ℃; Using acetonitrile (A) - 0.1% formic acid aqueous solution (B) as the mobile phase, gradient elution (0 ～ 20min, 5% ～ 15% A; 20 ～ 50min, 15% ～ 30% A; 50 ～ 60min, 30% ～ 50% A; 60 ～ 70min, 50% ～ 95% A); Flow rate: 1.0mL · min-1, injection volume: 10 μ L。
2.4 Mass spectrum conditions
Capillary temperature 350 ℃; Capillary voltage 3500V; Jacket gas (nitrogen) flow rate 12.0L · min-1; Auxiliary gas (nitrogen) pressure 206.8kPa; The full scanning range of mass spectrometry is m/z 50~1000; Collision gas helium, collision energy is automatically selected during the test; Collect 1 needle for each positive and negative ion mode.
2.5 Test of a reference substance
The 39 reference solutions prepared under 2.1 were tested under 2.3 chromatographic conditions and 2.4 mass spectrometric conditions, and their retention time and relative molecular weight were recorded (Table 1).
2.6 Determination of test solution
Take the medicinal material Cistanche deserticola collected from the desert area of Tazhong, Xinjiang, and prepare the test solution according to the method described in 2.2. Then, perform the determination according to the chromatographic conditions described in 2.3 and the mass spectrometry conditions described in 2.4, and determine its HPLC chromatogram and mass spectrometry in the positive and negative ion mode (Figures 1-3).
Fig． 1 HPLC chromatogram of 60% methanol extract of Cistanches Herba
Fig． 2 Positive TIC MS chromatogram of 60% methanol extract of Cistanches Herba
Fig． 3 Negative TIC MS chromatogram of 60% methanol extract of Cistanches Herba
2.7 Identification of HPLC chromatogram of medicinal materials
Under the same conditions (2.3 chromatographic conditions and 2.4 mass spectrometry conditions), the components were identified by comparing the retention time and relative molecular weight of the test sample and the control sample. The specific identification process of the chromatogram peak of the test sample (Figure 1) is as follows.
2.7.1 Identification of chromatographic peak 1
The retention time of chromatographic peak 1 is 16.67 minutes. In the positive ion mode, the excimer ion peak m/z365 is given as [M+Na]+, while in the negative ion mode, the excimer ion peak m/z387 [M+COOH] - is given. The relative molecular weight of peak 1 is determined to be 342. The fragment ion m/z179 is also given, which originates from the loss of 1 molecule of glucose 163 Da due to the cleavage of the glycoside bond. According to the retention time and relative molecular weight of the reference substance, chromatographic peak 1 was identified as coniferin.
2.7.2 Identification of chromatographic peak 2
The retention time of chromatographic peak 2 is 17.36min. Under the negative ion mode, the excimer ion peaks m/z357 [M – H] -, 393 [M+Cl] - are given, and the relative molecular weight of peak 2 is determined to be 358. At the same time, the fragment ion m/z195 is derived from the loss of 1 molecule of glucose 163 Da due to the glycoside bond breaking. The chromatographic peak 2 was identified as demethylsyringin according to the retention time and relative molecular weight of the reference substance.
2.7.3 Identification of chromatographic peak 3
The retention time of chromatographic peak 3 is 19.04min, the excimer ion peak m/z395 [M+Na]+is given in the positive ion mode, and the excimer ion peak m/z417 [M+HCOO] - is given in the negative ion mode, and the relative molecular weight of peak 3 is determined to be 372. At the same time, the fragment ion m/z209 is derived from the loss of 1 molecule of glucose 163 Da due to the glycoside bond breaking. The chromatographic peak 3 was identified as syringin according to the retention time and relative molecular weight of the reference substance.
2.7.4 Identification of chromatographic peak 4
The retention time of chromatographic peak 4 is 27.66 min. The excimer ion peak m/z804 [M+NH4]+is given in the positive ion mode, and the excimer ion peak m/z785 [M – H] - is given in the negative ion mode. The relative molecular weight of peak 4 is determined to be 786. At the same time, the fragment ion m/z623 is derived from the ester bond-breaking loss of 1 molecule of caffeine 163 Da. Combined with the retention time and relative molecular weight of the reference substance, chromatographic peak 4 was identified as echinacea.
2.7.5 Identification of chromatographic peak 5
The retention time of chromatographic peak 5 is 31.48min, the excimer ion peak m/z818 [M+NH4]+is given in the positive ion mode, and the excimer ion peak m/z799 [M – H] - is given in the negative ion mode, and the relative molecular weight of peak 5 is determined to be 800. At the same time, the fragment ion m/z637 is derived from the ester bond-breaking loss of 1 molecule of caffeine 163Da. Combining the retention time and relative molecular weight of the reference substance, it was confirmed that chromatographic peak 5 was cistanche glycoside A.
