Introduction Schisandra chinensis is a traditional Chinese medicine, of which fruits of S. chinensis (Turcz.) Baill and Schisandra sphenanthera Rehd. et Wils have been recorded as "S. chinensis Fructus" and "S. sphenanthera Fructus" in the Chinese pharmacopoeia, with functions such as astrin- ging the lungs, intestines and sweat, arresting the seminal emission, soothing the nerves, anti-diarrhea, and coagulation. Schisandrol A and schisantherin con- tents are respectively used for the quality control of S. chinensis Fructus and S. sphenanthera Fructus in the Chinese Pharmacopoeia (The State Pharmacopoeia Commission of PR China, 2010), but the selected indexes are too simple and short of a specificity, so that they can not really reflect their inherent quality. A large number of chemical and pharmacological studies have shown that the main active constituents of S. chinensis include schizandrol A, schisandrol B, schisantherin, schisandrin A, schisandrin B, etc, those of S. sphenan- therae include schisantherin, schisandrin A, anwulignan and so on, and these components have some important pharmacological effects (Chen et al., 1997). There have been many reports on the content determi- nation of lignan components in S. chinensis and S. sphenanthera. Most of them focus on several main lignans, such as schisandol A, schisandrol B, schisan- therin, schisandrin A and schisandrin B. Fewer reports on the lignans with lower contents but have some important pharmacological activities (such as schisan- henol and anwulignan). In this study, a HPLC method (Huang et al., 2006; Li et al., 2011) was used to simultaneously separate and determine the content of eight active lignan compo- nents (schisandrol A, schisan-drol B, schisantherin, schisanhenol, anwulignan, schi-sandrin A, schisandrin B and schisandrin C) in S. chinensis and S. Sphenanthera. Based on the analysis on the results of content determination, a quality standard was established, in order to provide a more comprehensive, scientific and A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2016; 11: S161-S167 Journal homepage: www.banglajol.info Abstracted/indexed in Academic Search Complete, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index; ISSN: 1991-0088 Abstract A high performance liquid chromatography method for the determination of eight lignans contents in Schisandra chinensis and Schisandra sphenanthera was developed. The chromatographic column was Agilent ZORBAX 300SB-C18 column (4.6 mm × 250 mm，5 μm). The mobile phase was methanol-water, a gradient elution was conducted and the detection wavelength was at 230 nm. The results showed that the recovery rate of eight lignans was 92.2-102.9% and RSD was 1.5-4.2%. The established content determination method was simple, sensitive, accurate and stable, and can be used to control the quality of S. chinensis and S. sphenanthera. Article Info Received: 10 January 2016 Accepted: 27 February 2016 Available Online: 11 March 2016 DOI: 10.3329/bjp.v11iS1.26844 Cite this article: Xu GY, Niu JM, Yuan GX, Bai Y, Li HY, Sheng Y, Li ZT. Determination of eight lignans in Schisandra Chinensis and Schisandra sphenanth era . Bangladesh J Pharmacol. 2016; 11: S161-S167. Determination of eight lignans in Schisandra chinensis and Schisandra sphenanthera Xu Guangyu1, Niu Jiamu2, Yuan Guangxin1, Bai Yu3, Li Hongyu1, Sheng Yu1 and Li Zitong1 1Pharmaceutical College, Beihua University, Jilin City, Jilin Province 132 013, China; 2Department of Research, Beihua University, Jilin City, Jilin Province 132 013, China; 3Pharmaceutical College, Jilin Medical College, Jilin City, Jilin Province 132 011, China. This paper was presented in the 3rd International Conference on Biomedicine and Pharmaceutics in Zhuhai, China, on December 11-13, 2015. http://www.bioxbio.com/if/html/BANGL-J-PHARMACOL.html http://www.asiajol.info/index.php/record/view/50673 efficient method for the evaluation and quality control of S. chinensis and S. Sphenanthera medicinal materials. Materials and Methods Instruments and reagents LC-2010A liquid chromatograph, including LC solution