Chinese Journal of
Natural Chinese Journal of Natural Medicines 2018, 16(12): 09510960 Medicines
Flavonoids rather than alkaloids as the diagnostic constituents to distinguish Sophorae Flavescentis Radix from Sophorae Tonkinensis Radix et Rhizoma: an HPLC fingerprint study
DING Pei-Lan, HE Chang-Ming, CHENG Zhi-Hong*, CHEN Dao-Feng*
Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai 201203, China
Available online 20 Dec., 2018
[ABSTRACT] Sophorae Flavescentis Radix (Sophora flavescens Ait., SFR) and Sophorae Tonkinensis Radix et Rhizoma (S. tonkinensis Gapnep., STR) are two commonly used traditional Chinese medicines from Sophora (Leguminosae) plants, which are believed to possess similar bioactive components with entirely different clinical applications. In order to find out the characteristic chemical constituents potentially leading to the unique medicinal properties claimed for each of the two closely related TCMs, an HPLC fingerprint method was developed for analyses of the alkaloid and flavonoid constituents of SFR and STR, respectively, which were further evaluated and compared through similarity calculation and hierarchical clustering analysis (HCA). The results from the present study showed that the alkaloid fingerprints of the two herbs were similar, with many components co-existing in both drugs and various batches of samples from different species being mixed together in the HCA dendrogram. However, their flavonoid constituents were totally different with specific fingerprints being yielded for each herb, and further HCA analysis showed that the tested samples could almost be clearly divided into two groups based on their origins of species. The results from the present study indicated that the flavonoid constituents could serve as the differentially diagnostic constituents of SFR and STR and might potentially attributed to their distinct therapeutic effects.
[KEY WORDS] Sophora flavescens; Sophora tonkinensis; Alkaloids; Flavonoids; HPLC fingerprint; Hierarchical clustering analysis [CLC Number] R95, R917 [Document code] A [Article ID] 2095-6975(2018)12-0951-10
Introduction used internally to treat acute pharyngolaryngeal infections and sore throat [1-2]. Therefore, it is of great interest to find out the Sophorae Flavescentis Radix (SFR, Sophora flavescens characteristic chemical constituents potentially leading to Ait.) and Sophorae Tonkinensis Radix et Rhizoma (STR, S. their unique clinical applications claimed for the two closely tonkinensis Gapnep.) are two commonly used traditional Chinese related herbs. medicines (TCMs) from Sophora (Leguminosae) plants [1]. Although the alkaloids in these two herbs have received Both the herbs were found to contain similar chemical consti- much attention, the holistic and component-based relation- tuents such as quinolizidine alkaloids, prenylated flavonoids, ships between their constituents and different clinical applica- and oleanane triterpenoids, while their clinical applications tions are still unknown. In the Chinese Pharmacopoeia [1], are entirely different [2-3]. SFR is mostly used externally for three alkaloids (matrine, oxymatrine, and sophoridine) widely the treatments of skin diseases and gynaecological diseases, present in Sophora plants [4], are chosen as qualitative mark- such as eczema, dermatitis, and colpitis, while STR is frequently ers for identification of these two herbs in the thin layer chromatography tests, which is known to lack specificity in distinguishing SFR and STR. A similar situation exists in the [Received on] 29- Sep.-2018 quantitative tests for the two herbs recorded in the Chinese [Research funding] This work was supported by the Natural Science Pharmacopoeia [1]. These problems may arise from the vari- Foundation of China (Nos. 30672597 and 81330089). ous pharmacological findings of alkaloids that are tentatively [*Corresponding author] Tel: 86-21-51980157; E-mail: chengzhh@ fudan.edu.cn (CHENG Zhi-Hong); Tel: 86-21-51980135; E-mail: assigned as the unique active constituents of the two herbs. It [email protected] (CHEN Dao-Feng) is well known that the therapeutic efficacy of TCMs is often These authors have no conflict of interest to declare. attributed to the synergic effect of their multiple bioactive Published by Elsevier B.V. All rights reserved components and multi-targets, and thus analysis of merely
