Authentication of Schisandra chinensis and Schisandra sphenanthera in Chinese patent medicines

Authentication of Schisandra chinensis and Schisandra sphenanthera in Chinese patent medicines

Journal of Pharmaceutical and Biomedical Analysis 131 (2016) 263–271 Contents lists available at ScienceDirect Journal of Pharmaceutical and Biomedi...

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Journal of Pharmaceutical and Biomedical Analysis 131 (2016) 263–271

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba

Authentication of Schisandra chinensis and Schisandra sphenanthera in Chinese patent medicines Pin Jiang a,b,1 , Yan Lu b,1 , Daofeng Chen a,b,∗ a b

State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai 201203, China

a r t i c l e

i n f o

Article history: Received 4 July 2016 Received in revised form 29 August 2016 Accepted 30 August 2016 Available online 31 August 2016 Keywords: Herbal authentication Schisandra chinensis Schisandra sphenanthera Chinese patent medicine TLC HPLC

a b s t r a c t Authentication of species is crucial for ensuring the safety and efficacy of herbal medicines. The fruits of Schisandra chinensis and S. sphenanthera have been used for the same traditional Chinese drug, Wuweizi, but are found to be quite different according to their constituents, pharmacological effects, and qualities. These two fruits have been recorded as Schisandrae Chinensis Fructus (Wuweizi) and Schisandrae Sphenantherae Fructus (Nan-wuweizi), respectively, by Chinese Pharmacopoeia, 2000 edition. However, Nan-wuweizi is often found to be taken as Wuweizi in some Chinese patent drugs intentionally or by mistake because of its lower price and similar characteristics to Wuweizi. In this study, the selection and validation of special chemical markers for the identification of Schisandra species were established by HPLC-DAD-MS profiling analysis. Simple TLC and HPLC methods were proposed for the accurate determination of Nan-wuweizi from Wuweizi in Chinese patent medicines, using schisandrin and anwulignan as the identifying markers for Wuweizi and Nan-wuweizi, respectively. Through the establishment of a statistical model, adulterated or misused ratios of Nan-wuweizi in Wuweizi (w/w), as well as in Fenghan Kesou pills, can be determined. The limit of detection of Nan-wuweizi in a mixture (w/w) using both TLC and HPLC methods is 5% (mixed crude drugs of 50 mg and 5 g in a 1000 g prescribed amount). The constructed statistical model relating the HPLC peak area ratio (anwulignan/schisandrin) and adulteration ratio is suitable for mixed crude drugs and Fenghan Kesou pills, and the two fitting equations have a good correlation (r = 0.9979). Furthermore, 36 commercial Chinese patent medicines containing Wuweizi or Nan-wuweizi according to their labels were checked by these methods, and Nan-wuweizi was detected in Renshen Wuweizi Granules and Fenghan Kesou Pills. The ratios of Nan-wuweizi in these mixtures (w/w) were 100:0 for both, which does not comply with the statutory prescription. This study provided a simple and reliable method to prevent the adulteration or misuse of Nan-wuweizi in crude drugs and patent medicines of Wuweizi. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Herbal medicines have become increasingly popular throughout the world and have been commonly used for preventive and therapeutic purposes since ancient times [1]. More than 2000 herbal products have been included in the Chinese pharmacopoeia (State Pharmacopoeia Committee, 2015) [2]. The authentication of materia medica, usually dried or processed plant parts, is important to ensure their safety and efficacy. Some herbs belonging to closely related species, but differing in medicinal properties, are

∗ Corresponding author at: Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai 201203, China. E-mail address: [email protected] (D. Chen). 1 These two authors contributed equally to this work. http://dx.doi.org/10.1016/j.jpba.2016.08.040 0731-7085/© 2016 Elsevier B.V. All rights reserved.

