FITOTE-02841; No of Pages 10 Fitoterapia xxx (2013) xxx–xxx
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Fitoterapia
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Screening of osteoanagenesis-active compounds from Scutellaria baicalensis Georgi by hPDLC/CMC–online-HPLC/MS Jin Liu a,c, Sicen Wang c, Junyi Sun a, Jianfeng Shi b, Ye Li a, Jianzhong Gou a, Ang Li a,b,⁎, Langchong He c,⁎⁎ a b c
Department of Periodontology, Stomatological Hospital, College of Medicine, Xi'an Jiaotong University, Xi'an 710 004, China Research Center for Stomatology, Stomatological Hospital, College of Medicine, Xi'an Jiaotong University, Xi'an 710 004, China School of Medicine, Xi'an Jiaotong University, Xi'an 710 061, China
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journal homepage: www.elsevier.com/locate/fitote
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Article history: Received 22 October 2013 Accepted in revised form 24 December 2013 Available online xxxx
In present study, an online analytical method using human periodontal ligament cell/cell membrane chromatography (hPDLC/CMC) combined with high-performance liquid chromatography/mass spectrometry (HPLC/MS) was used for direct recognition, separation, and identification of compounds for the first time from Scutellaria baicalensis Georgi (SBG) that are active on hPDLCs. Baicalein (BAI) and wogonin (WOG), which were identified as the active compounds of the ethyl ether extract of S. baicalensis Georgi (SBGEE), could bind to the same membrane receptor of hPDLC for simvastatin (SIM). Moreover, BAI (0.15–0.6 mg/L) and WOG (0.015–0.6 mg/L) had the capability to enhance cell proliferation, matrix calcification, and formation of calcified nodules, which are comparable to the activities of SIM (0.1 mg/L) in vitro. These observations are consistent with the KA of the various drugs. It is very important for the development of SBG used to treat periodontitis. © 2013 Published by Elsevier B.V.
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Keywords: Human periodontal ligament cell membrane chromatography High-performance liquid chromatography/ mass spectrometry Scutellaria baicalensis Georgi Baicalein Wogonin
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1. Introduction
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Historically, plants are continual sources of drugs for discovery, as they provide compounds and derivatives that are invaluable sources of therapeutic agents. A wide variety of herbal medications for the skeletal system, in particular, have beneficial effects and minimal adverse effects [1]. Numerous reports on the pharmacological activities of
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Abbreviations: hPDLC, human periodontal ligament cell; CMC, cell membrane chromatography; HPLC, high performance liquid chromatogram; MS, mass spectrogram; TCM, traditional Chinese medicine; SBG, Scutellaria baicalensis Georgi; SBGEE, the ethyl ether extract of Scutellaria baicalensis Georgi; BAI, baicalein; WOG, wogonin; SIM, simvastatin. ⁎ Correspondence to: A. Li, Department of Periodontology, Stomatological Hospital, College of Medicine, Xi'an Jiaotong University, Xi'an 710 004, China. Tel.: +86 29 8721 6030; fax: +86 29 8727 3400. ⁎⁎ Corresponding author. Tel./fax: +86 29 8265 5451. E-mail addresses: [email protected] (A. Li), [email protected] (L. He).
flavonoids of Scutellaria baicalensis Georgi [2,3] indicate that these substances could significantly improve common cold, hyperlipidaemia and hypertension, ameliorate inflammation, and alleviate bone-related disorders, and promote reconstruction of the periodontium [4–6]. Our group has already screened baicalin as the active compounds acting on the epidermal growth factor receptor from S. baicalensis Georgi [7]. However, the constituents that bind to cell membrane receptors of human periodontal ligament cells (hPDLCs) from this traditional Chinese medicine (TCM) have not been investigated. Identification of active components in natural sources is very difficult. In this regard, cell membrane chromatography (CMC), a novel technique based on biological affinity chromatography [8,9], may be a viable approach, as it is effective at separating ingredients that are active toward specific membrane receptors [10–13]. In our previous investigations, CMC involved the use of a stationary phase consisting of a cell membrane enriched with specific receptors. This stationary phase was used to screen active constituents from complex samples [7,14–16]. In
0367-326X/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.fitote.2013.12.020
Please cite this article as: Liu J, et al, Screening of osteoanagenesis-active compounds from Scutellaria baicalensis Georgi by hPDLC/ CMC–online-HPLC/MS..., Fitoterapia (2013), http://dx.doi.org/10.1016/j.fitote.2013.12.020
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S. baicalensis Georgi was obtained from the Qinba mountains (Shannxi Province, China) and identified by the Department of Pharmacognosy, Xi'an Jiaotong University (Xi'an, China). The plant material was dried at room temperature and ground to powder. A reference sample has been deposited at the Specimen Laboratory, Research and Engineering Center for Natural Medicine, Xi'an Jiaotong University (Xi'an, China). Simvastatin (SIM), baicalein (BAI), and wogonin (WOG) (N 98% pure) were purchased from the National Institutes for Food and Drug Control of China (Beijing, China). Silica gel (ZEX-II, 5 μm, 200 Å) was obtained from Qingdao Meigao Chemical Co., Ltd. (Qingdao, China). hPDLCs were cultured in the Research Center, College of Stomatology, Xi'an Jiaotong University. Dulbecco's Modified Eagle Medium/F12 (DMEM/F12), fetal calf serum (FCS), and trypsin were purchased from Sigma (Saint Louis, MO, USA). Type I collagenase (Worthington Biochem, Freehold, NJ, USA), ascorbic acid 2-phosphate (Wako, Tokyo, Japan), glutamine, penicillin, and streptomycin were also used in the study. HPLC-grade methanol was acquired from Burdick & Jackson (NJ, USA). All other reagents used were of analytical grade. Stock solutions for analysis of SIM, BAI, and WOG (1 mg/mL each) were prepared by separately dissolving the standard drugs in methanol. Standard solutions (0.01 mg/mL) were
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2.2. Instruments
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A VICI AG 10G-0911V 10-port 2-pos valve (Valco Instrument Co, Inc., Houston, USA) was used as the column switcher, and two Shim-pack VP-ODS pre-columns (10 × 2.0 mm i.d., 5 μm, Shimadzu Corporation, Kyoto, Japan) were used as the enrichment columns. A hPDLC/CMC column (10 × 2.0 mm i.d.) was used as the first-dimension column and a Shimadzu Shim-pack VP-ODS column (150 × 2.0 mm i.d., 5 μm, Kyoto, Japan) was used as the second-dimension column. The HPLC/MS system (Shimadzu Corporation, Kyoto, Japan) consisted of three LC-20AD pumps, a DGU-20A3 degasser, an SIL-20A autosampler, a CTO-20A column oven, an SPD-20A UV–vis detector, an SPD-M20A diode array detector (DAD), an LCMS2010EV mass spectrometer, and an LC/MS Solution workstation. The data were acquired by LCSolution software (Shimadzu Corporation).
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2.3. Preparation of the extracts
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prepared by dilution of each stock solution with mobile phase (Ultrapure water). BAI, WOG, and SIM were diluted in dimethylsulfoxide (DMSO) stock solution prior to the addition to cell culture medium to form a mixture with a final DMSO concentration of b 0.1% (v/v).
