Simultaneous quantification of selected compounds from Salvia herbs by HPLC method and their application

Food Chemistry 130 (2012) 1031–1035

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Analytical Methods

Simultaneous quantification of selected compounds from Salvia herbs by HPLC method and their application Hai-Ting Cheng, Xiao-li Li, Xiao-rong Li, Yu-hang Li, Li-juan Wang, Ming Xue ⇑ Department of Pharmacology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, China

a r t i c l e

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Article history: Received 12 January 2010 Received in revised form 27 April 2011 Accepted 31 July 2011 Available online 6 August 2011 Keywords: Salvia przewalskii Maxim Salvia miltiorrhiza Bunge Active components Quantification HPLC

a b s t r a c t A simple and reliable reversed-phase high performance liquid chromatographic method was developed and validated for simultaneous determination of danshensu, protocatechuic aldehyde, rosemarinic acid, salvianolic acid B, dihydrotanshinone I, tanshinone I, cryptotanshinone and tanshinone IIA in Salvia herbs. The contents of these eight main components were compared between Salvia miltiorrhiza Bunge and Salvia przewalskii Maxim, which were used for treating coronary heart diseases, cerebrovascular disease, bone loss, hepatitis, hepatocirrhosis and chronic renal failure. The samples were successfully separated by the HPLC method. All the standard compounds showed a good linearity (R2 > 0.9994) in the relatively wide concentration range. The limit of detection of the eight compounds was in the range of 0.05–0.5 lg/ml and the limit of quantification was in the range of 0.25–1.50 lg/ml. The intra-day variability was in the range of 0.03–8.08% and the inter-day variability was in the range of 0.18–12.36%. The recoveries of the selected compounds were in the range of 96.2–108.1%. This method was accurate, precise and reproducible, and could be successfully applied to the quality control and stable experiment for the preparations consisted of these active components, and the content comparison of the herbs from Salvia species. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Salvia herbs belong to the Labiatae family plants, spreading all over the subtropical zone regions on the earth and including more than seven hundred species, and many of them have several bioactivities such as promoting circulation and improving blood flow (Xu, 1990). The dried root of Salvia miltiorrhiza Bunge, which is a well-known traditional Chinese medicine (TCM) named ‘danshen’ in the Orient countries, especially in China, is the representative of these materia medica in Salvia species. Salvia miltiorrhiza has been widely in TCM preparations and used for treating coronary heart diseases, cerebrovascular disease, bone loss, hepatitis, hepatocirrhosis and chronic renal failure (Liu et al., 2006; Su & Liu, 2009; Xu & Fu, 2006). In China, numerous pharmaceutical dosage forms of S. miltiorrhiza are commercially available. In the United States and the Europe, some products contained danshen can also be used for the nutritions or the alimentary substances in herbal shops. There are many TCM preparations-containing the dried roots of S. miltiorrhiza such as Fufang danshen tablet (FDT), Danshen injection (DSI) and Fufang danshen dripping pills (FDDP), including tablets, capsules, granules, injectables, oral liquids, sprays and dripping pills, etc (National Commission of Chinese

⇑ Corresponding author. Tel./fax: +86 10 83911520. E-mail address: [email protected] (M. Xue). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.07.126

Pharmacopoeia, 2005). FDDP are widely used products in China and also registered as a drug in several countries, including Russia, Korea and Cuba (Zhou, Zuo, & Chow, 2005). Amongst these preparations, danshen is the major active component. There is a large market of danshen in Asian countries as well as keen interest in the use and modernisation of herbal products throughout the world (Xu, 1990). The dried root of Salvia przewalskii Maxim is a sort of analogue of S. miltiorrhiza and has been widely used as the substitute of danshen in China (Shi, Xue, Cui, & Luo, 2002; Xiao et al., 1997; Xue et al., 2000; Zhang, Lv, & Lan, 2008). Although it has been demonstrated that S. miltiorrhiza and S. przewalskii products are similar in the effectiveness and safe for the treatment of cardiovascular diseases by some clinical trials, sufficient quality control is still lack. To ensure their clinical efficacy, the quality control, stable experiment of the preparations consisted of these active components and the comparisons of the herb contents from Salvia species are of great importance. According to the chemical structures and the pharmacological investigations, the major active constituents of Salvia are divided into two groups: the water soluble phenolic acids such as danshensu, protocatechuic aldehyde, rosemarinic acid and salvianolic acid B, and the lipophilic tanshinones such as tanshinone I, dihydrotanshinone I, cryptotanshinone, tanshinone IIA, tanshinone IIB and hydroxtanshinone (Kong, 1989; Liu et al., 2007; Zhang, Chen, & Li, 2007). With expanding the resource and controlling the quality of the herbs from different areas, it is very necessary to develop a

