Tissues-based chemical profiling and semi-quantitative analysis of bioactive components in the root of Salvia miltiorrhiza Bunge by using laser microdissection system combined with UPLC-q-TOF-MS
- Wenjian Xie1,
- Hongjie Zhang1,
- Jianguo Zeng2,
- Hubiao Chen1,
- Zhongzhen Zhao1Email author and
- Zhitao Liang1Email author
Received: 11 April 2016
Accepted: 20 June 2016
Published: 13 July 2016
Abstract
Background
The dry root of Salvia miltiorrhiza Bunge (Danshen in Chinese) is an used-widely traditional Chinese herbal medicine with and promising efficacy. This herbal plant has been extensively cultivated in China. Currently, people usually rely on its morphological features to evalaute its pharmaceutical quality. In this study, laser micro-dissection system (LMD) was applied to isolate single fresh tissue of root of S. miltiorrhiza. Under fluorescent microscopic model, five tissues namely cork, cortex, phloem, xylem ray and vessel were well recognized and isolated accurately by LMD, respectively and then the distribution pattern of the major bioactive compounds in various tissues was investigated by ultra-performance liquid chromatography-quadrupole/time of flight-mass spectrometry, which aims to validate the traditional experience on evaluating pharmaceutical quality of Danshen by morphological features.
Results
Total 62 chemical peak signals were captured and 58 compounds including 33 tanshinones, 23 salvianolic acids and 2 others were identified or tentatively characterized in micro-dissection tissues. Further semi-quantitative analysis indicated that the bioactive components such as tanshinones and salvianolic acids were mainly enriched in cork tissue.
Conclusion
Keywords
Background
The dry root of Salvia miltiorrhiza Bunge, namely Danshen in Chinese, which is an important traditional Chinese herbal medicine. Over two thousand years ago, Danshen has been categorized as a superior grade herbal medicine by The Divine Husbandman’s Classic of Materia Medica (Shen Nong Ben Cao Jing), which means that it can be beneficial to human’s health and it is safe, even it is taken for a long time [1]. Today, it has been used as a principal drug in many proprietary Chinese medicines for treating coronary heart disease, cerebrovascular disease, irregular menstruation and hepatosplenomegaly [2]. Around 20 kinds of proprietary Chinese medicines such as compound Danshen capsules, compound Danshen tablets, Danshen injection and compound Danshen dripping pills (CDDP) have been developed and some of its relative products have also been used as over the counter medicine (OTC) in Japan [3, 4]. Moreover, CDDP has been approved to carry out phase III clinical trial for preventing and treating stable angina and diabetic retinopathy by U.S. FDA [5].
Due to the increasing demands of this plant resources and extensive application in clinic, S. miltiorrhiza has been widely cultivated in Sichuan, Shangxi, Shanxi, Henan, Hebei, Shandong, Anhui, Hubei, Jiangsu and Zhejiang provinces of China and the supply of Danshen has been dominated by cultivated resource. According to the traditional experiences on morphological evaluation and classification of Danshen, it is divided into different grades by their size of main root and the color of outer bark for better transaction in the commercial markets [3]. As we know, however, the pharmaceutical quality of herbal medicines may be easily affected by some factors such as producing areas, harvest season and even cultivation technologies. Up to now, no objective evidences have been found to prove that the bigger size of main root and deeper brown–red of appearance of this medicinal plant could indicate the better pharmaceutical quality. It is no doubt that it is still unclear whether such simple quality classification criteria can really reflect its pharmaceutical quality or not. In addition, for quality evaluation of Danshen, although modern chromatographic methods involving HPLC fingerprint and determination of main components by HPLC have been established [4, 6], it is hard for medicinal vendors and consumers to equip with modern instruments to evaluate the quality of Danshen. On the other hand, it is well known that evaluating the quality of various grades of Chinese herbal medicines by morphological features is a convenient, quick and practical method compared with other methods that mostly rely on modern instruments.
