CC BY
17Rapid separation and identification analysis of Tilia amurensis
by fast HPLC-MS
Weiwei Ma
College of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin 150040, Heilongjiang
Province, China
Abstract
The flowers of Tilia amurensis (FTA) have been widely used in traditional Chinese medicine to relieve sleeplessness, headache, and nervous excitement. However, the chemical components are still not completely elucidated up to now. In this study, a rapid, sensitive and selective HPLC-MS has been developed for rapid separation and structural identification of constituents in FTA). The analysis was carried out on an AcQuityTM HPLC chromatographic
instrument and a mass spectrometer using positive and negative electrospray ionization.Using a fast HPLC system with an Acquity HPLC BEH Ci8 column, the total analysis time for this complex herb is less than 30 min, which used a column with 1.7 ^m particle packing which enabled higher speed of analysis, peak capacity, greater resolution and increased sensitivity. With various fragmentor voltages in MS, accurate mass measurements (less than 5 ppm error)
for molecular ions and characteristic fragment ions could represent reliable identification criteria for these compounds. The constituents of FTA were identified or tentatively characterized based on their retention time, mass fragmentation behaviors, MS/MS fragment ions, literature reports and the establishment of an in-house molecular formula database.
With this method, a total of 25 compounds of FTA were tentatively identified based on MS data and comparison with available databases. In conclusion, the fast HPLC with MS is a highly useful and efficient technique to separate and identify constituents in complex matrices of herbal medicines.
Keywords:
Astragali Radix; HPLC-MS; constituents; identification; traditional Chinese Medicine
1. Introduction
Tilia amurensis(Tiliaceae), is a tree with smooth cortex and a height of nearly 18m. The aerial
parts of this species have been used in traditional Chinese medicine (TCM) for treating nervous
disorders, insomnia, and headache. The flowers of Tilia amurensis (FTA), one of the most
commonly used traditional Chinese medicine (TCM), are used to treat colon spasm, menstrual
irregularities, as an emollient, and for rheumatism [1,2]. Pharmacological studies indicate that FTA has various bioactivities, such as hypotensive and anxiolytic effect, promoting the motility of human spermatozoa, protecting the myocardium in diabetic nephropathy, enhancing cardiovascular function, antiaging, hepatoprotective effect, inhibiting sterol biosynthesis, and antibacterial [3].
Clinical research shows that FTA can improve cardiovascular function, restore and strengthen immune response, and enhance vitality [4]. Some pharmacological reports concerning the biological properties of FTA indicated that the n-butanol extracts of this species induced an anxiolytic effect. To the best of our knowledge, however, the chemical components are still not completely elucidated up to now. It is widely accepted that a vast number of chemical constituents existing in TCM that
are responsible for the therapeutic effects, become challenging and the major obstacle for the further pharmacological investigation. Many analytical methods have been successfully applied in
analysis of TCM [5-8]. HPLC-MS could provide the accurate mass measurements and formulae of non-target compounds, and identify non-target compounds tentatively[9]. Recently, HPLC-MS have been playing an important role and demonstrated its great advantages for structural analysis of multiple-constituent in herbs with high sensitivity, short time and low consumption of samples [10-
13]. In the present study, a robust HPLC-MS system and BEH Ci8 column with a 1.7 ^m particle size was utilized for thorough analysis of the famous AR. To our knowledge, this is the first study on AR using hyphenated HPLC-MStechnology, and could provide a methodology for the identification of multiple-constituent of Chinese herb medicines.
2. Experimental
2.1 Chemicals and materials
Methanol and acetonitrile of HPLC grade were obtained from Tedia (Fairfield, OH, USA). formic acid was of FTA grade and from the Shanghai Reagent Company (Shanghai, China). Water for HPLC analysis was purified by the Millipore water purification system (Millipore, Milford, MA, USA) and filtered with 0.22 ^m membranes. Distilled water was used for the extract and for the preparation of samples. All the reference compounds were purchased from
the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China).
2.2. Plant material
FTA were collected in campus of Northeast Forestry University, China. Plant material was identified by Prof. LiqiangMu, Northeast Forestry University. Voucher specimens were stored at this site for future reference. Material wasselected and dried under dark conditions at room temperature for 1 weeks. Dry material was milled in an electricgrinder, obtaining particles of <3mm.