– 951 – DING Pei-Lan, et al. / Chin J Nat Med, 2018, 16(12): 951960 one or a few markers is not adequately representative for unique clinical applications claimed for SFR and STR, com- quality control of herbs. In the past two decades, more and prehensive HPLC fingerprint methods of flavonoids and al- more flavonoids were isolated and identified from the two kaloids in SFR and STR are needed with more samples col- herbs [3], some of which have been gradually revealed to pos- lected from different regions of China. The objectives of the sess significant bio-activities such as anticancer [5-6], anti-in- present study were as follows: 1) to establish HPLC finger- [7] [8] [9] flammatory , anti-diabetes and anti-virus activities . prints of the alkaloid and flavonoid constituents for the two These findings of the flavonoids prompted us to re-examine herbs, and 2) to compare and analyze the fingerprints by comprehensively the active components of the two herbs in similarity calculation and hierarchical clustering analysis terms of alkaloids and flavonoids. (HCA). HPLC fingerprinting has been used over the last decade for authentication and quality control of herbs and their Materials and Methods preparations. This method emphasizes the whole profile of Plant materials components in a complex system, and it is a strategy recom- The reference SFR and STR herbs were obtained from mended to assess the quality of botanical products by the US the National Institute for the Control of Pharmaceutical and Food and Drug Administration (FDA), the European Medi- Biological Products of China (Beijing, China). All the other cines Evaluation Agency (EMEA), and the State Food and botanical samples studied were obtained as commercial drugs [10] Drug Administration of China (SFDA) . To the best of our from 26 different districts in 19 provinces of China. All the knowledge, few reports have been published on systemic samples were authenticated by one of the authors (Dr. CHEN comparison of the alkaloid or flavonoid constituents between Dao-Feng). Among them, 24 samples were identified as the these two herbs using fingerprinting techniques, though many roots of S. flavescens, and 19 samples were the roots and rhi- quantitative studies on the major alkaloids in SFR and STR zomes of S. tonkinensis. The specimens were deposited in the [11-14] by various chromatographic techniques have been reported . Department of Pharmacognosy, School of Pharmacy, Fudan In 2013, Ma and co-workers analyzed flavonoids in 12 batches University, Shanghai, China. The identity, sampling part, of SFR and 4 batches of STR samples by HPLC fingerprinting collection source and collection time of the 43 tested samples and found preliminarily that these two herbs had different are summarized in Table 1. All the HPLC fingerprint data [15] flavonoid profiles . To investigate systematically the char- were obtained in 2004, and the phytochemical isolation and acteristic chemical constituents that mayt contribute to their statistical analysis were accomplished recently.
Table 1 Sample information of Sophorae Flavescentis Radix and Sophorae Tonkinensis Radix et Rhizoma used in the present study and their calculated similarity values for alkaloids and flavonoids Similarity No. Sample code Source Sampling part Collection time A B 1 SF1 NICPBP Roots Oct 2002 NT 0.90 2 SF2 Nanning, Guangxi Roots Oct 2001 0.95 0.80 3 SF3 Kunming, Yunnan Roots Oct 2001 0.93 0.88 4 SF4 Kunming, Yunnan Roots Jun 2003 NT 0.92 5 SF5 Guiyang, Guizhou Roots Aug 2001 0.94 0.89 6 SF6 Luoding, Guangdong Roots Oct 2001 0.97 0.96 7 SF7 Dujiangyan, Sichuan Roots Aug 2001 0.94 0.96 8 SF8 Nanchuan, Chongqing Roots Sep 2001 0.96 0.96 9 SF9 Chongqing Roots Oct 2001 0.95 0.94 10 SF10 Beibei, Chongqing Roots Jun 2003 NT 0.87 11 SF11 Fuzhou, Fujian Roots Oct 2001 0.96 0.85 12 SF12 Shanghai Roots Oct 2001 0.85 0.84 13 SF13 Tianshui, Gansu Roots Aug 2001 0.96 0.95 14 SF14 Lanzhou, Gansu Roots Jun 2003 NT 0.94 15 SF15 Yinchuan, Ningxia Roots Nov 2001 0.89 0.95 16 SF16 Hanzhong, Shaanxi Roots Jul 2001 0.94 0.38 17 SF17 Langao, Shaanxi Roots Aug 2001 0.98 0.94 18 SF18 Ziyang, Shaanxi Roots Jun 2003 NT 0.96 19 SF19 Wulumuqi, Xinjiang Roots Jun 2001 0.97 0.94 20 SF20 Nanchang, Jiangxi Roots Oct 2001 0.97 0.91