difficult to identify because they have similar morphological and microscopic characteristics [3]. However, they have different pharmacological effects and clinical applications according to the theory of traditional Chinese medicine [4]. In the global market for herbal medicines, adulterants and alternatives are traded for relatively lower costs [5]. Therefore, authentication and quality control of herbal species are challenging tasks for analysts [6]. Wuweizi (Schisandrae Fructus, Chinese Magnoliavine Fruit) has been used for thousands of years [7] because of its various biological activities that make it beneficial for the central nervous, endocrine, respiratory, digestive, and cardiovascular systems [8–13]. For a long time, Wuweizi was known as the ripe fruits of both Schisandra chinensis (Turcz.) Baill and Schisandra sphenanthera, Rehd. et Wils. [14]. However, research has confirmed that the two fruits are quite different regarding their pharmacological effects and constituents.

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Clinical studies have shown that the effects of Nan-wuweizi on the nervous, cardiovascular, and digestive systems are weaker than those of Wuweizi [15]. Wuweizi mainly contains lignans, organic acids, and glycosides, while Nan-wuweizi contains lignans and triterpenes [7]. Chinese Pharmacopoeia has recorded the two fruits as Wuweizi and Nan-Wuweizi, respectively, since 2000. According to the Chinese Pharmacopoeia, 2015 edition [2], the quality control indicators of Wuweizi are schisandrin and deoxyschisandrin, while those of Nan-wuweizi are schisantherin A and anwulignan. Currently, Nan-wuweizi is often referred to as Wuweizi in commercial drugs and some Chinese patent drugs, intentionally or by mistake, because of its lower price and similarities with Wuweizi. Although there are reports of the simultaneous determination of the main components of Wuweizi and Nan-wuweizi [16–27], few studies have reported the specific identification of Wuweizi from Nanwuweizi, especially in applications of traditional Chinese patent medicines. In addition, the accurate determination of the ratio of Schisandra mixed in crude drugs and Chinese patent medicines is a challenge. In this paper, simple TLC and HPLC methods are proposed for the accurate determination of Nan-wuweizi from Wuweizi in crude drugs and Chinese patent medicines using schisandrin and anwulignan as the identifying markers for Wuweizi and Nanwuweizi, respectively. Mixtures of various ratios of Wuweizi and Nan-wuweizi and Fenghan Kesou pills (FHKSP) made from these mixtures were used to validate the methods; FHKSP is a famous herbal preparation that is widely used in China for treating asthma, including symptoms such as headache, stuffy nose, phlegm, cough, tightness of the chest, and wheezing. Both TLC and HPLC methods are able to identify the two fruits from mixtures of at least 5% (w/w), either from crude drugs or Chinese patent medicines. Moreover, by establishing a statistical model for the peak area of Anwulignan/Schisandrin in HPLC, adulterations or mixtures of Nanwuweizi in Wuweizi (w/w) as well as in FHKSP can be determined. Finally, 31 commercial Chinese patent medicines that were labeled to contain Wuweizi, and 5 commercial Chinese patent medicines that were labeled to contain Nan-wuweizi were checked by the described methods.

2. Experimental 2.1. Materials and reagents Twenty batches of Wuweizi and Nan-wuweizi were collected from nine provinces in China (Table S1). Ten crude drugs were used to prepare Fenghan Kesou pills (FHKSP), including Citri Reticulatae Pericarpium, Pinelliae Rhizoma Praeparatum, Citri Reticulatae Pericarpium Viride, Armeniacae Semen Amarum, Ephedrae Herba, Perillae Folium, Schisandrae Chinensis Fructus, Mori Cortex, Glycyrrhizae Radix Et Rhizoma Praeparata Cum Melle, and Zingiberis Rhizoma Recens; were purchased from the Leiyunshang pharmacy (Shanghai, China); and were identified by Professor Dao-feng Chen. Thirty-six commercial Chinese patent drugs containing S. chinensis and S. sphenanthera were collected from different pharmaceutical manufacturers (Table S2). Seven chemical reference standards (CRS) (Fig. S1) and two reference drugs, including (1) schisandrin (batch no. 110857-201211), (2) schisandrol B (batch no. P0930267), (3) schisantherin A (batch no. 111529-200604), (4) schisantherin B (batch no. P0940045), (5) deoxyschisandrin (batch no. 110764-201111), (6) anwulignan (batch no. 111844-201102), and (7) schisandrin B (batch no. 110765-201311), Schisandrae Chinensis Fructus (Wuweizi) (batch no. 120922-201108), and Schisandrae Sphenantherae Fructus (Nan-wuweizi) (batch no. 121118-201003), were purchased from the National Institutes for Food and Drug Control (Beijing,