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Preparation of the various extracted flavonoids from S. baicalensis Georgi was as follows: Powdered plant material (50 g) was subjected to ultrasonic extraction with 50 mL methanol in a 100 mL triangular flask. Extraction was performed twice, each lasting 30 min. The filtered supernatant was concentrated to dryness by rotary evaporation to obtain the total flavonoids (SBGTF), which appeared as a dark-brown solid extract. The extract was subsequently suspended in 20 mL distilled water and thrice ultrasonically extracted successively with petroleum ether, ethyl ether, chloroform, and ethyl acetate to yield five different extracts, which were evaporated again to dryness. Sample solutions of all six extracts in methanol (1 mg/mL) were prepared and stored at 4 °C in the dark. Working solutions (0.1 or 0.01 mg/mL) were prepared by diluting the sample solutions with mobile phase on the day of the experiment.
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2.4. Cell culture and preparation for the hPDLC/CMC system
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According to the literature [17], extraction premolar teeth (n = 20) were collected from 10 individuals (ages 15–29 years) at the Stomatological Hospital of Xi'an Jiaotong University (Xi'an, China). The following approved guidelines, which were set by the National Institutes of Health Office of Human Subjects Research, were adopted in the study. The PDL was gently separated from the surface of the 1/3 root and then digested in a solution of 3 mg/mL type I collagenase for 30 min at 37 °C. The cell suspensions were seeded into dishes with DMEM/F12 medium supplemented with 15% FCS, 100 μmol/L ascorbic acid 2-phosphate, 2 mmol/L glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin, and then incubated at 37 °C in a humidified atmosphere with 5% carbon dioxide. Immunocytochemistry tests for keratin and vimentin gave negative and positive results, respectively, confirming that the cells were hPDLCs. Cells were subcultured every three days at a dilution of
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particular, we employed a human periodontal ligament cell/ cell membrane chromatography (hPDLC/CMC) system to screen the active ingredient berberine from Rhizoma Coptidis [17]. Periodontitis has high prevalence, a problem that may not be avoided in all countries [18]. The condition is characterized by the destruction of the periodontal attachment supporting the teeth, including the periodontal ligament (PDL), the cementum, and the alveolar bone. hPDLCs residing in the PDL could differentiate into either cementoblasts or osteoblasts that can produce mineralized tissues. Therefore, hPDLCs could play an important role in the regeneration of periodontal tissue [19,20]. The major goal of periodontal therapy is reconstructing periodontium destroyed by periodontopathy and maintaining healthy periodontium. However, purely mechanical treatment may not be effective for certain patients for whom additional antimicrobial and bone regeneration therapy would be essential for successful treatment [21,22]. Until now, there is no effective treatment for periodontitis. In the present study, a cell membrane was used to establish a hPDLC/CMC model, which was combined with a highperformance liquid chromatography/mass spectrometry (HPLC/ MS) system for screening active principles binding to cell membrane receptors of hPDLC from S. baicalensis Georgi. The active ingredients were recognized, separated, and identified directly through an online column-switching technique between these two systems. By in vitro tests, we verified that active constituents could promote osteogenesis by hPDLCs in a dose-dependent manner. We believe that results of this experiment could provide new and better ideas for using S. baicalensis Georgi in the treatment of periodontitis.
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Please cite this article as: Liu J, et al, Screening of osteoanagenesis-active compounds from Scutellaria baicalensis Georgi by hPDLC/ CMC–online-HPLC/MS..., Fitoterapia (2013), http://dx.doi.org/10.1016/j.fitote.2013.12.020
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2.6. Application of the hPDLC/CMC–online HPLC/MS system
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The hPDLC/CMC model was combined with the HPLC/MS system by means of an online 10-port column switcher. Any retention fraction which was “recognized” by the hPDLC/ CMC model was captured in the ODS enrichment column (EC1 or EC2), and then directly eluted into the HPLC/MS system for separation and identification. By using SIM as a positive control, this method was used to screen ingredients from S. baicalensis Georgi that are active toward hPDLC
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In order to verify whether SIM, BAI, and WOG were active on the same site of the hPDLC membrane receptor, we performed a competitive displacement test [10,11]. KA values in the hPDLC/CMC system were measured using a series of SIM solutions (0 to 16 × 10−7 mol/L) in the mobile phase. The chromatographic parameters were the same as above. The retention times of SIM at different concentrations were recorded. Next, standard solutions of BAI and WOG were injected into the hPDLC/CMC column. The capacity factor (k′) of the CMC chromatographic peak in the elution curve was calculated using Eq. (1): ′
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The HPLC conditions (the second dimension) were as follows: column, VP-ODS (150 × 4.6 mm i.d., 5 μm); mobile phase, methanol/0.4% glacial acetic acid—0.2% triethylamine (50:50, v/v); flow rate, 0.2 mL/min; column temperature, 37 °C; and DAD, 280 nm. MS conditions were set as follows: nebulizer gas, N2 (purity N 99.999%); flow rate, 1.5 L/min; drying gas, N2 (purity N 99.999%); pressure, 0.1 MPa; interface temperature, 250 °C; heat block temperature, 200 °C; detector voltage, 1.5 kV; scan range, 50–1000 m/z; and scan mode, positive ionization mode. VP-ODS enrichment columns were used for the first enrichment column (EC1) and the second enrichment column (EC2), which were combined by means of an online 10-port column switcher. EC1 and EC2 could be in either the first or the second-dimension system. Alternation of enrichment and elution could be achieved by the parallel connection of those two enrichment columns.
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where tR is the retention time of the ligand and t0 is the dead 249 250 time of non-retained solvent. The values of KA and KL could be 251 calculated from Eq. (2): 252
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2.7. Competitive displacement assay
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1 K ½R K ½R ¼ A S ½L þ A S VM k′ −X K L V M m
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membrane receptors. The standard solutions and six extracts 235 of flavonoids were analyzed separately. 236
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1:3 using a 0.25% aqueous solution of trypsin, and then resuspended to a single-cell suspension. Cells from exponentially growing cultures were used in all experiments. A cell count was performed to determine the cell density. The cell density was ≥1 × 107. The cells were washed thrice with phosphate-buffered saline (PBS). A Tris–HCl hypotonic solution (50 mmol/L, pH 7.4) was added to the washed cells to produce the signal cell suspension. The cells were subsequently ruptured by ultrasonication for 30 min, and the resulting homogenate was centrifuged at 1000 ×g for 10 min. The pellet was discarded and the supernatant was centrifuged at 12,000 ×g for 20 min at 4 °C. The resulting precipitate was resuspended in 10 mL Tris–HCl and the suspension was re-centrifuged at 12,000 ×g. A cell membrane suspension was obtained by adding 5 mL of PBS to the precipitate, and then stored until use. According to a previously reported method [8], 0.05 g silica was activated at 105 °C for 30 min and used as a carrier. It was then homogenized with the cell membrane suspension, by adding the mixture slowly to it under vacuum and with agitation at 4 °C to obtain the hPDLC membrane stationary phase (CMSP). The CMSP was then packed into a column (10 × 2.0 mm i.d.) through a wet-packing procedure to yield the hPDLC/CMC column. Ultrapure water, which was delivered at a flow rate of 0.2 mL/min, was used as the mobile phase in the CMC system (the first dimension). The detection wavelength was 280 nm, and the column temperature was 37 °C.
where KA and KL are the equilibrium dissociation constants for the analyte and marker in the mobile phase, respectively; [L]m, and [R]S are the molar concentrations of the ligands in the effluent and immobilized receptors at the surface of the stationary phase, respectively; and VM is the dead volume of the column. The term X was introduced to the denominator with the k′ value to eliminate the errors incurred during iterative testing. When SIM is used both as a marker and an analyte, KA and KL, which could be obtained from the ratio of intercept and slope, respectively, are equal. [R]S/VM is equal to the slope. Thus, upon substituting Eq. (2) in Eq. (3) the following is obtained:
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1 ½RS K ½R ¼ ½L þ A S : VM k′ V M m
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The KA values of the two ligands were obtained by linear 268 regression of the plot of 1/k′ versus [L]m. 269 2.8. 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium 270 bromide (MTT) assay 271 The MTT assay was used to measure the growth and viability of hPDLCs [23]. Cells were cultivated in 96-well plates (1 × 106 cells/well) and treated with the drugs at various concentrations for 1, 3, 5, 7, and 9 days. The absorbance values at 490 nm were determined using a microplate reader (Bio-Rad Instruments, USA).