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simple and effective HPLC method for simultaneously quantifying these two kinds of active components. Up to now, quantitative determination of these constituents in S. miltiorrhiza has been only focused on one or two compounds, just the single water soluble compound or the lipophilic compounds, which could not reflect the overall quality of S. miltiorrhiza (An et al., 2004; Ma, Zhang, Guo, & Gan, 2006; Shi, He, Yao, Chang, & Zhao, 2005). There are few reported methods about simultaneous determination of water soluble and lipophilic compounds. In our paper, a simple and reliable gradient reversed phase HPLC method has been developed for simultaneous determination of these eight main active and characteristic constituents. This method was accurate, precise and reproducible, and could be successfully applied to evaluate the quality of Salvia herbs. The method also provided the reference for the clinical medication and the nutrition application. 2. Materials and methods 2.1. Chemicals and reagents Danshensu, protocatechuic aldehyde, tanshinone I, cryptotanshinone, and tanshinone IIA were purchased from National Institute for Control of Pharmaceuticals and Biological Products (Beijing, China). Rosemarinic acid and dihydrotanshinone I were purchased from Shanghai Herb Medical Development Co. Ltd., (Shanghai, China), and salvianolic acid B were purchased from Shanghai Zerun Pharmacy Co. Ltd., (Shanghai, China). The purities of these compounds were more than 99%. Methanol used was of HPLC grade and obtained from Fisher Scientific Products (Fair Lawn, NJ, USA). Water was triply distiled. The other chemicals, reagents and solvents used were all of analytical grade. The roots of S. Przewalskii was purchased from the Huanghe Market of medicinal material (Lanzhou, Gansu, China), and the roots of S. miltiorrhiza produced from different areas in China were purchased from Beijing Sifang Chinese Traditional Medicine Electuary Co. Ltd., (Beijing, China). 2.2. Instrumentation and chromatographic conditions All analysis were performed on an Agilent high performance liquid chromatographic system (Series 1100, Agilent Technology, Palo Alto, CA, USA) which consisted of a G1310A quaternary solvent delivery pump, a G1379A on-line degasser, a G1313A autosampler and a G1314A ultraviolet wave detector. The chromatographic data were recorded and processed with HP chemstation software. The analytical column was an Agilent ZORBAX SB-C18 (5 lm, 150 mm  4.6 mm) column coupled with Agilent C18 guard column. The mobile phase was a mixture of solvent A (methanol) and B (water containing 0.5% acetic acid) employing gradient elution (from 20:80 to 80:20, v/v) at a flow rate of 1 ml/min. The gradient programme was as follows: initial 0–10 min, linear change from A– B (20:80) to A–B (35:65); 10–20 min, linear change to A–B (40:60); 20–40 min, linear change to A–B (70:30); and 40–50 min, linear change to A–B (80:20). Column temperature was maintained at 25 °C. The detection wavelength was set at 281 nm for DSS and PCA, at 330 nm for RAM and SAB, and at 254 nm for DTI, TSI, CT and TSA, respectively. The solvent was filtered through a 0.22 lm filter and degassed. The sample injection volume was 10 ll. The established method has been successfully applied to the simultaneous determination of DSS, PCA, RMA, SAB, DTI, TSI, CT and TSA from the Salvia herbs. The two categories of samples were prepared as described in the section of sample preparation. A volume of 10 ll of the filtered solution of each sample was injected into the HPLC instrument. Each sample was determined in