China) and Chengdu Push Bio-Technology (Chengdu, China). The purity of each compound was determined to be more than 98% by the normalization of the HPLC-UV peak areas. Methanol (HPLC grade) and acetonitrile (HPLC grade) were purchased from Adamas-beta (Shanghai, China). Formic acid (HPLC grade) was purchased from ROE Scientific, Inc. (Newark, NJ, USA). Deionized water was prepared by a Milli-Q reagent water purification system (Millipore, Bedford, MA, USA). Other reagents were of analytical grade. 2.2. Sample and reference standard solution preparations 2.2.1. Preparation of the reference standard solutions An adequate amount of each reference standard was dissolved in cyclohexane to prepare the reference standard test solution. Mixed solutions of all 7 reference standards were made using the same method. All of the test solutions were stored at 4 ◦ C prior to the TLC and HPLC analyses. 2.2.2. Preparation of the Wuweizi and Nan-wuweizi mixture solution Wuweizi and Nan-wuweizi were powdered and mixed at various ratios (w/w), including 100:0, 80:20, 60:40, 40:60, 20:80, 10:90, 5:95, and 0:100 (Nan-wuweizi:Wuweizi), to prepare eight Schisandrae mixtures. Twenty-five mL of cyclohexane was added to 1.0 g of each mixture, followed by ultrasonication for 30 min at room temperature. The mixture was filtered, and the filtrate was used as the mixture solution. The mixture solution was further filtered through a 0.45-␮m filter membrane for HPLC determination. Finally, the mixture solution was concentrated to 3 mL for TLC determination. 2.3. Preparation of the test solutions of Fenghan Kesou pills (FHKSP) and the negative control samples Eight laboratory-made FHKSP samples containing the above Schisandrae mixtures were prepared to validate the methods. Each Schisandrae mixture and the other 9 crude drugs were crushed into fine powders and mixed well to yield FHKSP according to the preparation process recorded in the Chinese Pharmacopeia [2]. The negative control (NC) FHKSP sample without Wuweizi was made by the same method. Five grams of the FHKSP sample was dispersed in 25 mL of cyclohexane and ultrasonicated for 30 min at room temperature. The mixture was filtered, and the filtrate was used as the mixture solution. The mixture solution was further filtered through a 0.45-␮m filter membrane for HPLC determination. Finally, the mixture solution was concentrated to 3 mL for TLC determination. The test solutions of NC samples were prepared by the same procedure. 2.4. Instrumentation and chromatographic conditions 2.4.1. TLC conditions for identification TLC analysis was carried out using a CAMAG HPTLC system (CAMAG, Muttenz, Switzerland) equipped with an automatic TLC sampler 4 (ATS 4), an automatic developing chamber (ADC 2), a chromatogram immersion device, a temperature control heater, a TLC visualizer and WinCATS 1.4.4 software. An aliquot of the standard or sample solution was directly deposited onto a TLC plate, which was developed in a presaturated solvent chamber with dichloromethane-methanol (12:0.1, v/v) as the developing reagent until the solvent front reached 1.5 cm from the top of plate. The developed TLC plate was then removed from the chamber and allowed to air-dry for 5 min. The air-dried TLC plate was observed under ultraviolet light at 254 nm and then sprayed with 5% phosphomolybdic acid in ethanol. The plate was then placed on a temperature-controlled heater at 105 ◦ C for 5 min until colored