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2.9. von Kossa staining
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After seeding in 12-well plates and treatment with different drugs for 21 days, cells were washed with PBS and fixed with 4% paraform for 30 min, and then incubated with 1% silver nitrate at room temperature under UV light (about
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Please cite this article as: Liu J, et al, Screening of osteoanagenesis-active compounds from Scutellaria baicalensis Georgi by hPDLC/ CMC–online-HPLC/MS..., Fitoterapia (2013), http://dx.doi.org/10.1016/j.fitote.2013.12.020
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In order to quantify the effects of promoting calcification secreted by cells, we evaluated the Ca2+ ion concentration in the matrix. Cells were treated in the same manner as above. After washing thoroughly with PBS three times, cells were incubated overnight at room temperature in 500 μL of 0.1 mol/L HCl to dissolve the calcium mineral from the calcified matrix. The supernatants were collected and the calcium level was determined using an XD-685 electrolyte analyzer [25]. The protein content of cells, which was used to normalize the calcium data, was determined through the bicinchoninic acid method (Sigma) [26].
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2.11. Statistical analyses
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All experiments were repeated three times. Statistical analysis was performed by using SPSS18.0. Data were evaluated through one-way analysis of variance (ANOVA) followed by Tukey's test as a post hoc comparison. All data are presented as mean ± standard error of the mean (SEM). The probability level (P) was considered significant at P b 0.05.
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3. Results
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3.1. Combined hPDLC/CMC–online HPLC/MS
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At position A (Fig. 1), the first retention fraction recognized by the hPDLC/CMC model was extracted though the ODS pre-column (EC1). At position B, the extracted components were pumped into an ODS analytical column (CODS) for qualitative analysis. At the same time, the second retention fraction was pumped alternately into another ODS pre-column (EC2) and into the CODS for analysis. In this alternative mode, online enrichment of retentive components was achieved and the sensitivity was improved accordingly. The chromatogram of SIM on hPDLC/CMC upon injection of it into the system is presented in Fig. 2-1A. The retentive fraction (R1) was then transferred to HPLC/MS for chromatographic separation and MS identification (Fig. 2-1B and C). The chromatogram indicates that R1–1 was SIM. Therefore, our online method could recognize, capture, and identify SIM.
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3.3. Interactions between BAI, WOG, and cell membrane 351 receptors 352
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Six different extracted flavonoids were studied by using the CMC system. The total extract (SBGTE) and ethyl ether extract (SBGEE) of S. baicalensis Georgi were significantly retained on the hPDLC/CMC. The petroleum ether extract was slightly retained, and the chloroform, ethyl acetate, and aqueous extracts were not retained. Thus, we selected SBGEE as the sample in the succeeding part of the experiment. Chromatograms of SBGEE obtained using our method reveal that R0 was the non-retentive fraction and R1 was the retentive fraction on hPDLC/CMC (Fig. 2-2A). Each fraction was assessed by capturing and switching on the online HPLC/ MS system for further separation and identification, as shown in Fig. 2-2C and D. From the UV and MS data, peaks R1–1 and R1–2 corresponding to the main components of the R1 fraction were identified as due to BAI and WOG, respectively. Comparison of results with Fig. 2-2B confirmed that BAI and WOG were present in the SBGEE, and were the major retained constituents at the same conditions. The chromatograms of BAI and WOG standard solutions in the online system are shown in Fig. 2-3 and 4. The main retention fractions on the hPDLC/CMC model were BAI (Fig. 2-3A–C) and WOG (Fig. 2-4A–C).
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3.2. Practical application of the hPDLC/CMC–online HPLC/MS 327 system 328
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1 h). Next, they were stained with 5% sodium thiosulfate for 2 min and finally counterstained with 1% neutral red for 10 min to ensure that calcified material turned black [24]. The images of the cellular layer morphology were captured under a phase-contrast microscope with a DP70 digital camera.
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The retention times of BAI and WOG continuously decreased with increasing concentration of SIM (from 0 to 16 × 10−7 mol/L) in the mobile phase. The corresponding plot of 1/k′ versus [L]m is presented in Fig. 3. Equations of the fitting lines for SIM, BAI, and WOG are YSIM = 39922X + 0.0483 (R2 = 0.9396), YBAI = 36633X + 0.1150 (R2 = 0.9166), and YWOG = 25644X + 0.1363 (R2 = 0.9124), respectively. The equilibrium dissociation constants obtained for SIM, BAI, and WOG are KL = 1.2098 × 10−6, KA = 1.11 × 10−6, and KA = 0.77 × 10−6, respectively.
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Similar to SIM, both BAI and WOG promoted hPDLC growth after incubation with both compounds. BAI (0.15–0.6 mg/L) and WOG (0.015–0.6 mg/L) screened from SBGEE were found to promote the proliferation of hPDLC on the fifth day. Similar to those of SIM, the retention activities of these ingredients were correlated to their pharmacological activities (Fig. 4-1 and 2).
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3.5. BAI and WOG stimulate mineralization
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In contrast to the control, BAI and WOG (0.15 mg/L) 372 markedly enhanced the formation of mineralized nodules in 373
Fig. 1. Brief scheme of the hPDLC/CMC–online HPLC/MS method. Position A: Affinity-recognition procedure using the hPDLC/CMC model. The first retention fraction from the complex sample (S1) is enriched onto an ODS pre-column (EC1) after the hPDLC/CMC column (C1). Establishing the equilibrium procedure of the HPLC/MS system from another ODS pre-column (EC2) to an analytical column (C2). Position B: Analytical identification procedure using the HPLC/MS system, with the first extracted fraction analyzed using an ODS column in HPLC/MS to identify the chemical structures. The second retention fraction from the same sample (S1) was enriched onto another extraction column (EC2) after the hPDLC/CMC column (C1). DUV, ultraviolet detector; DMS, mass spectrographic detector; P1 and P2, pumps.