working station, automatic sample injector and ultra- violet detector etc (Shimadzu Company, Japan); SHIM- PACK VP-ODS C18 chromatographic column (4.6 mm × 150 mm, 5 μm, Shimadzu Company, Japan), AL204 Electronic balance (Mettler-Toledo Instruments Co. Ltd, Switzerland); DHG-9245A Drier (Shanghai Hengke Science and Technology Co. Ltd, China ). Methanol and ethanol were of HPLC grade and pur- chased from Guoyao Biotechnology Co. (China). Water was purified using a Milli-Q system (Millipore, USA). All the reagents were of analytical grade. Reference substances Schisandrol A (batch number: 110857-200910), schisan- therin (batch number: 111529-200302) and wuanlinan (batch number: 111844-201002) were purchased from National Institutes for Food and Drug Control. Schisan- drol B, schizandrin A, schizandrin B, schizandrin C and schisanhenol were made in our laboratory. Their chemical structures all were verified by 1H-NMR and 13C-NMR, and the detection by high-performance liquid chromatography showed that they all had a single peak, with a purity greater than 98%. Experimental materials Fifteen batches of S. chinensis and 5 batches of S. Sphenanthera medicinal materials were collected from different places. All of them were identified by Prof. Zhang Lihua, College of Pharmacy, Beihua University. Samples of the medicinal materials were dried in nature and crushed (40 mesh), and then stored in a cool and dry place for later use. Types and sources of the samples are shown in Table I. Chromatographic conditions Chromtographic column: Agilent ZORBAX 300SB-C18 Chromtographic column (4.6 mm × 250 mm and 5 μm); Shimadzu GVP guard column (4.6 mm); flow phase: methanol (A), water (B), gradient elution (gradient conditions are shown in Table II); column temperature: 27°C; flow velocity: 0.8 mL•min-1; detection wave- length: 230 nm. Preparation of the reference substance solutions Schisandrol A (10 mg), schisandrol B (10 mg), schisantherin (8 mg), schisanhenol (8 mg), anwulignan (8 mg), schisandrin A (10 mg), schisandrin B (10 mg) and schisandrin C (8 mg) were precisely weighed and dissolved in appropriate amount of methanol in 10 mL constant volume bottles, respectively, to obtain 1.0, 1.0, 0.8, 0.8, 0.8, 1.0, 1.0 and 0.8 mg/mL stock solutions of S162 Bangladesh J Pharmacol 2016; 11: S161-S167 Table I Types and sources of the medicinal materials No. Types Sources No. Types Sources 1 FS Anguo, Hebei 11 FS Tonghua, Jilin 2 FS Shangzhi, Henlongjiang 12 FS Wangqing, Jilin 3 FS Mudanjiang, Henlongjiang 13 FS Changchun, Jilin 4 FS Hulin, Henlongjiang 14 FS JIFDC 5 FS Dandong, Liaoning 15 FS JIFDC 6 FS Benxi, Liaoning 16 KL Hengshan, Hunan 7 FS Tieling, Liaoning 17 KL Pingwu, Sichuan 8 FS Dunhua, Jilin 18 KL Songxian, Henan 9 FS Zuojia, Jilin 19 KL Yangcheng, Shanxi 10 FS Jiaohe, Jilin 20 KL Haozhou, Anhui FS: Fructus schisandrae (S. chinensis Fructus); KL: Kadsura longepedunculata (S. Sphenanthera Fructus); JIFDC: Jilin Institute for Food and Drug Control Table II Gradient elution conditions Time (min) A (%) B (%) 0-15 60-75 40-25 16-20 75 25 21-30 75-90 25-10 31-40 90-100 10-0 41-45 100 0 46-55 100-60 0-40 the reference substances. 1.0 mL of the reference subs- tance stock solutions were placed in 10 mL volumetric flasks, respectively, and methanol was added to the flasks to dilute the solution at the scale, for the prepa- ration of mixed reference solution. Preparation of test solutions 10 g dried sample powder weighed precisely was disso- lved in 300 mL methanol. The ultrasound extraction was conducted for 20 min. The solution was shaken and then left to stand for 10 min, and filtered. The residue was dissolved in 150 mL methanol, and the solution was extracted with ultrasound extraction for 20 min 2 times. Filtrates from three extraction were mixed and the mixed solution was dried with decompression evaporation. The residue was dissolved in methanol and set to the constant volume of 100 mL, and the solution was filtered with a 0.45 μm film membrane to obtain the sample solution. Results Analysis on the linearity 1.0 mL of each reference stock solution