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Analytical LettersPublication details, including instructions forauthors and subscription information:http://www.tandfonline.com/loi/lanl20
Chromatographic FingerprintAnalysis of Semen Ziziphispinosae by HPLC-DAD MethodWenzhi Zeng a , Yanjing Bai a , Qingying Zhang a &Yuying Zhao aa Department of Natural Medicines and State KeyLaboratory of Natural and Biomimetic Drugs , Schoolof Pharmaceutical Sciences, Peking UniversityHealth Science Center , P. R. ChinaPublished online: 19 Feb 2009.
To cite this article: Wenzhi Zeng , Yanjing Bai , Qingying Zhang & Yuying Zhao (2009)Chromatographic Fingerprint Analysis of Semen Ziziphi spinosae by HPLC-DAD Method,Analytical Letters, 42:2, 205-215, DOI: 10.1080/00032710802585642
To link to this article: http://dx.doi.org/10.1080/00032710802585642
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BIOANALYTICAL
Chromatographic Fingerprint Analysis of SemenZiziphi spinosae by HPLC-DAD Method
Wenzhi Zeng, Yanjing Bai, Qingying Zhang, and Yuying ZhaoDepartment of Natural Medicines and State Key Laboratory of Natural
and Biomimetic Drugs, School of Pharmaceutical Sciences,Peking University Health Science Center, P. R. China
Abstract: Reliable, reproducible and valid fingerprint analysis methods usinghigh-performance liquid chromatography-photodiode array detection (HPLC-DAD) for characteristic bioactive flavonoids and saponins of Semen Ziziphispinosae (SZS) were developed and validated. HPLC separation of the chemicalconstituents of SZS was performed on an YMC-PACK ODS-A RP-18 columnand detected at 270 and 204 nm for flavonoids and saponins, respectively.A mobile phase consisted of acetonitrile and 0.1% phosphoric acid aqueous solu-tion was used with linear gradient elution. Using spinosin and jujuboside B as thereference markers of flavonoids and saponins respectively, 9 common fingerprintpeaks of flavonoids and 10 common fingerprint peaks of saponins were specifiedbased on the fingerprint analysis of 10 batches of SZS from different regions inChina. The fingerprint analysis methods developed are reliable, reproducibleand valid, and might be used as a more convenient approach for the speciesidentification and quality monitoring and assessment of SZS.
Keywords: Chromatographic fingerprint, HPLC, Semen Ziziphi spinosae,Ziziphus, Ziziphus jujuba var. spinosa
Received 26 September 2008; accepted 28 October 2008.This work was financially supported by the Program for Changjiang Scholar
and Innovative Team in University (grant number: 985-2-063-112) and theNational Natural Science Foundation of China (grant no. 20432030, 20742005).
Address correspondence to Qingying Zhang, Tel.: +861082801725; Fax:+861062015584. E-mail: [email protected]
Analytical Letters, 42: 205–215, 2009Copyright # Taylor & Francis Group, LLCISSN: 0003-2719 print=1532-236X onlineDOI: 10.1080/00032710802585642
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INTRODUCTION
Unlike the Western medicine, which is usually composed of a singlechemical entity, the traditional Chinese herbal medicine (TCHM) usuallycontains hundreds and thousands of complicated components. And it isbelieved that it is the multi-compounds that are responsible for the phar-macological efficacy of TCHM. Moreover, the entity and contents ofbioactive components of TCHM are highly variable depending on theplant species, geographical and climatic conditions, cultivation and har-vesting times, post-harvesting formulation processes, storage time andso on. Therefore, the quality assessment of TCHM should be differentfrom that of Western medicines. However, the traditional quality controlof TCHM, mainly focusing on the determination of one or several indi-vidual marker compounds, is obviously contrary to traditional Chinesemedicine (TCM) theories and thus faces many challenges in the standar-dization of TCHM. Chromatographic fingerprint, a comprehensive andquantifiable identification method that is able to reveal the overall chemi-cal information of herbal medicines with chromatograms, spectrogramsand other graphs by analytical and chemical techniques, is used andaccepted by more and more people for the species identification and qual-ity assessment of herbal medicines. Chromatographic fingerprinting iscurrently required by the Chinese State Food and Drug Administration(CSFDA) for the quality control of injections made from herbal medi-cines and is promoted for use in the manufacture of oral preparations(State Food and Drug Administration of China. 2000). Currently, finger-print analysis has also been introduced and accepted by World HealthOrganization (WHO) as a strategy for the assessment of herbal medicines(World Health Organization, WPR=RC52=7. 2001). Furthermore, bothFood and Drug Administration (FDA) and European Medicines Agency(EMEA) have clearly denoted that the appropriate fingerprint chro-matogram should be applied to assess the consistency of the botanicaldrugs (US Food and Drug Administration. 2000; European MedicinesAgency. 2001).