Please cite this article as: Liu J, et al, Screening of osteoanagenesis-active compounds from Scutellaria baicalensis Georgi by hPDLC/ CMC–online-HPLC/MS..., Fitoterapia (2013), http://dx.doi.org/10.1016/j.fitote.2013.12.020
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Please cite this article as: Liu J, et al, Screening of osteoanagenesis-active compounds from Scutellaria baicalensis Georgi by hPDLC/ CMC–online-HPLC/MS..., Fitoterapia (2013), http://dx.doi.org/10.1016/j.fitote.2013.12.020
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Fig. 2. Chromatograms of SIM, SBGEE, BAI, and WOG solutions obtained by using the combined system. (1). Chromatograms of SIM. (A) CMC chromatogram of SIM standard; (B) HPLC/MS chromatogram of the SIM solution; (C) HPLC/MS chromatogram of the retention fraction (R1) retained in the loops. The retention fraction (R1–1) was identified as SIM. (2). Chromatograms of SBGEE. (A) hPDLC/CMC chromatogram of SBGEE (including R0 and R1 fractions); (B) HPLC/MS chromatogram of the SBGEE solution; (C) HPLC/MS chromatograms of the R1 fraction with two main retention peaks (R1–1 and R1–2), identified as BAI and WOG, respectively; (D) HPLC/MS chromatograms of R0 retained in the loops. (3). Chromatograms of BAI. (A) hPDLC/CMC chromatogram of the BAI standard solution, including the R1 fraction; (B) HPLC/MS chromatogram of the BAI standard solution; (C) HPLC/MS chromatogram of the R1 fraction with only one retention peak (R1–1) identified as BAI. (4). Chromatograms of WOG. (A) hPDLC/CMC chromatogram of the WOG standard solution, including the R1 fraction; (B) HPLC/MS chromatogram of the WOG standard solution; (C) HPLC/MS chromatogram of the R1 fraction with only one retention peak (R1–2) identified as WOG.
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hPDLCs. SIM (0.1 mg/L) produced similar results, as demonstrated in Fig. 4-3.
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3.6. The effect of BAI and WOG on hPDLC matrix calcification
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After treatment with 0.15–0.6 mg/L BAI or WOG or 0.1 mg/L SIM for 21 days, matrix calcification increased, as compared with control cultures (Fig. 4-4). In the presence of
0.15 mg/L BAI or WOG, or 0.1 mg/L SIM, the matrix 419 calcification in particular was conspicuously increased by 420 approximately 2.5-fold. 421 4. Discussion
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Traditional Chinese medicine (TCM) is an ancient and 423 traditional medical practice system that differs from modern 424
Please cite this article as: Liu J, et al, Screening of osteoanagenesis-active compounds from Scutellaria baicalensis Georgi by hPDLC/ CMC–online-HPLC/MS..., Fitoterapia (2013), http://dx.doi.org/10.1016/j.fitote.2013.12.020
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medicine in terms of the substances used and philosophy. It is globally becoming increasingly popular and important to health maintenance and disease treatment. However, the use of TCM has been greatly restricted in clinical practice because of the lack of scientific and technological approaches in application. The most important reasons for this restriction are its high complexity, and the lack of knowledge of targets in the body, chemical composition, and mechanisms of action. Furthermore, the holistic and dynamic nature of disease, and the interaction among various biological components could increase the complexity of TCM studies. Therefore, we used the hPDLC/CMC–online-HPLC/MS method to raise the possibility of elucidating the active ingredients that specifically bind to the receptors of the cell membrane.
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Fig. 3. Retention times on the hPDLC/CMC column and fitting curves constructed by plotting 1/k′ versus [L]m for SIM (1), BAI (2), and WOG (3). The six concentrations of SIM were 0, 1, 2, 4, 8, and 16 × 10−7 mol/L. The retention times of BAI and WOG decreased with increasing SIM concentration in the mobile phase. From the fitting lines, the equilibrium dissociation constant for SIM was found to be KL = 1.2098 × 10−6, and those for BAI and WOG were found to be KA = 1.11 × 10−6 and 0.77 × 10−6, respectively.
The dried root of S. baicalensis Georgi (Chinese name Huang-qin) is a long-known and multipurpose medicine in oriental countries. The observed biological activities of S. baicalensis Georgi in health promotion and disease prevention [27] are credited to its abundant flavonoids [28,29]. More than 40 flavonoid derivatives from the plant, such as baicalin, baicalein (BAI) and wogonin (WOG) have been identified. Over the years, studies have mainly focused on the pharmacological effects of baicalin in the treatment of periodontitis [6], but few have reported on the effects of BAI and WOG. As a mature and creditable approach, online combined CMC-HPLC/MS method, which was used for the research of drugs acting on specific receptor, could preliminarily identify
Please cite this article as: Liu J, et al, Screening of osteoanagenesis-active compounds from Scutellaria baicalensis Georgi by hPDLC/ CMC–online-HPLC/MS..., Fitoterapia (2013), http://dx.doi.org/10.1016/j.fitote.2013.12.020
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the drugs with mass-spectrometer [7,14–16]. In this study, we used a hPDLC/CMC–online-HPLC/MS system to qualitatively screen and identify the active components from the complex samples of S. baicalensis Georgi. The SIM standard solution was used to demonstrate that the system established by using the primary cultured hPDLC was suitable for recognition, analysis, and identification of the active components acting on the hPDLC membrane receptor. Fig. 2-2A was the chromatogram of SBGEE on hPDLC-CMC, which had two retained fractions (R0 and R1). It was supposed that R0 could not interact with hPDLC. Each fraction was assessed by capturing and switching on the online HPLC/MS system for further separation and identification (showed in Fig. 2-2C and D). The eluted fraction from the CMC system was preserved in the first-dimension enrichment column due to the better retentive ability of the C18 VP-ODS than the CMC column. The retention fraction could be easily eluted into the analytical column for analysis in the second dimension. The HPLC
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Fig. 4. Effect of BAI, WOG, and SIM on hPDLC proliferation, von Kossa staining, and matrix calcification. (1). The proliferation of hPDLC by BAI and SIM in the entire period (1, 3, 5, 7, and 9 days) was determined using the MTT assay. hPDLC proliferation was increased by BAI (0.15–0.6 mg/L) and SIM (0.1 mg/L) at the fifth day; (2). Enhanced proliferation of hPDLC by WOG and SIM. The cell activity was increased by WOG (0.015–0.6 mg/L, especially 0.15 mg/L); (3). The effect of BAI 24 and WOG on the formation of calcified nodules was measured by von Kossa staining. There were some mineralization tubercles formed after 21 day incubation with 0.15 mg/L BAI or WOG or 0.1 mg/L SIM. (4). The calcium content of the matrix was determined as described in the text, and normalized to the protein content. The normalized value obtained from the cells incubated under the control conditions was set at 100%. The matrix calcification was markedly increased by 2.5-fold after 21 day treatment with 0.15 to 0.6 mg/L BAI or WOG or 0.1 mg/L SIM, compared with the control group. Data represent mean ± SEM. Statistical comparisons were made using one-way ANOVA followed by Tukey's test as a post hoc comparison. *P b 0.05, **P b 0.01, and ***P b 0.001, compared with the control group.
chromatograms and mass-spectrograms were obtained and used to identify R1–1 as BAI and R1–2 as WOG. The chromatograms of BAI and WOG standard solutions in the online system are shown in Fig. 2-3 and -4. Fig. 2-2B was the chromatogram of SBGEE analyzed by being injected directly into HPLC/MS. The comparison of Fig. 2-2B and C could prove that BAI and WOG were the major retained components in SBGEE on hPDLC/CMC column. Through our system, we screened the SBGEE as the active extracted part. The active ingredients were BAI and WOG, instead of the typically identified compound baicalin. This finding indicates that S. baicalensis Georgi could interact with the hPDLC membrane receptor via two main flavonoids BAI and WOG. Do other types of flavonoids would turn out positive results in our assay? In order to solve this problem, we chose Baicalin as the testing object for analyzing. The chromatogram result of baicalin standard solution which was analyzed by being injected into hPDLC/CMC directly shows
Please cite this article as: Liu J, et al, Screening of osteoanagenesis-active compounds from Scutellaria baicalensis Georgi by hPDLC/ CMC–online-HPLC/MS..., Fitoterapia (2013), http://dx.doi.org/10.1016/j.fitote.2013.12.020
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This work was supported by the National Natural Science Foundation of China (no. 81173311) and Shaanxi Province “13115” Science and Technology Innovation Engineering Major Projects (no. 2009ZDKG-77).