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Continued Similarity No. Sample code Source Sampling part Collection time A A 21 SF21 Tianjin Roots Mar 2002 0.94 0.92 22 SF22 Huaiyin, Jiangsu Roots Oct 2001 0.80 0.83 23 SF23 Bozhou, Anhui Roots Sep 2001 0.84 0.86 24 SF24 Shanghai Roots Feb 2001 NT 0.86 25 ST1 NICPBP Roots and rhizomes Oct 2002 NT 0.83 26 ST2 Nanning, Guangxi Roots and rhizomes Oct 2002 NT 0.96 27 ST3 Nanning, Guangxi Roots and rhizomes Oct 2001 0.47 0.96 28 ST4 Kunming, Yunnan Roots and rhizomes Oct 2001 0.87 0.68 29 ST5 Guiyang, Guizhou Roots and rhizomes Aug 2001 0.96 0.95 30 ST6 Luoding, Guangdong Roots and rhizomes Oct 2001 0.95 0.95 31 ST7 Zhaoqing, Guangdong Roots and rhizomes Dec 2001 0.94 0.83 32 ST8 Dujiangyan, Sichuan Roots and rhizomes Aug 2001 0.83 0.94 33 ST9 Chongqing Roots and rhizomes Oct 2001 0.95 0.94 34 ST10 Fuzhou, Fujian Roots and rhizomes Oct 2001 0.97 0.97 35 ST11 Shanghai Roots and rhizomes Oct 2001 0.88 0.94 36 ST12 Lanzhou, Gansu Roots and rhizomes Oct 2001 0.91 0.94 37 ST13 Lanzhou, Gansu Roots and rhizomes Jun 2003 NT 0.91 38 ST14 Yinchuan, Ningxia Roots and rhizomes Nov 2001 0.89 0.95 39 ST15 Ankang, Shaanxi Roots and rhizomes Aug 2001 0.94 0.84 40 ST16 Shijiazhuang, Hebei Roots and rhizomes Oct 2001 0.87 0.97 41 ST17 Quzhou, Zhejiang Roots and rhizomes Nov 2001 0.94 0.93 42 ST18 Shimen, Hunan Roots and rhizomes Jul 2001 0.92 0.27 43 ST19 Shanghai Roots and rhizomes Mar 2000 NT 0.88 NT: not tested; SF: Sophora flavescens; ST: S. tonkinensis; NICPBP: the National Institute for the Control of Pharmaceutical and Biological Products of China. A and B represented the calculated similarity values for alkaloids and flavonoids, respectively.
Chemicals and reagents ultrasonication at room temperature for 45 min. The extrac- Cytisine, sophocarpine, matrine, sophoranol, oxymatrine, tion was cooled to ambient temperature and then made up to and oxysophocarpine were isolated from the roots of S. fla- its original weight with chloroform, followed by filtration vescens or S. tonkinensis in our laboratory, and their structures with paper filters. An accurate aliquot (10.0 mL) of the suc- were confirmed by various spectral analyses [16-18]. Their puri- cessive filtrate was collected and evaporated to dryness ties were greater than 98% by HPLC-PDA analysis detected under reduced pressure, and the residue was then re-dissolved at 220 nm. Methanol and acetonitrile were of HPLC-reagent in 5 mL of 50% aqueous methanol. The sample solution was grade (Tedia, Fairfield, OH, USA). Triethylamine, phosphoric filtered with a 0.45 μm membrane filter prior to HPLC acid, chloroform, 25% aqueous ammonia, and glacial acetic analysis. Five microliters of each solution were injected for acid were of analytical-reagent grade and obtained from Sino- analysis. pharm Chemical Reagent Co., Ltd. (Shanghai, China). Dis- The procedure for the sample preparation of flavonoids tilled water was obtained from a Milli-Q system (Millipore, was the same as that for alkaloids, except that the extraction Bedford, MA, USA). solvent of methanol (40 mL) was used, and the volumes of Preparation of sample solutions the successive filtrate and injection were 20 mL and 20 L, Sample preparation for the alkaloid analysis was con- respectively. ducted as follows: herbal samples (0.5 g) were pulverized, Apparatus and HPLC conditions sieved through a No. 50 mesh, transferred into a 50-mL All chromatographic separations were conducted on an flask with stopper, and weighed accurately. Twenty milli- Agilent 1100 series HPLC instrument (Palo Alto, CA, USA) liter of chloroform and 0.2 mL of 25% aqueous ammonia equipped with a photodiode array detector (PDA), and the were accurately added and the flask was re-weighed. The data were recorded and analyzed by Hewlett Packard Chem- sample was kept in dark overnight, and then extracted under Station software (Palo Alto, CA, USA).