Semen Ziziphi spinosae (SZS), the dried ripe seed of Ziziphus jujubaMill. var. spinosa Hu ex H. X. Chou (Bunge), has been widely used as asedative medicine in China. It has been shown to possess a variety ofbiological activities, such as sedative, immunostimulant, antioxidant,antiinflammatory and antimyocardial ischemia effects (Zhu et al. 1990;Guo et al. 1998; Peng et al. 1995; Wu et al. 2004), and the flavonoids andsaponins isolated from SZS are considered as the major bioactive consti-tuents related to the above activities (Bai et al. 2003; Cheng et al. 2000).
In this paper, high-performance liquid chromatography-photodiodearray detection (HPLC-DAD) was used to develop specific, practical
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and reliable chromatographic fingerprint analysis methods of thebioactive flavonoids and saponins for the species identification andquality assessment of SZS.
EXPERIMENTAL
Apparatus
A Jasco HPLC system (Tokyo, Japan), equipped with a MD-1580quaternary pump, a PU-1510 photodiode array spectrophotometricdetector (DAD) and a column oven, was used for chromatographicanalysis. Chromatographic separations were carried out on an YMC-PACK ODS-A RP-18 (250� 4.60 mm ID; particle size 5 mm) analyticalcolumn. The column temperature was maintained at 35�C. An ultrasoniccleaner KQ-500DB (KunShan, PR China) was used for extraction.
Reference Standards and Reagents
Reference standards, spinonsin (1), 6000-feruloylspinonsin (2), jujubosideA (3), and jujuboside B (4) (Fig. 1), were obtained from SZS and theirstructures were identified by comparing their UV and NMR data withthe published values [9, 10]. HPLC grade acetonitrile (CH3CN) was pur-chased from Fisher Scientific (Fair Lawn, NJ). Deionized water (H2O)was purified by Milli-Q system (Bedford, MA). Methanol (MeOH), phos-phoricacid (H3PO4), n-butanol (BuOH), methylene chloride (CH2Cl2)and sodioum hydroxide (NaOH) were of analytic grade from BeijingReagent Company (Beijing, PR China). 0.45 mm membrane filter forfiltering HPLC used solvent were bought from Millipore (Bedford,MA, USA).
The samples of SZS were purchased from 10 different regions inChina (Table 1) and authenticated by Professor Junhua Zheng, Schoolof Pharmaceutical Sciences, Peking University Health Science Center.The voucher specimens were stored at the Herbarium of Departmentof Natural Medicines, School of Pharmaceutical Sciences, PekingUniversity Health Science Center.
Preparation of Solutions
Stock standard solutions (1 mg=mL) of 1–4 were prepared by dissolvingcompounds 1–4 (10 mg) in 10 mL 50% methanol. A working solution wasthen prepared by dilution of each stock solution with 50% methanol.
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Table 1. Relative retention time of fingerprint analysis of flavonoids of 10 batchesof SZS
Peak no.
Sample 1 2 3 4 5 6 7� 8 9
Yanan, Shanxi 0.49 0.69 0.79 0.96 1 1.21 1.23 1.38Xingjiang 0.48 0.69 0.78 0.96 1 1.21 1.23 1.39Baoji, Shanxi 0.50 0.72 0.79 0.96 1 nd nd ndJiangxi 0.47 0.68 0.78 0.96 1 1.21 1.24 1.39Hunan 0.48 0.69 0.78 0.96 1 1.21 1.24 1.39Hubei 0.48 0.69 0.79 0.96 1 1.21 1.23 1.39Hebei 0.47 0.68 0.78 0.96 1 1.21 1.24 1.39Guizhou 0.47 0.68 0.78 0.96 1 1.21 1.24 1.39Fujian 0.49 0.70 0.78 0.96 1 1.21 1.23 ndBeijing 0.47 0.68 0.78 0.96 1 1.22 1.24 1.40Average value 0.48 0.69 0.78 0.96 1 1.21 1.24 1.39% RSD 2.29 2.02 0.27 0.14 0.25 0.26 0.45
nd: not detected.
