張亦琳 潘高 延永 葛穎 王茜
摘 要:該研究采用ESI-Q-TOF-MS直接進(jìn)樣分析法對陜西商州、洛南、大荔、丹鳳和銅川等五個地區(qū)丹參的化學(xué)成分進(jìn)行了比較分析,通過丹參水溶性和脂溶性成分的質(zhì)譜豐度差異評價了不同地區(qū)丹參化學(xué)成分含量的變化,對不同產(chǎn)地丹參的化學(xué)成分進(jìn)行了鑒定,綜合分析選出了最優(yōu)的丹參種植產(chǎn)地。結(jié)果表明:五地丹參均含有丹參酮ⅡA、隱丹參酮、丹參酮Ⅰ、二氫丹參酮、丹參酚酮、次丹參醌、丹參醌Ⅱ等9種脂溶性化學(xué)成分和丹參素、咖啡酸、阿魏酸、迷迭香酸、原紫草酸、紫草酸、原兒茶醛、丹酚酸A、丹酚酸B等9種水溶性化學(xué)成分,其中丹參素鈉、紫草酸、丹酚酸B和隱丹參酮的含量普遍較高,質(zhì)譜豐度均大于30%。但是,隨著種植環(huán)境不同,不同地區(qū)丹參的化學(xué)成分含量差異很大,如商州產(chǎn)丹參中含有較多具有生物活性的丹參酮類物質(zhì),其中丹參酮Ⅰ含量遠(yuǎn)遠(yuǎn)高于其他四個產(chǎn)地,質(zhì)譜豐度達(dá)到72.6%,而其他地區(qū)丹參酮Ⅰ的質(zhì)譜豐度為1.8%~11.3%。這表明丹參質(zhì)量按照地區(qū)排序為商州>銅川>大荔>洛南>丹鳳。該方法為中藥藥材質(zhì)量評價提供了科學(xué)、可靠、便捷的途徑,為藥材規(guī)格等級的制定提供了新途徑,為陜西丹參種植區(qū)域的選擇提供了重要信息。
關(guān)鍵詞:丹參, ESI-Q-TOF-MS, 化學(xué)成分, 豐度, 藥材質(zhì)量
Alcohol extract:The roots were smashed after fullydried through a warm air (50 ℃), and then sieved through a no. 60 mesh. Each of the fine powdered samples (1.000 0 g) was accurately weighed and extracted with 50 mL ethanol for 30 min using microwave method. After cooling, the extracting solution was filtered through a paper filter and ethanol was removed by reduced pressuredistillation. The evaporated residue wasdissolved in methanol and made up to volume in a 5.0 mL volumetric flask. The solutions were filtered through a membrane filter (0.45 μm) and then injected into the ESI-Q-TOF-MSdirectly.
Water extract:The roots were smashed after fullydried through a warm air (50 ℃), and then sieved through a no. 60 mesh. Each of the fine powdered samples (1.000 0 g) was accurately weighed and extracted with 50 mL water for 3 h in 90 ℃ water bath. After cooling, the extracting solution was centrifuged at 12 000 r·min-1 for 15 min, and then removed water by freezedrying. The crude extract solution was made up in a certain concentration in water with 0.5 mg·mL-1, and filtered through a 0.45 μm membrane filter.
1.3 Instrumentation and analytical conditions
All analytes were performed on a Bruker micrOTOF-Q II ESI-Q-TOF-MS system in tune low method. Sodium formate solution was used as a calibration standard liquid in Enhanced Quadratic model. The sample injection volume was 10.0 μL anddetected in tune_low method from 50 m/z to 3 000 m/z. The capilary parameters were set to 4 000 V for alcohol extract samples and 3 500 V for water extract samples. Nebulizer pressure was set to 0.04 MPa. Carrier gas was nitrogen and helium, which haddry gas flow rate of 4.0 L·min-1 anddry heater at 180 ℃.
Samples ofdanshen, extracted fromdifferent regions in Shaanxi Province, were prepared. A volume of 10.0 μL of filtered solution was injected into the instrumentdirectly and each sample wasdetermined parallelly three times.
1.4data analysis
The chromatographicdata were recorded and processed with a Bruker CompassdataAnalysis 4.0 software. The ion abundance refers to the ion signal strength which was taken as the vertical coordinate in the standard apectrum. The ion abundance at the peak of ionic strength was set to be 100% in specified charge ratio, and the ratio between other ionic peak strength and the maximum peak strength was the ionic abundance at each peak.
A=IImax×100%
A is the ion abundance, I is the ion strength, Imax is the ion strength of the maximum peak.
2 Results and Analysis
ESI-Q-TOF-MS parameters such as fragmentor, spray pressure and capilary voltage which could influence the ion peak of corresponding molecular and the relative abundances were optimized fordetecting to alcohol extract and water extract ofdanshen.? Fig. 1 is a typicaldirect injection ESI-Q-TOF-MS spectrum.
There were altogether 18 known chemical consti-tuents obtained from the fragment ions information which effectively reflect the structures (Fig. 2) ofdanshen constituent.
The spectrums showed concrete MS analysis results and 18 compounds were identified (Table 1). In the positive ion mode, the adduct ion of? [M+Na]+ was the mainly observed peaks. For instance, peak at m/z 317.112 8 corresponded to the molecule of Tanshinone ⅡA with the adduct ion of? [M+Na]+. In the negative ion mode, the adduct ion of? [M-H]- was observed as common fragment peak, for example, peak at m/z 717.154 0 was the molecule of salvianolic acid B. It was nearly uniform in compounds classification amongdanshen samples from five regions, but a greaterdistinction was existed in some specific constituent.