Table 1 Molecular weight and retention time ( tＲ ) of 39 standard compounds
2.7.6 Identification of chromatographic peak 6
The retention time of chromatographic peak 6 is 34.17min. Two excimer ion peaks m/z846642 [M+NH4]+are given in the positive ion mode, and two excimer ion peaks m/z827623 [M – H] - are also given in the negative ion mode. It is speculated that peak 6 may contain two compounds with a relative molecular weight of 828624. At the same time, the fragment ion m/z665461 is derived from the ester bond breaking loss of 1 molecule of caffeine 163 Da, and the fragment ion m/z622 is derived from the acetyl ester bond breaking. These two compounds were isolated and combined with the retention time and relative molecular weight of the reference substance, the chromatographic peak 6 was a mixture of Cimicifugarin and anthocyanin A.
2.7.7 Identification of chromatographic peak 7
The retention time of chromatographic peak 7 is 35.34 min. The excimer ion peak m/z832 [M+NH4]+, 837 [M+Na]+is given in the positive ion mode, and the excimer ion peak m/z813 [M – H] - is given in the negative ion mode, and the relative molecular weight of peak 7 is 814. At the same time, the fragment ion m/z637 is derived from the ester bond-breaking loss of 1 molecule of ferulic acid 177Da. Combined with the retention time and relative molecular weight of the reference substance, chromatographic peak 7 was identified as cistanche glycoside B.
2.7.8 Identification of chromatographic peak 8
The retention time of chromatographic peak 8 is 36.17min, the excimer ion peak m/z647 [M+Na]+is given in the positive ion mode, and the excimer ion peak m/z623 [M – H] - is given in the negative ion mode, and the relative molecular weight of peak 8 is determined to be 624. At the same time, the fragment ion m/z461 is given, which originates from the ester bond breaking and loss of 1 molecule of caffeine group 163 Da. Combined with the retention time and relative molecular weight of the reference substance, chromatographic peak 8 was identified as heterocoenoside.
2.7.9 Identification of chromatographic peak 9
The retention time of chromatographic peak 9 is 38.20 minutes. The excimer ion peaks m/z860 [M+NH4]+, 865 [M+Na]+are given in the positive ion mode, and the excimer ion peaks m/z841 [M – H] -, 877 [M+Cl] - are given in the negative ion mode. The relative molecular weight of peak 9 is determined to be 842. At the same time, the fragment ion m/z679636 is derived from the ester bond-breaking loss of 1 molecule of caffeine 163 Da and 1 molecule of acetyl 43 Da. The retention time and relative molecular weight of the reference substance are consistent, and the identification peak 9 is 2 '- acetyl cistanche glycoside A.
2.7.10 Identification of chromatographic peak 10
The retention time of chromatographic peak 10 is 39.04min. The excimer ion peak m/z656 [M+NH4]+, 661 [M+Na]+is given in the positive ion mode, and the excimer ion peak m/z637 [M – H] -, 673 [M+Cl] - is given in the negative ion mode. The relative molecular weight of peak 10 is determined to be 638. At the same time, the fragment ion m/z475 is derived from the ester bond breaking loss of 1 molecule of caffeine 163 Da. The retention time and relative molecular weight of the reference substance are consistent, and the identification peak 10 is Cistanche C.
2.7.11 Identification of chromatographic peak 11
The retention time of chromatographic peak 11 is 40.82 min. The excimer ion peak m/z661 [M+Na]+, 677 [M+K]+is given in the positive ion mode, and the excimer ion peak m/z637 [M – H] -, 673 [M+Cl] - is given in the negative ion mode. It can be determined that the relative molecular weight of peak 11 is 638. At the same time, the fragment ion m/z475 is derived from the ester bond-breaking loss of 1 molecule of caffeine 163 Da. Combining the retention time and relative molecular weight of the reference substance, chromatographic peak 11 was identified as isocistanche glycoside C.
2.7.12 Identification of chromatographic peak 12
The retention time of chromatographic peak 12 is 41.48 min. The excimer ion peak m/z684 [M+NH4]+, 689 [M+Na]+is given in the positive ion mode, and the excimer ion peak m/z665 [M – H] - is given in the negative ion mode. It can be determined that the relative molecular weight of peak 12 is 666. At the same time, it is shown that the fragment ion m/z503460 comes from the ester bond breaking loss of 1 molecule of caffeine 163 Da and 1 molecule of acetyl 43 Da. According to the retention time and relative molecular weight of the reference substance, chromatographic peak 12 was identified as 2 '- acetyl coeliaoside.
Cistanche deserticola experiment
2.7.13 Identification of chromatographic peak 13
The retention time of chromatographic peak 13 is 44.29min, the excimer ion peak m/z689 [M+Na]+is given in the positive ion mode, and the excimer ion peak m/z665 [M – H] - is given in the negative ion mode, and the relative molecular weight of peak 13 is 666. At the same time, it is shown that the fragment ion m/z503460 comes from the ester bond breaking loss of 1 molecule of caffeine 163 Da and 1 molecule of acetyl 43 Da. Combined with the retention time and relative molecular weight of the reference substance, chromatographic peak 13 was identified as anthocyanin B.