Figure 1. Structures of reference standards 1–4.
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Chromatographic Conditions
The mobile phase was consisted of acetonitrile (A) and 0.1% H3PO4
aqueous solution (B) with a linear gradient elution procedure (0–60 min,20% A! 40% A) and filtered through a 0.45 mm membrane filter beforeuse. The flow rate of the mobile phase was 1 mL=min. The UV detectionwavelength was set at 270 nm for flavonoids, while 204 nm for saponins.
Sample Preparation for Fingerprint Analysis of Flavonoids
The powdered sample of SZS (1.0 g) was accurately weighted andextracted with 10 mL of methanol-water (50:50, v=v) in an ultrasonicwater bath sonication at 40�C for 40 min, then the solutions were filteredthrough a 0.45 mm cellulose acetate filter and 10 mL sample were injectedinto the HPLC system for analysis.
Sample Preparation for Fingerprint Analysis of Saponins
The powdered sample of ZJS (5.0 g) was accurately weighted and extractedwith methanol for 4 hrs using Soxhlet’s extractor after being defatted withCH2Cl2 firstly and the methanolic extract was concentrated under reducedpressure. The residue obtained was suspended in 30 mL water andextracted with n-butanol (2� 20 mL). The combined n-butanol layer, afterbeing extracted with 5% NaOH (2� 4 mL) to remove the flavonoids andsuccessively washed with water (4� 15 mL), was concentrated to drynessunder reduced pressure. The dried extract was then made up to exactly2 mL of solution with methanol using a volumetric flask and filteredthrough a 0.45mm membrane filter before injecting a 10mL sample.
RESULTS AND DISCUSSION
Optimization of HPLC Conditions
Flavonoids and saponins, the major bioactive constituents of SZS, wereusually chosen as the marker compounds for the quality assessment ofSZS, and the HPLC-DAD was the most frequently employed methodbecause of its high sensitivity and convenience. However, flavonoids andsaponins showed UV absorption maxima at different wavelengths becauseof the difference in their structures, it is rather difficult to analyze them atthe same detection wavelength. Therefore, different detector wavelengthsof 270 and 204 nm were chosen for flavonoids and saponins respectively
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based on their UV spectra. In order to obtain better chromatogram,several kinds of mobile phase systems, including acetonitrile-water,acetonitrile-H3PO4 aqueous solution, methanol-water and methanol-H3PO4 aqueous solution, were investigated. Finally, linear gradient eluentsystem consisting of acetonitrile-0.1% H3PO4 aqueous solution was chosenas the mobile phase for both fingerprint analysis of flavonoids and sapo-nins due to the excellent separation and durable analysis times.
Optimization of Sample Preparation
In the preparation of samples, the effects of four major factors, includingextraction solvent, extraction method, extraction time and purificationmethod, were investigated, and the optimal sample preparation condi-tions were presented in detail in ‘‘Sample Preparation for FingerprintAnalysis of Flavonoids’’ and ‘‘Sample Preparation for FingerprintAnalysis of Saponins.’’
What is worth to note is that the developed preparation methodfor flavonoids and saponins were quite different from each other. It isimpossible to develop a sample preparation method that can fulfill thesuccessively HPLC fingerprint analysis of flavonoids and saponinsbecause of the large differences in their response factors to UV andcontents in SZS. It is hard to detect saponins effectively when impurities,such as flavonoids and oils, were presented, and so CH2Cl2 and 5%NaOH was employed to remove the oils and flavonoids from thesaponins in order to get better HPLC-UV chromatogram of saponins.
Stability, Precision and Reproducibility
The stability of the fingerprint analysis was assessed by analyzing thesame sample at 0, 2, 4, 8, 12, 24, 48 h. The precision of the fingerprintanalysis was assessed by analyzing the same sample five times. The datafor studies of the reproducibility of the fingerprint analysis were evalu-ated by assaying five sample extracts prepared as described. The resultsshowed that all the relative standard deviations (% RSD) of 9 flavonoidcommon peaks and 10 saponin common peaks assays in stability, preci-sion and reproducibility tests were less than 1.34% for relative retentiontime and less than 5.00% for relative peak area, respectively.