Combined predecessors work (Pan et al., 2002), the special fragmentation of Tanshinone ⅡA as a representative Lipid soluble chemical constituent wasdisplayed in Fig.3. Tanshinone ⅡA lost neutral molecular fragments-CH3, H2O and -CO to form the peak at m/z 303.123 7, 300.103 2 and 289.112 4. Fragment peak at m/z 285.138 3 was obtained not only by losing a H2O molecule from m/z 303.123 7, but also via splitting a molecule -CH3 from the fragment m/z 300.103 2 which can split into fragment m/z 195.044 1 and 272.116 2. In brief, the fragmentation of Tanshinone ⅡA occurred through a loss of neutral small molecular fragments -CH3, -CO, H2O and so on.
Through the abundance analysis, it varied quitedrastically among the 18 chemical constituents from five regions at the same conditions. The reason for the variation might bedue todifferent planting environment.
For lipid soluble chemical constituents ofdanshen from fivedifferent regions (Table 2, Fig. 4), it was obvious that the abundance of some chemical constituents, such as Tanshinone I, Cryptotanshinone,danshenxinkun A anddanshenxinkund, varied greatly with the change ofdanshen planting area.danshen from Shangzhou had overwhelming superiority in the content of Tanshinone I,danshenxinkun A anddanshenxinkund with the abun-
Table 1 Identificationdata of chemical constituents ofdanshen bydirect injection ESI-Q-TOF-MSNote:1. Tanshinone I; 2.? Tanshinone ⅡA; 3. Tanshinone ⅡB; 4. dihydrotanshinone; 5. Cryptotanshinone; 6. danshenxinkun A; 7. danshenxinkund; 8. 2-Isopropyl-8-methylphenanthrene-3,4-dione; 9. 7β-hydroxy-8-13-abietadiene-11,12-dione.
Note:10.danshensu; 11. Caffeic acid; 12.? Ferulic acid; 13.? Rosmarinic acid; 14.? Lithospermic acid; 15. Protocatechuic acid; 16. Prolithospermic acid; 17. Salvianolic acid A; 18.? Salvianolic acid B.
tanshinone with a value of 100%, followed by 63.8% indanfeng.
Compared to the previous,other five lipid soluble chemical constituentsdid not possess bigdifferent gap or obvious abundancedegree. It was alike among the trends among Tanshinone ⅡA, Tanshinone ⅡB,dihydrotanshinone, 2-Isopropyl-8-methylphenanthrene-3,4-dione and 7-β-hydroxy-8-13-abietadiene-11,12-dione. They respectively reached the maximum abundance value of 20.7% (Dali), 7.5% (Dali), 32.1% (Dali), 6.2% (Shangzhou) and 16.7% (Tongchuan).
In brief, the abundance value of Cryptotanshinone was the highest in all samples, the followed wasdanshenxinkun A with a range from 4.0% to 100%, and other lipid soluble constituents also owned good abundance response except for Tanshinone ⅡB and 2-isopropyl-8-methylphenanthrene-3,4-dione. The rank of the content of lipid soluble constituents in five regions was Shangzhou >dali >Tongchuan > Luonan >Danfeng.
For water soluble constituents ofdanshen from fivedifferent regions (Table 2, Fig. 5), the tendency of abundance variation is nearly the same. All samples contain moredanshensu, lithospermic acid, prolithospermic acid and salvianolic acid B than other chemical constituents. The four constituents reached the respective maximum abundance value of 100.0% (Shangzhou & Tongchuan), 100.0% (Luonan), 51.9% (Shangzhou) and 65.1% (Luonan). It was well to be reminded thatdanshensu and lithospermic acid had higher abundance signal response with a value more than 45.0%, and abundance gap (55.2% fordanshensu, 41.6% for lithospermic acid) was obvious from the maximum to the minimum value. In the meantime, ferulic acid, rosmarinic acid and salvianolic acid A showed nearly the common variation tendency with low abundance value. They reached the maximum of 6.6%, 6.5% and 15.9% indanshen from Shangzhou. There was no obviousdistribution indifferent regions.
Salvianolic acid B is important as active medical constituent in clinic.danshen from luonan owned the highest abundance value of 65.1%, and the lowest level was also achieved a abundance value of 34.1% in Shang-zhou.
In brief, the abundance values ofdanshensu and lithospermic acid were the highest in all samples, the followed is salvianolic acid B with a range from 34.1% to 65.1%, and other water soluble constituents also owned good abundance response except for ferulic acid and rosmarinic acid. The rank of the content of water soluble constituents in five regions was Shangzhou>Tongchuan>Luonan>Dali>Danfeng.
3 Conclusion
This paperdeveloped a scientific, reliable and convenient identification method to compare the chemical constituent indanshen fromdifferent regions bydirect injection ESI-Q-TOF-MS. Eighteen chemical constituent were identified in alldanshen samples. The chemical compunds are strongly correlated with geographicaldistinctions and varied quitedrastically at the same conditions. By comprehensive comparison, the quality ofdanshen ranked by region is Shangzhou>Tongchuan>Dali>Luonan>Danfeng. Cryptotanshinone,danshensu, lithospermic acid and salvianolic acid B can help to carry on quality control as potential markers for the evaluation. Meanwhile, the method and results can be a guidance ofdanshen in effective cultivation and medicinal collection in Shaanxi Province.
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