Several pharmacological studies have demonstrated that bioactive effects of Danshen are mainly attributed to its secondary metabolites including diterpene quinones and salvianolic acids such as tanshinone I (Tan I), dihydrotanshinone I (DHTan I), tanshinone IIA (Tan IIA), cryphtotanshinone (CTan) and salvianolic acid B (SaB) [7–9]. Mapping the distribution of these bioactive components and carrying out semi-quantitative analysis in various herbal tissues can help to evaluate pharmaceutical quality of herbal medicine. Laser micro-dissected system (LMD) plus with ultra-performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UPLC-Q-TOF-MS) has been demonstrated as a powerful tool to establish an objective relationship between major bioactive second metabolites and morphological features of herbal medicine [10–13]. Here, this strategy was firstly applied to validate the traditional experience and judge them as true or false views, with regard to pharmaceutical quality, which is important for the quality evaluation and classification of different grades of Danshen.
Experiment section
Plant materials
Sample information of S. miltiorrhiza in the present study
Sample no. | Colour of outer barka | Sizeb (cm) | Sources | Collection date |
---|---|---|---|---|
S1 | Brownish–red | 0.8 | Cultivation, Zhongjiang County, Sichuan province | 2014.11.19 |
S2 | Dark brownish–red | 1.3 | Cultivation, Shangluo City, Shanxi province | 2014.11.19 |
S3 | Dark brownish–red | 0.75 | Cultivation, Fangcheng County, Henan province | 2014.11.19 |
S4 | Brownish–red | 1.4 | Wild, Henan province | 2014.11.19 |
S5 | Brownish–red | 0.7 | Cultivation, Linqu County, Shandong province | 2014.11.19 |
S6 | Brownish–red | 1.0 | Cultivation, Beijing | 2014.11.19 |
S7 | Brownish–red | 0.5 | Cultivation, Beijing | 2014.11.19 |
S8 | Brownish–red | 0.65 | Cultivation, Beijing | 2014.11.19 |
S9 | Brownish–red | 1.0 | Cultivation, Nanjing, Jiangsu province | 2015.05.31 |
Chemicals and reagents
Chemical structures of 5 chemical markers
Materials and instruments
Cryotome (Thermo Shandon As620 Cryotome, Cheshire, UK), Cryogen (Thermo Shandon, Cheshire, UK), Non-fluorescent polyethylene terephthalate (PET) microscope steel frame slide (76 × 26 mm, 1.4 μm, Leica Microsystems, Bensheim, Germany), Leica Laser microdissection 7000 system, 500 μL micro-centrifuge tube (Leica), Centrifuge (Centrifuge 5417R, Eppendorf, Hamburg, Germany), Ultrasonic instrument (CREST 1875HTAG Ultrasonic Processor, CREST, Trenton, NJ), HPLC grade vial (1.5 mL, Grace, Hong Kong), Glass-lined pipe with plastic ring (400 μL, Grace, Hong Kong), Electronic balance (Mettler Toledo MT5 style), Agilent 6540 ultra-definition accurate mass quadrupole time-of-flight spectrometer equipped with a mass hunter workstation software (Agilent version B.06.00 series, Agilent Technologies, USA), Acquity UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm) coupled with a C18 pre-column (2.1 mm × 5 mm, 1.7 μm, Waters, USA).