2.3. Sample preparation for HPLC-MS analysis Extracts preparation
The dried and milled material (50 g) was initially macerated with ethanol (500ml) for 10 h. and then reflux extractionfor 2h(two times). The extract was concentrated under low pressure at 40 °C. After drying, the plant material wasextracted with n-butanol (3*1L). Finally, the extract was vacuum-concentrated to produce the n-butanol extract.Samples of the n-butanol extract (3 g) were extracted with 95% EtOH (50 mL) by ultrasonication for 30 min under
ambient temperature, and centrifuged at 3000 rpm for 10min. The supernatant 10 ^L was filtered through a 0.45 ^mmembrane before injection and was injected to HPLC-MS for the determination of multiple-constituent in herbs.
2.4 Apparatus and chromatographic conditions
Chromatographic separation was performed using an AcQuity™ Liquid Chromatograph system (Milford, MA,U.S.A.), equipped with a binary solvent delivery system, an auto-sampler, diode-array detector. An Acquity HPLCBEH C18 column (1.7m m, 2.1*100 mm) together with an Acquity HPLC BEH C18 column guard column (Milford,MA, U.S.A.) were used to separate the components. The column temperature was kept constant at 40 C. The mobilephase consisted of 0.1% formic acid water (A) and 0.1% formic acid acetonitrile (B) using a gradient elution of 0-1min, 10-20% B; 1-2.5 min, 20-100% B; 2.5-4.5 min, 100-10% B; 4.5-7 min, 10% B. The injection volume was 5^L and the flow rate of the LC system was 0.5 mL/min. The eluent was introduced to the mass spectrometry directly,i.e. without a split, and the re-equilibration time of gradient elution was 1 min.
2.5 MS condition
HPLC system equipped with an ESI ion source operating in both positive and negative ion mode. The full-scan datawere acquired from 50 to 1000Da with a 0.3 s scan time, using a capillary voltage of 2.0 kV, desolvation temperatureof 300 C, sample cone voltage of 30V, extraction cone voltage of 8V, source temperature of 120 C, cone gas flow of55 L/h and desolvation gas flow of 400 L/h, collision energy of 20-50 eV, collision gas pressure of ~1.6*10- mbarargon. Data were centroided and reference mass correction on each sample was performed with a continuous infusionof a 0.1 ng/mL solution of leucine enkephalin at a flow rate of 50^lmin-1, generating a reference ion for negative ionmode ([M-H]- = 554.2614) to ensure accuracy during the MS
analysis. The data were collected in the centroid mode,and the LockSpray frequency set at 10s and averaged over 10 scans for correction.
2.6 Establishment of an in-house molecular formula database
By comprehensively searching such databases as Chemspider, PubMed of the U.S. National Library Medicine and theNational Institutes of Health, and, Pubchem compound an in-house molecular formula database of all compoundsreported in the literatures on AR and its related products was established by use of MS software which includes thecompound name, molecular formula, accurate molecular mass, chemical structure and literature citations of eachpublished known compound.
3. Results and discussion
3.1 Optimization of HPLC and MS conditions
HPLC and MS conditions were optimized to obtain better detection. HPLC chromatograms with good separation,different mobile phase compositions were screened and found that methanol was the most suitable eluting solventsystem. Considering sensitivity and resolution, the ultimate flow rate was optimized at 0.5 mL/min. Furthermore, theliquid chromatography column temperature was set at 40°Cto reduce the column pressure resulting from a higherflow rate. To acquire maximum sensitivity for most constituents, TOF/MS parameters such as desolvationtemperature, source temperature, capillary voltage, and desolvation gas flow were optimized by flow injectionanalysis. The experiments were performed: desolvation temperature (200C, 250C, 300C), source temperature(100C, 120C, 130C), capillary voltage (1.5 kV, 2.0 kV, 2.5 kV), desolvation gas flow (300L h-1, 4000 L h-1, 600
L h-1), and the peak area was taken as criteria for optimization. The optimum conditions were decided as follows:desolvation temperature 3000C, source temperature 120C, desolvation gas (N2) 400 L h-1, capillary voltage 2.0kV.