triplicate. Peaks in the chromatograms were identified by comparing the retention times and on-line UV spectra with those of the standards. Retention time of DSS, PCA, RMA, SAB, DTI, TSI, CT and TSA were 3.42, 6.43, 18.00, 19.19, 33.71, 40.37, 43.32 and 49.69 min, respectively. 2.3. Preparation of standard solutions The standard stock solutions of DSS, PCA, RMA and SAB were separately prepared in 1% (v/v) acetic acid solutions at a concentration of 1 mg/ml, and the standard stock solutions of DTI, TSI, CT and TSA were separately prepared in methanol at a concentration of 1 mg/ml. These stock solutions were diluted to appropriate concentration range for the establishment of calibration curves. Calibration standards were prepared from the mixture of above stock solutions or dilutions with methanol. 2.4. Sample preparation The dried roots of S. miltiorrhiza and S. Przewalskii were comminuted and passed through 100 mesh sieves, respectively. After accurately weighed, 0.5 g of sample powder was transferred to a 50 ml conical flask; then the powdered drug was extracted with 5 ml amount of 80% alcohol for ultrasonic extraction 60 min. Alcohol (80%) was added to compensate to the final volume. After centrifugation, the upper layer liquid was collected and stored at 4 °C. The herb residue was immersed in 5 ml deionized water overnight and treated by the ultrasound extraction at 80 °C for 60 min. Deionized water was added to compensate to the volume. The combined sample solution was filtrated through a syringe membrane filter (0.22 lm), and then 10 ll of sample was injected into HPLC system. 2.5. Method validation 2.5.1. Linearity In this study, each calibration curve was analysed three to four times with six to seven different concentrations using the same HPLC condition as described above. The calibration graphs were plotted based on linear regression analysis of the integrated peak areas (y) versus concentrations (x). The regression equation was calculated in the form of y = ax + b, where y and x were the values of peak area and sample amount, respectively. The standard solution was diluted with methanol to provide appropriate concentrations. The limit of detection (LOD) was defined as the lowest concentration level resulting in a peak area of three times the baseline noise (S/N > 3). The limit of quantification (LOQ) was defined as the lowest concentration level resulting in a peak area of ten times the baseline noise (S/N > 10) (Li, Li, Wang, Li, & Xue, 2008). 2.5.2. Precision The precision test was evaluated by the intra-day and inter-day variability. Three different concentration solutions (low, medium and high) of the standards were prepared in methanol. Five replicates of the samples at each concentration were evaluated on the same day for intra-day precision, whilst repeated analysis at each concentration of the samples five times per day over five consecutive days for inter-day precision. The quantity of each analyte was obtained from corresponding calibration curve. The relative standard deviation (R.S.D.) was taken as a measure of precision. 2.5.3. Recovery In order to check the accuracy of the developed method, the recovery experiments were carried out as follows: three different quantities (low, medium, and high) of the authentic standards were spiked into the samples in form of solution. The quantity of

H.-T. Cheng et al. / Food Chemistry 130 (2012) 1031–1035

each analyte was subsequently obtained from the corresponding calibration curve. 2.6. Statistical analysis All contents of the components from Salvia herbs were presented as the mean value ± standard deviation (S.D.). Within group comparisons were performed by the analysis of variance using one-way ANOVA test. Significant differences amongst these groups were assessed by LSD t-test. The values of p < 0.05 were considered statistically significant difference. 3. Results and discussion 3.1. Chromatographic and sample extraction conditions A good quantification method should satisfy the requirement that the analysed peaks have baseline separation with other adjacent peaks, and it is need that a relatively short analysis time. In our study, ratio of methanol and water containing acetic acid in mobile phase, column temperature, detection wavelength and flow rate were carefully investigated for the good separation. A simple gradient programme was used to elute the eight active markers in a single run within a reasonable period of time. The most efficient detection of these constituents was obtained by selecting the maximal UV absorption wavelength of these compounds. In this study, three different detection wavelengths were set according to the UV absorption maxima of the compounds. Danshensu and protocatechuic aldehyde were detected at 281 nm, rosemarinic acid