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Fig. 1. Selection of chemical markers differentiating Wuweizi and Nan-Wuweizi using LC–DAD–MS profile analysis. A: Reference Schisandrae Chinensis Fructus (Wuweizi); B: Reference Schisandrae Sphenantherae Fructus (Nan-wuweizi). Peak 1, schisandrin; 2, gomisin D; 3, schisandrol B; 4, gomisin O; 5, schisantherin A; 6, schisantherin B; 7, schisantherin C; 8, gomisin L1; 9, deoxyschisandrin; 10, schisantherin P; 11, anwulignan; 12, gomisin G; 13, ␥-schisandrin; 14, schisandrin B.

spots appeared on it. The plate was also observed under visible light in a TLC visualizer and documented by a digital camera. 2.4.2. HPLC conditions for identification HPLC analysis was carried out using an Agilent 1200 series HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with an online vacuum degasser, a quaternary pump, an autosampler, a thermostatic column compartment, a diode array detector (DAD, wavelength range from 190 to 400 nm), and B. 04. 02 Chemstation software. Chromatography was conducted on a Phenomenex Luna-C18 column (250 mm × 4.6 mm, 5 ␮m) (Torrance, CA, USA) at a column temperature of 35 ◦ C. Optimal separation was obtained under isocratic elution with a mixture of 0.1% formic acid and water-methanol-acetonitrile (28:60:12, v/v/v) as the mobile phase. The flow-rate was set at 1.0 mL/min, and the injection volume was 1 ␮L. The samples were detected at a wavelength of 300 nm. 2.4.3. LC–MS conditions The chromatographic peaks were confirmed by LC–MS, using an Agilent 1200 LC–MSD equipped with electrospray ionization (ESI) in positive ion mode. The following operating parameters were obtained: capillary voltage, 3500 V; drying gas, N2 ; flow rate, 10 L/min; drying gas temperature, 350 ◦ C; and nebulizer pressure, 35 psig. Mass spectra were recorded across the range m/z 100–1000. The HPLC conditions were the same as described above. 3. Results and discussion 3.1. Selection and validation of chemical markers for the identification of Schisandra species using LC-DAD-MS profile analysis The LC-DAD-MS characteristic chromatograms of Wuweizi and Nan-wuweizi reference drugs are shown in Fig. 1. The chromatogram of Wuweizi showed schisandrin, gomisin D, schisandrol

B, schisantherin A, schisantherin B, schisantherin C, gomisin L1, deoxyschisandrin, gomisin G, ␥-schisandrin and schisandrin B, while Nan-wuweizi exhibited peaks for gomisin O, schisantherin A, gomisin L1, deoxyschisandrin, schisantherin P, anwulignan, gomisin G, and ␥-schisandrin (Table S3). Schisandrin, gomisin D, schisandrol B, gomisin O, schisantherin P, anwulignan, and schisandrin B were the key compounds that distinguished the two Schisandra species. Schisandrin, gomisin D, schisandrol B, and schisandrin B were the main components of Wuweizi, while gomisin O, schisantherin P, and anwulignan were the main components of Nan-wuweizi. These differences were also exhibited in the chromatograms of 20 batches of Wuweizi and Nan-wuweizi samples of different origins (Fig. 2). Taking into account the higher contents, strong bioactivities, universality, and specificity of the chemical markers, schisandrin and anwulignan were used as characteristic indicators to identify Wuweizi and Nan-wuweizi, respectively.

3.2. Optimization of identification conditions of TLC and HPLC To obtain a higher extraction efficiency for the Schisandrae mixture and FHKSP, various extractive methods (ultrasonication and reflux), extraction solvent systems (methanol, ethanol, and cyclohexane), and extraction times (15, 30, 45, and 60 min) were assessed on the basis of chromatograms. Extraction with a cyclohexane solution in an ultrasonic water bath for 30 min was found to be the optimal sample preparation method yielding the best extraction efficiency. The sample solutions were optimized according to the TLC and HPLC chromatographic conditions. Different developing systems, including dichloromethane/methanol, chloroform/acetone, and petroleum ether/ethyl formate/formic acid, and different mobile phase systems, including acetonitrile/water, methanol/water, methanol/acetonitrile/water, methanol/acetonitrile/water with 0.1% formic acid, were investigated. Additionally, different TLC