2.7.14 Identification of chromatographic peak 14
The retention time of chromatographic peak 14 is 46.67 min. The excimer ion peak m/z675 [M+Na]+is given in the positive ion mode, and the excimer ion peak m/z651 [M – H] - is given in the negative ion mode. The relative molecular weight of peak 14 is 652. At the same time, the fragment ion m/z475 is derived from the ester bond-breaking loss of 1 molecule of ferulic acid group 177Da. Combined with the retention time and relative molecular weight of the reference substance, chromatographic peak 14 was identified as epimeridinosideA.
2.7.15 Identification of chromatographic peak 15
The retention time of chromatographic peak 15 is 49.90 min. The excimer ion peak m/z698 [M+NH4]+, 703 [M+Na]+is given in the positive ion mode, and the excimer ion peak m/z679 [M – H] -, 715 [M+Cl] - is given in the negative ion mode. The relative molecular weight of peak 15 is 680. At the same time, the fragment ion m/z517474 is derived from the ester bond-breaking loss of 1 molecule of caffeine 163 Da and 1 molecule of acetyl 43 Da. The chromatographic peak 15 was identified as cistanche glycoside K based on the retention time and relative molecular weight of the reference substance.
2.7.16 Identification of chromatographic peak 16
The retention time of chromatographic peak 16 is 55.36 minutes. In the positive ion mode, the excimer ion peak m/z717 [M+Na]+is given, while in the negative ion mode, the excimer ion peaks m/z693 [M – H] -, 729 [M+Cl] - are given, resulting in a relative molecular weight of 694 for peak 16. At the same time, it is shown that the fragment ion m/z517474 comes from the ester bond breaking loss of 1 molecule of ferulic acid group 177 Da and 1 molecule of acetyl group 43 Da. Combined with the retention time and relative molecular weight of the reference substance, chromatographic peak 16 was identified as cistanche glycoside J. The chromatographic peaks of the 60% methanol extract of Cistanche deserticola were identified by HPLC-MS combined with the relative molecular weight, retention time, and fragment ion of the separated reference substance. The results showed that 17 components of the 16 chromatographic peaks were accurately identified (Table 2).
Table 2 Identified chromatographic peaks of Cistanches Herba and reference standard compounds
3 .Discussion
3.1 Selection of test article preparation method
The main purpose of this study is to identify the HPLC peaks of Cistanche deserticola. Therefore, the selection of extraction solvent and extraction method is based on the ability to obtain more HPLC peaks. The research team has carried out long-term research on Cistanche deserticola and has done a lot of work in quality analysis. In this paper, the extraction solvent and extraction method of Cistanche deserticola were also selected based on the previous analysis work of the research group and the Chinese Pharmacopoeia (2015 edition). Finally, 60% methanol solvent ultrasonic extraction for 40 min was selected as the extraction method.
3.2 HPLC-MS condition selection
The selection of chromatographic column in this paper is based on the quality analysis of Cistanche deserticola in the early stage of the research group, and the investigation of AgilentZORBAXEclipsePlusC18 (4.6mm × 250mm，5 μ m)， DionexC18(4.6mm × 250mm，5 μ m)， ThermoC18(4.6mm × 250mm，5 μ m) Chromatographic column, finally select AgilentZORBAXEclipsePlusC18 chromatographic column (4.6mm × 250mm，5 μ m) It has a good separation effect on Cistanche deserticola; The 39 reference substances include phenylethanoid, lignans, and phenylpropanoid, so the detection wavelength is 254nm; At the same time, the effects of mobile phase such as methanol-water, acetonitrile-water, methanol-0.1% phosphoric acid water, acetonitrile-0.1% formic acid water on chromatographic separation and mass spectrometry detection were investigated, and acetonitrile-0.1% formic acid water was finally selected as the mobile phase.
3.3 Summary
The method established in this study accurately identified 17 components from 16 chromatographic peaks of 60% methanol extract of Cistanche deserticola.
Cistanche extract powder
The research results not only have a certain reference value for the further quality analysis of the medicinal material but also can avoid some repetitive work on the structure identification of the known compounds of the medicinal material. However, in general, there are relatively few identified chromatographic peaks, and many chromatographic peaks have not been identified because they have not been assigned to the reference substance. Most identified components are phenylethanoid glycosides, while lignans are rare. This may be because the extraction method referred to in the 2015 edition of the Chinese Pharmacopoeia is mainly aimed at phenylethanoid glycosides, or because the content of echinacoside and coliform-like compounds in the extract of this medicinal material is too high, which makes it difficult to detect other compounds with small content. The specific reasons need to be further clarified.
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