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The separation of alkaloids was performed on a YMC- (SMFC) were then produced for SFR and STR, respectively, pack ODS-AQ column (4.6 mm × 250 mm, 5 μm; YMC, Kyoto, by the fingerprint software. The similarity value of each chro- Japan), and the mobile phase consisted of water, 0.1% triethy- matograph against the corresponding SMFC was thus calculated. lamine in methanol (V/V) and 25 mmol·L−1 phosphoric acid As shown in Fig. 1A, the alkaloid profiles of 18 batches aqueous solution (44 : 46 : 10) at a flow rate of 0.8 mL·min−1. of SFR samples were generally consistent, and 15 peaks were The column temperature was kept at 40 C, and the detection assigned as the common peaks in SMFC (Fig. 1B). The simi- wavelength was set at 220 nm. larity values of these samples were found in the range of The separation of flavonoids was carried out on a Sphere- 0.80–0.98 (Table 1), indicating that similar alkaloid compo- clone Luna C18 column (4.6 mm × 250 mm, 5 μm; Phenomenex, nents were present in these samples regardless of the collec- Torrance, CA, USA). The mobile phase was 0.2% acetic acid tion sources. In addition, Peaks 5, 6, 8, 13, and 14 were iden- aqueous solution (V/V, A) and 0.2% acetic acid in acetonitrile tified as oxysophocarpine, oxymatrine, sophoranol, sopho- (V/V, B) with a gradient program as follows: 0–30 min, linear carpine, and matrine, respectively, by comparison of their gradient 0%–45% B; 30–60 min, linear gradient 45%–85% B; retention times and UV profiles with those of the authentic 60–70 min, linear gradient 85%–96% B; 70–90 min, isocratic standards. Peaks 5–7 and 14 were the principal alkaloids in 96% B at a flow rate of 1.2 ml·min−1. The column tempera- SFR based on their peak areas, among which Peak 6 (oxyma- ture was kept at 40 C, and the PDA detector was set at trine) was of the highest content in all samples, followed by 280 nm. The UV spectra were recorded on-line in the range of Peaks 5 and 7 in most of the samples. 200– 400 nm. Data processing and analysis Similar to SFR, the alkaloid profiles of 15 batches of Similarity analysis was performed with a professional STR samples were also fingerprinted (Fig. 2A) and analyzed. software named ‘Similarity Evaluation System for Chroma- As a result, 15 common peaks were also assigned with Peaks tographic Fingerprint (Version 2004A)’ which was developed 3, 4, 5, 8, 12, and 13 being identified as cytisine, oxysopho- by Chinese Pharmacopoeia Commission. In the present study, carpine, oxymatrine, sophoranol, sophocarpine and matrine, the software was employed to synchronize and generate a respectively (Fig. 2B). An intra-species similarity comparison simulative median fingerprint chromatogram, against which between STR samples was performed by the fingerprint soft- the similarity value of each chromatogram was calculated ware. Most of the similarity values of STR samples were in based on the approach of cosine value for vectorial angle. the range of 0.83–0.97 (Table 1), indicating that the samples The hierarchical cluster analysis (HCA) was carried out from the same species origin were closely similar to each with the Statistical Program for Social Sciences (SPSS) 11.5 other. The only exception was the sample ST-3 with a similar- software (SPSS Inc., Cary, NC, USA), applying the method ity value of 0.47. Together with the fingerprint results of SFR, of average linkage between groups in alkaloid analysis and these data indicated that the nature of the alkaloid compo- that of centroid linkage in flavonoid analysis, respectively. nents were very similar between these two species, both of Squared euclidean distance was selected as similarity meas- which possessed oxysophocarpine, oxymatrine, sophoranol, urement in HCA. sophocarpine, matrine and some unidentified peaks with the same retention times and UV spectra. However, the peak in- Results and Discussion tensities of some peaks were slightly different. For example, HPLC fingerprints