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Fig. 2. Validation of the chemical markers using HPLC analysis in 20 batch samples of Wuweizi and Nan-Wuweizi from different origins.

detection methods (ultraviolet light and spraying color) and HPLC detection wavelengths (254 nm, 280 nm, and 300 nm) were assessed on the basis of chromatograms. The results showed that the developing reagent, dichloromethane-methanol (12:0.1, v/v), showed good separation of spots, while the mobile phase consisting of 0.1% formic acid and water-methanol-acetonitrile (28:60:12, v/v/v) showed good separation and sharp peaks. The developed TLC plate was observed under ultraviolet light at 254 nm (schisandrin) and then sprayed with 5% phosphomolybdic acid in ethanol (anwulignan). In addition, the HPLC samples were detected at a wavelength of 300 nm.

3.3. Analysis to distinguish Wuweizi and Nan-wuweizi in mixed crude drugs Simple TLC and HPLC methods were proposed for the accurate recognition of Nan-wuweizi from crude drugs of Wuweizi. To assess the LOD of the methods, various ratios of Nan-wuweizi in Wuweizi were selected to prepare eight Schisandra mixtures. Schisandrin

and anwulignan were used as markers for the identification of Nanwuweizi and Wuweizi, respectively. In the TLC chromatograms, spots of schisandrin were visible under ultraviolet light at 254 nm, while the spots of anwulignan were colored by 5% phosphomolybdic acid in ethanol (Fig. 3A). The comparison of TLC images showed differences between the mixtures of various designated concentrations. The proportion of Nan-wuweizi increased in Schisandra mixtures, and the intensity of schisandrin spots gradually decreased, while the intensity of anwulignan spots increased gradually, but significantly. At least 5% (w/w) of Nan-wuweizi in the mixture could be detected accurately, but the LOD was no less than 50 mg. In HPLC analysis, characteristic peaks in the chromatograms were compared to discriminate the Schisandra mixtures (Fig. 3B). The trends in the peak areas of the main chemical markers, schisandrin, schisantherin A, deoxyschisandrin, anwulignan, and schisandrin B, in the mixture samples are shown in Fig. S2. When the proportion of Nan-wuweizi in the Schisandra mixtures was increased, the peak areas of schisandrin and schisandrin B gradually became weaker, while those of schisantherin A, deoxyschisandrin,

Fig. 3. TLC (A) and HPLC (B) results of species identification in mixed crude drugs consisting of Wuweizi and Nan-wuweizi. A: 254 nm (a) and spray color under visible light (b); R: Reference standards, R1, Wuweizi, R2, Nan-wuweizi, R3, anwulignan, R4, schisandrin; No. 1–8: Mixtures with 0%, 5%, 10%, 20%, 40%, 60%, 80%, 100% Nan-wuweizi and 100%, 95%, 90%, 80%, 60%, 40%, 20%, 0% Wuweizi, respectively. B: W, Wuweizi; N, Nan-wuweizi.

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Fig. 4. TLC (A) and HPLC (B) results of species identification in Fenghan Kesou pills (FHKSP) consisting of Wuweizi and Nan-wuweizi. A: 254 nm (a) and spray color under visible light (b); R: Reference standards, R1, Wuweizi, R2, Nan-wuweizi, R3, anwulignan, R4, schisandrin; No. 1–9: Mixtures with 0%, 0%, 5%, 10%, 20%, 40%, 60%, 80%, 100% Nan-wuweizi and 0%, 100%, 95%, 90%, 80%, 60%, 40%, 20%, 0% Wuweizi, respectively. B: W, Wuweizi; N, Nan-wuweizi.