was precisely weighed, respectively. The solutions were prepared into solutions of series mass concentrations by multiple dilution method. In line with the chromatographic conditions described in section 2.4, 10 μL of the prepared solutions were injected and the peak areas were recorded. The peak area y was taken as the ordinate and the mass concentration of reference substances x (μg) was taken as the abscissa to obtain regression equations, linear range and correlation coefficients of each component for the analysis on the linear regression. As shown in Table III, the results showed that each component exhibited a good linearity in the experimental range. Limit of quantitation (LOQ) and limit of detection (LOD) 1.0 mL of each reference stock solution were precisely weighed, respectively. The solutions were prepared into solutions of series mass concentrations by multiple dilution method. In line with the chromatographic conditions described in section 2.4, 10 μL of the prepared solutions were injected and the peak areas were recorded. SNR (signal to noise ratio) of 10:1 was used as the limit of quantitation and SNR of 3:1 as the limit of detection. It was estimated that the LOQ of schisandrol A, schisandrol B, schisantherin, schisan- henol, anwulignan, schisandrin A, schisandrin B and schisandrin C in schisandra was 25, 25, 20, 10, 40, and 12.5, 25 and 20 μg/mL, respectively; the LOD of them was 5, 5, 8, 2, 8, and 2.5, 5 and 4 μg/mL, respectively. Precision test 10 μL of each of the mixed reference solution were injected in line with the chromatographic conditions described in section 2.1 and continuously measured 6 times. Peak area RSD values of schisandrol A, schisandrol B, schisantherin, schisanhenol, anwulignan, schisandrin A, schisandrin B and schisandrin C in schisandra were 1.43%, 1.89%, 2.39%, 2.41%, 2.87%, 3.49%, 3.58% and 3.9%, respectively, indicating that the precision of this method used for the content determi- nation of these eight lignans should be good. Reproducibility test In order to make sure that the results could more accurately reflect methodological parameters, samples with higher contents of 8 lignans (No. 20) were chosen for the analysis, and the reproducibility, stability and recovery of the method were investigated. The medi- cinal material powders (No. 20) were simultaneously prepared into 5 copies of the test sample solution according to the method described in section 2.6, and 10 Table III Regression equations, linearity and correlation coefficients Regression equation Linear range μg/mL R Schisandrol A y=2192.5x + 586.4 25-500 0.9997 Schisandrol B y=2288.5x + 300.3 25-500 0.9997 Schisantherin y=2642.2x + 336.8 20-400 0.9996 Schisanhenol y=1142.9x + 108.5 20-400 0.9994 Anwulignan y=2032.8x + 85.29 20-400 0.9995 Schisandrin A y=2473.6x + 35.86 25-500 0.9995 Schisandrin B y=1278.9x + 123.3 25-500 0.9996 Schisandrin C y=2298.5x + 578.5 20-400 0.9997 Bangladesh J Pharmacol 2016; 11: S161-S167 S163 μL of samples were injected in line with the chromato- graphic conditions described in section 2.4, to measure peak area RSD values of the eight lignans, respectively. Peak area RSD (relative standard deviation) values of the eight lignans were 1.47%, 2.31%, 2.47%, 6.86%, 2.82%, 2.12%, 3.86% and 4.55%, respectively, showing a good reproducibility of this method although a poor reproducibility of the method could be seen in the content determination of schisanhenol and schisandrin C due to their lower contents in the test samples. Stability test The medicinal material powders (No. 20) were prepar- ed into the test sample solutions according to the method described in section 2.6. The test sample solutions were injected and measured at 1, 2, 4, 8, 16 and 24 hours respectively, relative peak areas of the chromatographic peaks were recorded, and RSD values were calculated based on the relative peak areas measured at different time points. The results showed that contents of the eight lignans were stable within 24 hours and RSD values of them were 1.6, 2.9, 2.9, 5.7, 2.9, 1.8 and 4.2%, respectively. Recovery test A sample recovery method was used in this test. 9 copies of 0.1 g medicinal material powders containing the above 8 lignans (No. 20) were precisely weighed. They were added to the reference solutions according to the high, medium and low concentrations, respectively, and three copies of them were added to the solution at each concentration in parallel, which were prepared into sample solutions according to the method describe- ed in section 2.6. 