3.2 HPLC-MS analysis of AR
The developed HPLC-MS method was applied to analyze and identify the chemical components of FTA. The total ioncurrent chromatogram in negative ESI mode are shown in Fig.1 and the constituents in FTA were well separatedusing the developed HPLC method. 31 peaks were detected and 25 were identified by comparing the tR values, andthe MS fragments characteristics of the compounds as well as MS the fragments behaviors (Table 1) by HPLC- MSwith Masslynx™ in positive mode. For example, the information for mass spectra of quinic acid in the negative ionmode. The MS/MS of m/z showed fragments including m/z 173.0442, 127.0399, 93.0321, and 85.0288 ions, whichenabled us to discover more about the molecular weights and molecular formulae of the compounds. Differentcomponents could be determined by their specific molecular ions and fragment ions, along with theirchromatographic retention times, other compounds in positive were preliminarily identified. These results providedreliable information for confirming molecular weight and structure of the constituents. The 25 compounds whichcontained flavones, saponins, and terpene glycosides were tentatively characterized by matching the in-house formuladatabase within an error of 5 ppm. Compared with the previous publication [14], this report detected morecomponents and revealed the presence of many; the chemical information and pharmacological effect of theseconstituents require further investigation.Based on the complexity of the chemical constitutions in TCM, multi-components analysis used for the qualitycontrol of TCMs is more scientific and reasonable [15]. However, routine quality control of TCM bymulti-components analysis is limited for the shortage of various chemical reference substances. HPLC-MS canprovide a huge amountof information more rapidly and efficiently than other techniques [16]. High selectivity andsensitivity, and rapid characteristics have allowed the wide application of HPLC-MS for quantitative and qualitativeanalysis, as well as metabolite analysis and identification from bioassays of complex samples such as TCM [17].HPLC with minor particles (sub 1.7 ^m) have greatly improved the resolution, sensitivity and analytical speed,integrating with MS to form a new highperformance scientific platform for TCM research . Pharmacologicalstudies have demonstrated
FTA have cardioprotective, hepatoprotective, hypotensive, immunostimulant, anti-ageing,antioxidant, anti-diabetic, and anti-inflammatory activities [19]. It has been widely used in China and East Asia areafor many years to treat myocardial ischemia, liver fibrosis, chronic nephritis, diabetes, etc [20,21]. Although the herbis commonly used in treating various diseases in Chinese herbal formula, scientific reports on its major constituentsare limited. 4. Conclusion
A rapid and robust HPLC-MS method was developed to separate and determinate the constituents of FTA, awell-known TCM herb that has been used clinically in China for centuries to cure various diseases. Herein, thedeveloped method was simple, reliable and sensitive, which revealed that HPLC-MS could separate the complexconstituents in a shorter time and be appropriate for rapid analysis and identification of main components in FTA. The
results showed that a total of 25 compounds were characterized tentatively using HPLC-MS and database-matchingtechniques. It is concluded that a rapid and robust platform based on HPLC-MS was established, which is useful foridentifying multiple-constituent of TCM. This identification and structural elucidation of the chemical constituentsprovided essential data for further pharmacological studies of FTA. Furthermore, with the introduction of thehyphenated HPLC-MS analytical system to the TCM, this will provide a type of validated rapid and higherthroughput methodology for the identification of constituents for Chinese herbs; we expected that approach would beuseful for the screening and characterization of compounds in other famous herbs. References
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1. TOF MS ES-
l "i " | " I T 'I-1 I I I I | I I I I | I I I I | I I I I | I I I I | I I I I | I I i I | I I I I | 1 f I l ï 'I ¡'I l'I'l'l I I | 'I
1.00 2.00 3.00 4.00 5.00 6.00 7.00
Fig.1. The HPLC/MS total ion chromatogram in negative ESI mode; Peak numbers are consistent with those in table1.
Table 1. MS/MS data of compounds detected in Tilia amurensis by HPLC-MS with MasslynxTM in negative mode.