Samples preparation
Total micro-dissected area in different tissues
Sample no. | Special tissue/total micro-dissected area (μm2) | ||||
---|---|---|---|---|---|
Cork | Cortex | Phloem | Xylem ray | Vessel | |
S1 | 1,006,611 | 1,003,330 | 1,063,204 | 1,022,559 | 1,020,931 |
S2 | 1,000,990 | 1,000,072 | 1,000,320 | 1,000,791 | 1,000,276 |
S3 | 1,000,160 | 1,003,816 | 1,000,051 | 1,000,830 | 1,000,686 |
S4 | 1,000,011 | 1,000,962 | 1,000,249 | 1,000,343 | 1,000,589 |
S5 | 1,000,583 | 1,000,699 | 1,000,983 | 1,000,300 | 1,000,349 |
S6 | 1,000,736 | 1,001,599 | 1,000,860 | 1,001,172 | 1,000,058 |
S7 | 1,003,180 | 1,000,194 | 1,000,901 | 1,000,148 | 1,001,122 |
S8 | 1,000,609 | 1,000,606 | 1,000,728 | 1,000,407 | 1,000,354 |
S9 | 1,000,402 | 1,000,365 | 1,000,629 | 1,000,310 | 1,000,291 |
Standard solution preparation
Each standard including Tan I, DHTan I, Tan IIA, CTan and Sa B was accurately weighed and dissolved individually in methanol to produce mixed stock solution with concentrations at 0.96 mg/mL of Sa B, 0.992 mg/mL of DHTan I, 0.954 mg/mL of Tan I, 0.991 mg/mL of CTan, 1.028 mg/mL of Tan IIA. The series concentrations of mixed working solution were prepared by diluting the mixed stock solution with methanol. In addition, due to the high sensitive requirement in UPLC-QTOF-MS, here a blank control containing solvent was set to exclude the negative impact on analyzing process.
Method of UPLC-QTOF-MS
According to the results of preliminary experiment, the optimal running parameters of UPLC were set as follows: the mobile phase consisted of water with 0.1 % formic acid (A) and acetonitrile with 0.1 % formic acid (B) with an procedure of linear gradient elution: 0-8 min (40 % B), 8–20 min (40–75 % B), 20–22 min (75–100 % B), 23–25 min (100 % B), the injection volume was 3 μL and the flow rate was set at 0.35 mL/min. Salvianolic acids were more sensitive in negative ion scanning mode while tanshinones were more sensitive in positive ion scanning mode, so the mass spectra were acquired in both positive and negative modes by scanning from 100 to 1700 in mass to charge ratio (m/z), the scanning of MS was performed under the following operation parameters: dry gas temperature of 325 °C, dry gas (N2) flow rate of 8 L/min, nebulizer pressure of 45 psi, V-cap of 4500, nozzle voltage 500 V, and fragmentor 150 V.
Results and discussion
Microscopic characteristics and separation of tissues
Cross-sections of the root of S. miltiorrhiza (S7) a observed under the bright filed mode b observed under the fluorescent mode
Identification of chemicals in various tissues
The represent BPC chromatograms from cork tissue of S1 and S2 detected under positive mode (a), cork tissue of S1 and cortex tissue of S5 (b) as well as cork tissue of S2 and S5 (c) detected under negative mode.1 SP solvent peak