and anwulignan showed a gradual but significant increase. It was easy to detect sample adulteration or misuse. At least 5% (w/w) of Nan-wuweizi in the mixture could be detected accurately, but the LOD was no less than 50 mg. The TLC and HPLC methods proved the reliability and specificity of the selected markers. Schisandrin and anwulignan peaks were detected in the mixture samples, indicating the presence of Wuweizi and Nan-wuweizi, respectively. Both TLC and HPLC showed consistent results for the identification of Wuweizi and Nan-wuweizi. Both methods were able to identify at least 5% (w/w) of Nan-wuweizi in the mixture, indicating a high discriminating power and enabling differentiation based on chemical markers. The results of the TLC images and HPLC peaks can be used to assist in determining the presence or absence of different Schisandra species in crude drugs. Thus, this work can serve as a reference for the identification of Nan-wuweizi and Wuweizi in traditional Chinese patent medicines. 3.4. Analysis to distinguish the model Fenghan Kesou pills (FHKSP) consisting of Schisandra mixtures As an illustrative case study, FHKSP was used as the model patent drug in which Wuweizi is one of the ten prepared slices in the prescription of the Chinese crude drugs. The laboratory-made FHKSP and NC preparations were used to validate the method. One of the critical steps for the analysis of the targeted chemical markers is sample preparation to account for interference from non-target components. In addition, simple extraction conditions were applied to reduce the complexity of the extracted markers and to focus on the difference of various ratios of FHKSP. Fig. 4A gives an overview of the samples, demonstrating the excellent specificity of the TLC method. As expected, the schisandrin and anwulignan markers were not detectable in the NC sample. On the other hand, the markers were observed in FHKSP samples of various ratios and showed spots that matched CRS and matched the results for the Schisandra mixtures. At least 5% (w/w) of Nan-wuweizi could be detected in FHKSP, for which the LOD was no less than 5 g in a prescribed amount (1000 g). Similar results were observed for the HPLC method, indicating that the selected chemical markers generally do not interfere with our methods (Fig. 4B). The main chemical marker peaks of schisan-

drin and anwulignan in the mixture samples are shown in Fig. 4B. With the increasing proportion of Nan-wuweizi in the Schisandra mixtures, the schisandrin peak areas gradually decreased, while the anwulignan peak areas increased gradually but significantly. It was easy to determine sample adulteration or misuse. At least 5% (w/w) of Nan-wuweizi could be detected in the mixture in FHKSP, for which the LOD was no less than 5 g in a prescribed amount (1000 g). At present, no official threshold levels are in place for undeclared Schisandra species in Chinese patent medicines. Therefore, the minimal limit of our methods and the threshold level of 5% (w/w) of Nan-wuweizi in Schisandra mixtures were set as the orientation for checking items regarding quality standards. 3.5. Statistical model optimization and validation for the determination of the mixed ratio in crude drugs and FHKSP The present method can identify the presence or absence of Nan-wuweizi in a mixture. However, the mixed ratio in crude drugs and FHKSP is a more pertinent issue. The changes in peak areas of HPLC provided a basis for the establishment of a mathematical model to determine the extent of adulteration or misuse in samples. A statistical model was established to determine the composition of samples. Wuweizi and Nan-wuweizi were powdered and mixed at various ratios (w/w) of 90:10, 80:20, 60:40, 40:60, 20:80, 10:90, 5:95, and 0:100 (Nan-wuweizi:Wuweizi) to prepare eight Schisandrae mixtures for crude drugs and FHKSP. To establish the HPLC mathematical model, eight Schisandrae mixtures were made and each working solution was injected twice. Then, a model function curve was constructed for each mixture by plotting the peak area ratio versus the corresponding mixture ratio of Nan-wuweizi and Wuweizi. As displayed in Fig. 5, four different peak area ratios (anwuligan/schisandrin, anwuligan/schisandrin B, schisantherin A/schisandrin, schisantherin A/schisandrin B) were compared for model optimization, for which the correlation coefficient of mixtures of crude drugs and FHKSP were 0.9979, 0.9976, 0.9720, and 0.9887. The fit coefficient (R) for each linear regression equation was no less than 0.90. To validate the performance of the established model, the remaining six samples (three mixed crude drugs and three FHKSP) were used as an external prediction set. The relationships between the actual value and model-determined

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Fig. 5. Model optimization in crude drugs and FHKSP. A: anwuligan/schisandrin; B: anwuligan/schisandrin B; C: schisantherin A/schisandrin; D: schisantherin A/schisandrin B.