of alkaloids matrine was found to be the second highest alkaloid in most Quinolizidine alkaloids of SFR and STR are well studied of STR samples instead of oxysophocarpine in SFR. It is and long regarded as their main bioactive components, which worthy noting that the reason for the low similarity value of have reported to exhibit significant pharmacological activities [2-4]. the reference drug (ST-3) remained unknown, but this excep- It is therefore of great interest to investigate the potential tional sample did not significantly affect the overall conclu- difference in nature of alkaloids present in these two herbs by sion of the fingerprints. fingerprint analysis. Furthermore, the hierarchical clustering analysis (HCA) In order to extract the alkaloids from the herbs effectively, was conducted to compare the alkaloid profiles of these two a mixture of chloroform and 25% aqueous ammonia was used herbs. Based on 21 alkaloid constituents recognized auto- as the extraction solvent in the present study. This extraction matically in the HPLC chromatograms of 18 batches of SFR solvent has been found to extract the alkaloids from the herbs and 15 of STR samples, a 21 × 33 matrix was formed from well in our recent reports [11, 13]. After optimization of the HCA using SPSS 11.5 software and the results are shown in HPLC conditions, the fingerprint methods of the two herbs Fig. 3. It was found that the 33 tested samples could be classi- were validated for precision, repeatability, and stability. The fied into two groups. Group I, with a relatively low content of RSD values of all the validation tests were less than 1.65%. oxymatrine (peak intensity: 629–4005 mAU), covered 9 SFR Then the chromatographic fingerprints of the alkaloids were and 12 STR samples. Group II, with a much higher content of generated for 18 batches of SFR and 15 of STR samples, oxymatrine (peak intensity: 4859–8108 mAU), consisted of 9 respectively. Two simulative median fingerprint chromatograms SFR and 3 STR samples. Further HCA analysis showed that
– 954 – DING Pei-Lan, et al. / Chin J Nat Med, 2018, 16(12): 951960 group I could be divided into three subgroups I-A, I-B, and the hierarchical clustering dendrogram (Fig. 3). Therefore, it I-C, and each subgroup was made up of a single species ori- was concluded that the alkaloid constituents of these two gin. Subgroup I-A covered 11 STR samples with a relatively herbs were closely similar, with most of the alkaloids co-existed, higher content of oxymatrine; subgroup I-B consisted of 9 and cannot be used as diagnostic markers to distinguish these SFR samples with a particularly high content of oxysopho- two drugs. More importantly, the apparent lack of specificity carpine; and subgroup I-C contained only one STR sample of alkaloids may indicate other potential relations between (ST-3), with the characteristic of an extremely high content of the overall therapeutic effect and components in these two matrine but low content of oxymatrine. Group II was slightly herbs. However, due to their various and notable pharma- more complicated. One SFR sample (SF-16) and one STR cological effects [2-4], it is still reasonable to assess their sample (ST-16) were dispersed from the others, and were quality with the alkaloid fingerprint. In addition, our results assigned as subgroups II-C and II-D, respectively, because of from the alkaloid fingerprints also indicated that oxymatrine their corresponding high contents of matrine and oxymatrine. together with matrine, and oxymatrine together with oxyso- The remaining 10 samples were divided into subgroups II-A phocarpine, could be served as the dominant quantitative and II-B mainly based on their differences in oxysopho- markers for quality assessments of SFR and STR, respec- carpine contents. These HCA results of the alkaloids profiles tively. This was in good agreement with those reported in suggested that the species of SFR and STR mixed together in our previous studies [11, 13].