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compounds from medicinal herbs. The beneficial influence of the active ingredients BAI and WOG on bone metabolism might contribute to their use in new and effective therapeutic drugs for treating periodontitis.
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no retained peak (the Chromatograms have not shown). In other words, baicalin does not show to be positive in this system because it could not be combined with the receptor of hPDLC. However, another experiment has already screened baicalin as the active compounds acting on the epidermal growth factor receptor from S. baicalensis Georgi [7]. So it is conjectured that baicalin may have a good effect in the treatment of periodontitis through other approaches, such as direct stimulation of intracellular structure of hPDLC. Therefore, we can explain that the advanced technique is specific for screening the active components from Chinese herbal medicines. Competitive displacement experiments were employed in investigating whether BAI and WOG share a common binding receptor site with SIM on the hPDLC membrane. The retention times of BAI and WOG continuously decreased with increasing concentration of SIM. The degree of shift of the retention times is relevant to the affinity. The ligands interact with the membrane receptor and the effective concentration of the binding sites that both the markers and the ligands occupy. Therefore, the trend suggests that the two molecules acted on the same site as that of SIM on the hPDLC receptor. The KA values of the various drugs indicate that BAI and WOG had low affinity to hPDLC compared with SIM. Our study confirmed that the active ingredients of S. baicalensis Georgi, BAI, WOG, and SIM, combined with the cell membrane receptor on hPDLC, and that they bound to the same receptor to exert their pharmacodynamic effect. Therefore, our next research direction is finding the membrane protein receptors on hPDLC. Although the ingredients screened from S. baicalensis Georgi interact with hPDLC, it remains to be determined whether this interaction is positive regulation. Skeletal tissues, which consist of cells and matrixes, undergo uninterrupted bone remodeling, including old bone absorption and new bone formation. Bone mineralization is a process that requires inorganic minerals such as calcium and phosphorus deposited in the bone matrix in the form of crystals. Thus, cells secrete calcium salt crystals into the extracellular matrix to promote matrix mineralization, which begins bone reconstruction. Our study illustrates this process by the detection of the concentration of Ca2+ ion in the matrix, and staining of the mineralized nodules. The retention times of BAI and WOG in the hPDLC/CMC system were shorter than that of SIM because the interactions of BAI and WOG with hPDLC were weaker. The in vitro activity experiments clearly showed that BAI (0.15–0.6 mg/L) and WOG (0.015–0.6 mg/L) could increase the proliferative activity of hPDLC, promote cell matrix Ca2+ ion secretion, and enhance bone mineral nodule formation. According to this, we could confirm that the screened active substances BAI and WOG induced hPDLC to undergo osteogenic conversion and further osteoanagenesis. The Department of Orthopedics and Traumatology confirmed that BAI could influence bone metabolism by promoting osteoblast differentiation [30], inhibiting osteoclast formation, and increasing osteoclast apoptosis [31] through different pathways. All these reports and our findings are in good agreement with the concept that the active substances may promote bone formation. In summary, we directly and rapidly screened osteoanagenesis-active compounds from the flavonoids of S. baicalensis Georgi that bind to hPDLC membrane receptors by the hPDLC/CMC–online-HPLC/MS method. This could efficiently drive the screening process in the discovery of lead drug
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[25] Baek KH, Lee WY, Oh KW, Tae HJ, Lee JM, Lee EJ, et al. The effect of simvastatin on the proliferation and differentiation of human bone marrow stromal cells. J Korean Med Sci 2005;20:438–44. [26] Shihabi Z, Dyer R. Protein analysis with bicinchoninic acid. Ann Clin Lab Sci 1988;18:235–9. [27] Chan E, Wong CY, Wan C, Kwok C, Wu J, Ng K, et al. Evaluation of antioxidant capacity of root of Scutellaria baicalensis Georgi, in comparison with roots of Polygonum multiflorum Thunb and Panax ginseng CA Meyer. Am J Chin Med 2010;38:815–27. [28] Bochořáková H, Paulova H, Slanina J, Musil P, Táborská E. Main flavonoids in the root of Scutellaria baicalensis cultivated in Europe and their comparative antiradical properties. Phytother Res 2003;17:640–4. [29] Li C, Lin G, Zuo Z. Pharmacological effects and pharmacokinetics properties of Radix Scutellariae and its bioactive flavones. Biopharm Drug Dispos 2011;32:427–45. [30] Kim JM, Lee SU, Kim YS, Min YK, Kim SH. Baicalein stimulates osteoblast differentiation via coordinating activation of MAP kinases and transcription factors. J Cell Biochem 2008;104:1906–17. [31] Kim MH, Ryu SY, Bae M, Choi J-S, Min YK, Kim SH. Baicalein inhibits osteoclast differentiation and induces mature osteoclast apoptosis. Food Chem Toxicol 2008;46:3375–82.
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[18] Desvarieux M, Demmer RT, Rundek T, Boden-Albala B, Jacobs DR, Papapanou PN, et al. Relationship between periodontal disease, tooth loss, and carotid artery plaque The Oral Infections and Vascular Disease Epidemiology Study (INVEST). Stroke 2003;34:2120–5. [19] Lekic P, Rojas J, Birek C, Tenenbaum H, McCulloch C. Phenotypic comparison of periodontal ligament cells in vivo and in vitro. J Periodontal Res 2001;36:71–9. [20] Seo B-M, Miura M, Gronthos S, Mark Bartold P, Batouli S, Brahim J, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet 2004;364:149–55. [21] Shimono M, Ishikawa T, Ishikawa H, Matsuzaki H, Hashimoto S, Muramatsu T, et al. Regulatory mechanisms of periodontal regeneration. Microsc Res Tech 2003;60:491–502. [22] Winkelhoff AJV, Rams TE, Slots J. Systemic antibiotic therapy in periodontics. Periodontol 1996;2000(10):45–78. [23] Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55–63. [24] Puchtler H, Meloan SN. Demonstration of phosphates in calcium deposits: a modification of von Kossa's reaction. Histochemistry 1978;56:177–85.
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Fitoterapia
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Screening of osteoanagenesis-active compounds from Scutellaria baicalensis Georgi by hPDLC/CMC–online-HPLC/MS Jin Liu a,c, Sicen Wang c, Junyi Sun a, Jianfeng Shi b, Ye Li a, Jianzhong Gou a, Ang Li a,b,⁎, Langchong He c,⁎⁎ a b c
Department of Periodontology, Stomatological Hospital, College of Medicine, Xi'an Jiaotong University, Xi'an 710 004, China Research Center for Stomatology, Stomatological Hospital, College of Medicine, Xi'an Jiaotong University, Xi'an 710 004, China School of Medicine, Xi'an Jiaotong University, Xi'an 710 061, China
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Article history: Received 22 October 2013 Accepted in revised form 24 December 2013 Available online xxxx
In present study, an online analytical method using human periodontal ligament cell/cell membrane chromatography (hPDLC/CMC) combined with high-performance liquid chromatography/mass spectrometry (HPLC/MS) was used for direct recognition, separation, and identification of compounds for the first time from Scutellaria baicalensis Georgi (SBG) that are active on hPDLCs. Baicalein (BAI) and wogonin (WOG), which were identified as the active compounds of the ethyl ether extract of S. baicalensis Georgi (SBGEE), could bind to the same membrane receptor of hPDLC for simvastatin (SIM). Moreover, BAI (0.15–0.6 mg/L) and WOG (0.015–0.6 mg/L) had the capability to enhance cell proliferation, matrix calcification, and formation of calcified nodules, which are comparable to the activities of SIM (0.1 mg/L) in vitro. These observations are consistent with the KA of the various drugs. It is very important for the development of SBG used to treat periodontitis. © 2013 Published by Elsevier B.V.