10 μL of the sample solutions were injected in line with the chromatographic conditions described on section 2.4 to calculate the recovery and the results are shown in Table IV. The results showed that recoveries of the eight lignans at the high, medium and low concentrations was within 92.2-102.9%, and RSD values of their recoveries were within 1.5-4.2%. Sample determination Test solutions of S. chinensis and S. sphenanthera samples (Table I) were prepared in line with the method described in section 2.6. 10 μL (n = 3) of the sample solutions were injected in line with the chromatographic conditions. The chromatograhic peak Table IV Recovery test results n = 6 Blank (μg) Addition (μg) Detection (μg) Recovery (%) RSD (%) Schisandrol A 780.2 1000 1764.6 98.0 2.0 500 1259.1 97.3 2.3 250 1018.5 98.5 2.5 Schisandrol B 425.5 1000 1431.5 101.4 2.3 500 928.9 100.8 2.7 250 686.1 102.5 3.1 Schisantherin 840.5 800 1619.5 97.5 3.3 400 1193.4 94.4 2.4 200 998.5 95.0 2.7 Schisanhenol 20.2 80 98.8 93.1 3.2 40 59.3 95.6 2.8 20 38.8 93.2 4.2 Anwulignan 1080.6 800 1878.4 99.8 1.5 400 1466.6 98.7 1.7 200 1268.7 98.9 1.9 Schisandrin A 1170.3 1000 2152.7 98.5 2.0 500 1642.2 97.6 2.8 250 1435.5 101.3 2.6 Schisandrin B 670.9 1000 1673.6 100.4 1.8 500 1185.0 102.1 2.2 250 940.4 102.9 2.4 30.1 80 108.1 93.5 2.6 Schisandrin C 40 69.0 96.3 2.9 20 47.8 92.2 3.9 S164 Bangladesh J Pharmacol 2016; 11: S161-S167 areas were recorded, and based on the regression equations, schisandrol A, schisandrol B, schisantherin, schisanhenol, anwulignan, schisandrin A, schisandrin B and schisandrin C contents in the schisandra samples were calculated. The results are shown in Table V. Representative chromatogram of the reference substances, S. chinensis and S. Sphenanthera are shown in Figure 1. A: mixed reference substances, B: S. chinensis (No. 5), C: S. Sphenanthera (No. 20). 1: schisandrol A, 2: schisan- drol B, 3: schisantherin, 4: schisanhenol, 5: anwulignan, 6: schisandrin A, 7: schisandrin B, 8: schisandrin C. Discussion Many studies have indicated that although the main ingredients of S. chinensis Fructus and S. sphenanthera Fructus are lignans (Opletal et al., 2004; Lu and Chen, 2009; Slanina et al., 1997), their kinds and contents are markedly different (Yuan et al., 2011). In this study, a simple, rapid and sensitive HPLC method has been developed for the simultaneous quantitative analysis of eight lignans in S. chinensis and S. sphenanthera, and the content of eight active lignan components in 15 batch of S. chinensis and 5 batch of S. Sphenanthera were determined in this method. The results of samples determination showed that types of lignans contained in schisandra from different origins were the same, namely, they all contained schisandrol A, schisandrol B, schisantherin, schisanhenol, anwulignan, schisandrin A, schisandrin B and schisandrin C, and no anwulignan was detected in 15 batches of schisandra. There was a great difference in the lignan constituents content of schisandra from different origins, suggesting that although lignan constituents contained in schisandra from different rigins were similar, their quality should be quite different, and appropriate methods should be established to control the quality of them. The type and content of 5 batches of S. chinensis were quite different, showing a great difference in the quality of S. Sphenanthera from different origins; the compari- son of them showed that 5 batches of the samples all contained schisantherin, anwulignan and schisandrin A, and their contents were higher compared with the others (accounting for 61.92-96.88% of total content of above 8 lignans), suggesting