Table 1 Validation of mixed ratio in crude drugs and FHKSP. Area Ratio Actual value Determined value

anwuligan/schisandrin anwuligan/schisandrin B schisantherin A/schisandrin schisantherin A/schisandrin B

Mixtures of wuweizi and nan-wuweizi

Fenghan Kesou Pills

15%

50%

85%

15%

50%

85%

15.84% 14.66% 17.19% 15.16%

51.47% 52.31% 53.15% 54.73%

86.13% 82.07% 86.75% 88.42%

16.76% 12.28% 14.32% 19.20%

52.39% 45.22% 47.31% 53.60%

87.06% 80.85% 90.17% 86.49%

value are shown in Table 1, which shows that the results for anwuligan/schisandrin were close to the actual value. Taking into account the fit coefficient, correlation coefficient, and model-determined value, anwuligan/schisandrin is a suitable model to determine mixed ratios in crude drugs and FHKSP. 3.6. Applicability to authenticate 36 commercial Chinese patent medicines The applicability of the detection methods was assessed over a broad range of commercially available herbal patent drugs collected from a pharmacy. All of the drugs included species of S. chinensis or S. sphenanthera. Samples were prepared and analyzed according to the methods describe above. The identification methods using schisandrin and anwulignan as markers proved to be feasible for Schisandra mixtures and laboratory-made FHKSP. An overview of the results of the 36 commercial samples in this study is given in Table 2. The results of the identification and labeling of the species were consistent for all of the analyzed samples except Renshen Wuweizi Granules and Fenghan Kesou Pills. These two samples were labeled to contain Wuweizi, but Nan-wuweizi markers were detected using TLC and HPLC methods, indicating unlabeled amounts of Schisandra mixtures in these samples (Fig. 6A/B). According to the ratio of the HPLC peak areas (anwulignan/schisandrin), these assays were both close to +∞. The ratio

indicated 100% Nan-wuweizi in the collected Renshen Wuweizi Granules and Fenghan Kesou pill samples. On the other hand, the labeled species were correct for Dengzhan Shengmai capsules and Fufang chuanbeijing tablets, as indicated by the results shown in the figures (Fig. 6A/B). The LC–MS method was also evaluated for the characteristic peaks to confirm the chemical markers. Average mass spectra of schisandrin and anwulignan in samples of two species, samples within m/z 100–1000 are shown in Fig. 6B. The ions at m/z 432.8 ([M+H]+ ) and 454.8 ([M+Na]+ ) indicate the presence of schisandrin, while the ions at m/z 329.2 ([M+H]+ ) and 351.2 ([M+Na]+ ) indicate the presence of anwulignan. From 36 patent drugs collected from the pharmacy, the use of Schisandra species as raw materials in Chinese patent drugs, including S. sphenanthera, is correct. Additionally, drugs containing S. chinensis were checked for mixed contents of species. Moreover, when the preparation processes of Chinese patent medicines use a water-extraction process, the authentication methods are limited for Schisandrae Fructus. 4. Conclusions In the present study, simple TLC and HPLC methods were established for the authentication of Wuweizi and Nan-wuweizi in crude drugs and herbal patent drugs using schisandrin and anwulignan as the identifying markers, respectively, without mutual interference.

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Fig. 6. (A) TLC test results of typical commercial herbal patent drugs. A: 254 nm; B: spray color under visible light. 1, Renshen Wuweizi Granules (RSWWZG); 2, Fenghan Kesou pills (FHKSP); 3, Dengzhan Shengmai Capsules (DZSMC); 4, Fufang chuanbeijing Tablets (FFCBT) as cases. R: Reference standards, R3, anwulignan; R4, schisandrin. (B) HPLC and LC–MS test results of typical commercial herbal patent drugs: 1, RSWWZG; 2, FHKSP; 3, DZSMC; 4, FFCBT as cases. R: Reference standards, R3, anwulignan; R4, schisandrin.

