Chinese Medicine Cistanche Chemical Composition And Analysis Of Components In The Body Ⅲ

Chapter 2 Qualitative study on the chemical composition of Cistanche deserticola

Cistanche is the dry scaly leaf fleshy stem of Cistanche deserticola YC.Ma and  Cistanche tubulosa (Schenk) R.Wight. It is one of the famous kidney-yang nourishing traditional Chinese medicines. It has the functions of nourishing kidney-yang, replenishing essence and blood, and moistening It has a laxative effect and is often used to treat impotence, infertility, soreness of waist and knees, weakness of muscles and bones, intestinal dryness and constipation, etc. It has pharmacological effects such as anti-dementia, anti-Parkinson's disease, protection from cerebral ischemia, and improvement of learning and memory abilities. In order to clarify the material basis for the effectiveness of Cistanche deserticola, this chapter uses HPLC and LC-IT-TOF-MS to comprehensively characterize the chemical components of Cistanche. Based on a systematic summary of the cracking rules of various types of chemical components, the properties of Cistanche deserticola are systematically analyzed. Chemical composition, to provide rich data from reliable sources for in-depth elucidation of the medicinal properties of Cistanche deserticola in vivo.

HOW LONG DOES IT TAKE FOR CISTANCHE TO WORK?

Section 1 Experimental Materials

1.Instrument

Shimadzu HPLC-IT-TOF-MS liquid mass spectrometer (equipped with 2 LC-20ADXR pumps, SIL-20AC automatic sampler, CTO-20AC column oven, SPD-M20A UV detector, DGU-20A3R decontamination Gas machine, CBM-20A controller, ESI ion source, Shimadzu Company, Japan), Milli-Q ultrapure water purification system (Milipore Company, USA): Mettler ME204 electronic analytical balance (Mettler Toledo Company, Switzerland).

2. Reagents

Chromatography grade and mass spectrometry grade methanol, acetonitrile and mass spectrometry grade formic acid were purchased from Thermo-Fisher Company (Pittsburgh, PA, USA). The water was Milli-Q ultrapure water made in the laboratory (Millipore, Bedford, MA, USA). The rest Reagents are all of the analytical grade.

Salidroside, 6-deoxycatalpol, 8-epi-loganic acid, echinacoside, acteoside, isoside Verbascoside (isoacteoside,) and pinoresinol-β-D-glucopyranoside (pinoresinol-β-D-glucopyranoside) were both provided by Professor Tu Pengfei's research group at the Key Laboratory of Natural and Biomimetic Drugs at Peking University. Sarcoside F ( cistanosideF) was purchased from Chengdu Mansite Biotechnology Co., Ltd., cistanoside A (cistanoside A), 2'-acetylacteoside (2'-acetylacteoside), 2'-acetylpoliumoside (2'-acetylpoliumoside), cistanoside Polyglucoside (poliumoside), cistanoside E (cistanosideE), isocistanoside E (isocistanosideE), cistanoside C (cistanosideC), isocistanoside C (isocistanosideC) and cistanoside D (cistanosideD) were all purchased from Shanghai Shidande Biotechnology Co., Ltd., tubuloside A (tubulosideA) and tubuloside B (tubulosideB) were purchased from Chengdu Weikeqi Biotechnology Co., Ltd.

Section 2 Experimental content

1. Sample preparation

1.1 Preparation of reference substance

Precisely weigh appropriate amounts of various reference substances in the above "Section 1 Experimental Materials" and prepare 10 mM stock solutions with DMSO. Precisely pipette 2 mL of each stock solution and dilute it with 25% acetonitrile to make a 2 μM mixed control solution. 1.2 Preparation of test solution

The aqueous extract of Cistanche deserticola was made by our laboratory, and the sample is stored in the Modernization Research Center of Traditional Chinese Medicine, School of Traditional Chinese Medicine, Beijing University of Chinese Medicine. Take 20 mg of Cistanche deserticola powder, add 40 mL of water to dissolve, vortex and mix, centrifuge at 10000 rpm for 10 min, and take the supernatant. That is, the test solution is obtained.