Characteristics of bioactive components in various tissues
Peak no.a | Rt (min) | Polarity | Formula | Identification |
---|---|---|---|---|
1 | 2.63 | 313.0718 [M−H]− | C17H14O6 | Salvianolic acid Fb |
2 | 3.59 | 535.1818 [M−H]− | C26H32O12 | (+)1-hydroxypinoresinol-1-O-β-D-glucosideb |
3 | 3.96 | 359.0732 [M−H]− | C18H16O8 | Rosmarinic acidb |
4 | 4.43 | 717.1406 [M−H]− | C36H30O16 | Salvianolic acid Bc |
5 | 4.56 | 137.0242 [M−H]− | C7H6O3 | Protocatechualdehydeb |
6 | 4.98 | 193.0479 [M−H]− | C10H10O4 | Ferulic acidb |
7 | 6.04 | 335.0894 [M+Na]+, 313.1071 [M+H]+ | C18H16O5 | Tanshindiol Cb |
8 | 7.18 | 297.1118 [M−H]− | C19H22O3 | Arucadiolb |
9 | 7.40 | 319.0944 [M+Na]+, 297.1124 [M+H]+ | C18H16O4 | Danshenxinkunb |
10 | 7.63 | 117.0193 [M−H]− | C4H6O4 | Succinic acidb |
11 | 7.77 | 357.0588 [M−H]− | C18H14O8 | Prolithospermic acidb |
12 | 7.84 | 335.1252 [M + Na]+, 313.1432 [M+H]+ | C19H20O4 | Miltionone IIb |
13 | 8.99 | 317.0786 [M+Na]+, 295.0969 [M+H]+ | C18H14O4 | Trijuganone Ab |
14 | 9.12 | 319.0944 [M+Na]+, 297.1124 [M+H]+ | C18H16O4 | Tanshinone VIb |
15 | 9.27 | 383.9794 [M−H]− | Unknown | |
16 | 9.98 | 333.1097 [M+Na]+, 311.1279 [M+H]+ | C19H18O4 | Isotanshinoneb |
17 | 10.07 | 303.0996 [M+Na]+, 281.1162 [M+H]+ | C18H16O3 | Methylene dihydrotanshinoneb |
18 | 10.21 | 335.1252 [M+Na]+, 313.1434 [M+H]+ | C19H20O4 | Miltionone Ib |
19 | 10.35 | 491.1039 [M−H]− | C26H20O10 | Salvianolic acid Cb |
20 | 10.41 | 333.1098 [M+Na]+, 311.1282 [M+H]+ | C19H18O4 | Tanshinone II B b |
21 | 10.64 | 333.1100 [M+Na]+, 311.1282 [M+H]+ | C19H18O4 | 3α-hydroxytanshinone IIA/3β-hydroxytanshinone II A b |
22 | 10.87 | 333.1099 [M+Na]+, 311.1283 [M+H]+ | C19H18O4 | 3α-hydroxytanshinone IIA/3β-hydroxytanshinone II A b |
23 | 10.98 | 327.0872 [M−H]− | C18H16O6 | Methylsalvianolate Fb |
24 | 11.38 | 363.1202 [M+Na]+, 341.1380 [M+H]+ | C20H20O5 | Cryptomethyltanshinoateb |
25 | 11.57 | 295.0958 [M−H]− | C18H16O4 | Tanshinol Bb |
26 | 11.75 | 325.1079 [M−H]− | C14H14O9 | Monocaffeoyltartaric acidb |
27 | 12.01 | 285.1853 [M−H]− | C20H30O | Ferruginolb |
28 | 12.02 | 309.1125 [M+Na]+, 287.1642 [M+H]+ | C18H22O3 | Epicryptoacetalide/Cryptoacetalideb |
29 | 12.18 | 487.3401 [M−H]− | Unknown | |
30 | 12.24 | 309.1125 [M+Na]+, 287.2002 [M+H]+ | C18H22O3 | Epicryptoacetalide/Cryptoacetalideb |
31 | 12.35 | 313.1438 [M−H]− | C19H22O4 | Tanshinone Vb |
32 | 12.38 | 301.0838 [M+Na]+, 279.1016 [M+H]+ | C18H14O3 | Methylenetanshinquinoneb |
33 | 12.46 | 485.3274 [M−H]− | Unknown | |
34 | 12.57 | 537.1038 [M−H]− | C27H22O12 | Lithospermic acidb |
35 | 12.82 | 293.0819 [M−H]− | C18H14O4 | 3-hydroxymethylenetanshinoneb |