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Table 2 Screening identification results of commercial herbal patent drugs. Sample

C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 C-10 C-11 C-12 C-13 C-14 C-15 C-16 C-17 C-18 C-19 C-20 C-21 C-22 C-23 C-24 C-25 C-26 C-27 C-28 C-29 C-30 C-31 C-32 C-33 C-34 C-35 C-36

Pharmaceutical preparation

Wuweizi Keli Xiaoqinglong Keli Liganlong Keli Fenghan Kesou Keli Zaoren Anshen Keli Renshen Wuweizi Keli Yishenling Keli Fenghan Kesou Wan Bantu Wan Juhong Huatan Wan Sishen Wan Shihu Yeguang Wan Bushen Guchi Wan Wuzi Yanzong Wan Anshen Buxin Wan Ermu Ningsou Wan Baizi Yangxin Wan Anshen Jiaonang Dengzhan Shengmai Jiaonang Shengmai Jiaonang Yixinshu Jiaonang Jiannao Jiaonang Gengnian’an Jiaonang Zaoren Anshen Jiaonang Jianganling Jiaonang Guben Kecuan Jiaonang Fufang chuanbeijing Pian Hugan Pian Anshen Buxin Pian Biyan Pian Guben Kecuan Pian Wuzhi Jiaonang Wuzhi Ruanjiaonang Wuzhi Pian Shenqi Wuweizi Pian Xiaoke Wan

Species declared

S. chinensis S. chinensis S. chinensis S. chinensis S. chinensis S. chinensis S. chinensis S. chinensis S. chinensis S. chinensis S. chinensis S. chinensis S. chinensis S. chinensis S. chinensis S. chinensis S. chinensis S. chinensis S. chinensis S. chinensis S. chinensis S. chinensis S. chinensis S. chinensis S. chinensis S. chinensis S. chinensis S. chinensis S. chinensis S. chinensis S. chinensis S. sphenanthera S. sphenanthera S. sphenanthera S. sphenanthera S. sphenanthera

TLC

HPLC

Declaration confirmed

Schisandrin

Anwulignan

Schisandrin

Anwulignan

match match match match match n.d. match n.d. match match match match match match match match match match match match match match match match match match match match match match match n.d. n.d. n.d. n.d. n.d.

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Match = markers were detectable; n.d. = no markers were detectable.

At least 5% (w/w) of Nan-wuweizi in the mixture could be detected accurately. Through the establishment of a statistical model of peak areas of Anwulignan/Schisandrin in HPLC, adulteration or misuse of Nan-wuweizi in Wuweizi (w/w), as well as in FHKSP, can be determined. For the first time, 36 commercially available herbal patent drugs can be distinguished according to the contents derived different Schisandra species using the aforementioned methods. Two of the 36 patent drugs that were checked did not conform to the labeled Schisandra species. For a specific drug’s identification of, Schisandra species. First, TLC and HPLC methods are supplied for the preliminary determination of species, after which a statistical model can be used to further determine the adulteration or misuse ratio, and finally, LC–MS methods confirm the presence or absence of the chemical markers. By this approach, the presented methods are easily transferable and applicable in routine analytical laboratories for the rapid determination of the Schisandra species existing in commercially available herbal patent drugs. This work provides new insights into authentication and quality control to prevent the adulteration or misuse of Nan-wuweizi in crude drugs and patent medicines of Wuweizi. Acknowledgments This research was supported by a grant from Chinese Pharmacopoeia Commission, the improvement of national drug standards project. We deeply appreciate the valuable help provided by Prof. Zheng-Tao Wang and associate Prof. Li-Hua Gu of Shanghai University of Traditional Chinese Medicine, in the HPTLC analysis.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jpba.2016.08.040.

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