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Keywords: Human periodontal ligament cell membrane chromatography High-performance liquid chromatography/ mass spectrometry Scutellaria baicalensis Georgi Baicalein Wogonin
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1. Introduction
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Historically, plants are continual sources of drugs for discovery, as they provide compounds and derivatives that are invaluable sources of therapeutic agents. A wide variety of herbal medications for the skeletal system, in particular, have beneficial effects and minimal adverse effects [1]. Numerous reports on the pharmacological activities of
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Abbreviations: hPDLC, human periodontal ligament cell; CMC, cell membrane chromatography; HPLC, high performance liquid chromatogram; MS, mass spectrogram; TCM, traditional Chinese medicine; SBG, Scutellaria baicalensis Georgi; SBGEE, the ethyl ether extract of Scutellaria baicalensis Georgi; BAI, baicalein; WOG, wogonin; SIM, simvastatin. ⁎ Correspondence to: A. Li, Department of Periodontology, Stomatological Hospital, College of Medicine, Xi'an Jiaotong University, Xi'an 710 004, China. Tel.: +86 29 8721 6030; fax: +86 29 8727 3400. ⁎⁎ Corresponding author. Tel./fax: +86 29 8265 5451. E-mail addresses: [email protected] (A. Li), [email protected] (L. He).
flavonoids of Scutellaria baicalensis Georgi [2,3] indicate that these substances could significantly improve common cold, hyperlipidaemia and hypertension, ameliorate inflammation, and alleviate bone-related disorders, and promote reconstruction of the periodontium [4–6]. Our group has already screened baicalin as the active compounds acting on the epidermal growth factor receptor from S. baicalensis Georgi [7]. However, the constituents that bind to cell membrane receptors of human periodontal ligament cells (hPDLCs) from this traditional Chinese medicine (TCM) have not been investigated. Identification of active components in natural sources is very difficult. In this regard, cell membrane chromatography (CMC), a novel technique based on biological affinity chromatography [8,9], may be a viable approach, as it is effective at separating ingredients that are active toward specific membrane receptors [10–13]. In our previous investigations, CMC involved the use of a stationary phase consisting of a cell membrane enriched with specific receptors. This stationary phase was used to screen active constituents from complex samples [7,14–16]. In
0367-326X/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.fitote.2013.12.020
Please cite this article as: Liu J, et al, Screening of osteoanagenesis-active compounds from Scutellaria baicalensis Georgi by hPDLC/ CMC–online-HPLC/MS..., Fitoterapia (2013), http://dx.doi.org/10.1016/j.fitote.2013.12.020
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S. baicalensis Georgi was obtained from the Qinba mountains (Shannxi Province, China) and identified by the Department of Pharmacognosy, Xi'an Jiaotong University (Xi'an, China). The plant material was dried at room temperature and ground to powder. A reference sample has been deposited at the Specimen Laboratory, Research and Engineering Center for Natural Medicine, Xi'an Jiaotong University (Xi'an, China). Simvastatin (SIM), baicalein (BAI), and wogonin (WOG) (N 98% pure) were purchased from the National Institutes for Food and Drug Control of China (Beijing, China). Silica gel (ZEX-II, 5 μm, 200 Å) was obtained from Qingdao Meigao Chemical Co., Ltd. (Qingdao, China). hPDLCs were cultured in the Research Center, College of Stomatology, Xi'an Jiaotong University. Dulbecco's Modified Eagle Medium/F12 (DMEM/F12), fetal calf serum (FCS), and trypsin were purchased from Sigma (Saint Louis, MO, USA). Type I collagenase (Worthington Biochem, Freehold, NJ, USA), ascorbic acid 2-phosphate (Wako, Tokyo, Japan), glutamine, penicillin, and streptomycin were also used in the study. HPLC-grade methanol was acquired from Burdick & Jackson (NJ, USA). All other reagents used were of analytical grade. Stock solutions for analysis of SIM, BAI, and WOG (1 mg/mL each) were prepared by separately dissolving the standard drugs in methanol. Standard solutions (0.01 mg/mL) were
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A VICI AG 10G-0911V 10-port 2-pos valve (Valco Instrument Co, Inc., Houston, USA) was used as the column switcher, and two Shim-pack VP-ODS pre-columns (10 × 2.0 mm i.d., 5 μm, Shimadzu Corporation, Kyoto, Japan) were used as the enrichment columns. A hPDLC/CMC column (10 × 2.0 mm i.d.) was used as the first-dimension column and a Shimadzu Shim-pack VP-ODS column (150 × 2.0 mm i.d., 5 μm, Kyoto, Japan) was used as the second-dimension column. The HPLC/MS system (Shimadzu Corporation, Kyoto, Japan) consisted of three LC-20AD pumps, a DGU-20A3 degasser, an SIL-20A autosampler, a CTO-20A column oven, an SPD-20A UV–vis detector, an SPD-M20A diode array detector (DAD), an LCMS2010EV mass spectrometer, and an LC/MS Solution workstation. The data were acquired by LCSolution software (Shimadzu Corporation).
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prepared by dilution of each stock solution with mobile phase (Ultrapure water). BAI, WOG, and SIM were diluted in dimethylsulfoxide (DMSO) stock solution prior to the addition to cell culture medium to form a mixture with a final DMSO concentration of b 0.1% (v/v).
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Preparation of the various extracted flavonoids from S. baicalensis Georgi was as follows: Powdered plant material (50 g) was subjected to ultrasonic extraction with 50 mL methanol in a 100 mL triangular flask. Extraction was performed twice, each lasting 30 min. The filtered supernatant was concentrated to dryness by rotary evaporation to obtain the total flavonoids (SBGTF), which appeared as a dark-brown solid extract. The extract was subsequently suspended in 20 mL distilled water and thrice ultrasonically extracted successively with petroleum ether, ethyl ether, chloroform, and ethyl acetate to yield five different extracts, which were evaporated again to dryness. Sample solutions of all six extracts in methanol (1 mg/mL) were prepared and stored at 4 °C in the dark. Working solutions (0.1 or 0.01 mg/mL) were prepared by diluting the sample solutions with mobile phase on the day of the experiment.