36 | 12.88 | 321.1646 [M+Na]+, 299.1642 [M+H]+ | C19H22O3 | Miltiodiolb |
37 | 12.96 | 555.3268 [M−H]− | Unknown | |
38 | 13.00 | 301.0834 [M+Na]+, 279.1015 [M+H]+ | C18H14O3 | Dihydrotanshinone Ic |
39 | 13.11 | 301.0834 [M+Na]+, 279.1015 [M+H]+ | C18H14O3 | 1,2-dihydrotanshinone Ib |
40 | 13.16 | 329.1750 [M−H]− | C20H26O4 | Salviolb |
41 | 13.41 | 319.1306 [M+Na]+, 297.1491 [M+H]+ | C19H20O3 | Isocryptotanshinoneb |
42 | 13.84 | 303.0998 [M+Na]+, 281.1173 [M+H]+ | C18H16O3 | Danshenxinkun Bb |
43 | 14.25 | 361.1045 [M+Na]+, 339.1230 [M+H]+ | C20H18O5 | Methyl tanshinoateb |
44 | 14.32 | 357.0616 [M−H]− | C18H14O8 | Prolithospermic acidb |
45 | 14.62 | 333.1089 [M+Na]+, 301.1800 [M+H]+ | C19H24O3 | Miltipoloneb |
46 | 14.75 | 265.1470 [M−H]− | C18H18O2 | Methylenemiltironeb |
47 | 15.45 | 315.0846 [M−H]− | C17H16O6 | 5,3′-dihydroxy-7,4′-dimethoxyflavanoneb |
48 | 15.97 | 299.0684 [M+Na]+, 277.0867 [M+H]+ | C18H12O3 | Tanshinone Ic |
49 | 16.00 | 319.1307 [M+Na]+, 297.1488 [M+H]+ | C19H20O3 | Cryptotanshinonec |
50 | 16.15 | 315.1949 [M−H]− | C20H28O3 | 1-phenanthrenecarboxylic acidb |
51 | 16.35 | 297.1830 [M−H]− | C20H26O2 | 5-dehydrosugiolb |
52 | 16.64 | 299.2018 [M−H]− | C20H28O2 | Sugiolb |
53 | 16.79 | 299.0684 [M+Na]+, 277.0867 [M+H]+ | C18H12O3 | Isotanshinone Ib |
54 | 17.25 | 301.0834 [M+Na]+, 279.1015 [M + H]+ | C18H14O3 | Dihydroisotanshinone Ib |
55 | 17.75 | 315.1001 [M+Na]+, 293.1179 [M+H]+ | C19H16O3 | 1,2 -didehydrotanshinone II A b |
56 | 18.18 | 289.1204 [M+Na]+, 267.1386 [M+H]+ | C17H14O3 | Dihydrotanshinlactoneb |
57 | 18.87 | 303.1306 [M+Na]+, 281.1539 [M+H]+ | C19H20O2 | Δ1 -dehydromiltironeb |
58 | 19.13 | 325.1824 [M−H]− | C21H26O3 | 2-(7-Dihydroxyl)-benzofuranyl-,ferulic acidb |
59 | 19.30 | 317.1158 [M+Na]+, 295.1333 [M+H]+ | C19H18O3 | Tanshinone II A c |
60 | 20.01 | 317.1151 [M+Na]+, 295.1332 [M+H]+ | C19H18O3 | Isotanshinone II A b |
61 | 20.39 | 305.1515 [M+Na]+, 283.1700 [M+H]+ | C19H22O2 | Miltironeb |
62 | 21.31 | 683.4317 [M−H]− | C44H60O6 | 3,4-Dihydroxy-(1α,3α,4α,5β)-1-carboxy-4-hydroxy-1,3,5-cyclohexanetriyl ester-benzenepropanoicb |
The profile of chemicals in various tissues from S1 to S9
The distribution of bioactive components in various tissues from different samples
Sample no. | Herbal tissues/peak No.a | ||||
---|---|---|---|---|---|
Cork | Cortex | Phloem | Xylem ray | Vessel | |
S1 | 3, 4, 8, 9, 12–14, 16–22, 24–39, 41–57, 59–61 | 9, 17, 32, 36, 38, 39, 41, 46, 48, 49, 53 | 9, 17, 24, 32, 36, 38, 39, 41, 42, 46, 48, 49, 53, 54, 59 | 9, 17, 20, 32, 36, 38, 39, 41–43, 48, 49, 53, 54, 59, 61 | 10, 14, 32, 36, 38, 39, 41, 46, 48, 49, 53, 54, 59 |