Establishment of Chromatographic Fingerprint of Flavonoids of SZS
Spinosin (1) and 6000-feruloyl spinonsin (2), two of the most importantactive flavonoids from SZS, were often used as the indicator for species
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identifying and quality evaluating of this herbal drug. Under the optimalchromatographic conditions for flavonoid fingerprint analysis describedabove, the chromatographic fingerprints of SZS were obtained and 9main peaks were revealed, among which peaks 5 and 6 were designatedas spinosin and 6000-feruloyl spinonsin respectively by comparing theirretention times with the standards and further confirmed by their on-lineUV–vis spectra. The retention time and peak area of spinosin were stableand reproducible, so this compound was defined as reference peak andreference marker for calculating relative retention time and relative peakarea in this study.
Using the developed HPLC fingerprint analysis method of flavo-niods, the fingerprint of flavoniods of 10 samples of SZS purchased from10 different regions in China were analyzed. In the chromatographicfingerprint analysis, the chromatographic peaks whose relative retentiontime was same were defined as the common peak in different samples.The results of fingerprint analysis of flavonoids of 10 batches of SZSsamples showed that the fingerprints of 8 samples were very similar with9 common peaks being present and similar relative peak area ratios.However, the sample from Fujian province is a little different from thatof the 8 samples because of the absence of peak 9, while the sample fromBaoji of Shanxi Province is much different from others because of itsabsence of main peaks 6, 7, 8 and 9 and its relative small peak areasof all peaks, indicating that the sample from Baoji is of low quality.Moreover, peaks 2, 5, 6 were the three highest peaks with the peak areasof 5> 2> 6 in most cases, while in some cases with the peak areas of2> 5> 6 (e.g. sample from Yanan and Baoji). Based on the above results,9 common peaks were determined for flavonoid fingerprint, amongwhich peak 5 and 6 were spinosin and 6000-feruloyl spinonsin, respectively(Fig. 2 and Tables 1 and 2).
Establishment of Chromatographic Fingerprint of Saponins of SZS
Jujubosides A (3) and B (4), two of the most important characteristicsaponins from SZS, were also usually used as the indicator for speciesidentifying and quality evaluating of SZS. Under the chosen chromato-graphic fingerprint conditions of saponins, the chromatographic finger-prints of saponins of SZS were obtained and 10 main peaks wererevealed, among which peak 7 and 9 were identified as jujuboside Aand jujuboside B respectively by comparing the retention times withthe reference standards and further confirmed by their on-line UV–visspectra. The retention time and peak area of jujuboside B were stableand reproducible, and this compound was defined as reference peak
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and reference marker for calculating relative retention time and relativepeak area.
Using the developed HPLC fingerprint analysis method of saponins,the fingerprints of saponins of the 10 samples of SZS were analyzed. The
Table 2. Relative peak area of fingerprint analysis of flavonoids of 10 batchesof SZS
Peak no.
Sample 1 2 3 4 5 6 7� 8 9
Yanan, Shanxi 0.13 1.04 0.12 0.20 1 0.53 0.10 0.19Xingjiang 0.08 0.85 0.08 0.12 1 0.59 0.23 0.20Baoji, Shanxi 0.68 1.70 0.28 0.21 1 nd nd ndJiangxi 0.14 0.52 0.10 0.15 1 0.56 0.26 0.26Hunan 0.13 0.91 0.14 0.17 1 0.49 0.24 0.26Hubei 0.19 0.78 0.12 0.18 1 0.43 0.25 0.24Hebei 0.15 0.47 0.10 0.14 1 0.56 0.22 0.26Guizhou 0.26 0.84 0.20 0.18 1 0.44 0.22 0.12Fujian 0.20 0.99 0.16 0.20 1 0.44 0.20 ndBeijing 0.11 0.65 0.09 0.15 1 0.40 0.18 0.14Average value 0.13 1.04 0.12 0.20 1 0.53 0.10 0.19
nd: not detected.
Figure 2. Representative fingerprint chromatogram of flavonoids of SZS.
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results of fingerprint analysis of saponins of 10 batches of SZS samplesshowed that the fingerprints of 8 samples were very similar with ten com-mon peaks being present and similar relative peak area ratios. However,the sample from Jiangxi province is a little different from that of the 8samples because of the absence of peaks 2 and 8, while the sample from
Figure 3. Representative fingerprint chromatogram of saponins of SZS.