No. ifi(min) MW(Da) Fragmentions Identification Formula
1 0.596 191.0533 173.0442, 127.0399, 93.0321, 85.0288 Quinic acid C7H12O6
2 1.557 169.0058 125.0191 Gallic acid CvH6O5
3 1.689 165.0384 150.0182, 122.0361 -
4 2.078 305.0672 261.0738, 219.0677, 179.0302, Leucocyanidin C15H14O7
165.0067, 125.0278
5 2.178 153.0125 109.0262 Protocatechuic C7H6O4
10
11 12
13
2.52
2.678
2.767
2.985
3.117
3.271 3.295
3.395
14 3.627
577.1427
577.1292
865.1959
577.1272
4,011,429
289.0605 865.21
1153.2677
639.1456
451.1003, 339.0894, 161.0224, 451.1059, 289.0681, 125.0234 739.1699, 577.1375, 407.0751, 245.0738, 451.1025, 289.0673, 125.0232 269.0941, 101.0206 245.0782,
425.0890, 407.0744, 289.0711, 245.0795, 125.0240
425.0888, 407.0744, 245.0738, 161.0237,
713.1597, 695.1454, 451.1024, 425.0940, 289.0682, 287.0539, 161.0220, 125.0225 425.0384, 407.0704, 245.0759, 161.0261,
161.0390, 113.0173, 203.0693
739.1655, 713.1503, 695.1381, 577.1341, 451.1050, 425.0860, 407.0757, 289.0692, 287.0561, 245.0525, 161.0221, 125.0227 1027.2378, 1001.2226, 983.1995, 865.2052, 739.1766, 695.1248, 577.1368, 575.1211, 451.1025, 425.0871, 407.0790, 289.0687, 287.0549, 245.0453, 161.0235, 125.0203
314.0399, 299.0124, 271.0228, 255.0301
acid
Procyanidin dimers
Procyanidin dimers
Procyanidin dimers
Procyanidin dimers
Apigenin
arabinoside
Procyanidin
Procyanidin dimers
Procyanidin dimers
C30H26O12
C30H26O12
C45H38O18
C30H26O12
C20H18O9
C15H14O6
C45H38O18
C60H50O24
Isorhamnetin-3,7- C28H32Oi7 O-2-P-D-glucoside
6
7
8
9
15 3.778 785.2131 314.0437, 299.0175, 289.0605,
271.0250
16 3.857 609.1324 300.0197, 271.0192, 255.0255,
179,151
17 4.007 463.08 300.0179, 271.0181, 255.0263,
243.0273, 179.9672, 151.0017
18 4.207 593.1414 285.0370, 255.0286, 227.0363
19 4.245 447.0844 284.0274, 255.0273, 227.0289
20 4.378 447.084 284.0250, 255.0227, 227.0295
Isorhamnetin3,7-
O-2-P-D-glucose-
4'-O-a-L-
rhamnosidase
Rutin
Isoquercitrin
C41H70O14
C27H30O16
C21H20O12
Tiliroside C30H26O13
Astragalin C21H20O11
Kaempferol-7-O-P- C21H20O11 D-glucopyranoside
21 4.488 477.0616 301.0258
Quercetin-3-O-
C21H18O13
glucuronic acid glycosiders
22 4.745 405.2175 225.1488, 179.0974 - -
23 4.799 373.189 193.1218 - -
24 4.927 461.0755 285.038 Kaempferol-3- C21H18O12
beta-O-
glucuronide
25 5.149 338.1955 130.0827 - -
26 5.298 481.17 315.0763, 152.0110, 108.0152 - -
27 5.467 521.2597 503.2419, 389.2181, 371.2088, Alangionoside B C24H42O12
227.1548
28 5.546 301.0394 503.2419, 389.2181, 371.2088, Quercetin C15H10O7
227.1548
29 5.867 389.2169 371.2112, 227.1644, 209.1576, Rehmaionoside A C19H34O8
181.1403
30 6.399 299.0531 284.0296, 255.0330, 227.0333 3-methyl C16H12O6
keampferol
31 7.078 373.1759 355.2752, 275.2171, 96.9665, - -
79.9550
Study on the Mechanism of Sleep Improving Function of Root of Tall Oplopanax Effective Parts Based on the Regulation Mechanism of Cytokine-
theory
Yu Shuang Li Tingli Huang Lili
(College of Pharmacy of Heilongjiang University of Chinese Medicine, Harbin, China)
Abstracts: This experiment based on cytokine regulating mechanism to explore the mechani sm of sleep improving function of the effective parts of Root of Tall Oplopanax. In experiment, mice of ICR specie are chose, and enzyme-linked immunoassay method is applied to determine the sleep related cytokine content in ICR mice serum and brain and to research on the sleep improving mechanism of the effective parts of Root of Tall Oplopanax(64g/kg).
The results show that the mice, after have been given the effective parts of Root of Tall Oplopanax(64 g/kg) for 7 days, express an obvious increase of IL - 1 beta (p < 0.05) and TNF alpha (p < 0.01) content in hypothalamus. An increase of TNF alpha (p < 0.05) and IL - 4 (p < 0.05) in pallium, a significant decrease of IL-10 level in hypothalamus (p<0.01) and a decrease of IL-4 (p<0.05), IL-10 (p<0.05) level in serum are also found. The experimental results prove that the sleep improving function of the effective parts of Root of Tall Oplopanax is associated with cytokines levels in the brain. It plays a role of sleep improving by increasing the content of sleeping cytokines (IL-ip* TNF-a), and reducing the content of sleep inhibiting cytokines (IL-4* IL-10).
Key words: Root of Tall Oplopanax; Sleep; Cytokines