S2 | 4, 6, 7, 9, 17, 20, 24, 28, 31, 32, 36, 38, 39, 41–43, 45, 46, 48, 49, 52–56, 59, 61 | 9, 32, 36, 38, 39, 41, 48 49, 53, 54 | 9, 32, 36, 38, 39, 41, 48, 49, 53, 54 | 9, 17, 23, 28, 32, 36, 38, 39, 41, 48, 49, 53, 54 | 9, 17, 24, 28, 32, 36, 38, 39, 41, 48, 49, 53, 54 |
S3 | 4, 9, 12–14, 16–18, 20–22, 24, 28, 30, 32, 36, 38, 39, 41–43, 45, 48, 49, 51–55, 59, 61, 62 | 9, 17, 22, 32, 38, 39, 41 48, 49, 53, 54 | 9, 32, 38, 39, 41, 48, 49, 53, 54 | 9, 32, 38, 39, 41, 48, 49, 53, 54 | 9, 17, 24, 28, 32, 38, 39, 41, 48, 49, 53, 54 |
S4 | 4, 5, 9, 14, 20, 30, 32, 36, 38, 39, 41–43, 45, 48, 49, 53–55, 59, 61 | 9, 14, 20, 32, 38, 39, 41 48, 49, 53, 54 | 9, 17, 32, 38, 39, 41, 48, 49, 53, 54 | 9, 32, 38, 39, 41, 48, 49, 53, 54 | 9, 32, 38, 39, 41, 48, 49, 53, |
S5 | 4, 14, 16, 20, 23, 30, 32, 36, 38, 39, 41–46, 48, 49, 52, 54, 55, 59, 61, 62 | 1–5, 9–11, 14, 15, 17, 22–24, 28, 32, 36, 38, 39, 41, 48, 49, 53, 58 | 9, 17, 22, 24, 28, 32, 38, 39, 41, 46, 48, 49, 53 | 16, 24, 39 | 9, 10, 32, 38, 39, 41, 48, 49, 53, 54, 59 |
S6 | 4, 23, 30, 32, 38, 39, 41, 42, 46, 48, 49, 54, 59, 61 | 9, 23, 24, 30, 32, 36, 38, 39, 41, 49 | 23, 24, 30, 32, 38, 39, 41, 46, 48 | 9, 23, 24, 30, 32, 38, 39, 41, 46 | 23, 30, 32, 36, 38, 39, 41, 46 |
S7 | 4, 12, 14, 16, 18, 20, 23, 24, 30, 32, 36, 38, 41–43, 45, 46, 48–50, 52, 54, 59, 61 | 9, 16, 23, 24, 30, 32, 37–39, 41, 42, 45, 48, 49, 53 | 9, 23, 24, 30, 32, 38, 39, 41, 46, 49, 63 | 9, 23, 24, 30, 32, 38, 39, 41, 49 | 23, 30, 32, 38, 39, 41, 49 |
S8 | 4, 12, 14, 16, 18, 20, 23, 24, 30, 32, 36, 38, 41–43, 45, 48–50, 52, 54, 59, 61 | 4, 23, 24, 30, 32, 38, 39, 41, 49 | 23, 24, 30, 32, 36, 38, 39, 41, 46, 49 | 23, 30, 32, 38, 39 | 4, 23, 30, 32, 38, 39, 49 |
S9 | 4, 12, 17, 22–33, 35, 37–42, 48, 49, 59 | 23, 30, 32, 36, 38, 39, 41 | 23, 30, 32, 36, 38, 39, 41 | 23, 30, 32, 38, 39 | 23, 30, 32, 36, 38, 39, 41 |
Quantitative analysis of tanshinones and salvianolic acids in various tissues
Methodological validation data of chemical markers
Chemical markers | Calibration curve | R2 | LOD (ng/mL) | LOQ (ng/mL) |
---|---|---|---|---|
Sa B | Y = 34.82X−5199.5 | 0.9997 | 44.31 | 75.00 |
DHTan I | Y = 903.46X+2021.7 | 0.9996 | 3.88 | 12.90 |
Tan I | Y = 245.31X+1718.4 | 0.9997 | 3.73 | 12.42 |
CTan | Y = 1410.80X+1063.5 | 1.0000 | 3.87 | 12.89 |
Tan II A | Y = 1531.80X+12447 | 0.9998 | 8.03 | 26.74 |
Methodological validation data of chemical markers
The appearance of 9 research samples (S1–S9, from left to right)
A loading plot obtained from principal component analysis of the contents of major tanshinones contained in different tissues from all of samples
Conclusions
In conclusion, different tissues from the same sample and different samples have various chemical profiles. The total contents of salvianolic acid B and major tanshinones varied in samples from the same or different growing areas and different harvest seasons.