3. Mass spectrometry conditions

Electrospray ion source (ESI), the scanning mode is to scan positive and negative ions simultaneously and automatically trigger multi-level mass spectrometry. The mass scanning range of MS'~MS' for positive ions is m/z50~800, and for negative ions is m/z100~1000. The voltages of the spray chamber are 4.5kV and -3.5kV respectively: the atomizing gas (Nz) flow rate is 1.5Lmin; the drying gas pressure is 110MPa; the detection voltage is 1.55 kV; the temperatures of the curved desolventizer tube (CDL) and the heating module are both 200 ℃. The collision chamber pressure is 1.5e-Pa; the ion trap pressure is 2.8e-Pa; the number of repetitions of the positive and negative first stages is 1, and the ion accumulation time is 30 ms; the number of repetitions of the positive and negative second stages is 2, and the ion accumulation time is 20ms. ; Collision-induced dissociation (CID) energy is 50%.

Section 3 Results and Discussion

Column selection

In order to obtain better chromatographic separation, we extensively screened chromatographic columns from different manufacturers and specifications, such as ACEUltraCore 2.5 SuperC8 chromatographic column (3.0x150mm, 2.5 um, Advance Chromatography Technologies Ltd., Aberdeen, Scotland), Shiseido Capcell Core Ci8 ( 2.1 mm x 150mm, 2.7μm), Waters Acquity UPLC HSS T3 column (2.1 x 100 mm, 1.8 μm, Milford, MA, USA) and Capcell Core ADME column (2.1x150mm, 2.7μm, Shiseido Corporation, Japan). Through systematic comparison, it was found that when using the Capcell Core ADME chromatographic column (2.1x150mm, 2.7μm, Shiseido Co., Ltd., Japan), each chromatographic signal can be better separated (Figure 3-1).

2. Selection of liquid phase conditions

Since the main chemical components of Cistanche deserticola are phenylethanol glycosides, lignans, iridoids and other compounds, it generally responds better under negative ion conditions. In order to identify low-level chemical components in the water extract of Cistanche deserticola, it is speculated that acids may need to be added to the mobile phase to enhance the power. Therefore, this experiment used water-methanol, water-acetonitrile, 0.1% formic acid water-methanol and 0.1% formic acid water-acetonitrile as mobile phase systems for investigation. It was found that 0.1% formic acid water-acetonitrile had the best separation effect, with good peak shape and large peak capacity, so 0.1% formic acid water and acetonitrile were selected as the mobile phase. 3. Qualitative analysis of chemical components. Use 19 existing reference substances to explore the mass spectrometry fragmentation rules of various types of compounds in Cistanche deserticola. The mixed reference substances are PDA chromatograms at 340 nm on the ADME column and BPC charts in positive and negative ion modes. See Figure 3-1. We reviewed relevant literature through PubMed, RSC, CNKl, Springer, ScienceDirect, VIP, and Wanfang, etc., and constructed a self-made database of chemical components of Cistanche deserticola. Based on the comparison of mass spectrometry fragmentation rules and retention times of known compounds, we preliminarily identified 45 compounds in Cistanche deserticola. compounds, including 31 phenylethanol glycosides, 9 iridoids, 3 benzyl glycosides, and 2 lignans, as shown in Table 3-1.

By comparing the confidentiality time, MS' spectrum and MS' spectrum of the reference substance, compounds 11, 12, 13, 17, 21, 22, 24, 25, 26, 28, 29, 32, 33, 36, 37, 38 were sequentially Accurately identified as genipin, cistanchein F, cistancheoside E, echinaceaside, chrysantoside, cistanchein A, tuberosin A, larchresinol 4-0-β-D glucoside, mullein Glycosides, isocannabinoid glycoside, pinoresin monomethyl ether glycoside, 2'acetyl verbascoside, cistanche glycoside C, isocistancoside C, tuberosin B, and cistanche glycoside D.

This experiment uses HPLC-IT-TOF-MS technology to quickly and accurately systematically analyze the complex chemical components in the water extract of Cistanche deserticola, which provides a basis for comprehensive quality analysis of Cistanche deserticola and research on pharmacological substances in vivo.

Figure 3-1 Mixed reference substance chromatogram Top: BPC chart in positive ion mode: Bottom: BPC chart in negative ion mode

Figure 3-1 Mixed reference substance chromatogram Top: BPC chart in positive ion mode: Bottom: BPC chart in negative ion mode