Fig. 1 HPLC fingerprints of the alkaloid constituents of 18 Sophorae Flavescentis Radix samples (A) and the simulative median fingerprint chromatogram (SMFC) obtained by ‘Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine’ software (B). The chromatograms marked with SF-02, SF-03, SF-05–SF-09, SF-11–SF-13, SF-15–SF-17, SF-19–SF-23 and R represented 18 Sophorae Flavescentis Radix samples and the SMFC, respectively
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Fig. 2 HPLC fingerprints of the alkaloid constituents of 15 Sophorae Tonkinensis Radix et Rhizoma samples (A) and the simula- tive median fingerprint chromatogram (SMFC) obtained by ‘Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine’ software (B). The chromatograms marked with ST-03–ST-12, ST-14–ST-18 and R represented 15 Sophorae Tonkinensis Radix et Rhizoma samples and the SMFC, respectively
HPLC fingerprints of flavonoids comparison of the flavonoid profiles was conducted by simi- The above HPLC fingerprint studies of alkaloids showed larity value calculation. The results showed that the samples no obvious difference between SFR and STR, and it is rea- within the same species matched well (Table 1). For example, sonable to speculate that alkaloids might not be the major most of the similarity values of the SFR samples were in the components that lead to their different clinical applications. In range of 0.80–0.96 except for SF-16 (0.38) (Table 1). Similar the past two decades, plenty of flavonoids were reported in observation was also achieved in the STR samples, which all SFR and STR with various biological activities [3, 5-9]. This the similarity values were in the rage of 0.83–0.97 except ST-4 promoted us to investigate the potential difference in flavon- (0.68) and ST-18 (0.27). oids between these two herbs by HPLC fingerprinting. To better understand the nature of the flavonoids, 23 As shown in Figs. 4 and 5, the HPLC chromatograms of common peaks of STR samples were tentatively assigned by the two species demonstrated individual specific flavonoid comparison of their online UV profiles with literature data profiles. The chromatograms of SFR samples possessed 25 (Data not shown). Peaks 1, 6, and 10 might be pterocarpans common peaks dispersed in 20–52 min (Fig. 4B), while the exhibiting maximum absorptions at 310 and 285 nm with the STR samples showed a totally different profile with 23 common former stronger [19]; Peaks 2–5, 9, and 13 might be isoflavon- peaks congested in 25–65 min (Fig. 5B). An intra-species oids showing band II absorption at 260 nm due to benzoyl
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moieties, along with a shoulder on the right side [20]; Peaks 8, 11, 12, 18–20, 22, and 23 might be flavanones with a band II maximum absorption at 280–285 nm (Peak 22 at 295 nm) and a shoulder adjacent to the long-wave side [20]; Peak 7 was a chalcone exhibiting a characteristic strong band I absorption at 330–350 nm and a relative lower band II absorption at 280 nm [20]; and Peaks 14–17 and 21 might be aromatic compounds with maximum absorption at 260–280 nm. This deduction was quite consistent with our recent HPLC-ESI- MS/ MS study on STR flavonoids [21]. Thus, it could be con- cluded that the major sub-types of flavonoids in STR were fla- vanones and isoflavones, along with a few pterocarpins and chalcones. Furthermore, Peak 20 (flavanones, assigned as sophoranone [21]) was the dominant component, followed by Peaks 23 (flavanones), 1 (pterocarpins), 6 (pterocarpins) and 7 (chalcones) in all STR samples, except two with low similarity values (ST-4 and ST-18). Similarly, the common peaks present in SFR were also assigned (Data not shown). Peaks 3, 5 and 11 were assigned as pterocarpins; Peak 1 was identified as an isoflavonoid; Peak 22 was a typical chalcone exhibiting a strong band I absorption at 385 nm together with a relatively lower band II absorption at 255 nm [20]; and the remaining peaks except Peaks 23–26, were all flavanones/flavanols with a band II maximum absorption at 290–305 nm (Peak 2 at 280 nm) and a shoulder adjacent to the long-wave side [20]. Obviously, compared to STR, SFR was a much richer source of Fig. 3 Cluster dendrogram for 18 commercial samples of flavanones, while relative lack of isoflavonoids and chal- [21-22] Sophorae Flavescentis Radix and 15 commercial samples of cones . This is confirmed by that the most abundant Sophorae Tonkinensis Radix et Rhizoma based on their alka- peaks such as Peaks 12, 13 and 18 were all assigned as fla- loid chromatographic data vanones in all SFR samples.