that the 3 lignans may be the common components in S. chinensis from different origins, based on which the quality control of S. Sphenanthera can be achieved. Anwulignan is a lignan of Table V Determined contents of eight lignans in the samples (mg/g, n = 3) No. A B C D E F G H T 1 2.9 0.8 1.1 + a - b 0.6 4.0 0.3 9.8 2 6.0 4.1 2.5 0.4 - b 1.1 3.8 0.6 18.6 3 3.5 2.9 1.5 0.3 - b 0.4 2.6 0.2 11.4 4 4.7 4.6 1.6 0.5 - b 1.0 3.8 0.9 17.0 5 6.9 4.0 2.2 0.6 - b 1.4 3.2 0.7 18.9 6 4.5 3.4 1.1 0.4 - b 0.9 3.6 0.6 14.4 7 3.8 1.3 0.5 + a - b 0.3 2.2 + a 8.1 8 5.7 2.4 1.5 0.7 - b 1.9 3.0 0.6 15.8 9 6.6 2.5 1.6 0.7 - b 0.7 3.2 0.4 15.8 10 4.6 1.7 1.1 0.7 - b 0.7 2.4 0.3 11.6 11 7.2 2.9 1.9 0.8 - b 0.9 3.8 0.5 18.0 12 6.0 3.3 2.0 0.9 - b 1.3 4.0 1.4 18.9 13 4.0 1.4 0.9 0.5 - b 0.8 1.7 0.4 9.8 14 5.9 3.8 2.0 0.4 - b 1.1 4.3 1.3 18.7 15 5.2 1.8 1.2 0.4 - b 0.6 1.4 0.7 11.2 16 + a 0.5 0.5 0.7 1.2 6.4 - b - b 9.3 17 + a + a 2.6 0.2 1.6 2.5 + a 0.3 7.1 18 + a - b 3.5 0.5 2.4 4.6 + a 0.4 11.4 19 - b - b 3.8 0.4 2.9 4.8 + a + a 11.9 20 0.8 0.4 0.8 + a 1.1 1.2 0.7 + a 5.0 A: schisandrol A, B: schisandrol B, C: schisantherin, D: schisanhenol, E: anwulignan, F: schisandrin A, G: schizandrin B, H: schizandrin B, T: total contents of 8 Lignans. + a: ﹤ LOQ, - b: ﹤ LOD. Bangladesh J Pharmacol 2016; 11: S161-S167 S165 dibenzylbutanes, the other 7 lignans are all dibenzocyclooctenes lignans, and there are significant differences in the ultraviolet absorption spectra between the two types of lignans. Therefore, we need to select the appropriate detection wavelength to ensure that all lignans has a strong absorption. In this study, we investigated the absorption of eight lignans in 280 nm, 230 nm, 254 nm and 300 nm, respectively. The result showed that there was almost no absorption of the eight lignans at 300 nm wavelength; the absorption of anwulignan was strong and that of the other 7 lignan was weaker at 280 nm wavelength; at 254 nm wavelength, the situation was just opposite to that at 280 nm wavelength, showing an extremely weak 250 200 150 100 50 0 0 5 10 15 20 25 30 35 40 45 mAU 1 2 3 4 5 6 7 8 A 250 200 150 100 50 0 0 5 10 15 20 25 30 35 40 45 mAU B 1 2 3 4 6 7 8 5 250 200 150 100 50 0 0 5 10 15 20 25 30 35 40 45 mAU C 1 2 3 4 6 7 8 5 Min Min Min Figure 1: Chromatograms of reference substances, Fructus schisandrae and Kadsura longepedunculata S166 Bangladesh J Pharmacol 2016; 11: S161-S167 Author Info Niu Jiamu, Yuan Guangxin (Principal contact) e-mail: 17287598@qq.com; yuanguangxin2007@163.com wavelength was chosen as the detection wavelength in this experiment. Conclusion A simple, rapid and sensitive HPLC method has been developed for the simultaneous quantitative analysis of eight lignans in S. chinensis and S. sphenanthera. It was evident that this approach was a useful and rapid technique for identification of main lignans. Conflict of Interest The authors have no competing interests to disclose. Acknowledgements This work was financially supported by the Strategic New Industries and High-tech Development Project of Jilin Province (No. 2013G030), and National Natural Science Foundation of China (81401712), and the Jianfeng Teacher’s Research Foundation of Beihua University, and the science and technology research project of Education Department in Jilin province (2014-546, 2012-442). References Chen DF, Zhang SX, Xie L, Xie JX, Chen K, Kashiwada Y, Zhou BN, Wang P, Cosetino LM, Lee KH. Anti-AIDS agents- XXVI. Structure-activity correlations of gomisin-G-related anti-HIV lignans from Kadsura interior and of related synthetic analogues. 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Simultaneous and rapid determination of main lignans in different parts of Schisandra sphenanthera by Micellar electrokinetic capillary chromatography. Molecules 2011; 16: 3713-22. Bangladesh J Pharmacol 2016; 11: S161-S167 S167 Your feedback about this paper 1. Number of times you have read this paper 2. Quality of paper 3. Your comments Excellent Good Moderate Not good Text5: Group6: Off Dropdown7: [0] DatePrinted: This article was downloaded by you on: Mar 19, 2016