As mentioned before, traditional experience on quality evaluation of Danshen considers that the main root with bigger size and deeper brown–red has better pharmaceutical quality [23]. Now, the present study has revealed that its major active components such as tanshinones and salvia acids are mainly accumulated in cork tissue and higher amounts of tanshinones in cork would exhibit deeper brown–red. Thus, Danshen with thinner main root, more lateral roots and deeper brown–red of outer bark would contain higher tanshinone components. The results support one of the criteria of traditional pharmaceutical quality evaluation of Danshen that samples with deeper brown red of outer bark have better quality. However, it is contradicted with another criterion which samples with bigger size of main root have better quality. It is to say that bigger main root of this herbal medicine cannot ensure better pharmaceutical quality. Also, the factors of influencing the pharmaceutical quality involve production district, harvest season and cultivation technologies. For the quality evaluation by morphological features with size of main root and color of outer bark should be restricted to the samples from the same growing area with the same harvest season and cultivation technique. Therefore, comprehensive quality evaluation system of Danshen including morphological features as well as qualitative and quantitative analysis of chemicals should be established.
Declarations
Authors’ contributions
WX has carried out the experimental study and drafted the manuscript. ZL and ZZ initiated and have been significantly involved by contributing their intellectual content for the research work, analyzing the results and correcting the manuscript accordingly. HZ, JZ and HC have made their intellectual contributions in revising the manuscripts with their knowledgeable suggestions. All authors read and approved the final manuscript.
Acknowledgements
We acknowledge Mr. Alan Ho from the School of Chinese Medicine, Hong Kong Baptist University for his technical supports. This work is supported by the National Natural Science Foundation of the People’s Republic of China (Project No. 81303219) and Innovation and Technology Fund (ITS/185/13FX).
Competing interests
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Authors’ Affiliations
References
- Yan SX, Ji SF (1991) The divine husbandman’s classic of materia medica. Shan Xi Science Press, Tai Yuan, p 29Google Scholar
- State Pharmacopoeia Committee, Pharmacopoeia of the People’s Republic of China, China Medical Science and Technology Press, Beijing, China, 2010, pp 71Google Scholar
- Zhao ZZ, Xiao PG (2007) Encyclopedia on contemporary medicinal plants. World Publishing Corporation, Shang Hai, p 358Google Scholar
- Yuan D, Pan YN, Fu WW, Toshiaki M, Yoshihiro K (2005) Quantitative analysis of the marker compounds in Salvia miltiorrihiza root and its phytomedicinal preparations. Chem Pharm Bull 53:508–514View ArticleGoogle Scholar
- Clinical Trials.gov, This site provides a registry and results database of publicly and privately supported clinical studies of human participants conducted around the world. https://clinicaltrials.gov/ct2/results?term=Compound+Danshen+dripping+pills&Search=Search
- Liu AH, Lin YH, Yang M, Guo H, Guan SH, Sun JH, Guo DA (2007) Development of the fingerprints for the quality of the roots of Salvia miltiorrhiza and its related preparations by HPLC-DAD and LC-MSn. J Chromatogr B 846:32–41View ArticleGoogle Scholar