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Fig. 4 HPLC fingerprints of the flavonoid constituents of 24 Sophorae Flavescentis Radix samples (A) and the simulative me- dian fingerprint chromatogram (SMFC) obtained by ‘Similarity Evaluation System for Chromatographic Fingerprint of Tradi- tional Chinese Medicine’ software (B). The chromatograms marked with SF-01–SF-24 and R represented 24 Sophorae Flaves- centis Radix samples and the SMFC, respectively
Fig. 5 HPLC fingerprints of the flavonoid constituents of 19 Sophorae Tonkinensis Radix et Rhizoma samples (A) and the simu- lative median fingerprint chromatogram (SMFC) obtained by ‘Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine’ software (B). The chromatograms marked with ST-01–ST-19 and R represented 19 Sophorae Tonkinensis Radix et Rhizoma samples and the SMFC, respectively
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Taken together, the fingerprint analysis of flavonoids led us to conclude that both herbs contained flavanones as their principle flavonoid components, and STR was also a richer source of isoflavones. It still needs to point out that the fine structures of the flavanones in these two herbs were quite different from each other. The flavanones in STR exhibited band II absorption at 280–285 nm, while those of SFR showed a red shift with the maximum absorption moving to 290–305 nm, indicating that the flavanones in SFR might possess more auxochromes such as OH or OR groups in ring A than in STR [20]. This hypothesis was supported by our phytochemical study of the herbs and the previous reports [9, 23-29]. The flavanones obtained from SFR always have an OH or OMe group at- tached to C-5 in ring A, while those from STR were always absent of such groups. One further piece of evidence pre- sented to support this deduction is their retention times of the HPLC chromatograms, which most of the flavanones were eluted before 43 min in SFR instead of 45–63 min in STR, due probably to the increased polarity of the flavanones with more OH substitutes. Therefore, based on the above com- parison of the retention times and the online UV spectra of the flavonoid peaks in the two herbs, it was indicated that, in term of flavonoids, they had almost no individual components in common except for two minor pterocarpins at 24.46 min (Peak 3 of SFR and Peak 1 of STR) and 33.66 min (Peak 11 of SFR and Peak 6 of STR), as well as an unknown component at 50.90 min (Peak 24 of SFR and Peak 16 of STR). This result is consistent with the systematic identification of STR flavonoids [21] and SFR flavonoids [22] by HPLC-ESI-MS/MS methods. Again, an inter-species comparison of their flavonoid profiles was carried out by HCA. Based on 54 flavonoid com- ponents recognized automatically in the HPLC chromatograms of 24 batches of SFR and 19 of STR samples, a 54 × 43 matrix was formed for analysis. As shown in Fig. 6, except one SFR sample (SF-16) dispersed from the others because of its par- ticularly low flavonoid content, the remaining 22 SFR sam- Fig. 6 Cluster dendrogram for 24 commercial samples of ples and all 19 STR samples were clearly divided into two Sophorae Flavescentis Radix and 19 commercial samples of groups. Group I covered all 19 STR samples and merely one Sophorae Tonkinensis Radix et Rhizoma based on their fla- SFR sample (SF-16). And group II accounted for all the re- vonoid chromatographic data maining 22 SFR samples with the characteristic of a much clustering analysis. The results showed that the alkaloid con- higher flavonoid content. The HCA results showed that these two herbs could be easily distinguished based on their flavonoid stituents were not a good marker to distinguish the two herbs constituents. In summary, the results of this study indicated due to similar alkaloids co-existed, though this type of con- that flavonoids rather than alkaloids could be the diagnostic stituents was long regarded as their major active components. constituents between SFR and STR, and might be a key factor However, their flavonoid constituents were entirely different attributed to their different clinical applications. Quantification with few components in common, which could serve as the and biological evaluation of their flavonoids are currently qualitative markers for authentication of SFR and STR, even under investigation. when in absence of expected reference substances. Our study also indicated that the different flavonoids in the two herbs Conclusion might be another type of potential active components that In the present study, the chemical constituents of SFR lead to their corresponding unique clinical properties claimed and STR were comparatively studied by HPLC fingerprint for SFR and STR. This was the first report to systematically analysis in terms of alkaloids and flavonoids. Their fingerprints compare the chemical constituents of SFR and STR by HPLC were evaluated by similarity calculation and hierarchical fingerprints.
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Cite this article as: DING Pei-Lan, HE Chang-Ming, CHENG Zhi-Hong, CHEN Dao-Feng. Flavonoids rather than alkaloids as the diagnostic constituents to distinguish Sophorae Flavescentis Radix from Sophorae Tonkinensis Radix et Rhizoma: an HPLC fingerprint study [J]. Chin J Nat Med, 2018, 16(12): 951-960.
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