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According to the literature [17], extraction premolar teeth (n = 20) were collected from 10 individuals (ages 15–29 years) at the Stomatological Hospital of Xi'an Jiaotong University (Xi'an, China). The following approved guidelines, which were set by the National Institutes of Health Office of Human Subjects Research, were adopted in the study. The PDL was gently separated from the surface of the 1/3 root and then digested in a solution of 3 mg/mL type I collagenase for 30 min at 37 °C. The cell suspensions were seeded into dishes with DMEM/F12 medium supplemented with 15% FCS, 100 μmol/L ascorbic acid 2-phosphate, 2 mmol/L glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin, and then incubated at 37 °C in a humidified atmosphere with 5% carbon dioxide. Immunocytochemistry tests for keratin and vimentin gave negative and positive results, respectively, confirming that the cells were hPDLCs. Cells were subcultured every three days at a dilution of
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particular, we employed a human periodontal ligament cell/ cell membrane chromatography (hPDLC/CMC) system to screen the active ingredient berberine from Rhizoma Coptidis [17]. Periodontitis has high prevalence, a problem that may not be avoided in all countries [18]. The condition is characterized by the destruction of the periodontal attachment supporting the teeth, including the periodontal ligament (PDL), the cementum, and the alveolar bone. hPDLCs residing in the PDL could differentiate into either cementoblasts or osteoblasts that can produce mineralized tissues. Therefore, hPDLCs could play an important role in the regeneration of periodontal tissue [19,20]. The major goal of periodontal therapy is reconstructing periodontium destroyed by periodontopathy and maintaining healthy periodontium. However, purely mechanical treatment may not be effective for certain patients for whom additional antimicrobial and bone regeneration therapy would be essential for successful treatment [21,22]. Until now, there is no effective treatment for periodontitis. In the present study, a cell membrane was used to establish a hPDLC/CMC model, which was combined with a highperformance liquid chromatography/mass spectrometry (HPLC/ MS) system for screening active principles binding to cell membrane receptors of hPDLC from S. baicalensis Georgi. The active ingredients were recognized, separated, and identified directly through an online column-switching technique between these two systems. By in vitro tests, we verified that active constituents could promote osteogenesis by hPDLCs in a dose-dependent manner. We believe that results of this experiment could provide new and better ideas for using S. baicalensis Georgi in the treatment of periodontitis.
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2.6. Application of the hPDLC/CMC–online HPLC/MS system
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The hPDLC/CMC model was combined with the HPLC/MS system by means of an online 10-port column switcher. Any retention fraction which was “recognized” by the hPDLC/ CMC model was captured in the ODS enrichment column (EC1 or EC2), and then directly eluted into the HPLC/MS system for separation and identification. By using SIM as a positive control, this method was used to screen ingredients from S. baicalensis Georgi that are active toward hPDLC
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In order to verify whether SIM, BAI, and WOG were active on the same site of the hPDLC membrane receptor, we performed a competitive displacement test [10,11]. KA values in the hPDLC/CMC system were measured using a series of SIM solutions (0 to 16 × 10−7 mol/L) in the mobile phase. The chromatographic parameters were the same as above. The retention times of SIM at different concentrations were recorded. Next, standard solutions of BAI and WOG were injected into the hPDLC/CMC column. The capacity factor (k′) of the CMC chromatographic peak in the elution curve was calculated using Eq. (1): ′
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The HPLC conditions (the second dimension) were as follows: column, VP-ODS (150 × 4.6 mm i.d., 5 μm); mobile phase, methanol/0.4% glacial acetic acid—0.2% triethylamine (50:50, v/v); flow rate, 0.2 mL/min; column temperature, 37 °C; and DAD, 280 nm. MS conditions were set as follows: nebulizer gas, N2 (purity N 99.999%); flow rate, 1.5 L/min; drying gas, N2 (purity N 99.999%); pressure, 0.1 MPa; interface temperature, 250 °C; heat block temperature, 200 °C; detector voltage, 1.5 kV; scan range, 50–1000 m/z; and scan mode, positive ionization mode. VP-ODS enrichment columns were used for the first enrichment column (EC1) and the second enrichment column (EC2), which were combined by means of an online 10-port column switcher. EC1 and EC2 could be in either the first or the second-dimension system. Alternation of enrichment and elution could be achieved by the parallel connection of those two enrichment columns.
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where tR is the retention time of the ligand and t0 is the dead 249 250 time of non-retained solvent. The values of KA and KL could be 251 calculated from Eq. (2): 252
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membrane receptors. The standard solutions and six extracts 235 of flavonoids were analyzed separately. 236
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1:3 using a 0.25% aqueous solution of trypsin, and then resuspended to a single-cell suspension. Cells from exponentially growing cultures were used in all experiments. A cell count was performed to determine the cell density. The cell density was ≥1 × 107. The cells were washed thrice with phosphate-buffered saline (PBS). A Tris–HCl hypotonic solution (50 mmol/L, pH 7.4) was added to the washed cells to produce the signal cell suspension. The cells were subsequently ruptured by ultrasonication for 30 min, and the resulting homogenate was centrifuged at 1000 ×g for 10 min. The pellet was discarded and the supernatant was centrifuged at 12,000 ×g for 20 min at 4 °C. The resulting precipitate was resuspended in 10 mL Tris–HCl and the suspension was re-centrifuged at 12,000 ×g. A cell membrane suspension was obtained by adding 5 mL of PBS to the precipitate, and then stored until use. According to a previously reported method [8], 0.05 g silica was activated at 105 °C for 30 min and used as a carrier. It was then homogenized with the cell membrane suspension, by adding the mixture slowly to it under vacuum and with agitation at 4 °C to obtain the hPDLC membrane stationary phase (CMSP). The CMSP was then packed into a column (10 × 2.0 mm i.d.) through a wet-packing procedure to yield the hPDLC/CMC column. Ultrapure water, which was delivered at a flow rate of 0.2 mL/min, was used as the mobile phase in the CMC system (the first dimension). The detection wavelength was 280 nm, and the column temperature was 37 °C.
where KA and KL are the equilibrium dissociation constants for the analyte and marker in the mobile phase, respectively; [L]m, and [R]S are the molar concentrations of the ligands in the effluent and immobilized receptors at the surface of the stationary phase, respectively; and VM is the dead volume of the column. The term X was introduced to the denominator with the k′ value to eliminate the errors incurred during iterative testing. When SIM is used both as a marker and an analyte, KA and KL, which could be obtained from the ratio of intercept and slope, respectively, are equal. [R]S/VM is equal to the slope. Thus, upon substituting Eq. (2) in Eq. (3) the following is obtained:
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The KA values of the two ligands were obtained by linear 268 regression of the plot of 1/k′ versus [L]m. 269 2.8. 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium 270 bromide (MTT) assay 271 The MTT assay was used to measure the growth and viability of hPDLCs [23]. Cells were cultivated in 96-well plates (1 × 106 cells/well) and treated with the drugs at various concentrations for 1, 3, 5, 7, and 9 days. The absorbance values at 490 nm were determined using a microplate reader (Bio-Rad Instruments, USA).
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2.9. von Kossa staining
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After seeding in 12-well plates and treatment with different drugs for 21 days, cells were washed with PBS and fixed with 4% paraform for 30 min, and then incubated with 1% silver nitrate at room temperature under UV light (about
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Please cite this article as: Liu J, et al, Screening of osteoanagenesis-active compounds from Scutellaria baicalensis Georgi by hPDLC/ CMC–online-HPLC/MS..., Fitoterapia (2013), http://dx.doi.org/10.1016/j.fitote.2013.12.020
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In order to quantify the effects of promoting calcification secreted by cells, we evaluated the Ca2+ ion concentration in the matrix. Cells were treated in the same manner as above. After washing thoroughly with PBS three times, cells were incubated overnight at room temperature in 500 μL of 0.1 mol/L HCl to dissolve the calcium mineral from the calcified matrix. The supernatants were collected and the calcium level was determined using an XD-685 electrolyte analyzer [25]. The protein content of cells, which was used to normalize the calcium data, was determined through the bicinchoninic acid method (Sigma) [26].