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and salvianolic acid B at 320 nm, and dihydrotanshinone I, tanshinone I, cryptotanshinone and tanshinone IIA were detected at 254 nm. Under the proposed analytical conditions, baseline resolution was obtained for all of the analytes. Typical chromatograms of the authentic standards solution and the sample solution are depicted in Fig. 1. In order to obtain quantitative extraction of the sample, variables involved in the procedure such as solvent, extraction method and extraction time were optimised. Alcohol (100%, 80%, 50% and 20%) and water were employed as extraction solvents. Pure water could not extract SAB and tanshinones completely, whilst DSS and PCA could not be efficiently extracted by pure alcohol. From the extraction efficiency of the different solvents, when 80% alcohol was employed, the peak areas of the eight markers reached the highest values. Therefore, 80% alcohol was selected as the extraction solvent. After comparing several extraction methods such as ultrasonic and soaking, ultrasonic extraction was found to be suitable extraction method. 3.2. Method validation The method validation including sensitivity, linearity, precision and recovery were examined as described above. The external standard method was used to get the regression equations. The calculated results were listed in Table 1. The R2 in Table 1 was referred to the correlation coefficient of the equation. All the standard compounds showed a good linearity (R2 > 0.9994) in the relatively wide concentration range. LOD was in the range of 0.05–0.5 lg/ml. LOQ was in the range of 0.25–1.50 lg/ml.

Fig. 1. Representative HPLC chromatograms of the eight components in standard solution (A) and the sample solution (B), Peaks: 1.danshensu, 2. protocatechuic aldehyde, 3. rosemarinic acid, 4. salvianolic acid B, 5. dihydrotanshinone I, 6. tanshinone I, 7. cryptotanshinone, 8. tanshinone IIA.

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Table 1 HPLC data for the calibration graphs and limit of quantification of the eight active compounds. Analyte (lg/ml)

Retention time (min)

Linear regression

R2

Linear range (lg/ml)

LOQ

Danshensu Protocatechuic aldehyde Rosemarinic acid Salvianolic acid B Dihydrotanshinone Tanshinone I Cryptotanshinone Tanshinone IIA

3.42 6.43 18.00 19.19 33.71 40.37 43.32 49.69

y = 5.053x 0.7711 y = 45.836x + 0.5872 y = 26.855x 1.700 y = 8.765x 6.787 y = 27.542x + 1.330 y = 63.145x + 7.159 y = 40.145x + 10.212 y = 62.950x 19.499

0.9996 0.9998 0.9998 0.9994 0.9998 0.9998 0.9994 0.9996

0.50–50 0.25–25 0.25–50 2–200 0.25–50 0.50–50 0.50–200 0.50–200

0.50 0.25 0.25 1.50 0.25 0.50 0.50 0.30

Table 2 Analytical results of intra-day and inter-day precision and accuracy. Analyte

Table 3 Analytical data of recoveries (n = 6).

Added (lg/ml)

Intra-day (n = 5)

Inter-day (n = 15)

R.S.D. (%)

Accuracy (%)

R.S.D. (%)

Accuracy (%)

Danshensu

0.5 10 50

8.08 0.67 0.20

106.49 104.43 97.83

12.36 0.99 0.73

104.91 104.42 97.93

Protocatechuic aldehyde

0.25 5 25 0.5 10 50

1.84 0.35 0.07 2.95 0.26 0.13

94.19 105.97 96.53 94.73 103.09 100.77

4.48 1.54 0.66 9.10 2.64 0.90

94.77 106.71 95.96 97.96 100.63 100.44

Salvianolic acid B

2 40 200

1.09 0.12 0.10

97.13 95.84 98.11

11.75 1.06 0.47

99.30 96.73 96.55

Dihydrotanshinone I

0.5 10 50

2.44 0.34 0.07

97.09 105.26 99.52

4.01 1.03 0.18

Tanshinone I

0.5 10 50

3.44 0.22 0.36

101.04 102.73 105.99

Cryptotanshinone

0.5 40 200

0.82 0.16 0.03

Tanshinone IIA

0.5 40 200

2.02 0.18 0.12

Rosemarinic acid

Analyte

Sample contents (lg/ ml)