Table 3. Relative retention time of fingerprint analysis of saponins of 10 batchesof SZS
Peak no.
Sample 1 2 3 4 5 6 7 8 9 10
Yanan, Shanxi 0.32 0.34 0.44 0.58 0.62 0.73 0.92 0.95 1.00 1.03Xingjiang 0.31 0.34 0.44 0.58 0.61 0.72 0.92 0.96 1.00 1.03Baoji, Shanxi 0.31 0.33 0.43 0.60 0.62 0.75 0.92 0.95 1.00 1.03Jiangxi 0.31 nd 0.43 0.58 0.61 0.71 0.92 nd 1.00 1.03Hunan 0.33 0.35 0.45 0.60 0.63 0.73 0.92 0.95 1.00 1.03Hubei nd nd nd 0.59 nd 0.72 nd nd 1.00 ndHebei 0.31 0.34 0.44 0.58 0.62 0.73 0.92 0.96 1.00 1.03Guizhou 0.31 0.33 0.43 0.58 0.61 0.73 0.92 0.95 1.00 1.03Fujian 0.32 0.34 0.45 0.59 0.62 0.73 0.92 0.95 1.00 1.03Beijing 0.34 0.37 0.48 0.62 0.64 0.76 0.92 0.95 1.00 1.03Average value 0.32 0.34 0.44 0.59 0.62 0.73 0.92 0.95 1.00 1.03% RSD 3.04 3.25 2.82 2.00 1.61 1.69 0.08 0.04 0.00 0.06
nd: not detected.
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Hubei Province is much different from others because of its absence ofalmost all the peaks except for the presence of peaks 4, 6 and 9, indicatingthat the sample from Hubei is of low quality. Moreover, peaks 4, 7 and 9were the three highest peaks with the peak areas of 9> 7> 4 in mostcases, while in rare cases with the peak areas of 7> 9> 4 (e.g. samplefrom Hebei). In addition, in the sample from Hubei, peaks 4, 6 and 9were the three highest peaks, while peaks 7, i.e. jujuboside A, were absent.Based on the above results, 10 common peaks were determined forsaponin fingerprint of SZS, among which peak 7 and 9 were identi-fied as jujuboside A and jujuboside B, respectively (Fig. 3 andTables 3 and 4).
CONCLUSIONS
In this paper, HPLC-DAD fingerprint analysis methods of characteristicbioactive flavonoids and saponins of SZS were developed and 10 batchesof SZS from different regions in China were analyzed using the developedHPLC-DAD methods. Using spinosin (1) and jujuboside B (3) as themarkers of flavonoids and saponins respectively, 9 common fingerprintpeaks of flavonoids and 10 common fingerprint peaks of saponins werespecified. The fingerprint analysis methods developed are reliable, repro-ducible and valid, and might be used as a more convenient approach forthe species identification and quality assessment of SZS.
Table 4. Relative peak area of fingerprint analysis of saponins of 10 batchesof SZS
Peak no.
Sample 1 2 3 4 5 6 7 8 9 10
Yanan, Shanxi 0.12 0.15 0.18 0.34 0.23 0.35 0.98 0.24 1.00 0.38Xingjiang 0.14 0.05 0.15 0.35 0.10 0.23 0.96 0.27 1.00 0.23Baoji, Shanxi 0.08 0.12 0.17 0.25 0.15 0.18 0.90 0.26 1.00 0.33Jiangxi 0.16 nd 0.16 0.37 0.11 0.31 0.55 nd 1.00 0.34Hunan 0.11 0.07 0.17 0.63 0.08 0.13 0.86 0.22 1.00 0.27Hubei nd nd nd 0.74 nd 3.78 nd nd 1.00 ndHebei 0.34 0.17 0.61 0.81 0.34 0.39 1.12 0.60 1.00 0.71Guizhou 0.07 0.04 0.18 0.25 0.07 0.11 0.50 0.16 1.00 0.36Fujian 0.08 0.10 0.12 0.42 0.11 0.22 0.50 0.22 1.00 0.30Beijing 0.14 0.21 0.28 0.62 0.26 0.39 0.73 0.44 1.00 0.52
nd: not detected.
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