- Chen CP, Yokozawa T, Chung HY (1999) Inhibitor effect of caffeic acid analogs isolated from Salviae miltiorrhizae radix against 1,1-diphenyl-2-picrylhydrazyl radical. Exp Toxicol Pathol 51:59–63View ArticleGoogle Scholar
- Liu GT, Zhang TM, Wang BE, Wang YW (1992) Protective action of seven natural phenolic compounds against peroxidative damage to biomembranes. Biochem Pharmacol 43:147–152View ArticleGoogle Scholar
- Yagi A, Fujimoto K, Tanonaka K, Hirai K, Takeo S (1989) Possible active components of tan-shen (Salvia miltiorrhiza) for protection of the myocardium against ischemia-induced derangements. Planta Med 55:51–54View ArticleGoogle Scholar
- Yi L, Liang ZT, Peng Y, Yao X, Chen HB, Zhao ZZ (2012) Tissue-specific metabolite profiling of alkaloids in Sinomenii Caulis using laser microdissection and liquid chromatography-quadrupole/time of flight-mass spectrometry. J Chromatogr A 1248:93–103View ArticleGoogle Scholar
- Liang ZT, Oh KY, Wang YQ, Yi T, Chen HB, Zhao ZZ (2014) Cell type-specific qualitative and quantitative analysis of saikosaponins in three Bupleurum species using laser microdissection and liquid chromatography-quadrupole/time of flight-mass spectrometry. J Pharm Biomed Anal 97:157–165View ArticleGoogle Scholar
- Liang ZT, Sham TT, Yang GY, Yi L, Chen HB, Zhao ZZ (2013) Profiling of secondary metabolites in tissues from Rheum palmatum L. using laser microdissection and liquid chromatography mass spectrometry. Anal Bioanal Chem 405:4199–4212View ArticleGoogle Scholar
- Chen YJ, Liang ZT, Zhu Y, Xie GY, Tian M, Zhao ZZ, Qin MJ (2014) Tissue-specific metabolites profiling and quantitative analyses of flavonoids in the rhizome of Belamcanda chinensis by combining laser-microdissection with UHPLC-Q/TOF-MS and UHPLC-QqQ-MS. Talanta 130:585–597View ArticleGoogle Scholar
- Yang M, Liu AH, Guan SH, Sun JH, Xu M, Guo DA (2006) Characterization of tanshinones in the roots of Salvia miltiorrhiza (Dan-shen) by high-performance liquid chromatography with electrospray ionization tandem mass spectrometry. Rapid Commun Mass Spectrom 20:1266–1280View ArticleGoogle Scholar
- Liu AH, Guo H, Ye M, Lin YH, Sun JH, Xu M, Guo DA (2007) Detection, characterization and identification of phenolic acids in Danshen using high-performance liquid chromatography with diode array detection and electrospray ionization mass spectrometry. J Chromatogr A 1161:170–182View ArticleGoogle Scholar
- Yasumasa I, Izumi M, Yutaka T (1989) Abietane type diterpenoids from Salvia miltiorrhiza. Phytochemistry 28:3139–3141View ArticleGoogle Scholar
- Asari F, Kusumi T, Zheng GZ, Cen YZ, Kakisawa H (1990) Cryptoacetalide and epicryptoacetalide, novel spirolactone diterpenoids from Salvia miltiorrhiza. Chem Lett 19:1885–1888View ArticleGoogle Scholar
- Ayhan U, Gulacti T, Nur T (1995) Diterpenoids from Salvia heldrichiana. Phytochemistry 40:1473–1475View ArticleGoogle Scholar
- Hui Y, Ip SP, Sun HD, Che CT (2003) Constituents of Salvia trijuga. Pharm. Biol. 41:375–378View ArticleGoogle Scholar
- Qian TX, Yan ZH, Li LN (1993) Mono-feruloyl-R, R-(+)-tartaric acid from Salvia chinensis. J Chinese Pharm Sci 2:148–150Google Scholar
- Yang Y, Zhu B, Sun LN, Wu ZJ, Chen WS (2013) Chemical constituents of Salvia przewalskii Maxim. Asian J Chem 25:1747–1748Google Scholar
- Lu YR, Foo LY (2002) Polyphenolics of Salvia-a review. Phytochemistry 59:117–140View ArticleGoogle Scholar
- Xu GJ (1996) Pharmacognosy of Chinese herbal medicine. Chinese Medical Science Press, Beijing, p 393Google Scholar