Added (lg/ml)

Recovery (%) (mean ± SD)

R.S.D%

Danshensu

6.05

1.0 5.0 25.0

97.66 ± 0.77 108.15 ± 0.63 96.15 ± 0.26

0.78 0.58 0.27

Protocatechuic aldehyde

0.88

0.5 2.5 12.5

99.68 ± 0.68 98.14 ± 0.80 100.15 ± 0.26

0.69 0.82 0.26

Rosemarinic acid

10.09

1.0 5.0 25.0

97.26 ± 0.71 106.39 ± 0.53 100.60 ± 0.25

0.73 0.50 0.25

Salvianolic acid B

73.99

4.0 20.0 100.0

98.74 ± 0.79 101.04 ± 0.66 102.59 ± 0.34

0.80 0.65 0.33

100.43 106.02 99.39

Dihydrotanshinone I

14.06

1.0 5.0 25.0

99.72 ± 0.92 106.04 ± 0.48 96.42 ± 0.57

0.93 0.45 0.59

4.84 5.10 1.16

100.53 102.51 104.58

Tanshinone I

14.48

1.0 5.0 25.0

105.06 ± 0.31 106.40 ± 0.36 99.36 ± 0.38

0.30 0.34 0.38

98.18 100.96 100.45

3.94 1.90 1.08

99.14 103.91 100.11

Cryptotanshinone

42.14

4.0 20.0 100.0

100.34 ± 0.38 103.71 ± 0.26 101.48 ± 0.28

0.37 0.25 0.28

99.44 98.49 103.22

6.72 3.36 0.27

100.50 101.56 103.20

Tanshinone IIA

66.08

1.0 20.0 100.0

102.96 ± 0.47 100.99 ± 0.10 100.77 ± 0.28

0.46 0.10 0.27

The relative standard deviation (R.S.D.) was taken as a measure of precision. The data showed that the R.S.D. of intra-day variability was in the range of 0.03–8.08% (Table 2) and the inter-day was in the range of 0.18–12.36% (Table 2). The recoveries of the selected eight components were 96.42–106.04% with the R.S.D. less than 0.93% (Table 3). From the results of precision test and recovery test, it indicated that the method manifested good precision and accuracy for the determination of these eight active components in Salvia herbs containing DSS and PCA, RMA, SAB, DTI, TSI, CT and TSA. For stability test, the same sample solution was analysed every 12 h in three days at the room temperature, and the analytes were found to be rather stable within three days (R.S.D. < 2%). 3.3. Sample analysis The content of each analyte was calculated from the corresponding calibration curve, the contents of these eight constituents from different herbs of Salvia are depicted in Table 4. For the data of these four kinds of the samples, which were S. przewalskii produced from Gansu province and S. miltiorrhiza

produced from Hebei, Henna and Shandong province in China, there were markedly differences in the contents of these eight representative constituents between S. przewalskii and S. miltiorrhiza. There were also relative differences in the contents of these most of constituents amongst the herbs of S. miltiorrhiza produced by the different provinces, especially amongst Hebei, Henan and Shandong province. There were markedly differences in the contents of DSS between S. przewalskii produced in Gansu province and S. miltiorrhiza produced in Henan and Hebei province except in Shandong province. Compared with the content of PCA, the content of PCA from S. miltiorrhiza was near four to five times lower than that from S. przewalskii. The contents of RMA from S. miltiorrhiza produced in Shandong, Henan and Hebei province were approximate two to four times higher than that from S. przewalskii. The level of SAB from S. miltiorrhiza was near ten times higher than that from S. przewalskii, whilst the contents of tanshinones such as DTI, TSI, CT and TSA in S. przewalskii were approximate four to ten times higher than that of these compounds in S. miltiorrhiza. It was obvious that the ratio of the contents of the eight compounds between S. miltiorrhiza and S. przewalskii varied drastically. The variation of the contents was mainly derived from the different quality of the raw material

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H.-T. Cheng et al. / Food Chemistry 130 (2012) 1031–1035 Table 4 Content comparison of the eight representative constituents obtained from different Salvia herbs. Analyte

S. Przewalskii Maxim/Gansua

DSS PCA RMA SAB DTI TSI CT TSA

517.0 ± 22.0 51.8 ± 1.8 857.1 ± 39.4 4546.4 ± 176.5 1106.0 ± 56.5 1098.6 ± 13.4 3674.7 ± 179.7 4813.5 ± 392.9

S. miltiorrhizaa Hebei

Henan

Shandong

714.4 ± 6.8**,NN 10.8 ± 1.2** 3212.7 ± 52.7**,NN 56558.3 ± 929.3**,NN 354.5 ± 13.6*,NN 129.7 ± 28.1**,N 563.4 ± 22.3*,NN 375.9 ± 16.0*,NN

731.6 ± 10.2**,NN 14.8 ± 1.2**,N 3675.4 ± 88.6**,NN 68368.5 ± 3178.8**,NN 499.3 ± 12.3*,NN 196.6 ± 21.1** 569.4 ± 19.7*,N 397.3 ± 61.4*

508.8 ± 21.1 11.7 ± 0.7** 1947.9 ± 66.3** 37340.5 ± 1644.4** 981.1 ± 11.7 180.6 ± 31.7** 1633.7 ± 46.4* 633.8 ± 17.1*

Abbreviations: DSS: Danshensu, PCA: Protocatechuic aldehyde, RMA: Rosemarinic acid, SAB: Salvianolic acid B, DTI: Dihydrotanshinone, TSI: Tanshinone I, CT: Cryptotanshinone, TSA: Tanshinone IIA. a Values are means ± S.D. (n = 3), lg/g dried medicinal herbs. * p < 0.05 compared with the group from S. przewalskii Maxim/Gansu. ** p < 0.001 compared with the group from S. przewalskii Maxim/Gansu. N p < 0.05 compared with the group from S.miltiorrhiza/Shangdong. NN p < 0.001 compared with the group from S.miltiorrhiza/Shangdong.

and the place of production, and also could be from the difference of production procedure, storage and transportation, etc. From the results, it was inferred that there could be various bioactivities in these TCM preparations that contained the different level of phenolic acids and tanshinones. If RMA or SAB is used for the main medicated effective and nutritive ingredient, it is better to choose the dried roots of S. miltiorrhiza; if tanshinone compounds are as the main effective constituents, the dried roots of S. przewalskii are the better selection; and if DSS need to be as the mainly medicated effective component, both the dried roots of S. miltiorrhiza and that of S. Przewalskii could be chosen. Generally, the roots of S. miltiorrhiza are replaced by the roots of S. przewalskii in the substitutive therapy or the nutritive supplement. 4. Conclusions A simple, reliable and accurate HPLC assay method of simultaneous determination of four phenolic acids and four tanshinones from Salvia herbs was successfully established. According to the data, generally, there were some differences amongst the contents of these eight active components from the Salvia herbs, which indicated that quality control and reasonable application of Salvia are very necessary. The contents of tanshinones in S. przewalskii were obviously higher than that in S. miltiorrhiza, whilst the content of salvianolic acid B in S. miltiorrhiza was markedly higher than that in S. przewalskii. The results suggested that this HPLC method could be considered as good quality criteria to control the quality of Salvia herbs such as S. miltiorrhiza and S. przewalskii and their related TCM preparations. Acknowledgements This work was supported by grant from the National Foundation of Natural Sciences of China (No. 30472057), Beijing Natural Science Foundation Program (No. 7052007), the Key Program of Beijing Municipal Commission of Education (KM201110025024) and Funding Project for Academic Human Resources Development in Institutions of High Learning under the Jurisdicrion of Beijing Municipality (PHR201007111).

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