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2.11. Statistical analyses
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All experiments were repeated three times. Statistical analysis was performed by using SPSS18.0. Data were evaluated through one-way analysis of variance (ANOVA) followed by Tukey's test as a post hoc comparison. All data are presented as mean ± standard error of the mean (SEM). The probability level (P) was considered significant at P b 0.05.
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3. Results
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At position A (Fig. 1), the first retention fraction recognized by the hPDLC/CMC model was extracted though the ODS pre-column (EC1). At position B, the extracted components were pumped into an ODS analytical column (CODS) for qualitative analysis. At the same time, the second retention fraction was pumped alternately into another ODS pre-column (EC2) and into the CODS for analysis. In this alternative mode, online enrichment of retentive components was achieved and the sensitivity was improved accordingly. The chromatogram of SIM on hPDLC/CMC upon injection of it into the system is presented in Fig. 2-1A. The retentive fraction (R1) was then transferred to HPLC/MS for chromatographic separation and MS identification (Fig. 2-1B and C). The chromatogram indicates that R1–1 was SIM. Therefore, our online method could recognize, capture, and identify SIM.
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3.3. Interactions between BAI, WOG, and cell membrane 351 receptors 352
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Six different extracted flavonoids were studied by using the CMC system. The total extract (SBGTE) and ethyl ether extract (SBGEE) of S. baicalensis Georgi were significantly retained on the hPDLC/CMC. The petroleum ether extract was slightly retained, and the chloroform, ethyl acetate, and aqueous extracts were not retained. Thus, we selected SBGEE as the sample in the succeeding part of the experiment. Chromatograms of SBGEE obtained using our method reveal that R0 was the non-retentive fraction and R1 was the retentive fraction on hPDLC/CMC (Fig. 2-2A). Each fraction was assessed by capturing and switching on the online HPLC/ MS system for further separation and identification, as shown in Fig. 2-2C and D. From the UV and MS data, peaks R1–1 and R1–2 corresponding to the main components of the R1 fraction were identified as due to BAI and WOG, respectively. Comparison of results with Fig. 2-2B confirmed that BAI and WOG were present in the SBGEE, and were the major retained constituents at the same conditions. The chromatograms of BAI and WOG standard solutions in the online system are shown in Fig. 2-3 and 4. The main retention fractions on the hPDLC/CMC model were BAI (Fig. 2-3A–C) and WOG (Fig. 2-4A–C).
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1 h). Next, they were stained with 5% sodium thiosulfate for 2 min and finally counterstained with 1% neutral red for 10 min to ensure that calcified material turned black [24]. The images of the cellular layer morphology were captured under a phase-contrast microscope with a DP70 digital camera.
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The retention times of BAI and WOG continuously decreased with increasing concentration of SIM (from 0 to 16 × 10−7 mol/L) in the mobile phase. The corresponding plot of 1/k′ versus [L]m is presented in Fig. 3. Equations of the fitting lines for SIM, BAI, and WOG are YSIM = 39922X + 0.0483 (R2 = 0.9396), YBAI = 36633X + 0.1150 (R2 = 0.9166), and YWOG = 25644X + 0.1363 (R2 = 0.9124), respectively. The equilibrium dissociation constants obtained for SIM, BAI, and WOG are KL = 1.2098 × 10−6, KA = 1.11 × 10−6, and KA = 0.77 × 10−6, respectively.
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Similar to SIM, both BAI and WOG promoted hPDLC growth after incubation with both compounds. BAI (0.15–0.6 mg/L) and WOG (0.015–0.6 mg/L) screened from SBGEE were found to promote the proliferation of hPDLC on the fifth day. Similar to those of SIM, the retention activities of these ingredients were correlated to their pharmacological activities (Fig. 4-1 and 2).
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In contrast to the control, BAI and WOG (0.15 mg/L) 372 markedly enhanced the formation of mineralized nodules in 373
Fig. 1. Brief scheme of the hPDLC/CMC–online HPLC/MS method. Position A: Affinity-recognition procedure using the hPDLC/CMC model. The first retention fraction from the complex sample (S1) is enriched onto an ODS pre-column (EC1) after the hPDLC/CMC column (C1). Establishing the equilibrium procedure of the HPLC/MS system from another ODS pre-column (EC2) to an analytical column (C2). Position B: Analytical identification procedure using the HPLC/MS system, with the first extracted fraction analyzed using an ODS column in HPLC/MS to identify the chemical structures. The second retention fraction from the same sample (S1) was enriched onto another extraction column (EC2) after the hPDLC/CMC column (C1). DUV, ultraviolet detector; DMS, mass spectrographic detector; P1 and P2, pumps.
Please cite this article as: Liu J, et al, Screening of osteoanagenesis-active compounds from Scutellaria baicalensis Georgi by hPDLC/ CMC–online-HPLC/MS..., Fitoterapia (2013), http://dx.doi.org/10.1016/j.fitote.2013.12.020
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Please cite this article as: Liu J, et al, Screening of osteoanagenesis-active compounds from Scutellaria baicalensis Georgi by hPDLC/ CMC–online-HPLC/MS..., Fitoterapia (2013), http://dx.doi.org/10.1016/j.fitote.2013.12.020
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Fig. 2. Chromatograms of SIM, SBGEE, BAI, and WOG solutions obtained by using the combined system. (1). Chromatograms of SIM. (A) CMC chromatogram of SIM standard; (B) HPLC/MS chromatogram of the SIM solution; (C) HPLC/MS chromatogram of the retention fraction (R1) retained in the loops. The retention fraction (R1–1) was identified as SIM. (2). Chromatograms of SBGEE. (A) hPDLC/CMC chromatogram of SBGEE (including R0 and R1 fractions); (B) HPLC/MS chromatogram of the SBGEE solution; (C) HPLC/MS chromatograms of the R1 fraction with two main retention peaks (R1–1 and R1–2), identified as BAI and WOG, respectively; (D) HPLC/MS chromatograms of R0 retained in the loops. (3). Chromatograms of BAI. (A) hPDLC/CMC chromatogram of the BAI standard solution, including the R1 fraction; (B) HPLC/MS chromatogram of the BAI standard solution; (C) HPLC/MS chromatogram of the R1 fraction with only one retention peak (R1–1) identified as BAI. (4). Chromatograms of WOG. (A) hPDLC/CMC chromatogram of the WOG standard solution, including the R1 fraction; (B) HPLC/MS chromatogram of the WOG standard solution; (C) HPLC/MS chromatogram of the R1 fraction with only one retention peak (R1–2) identified as WOG.
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hPDLCs. SIM (0.1 mg/L) produced similar results, as demonstrated in Fig. 4-3.
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After treatment with 0.15–0.6 mg/L BAI or WOG or 0.1 mg/L SIM for 21 days, matrix calcification increased, as compared with control cultures (Fig. 4-4). In the presence of
0.15 mg/L BAI or WOG, or 0.1 mg/L SIM, the matrix 419 calcification in particular was conspicuously increased by 420 approximately 2.5-fold. 421 4. Discussion
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Traditional Chinese medicine (TCM) is an ancient and 423 traditional medical practice system that differs from modern 424
Please cite this article as: Liu J, et al, Screening of osteoanagenesis-active compounds from Scutellaria baicalensis Georgi by hPDLC/ CMC–online-HPLC/MS..., Fitoterapia (2013), http://dx.doi.org/10.1016/j.fitote.2013.12.020
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medicine in terms of the substances used and philosophy. It is globally becoming increasingly popular and important to health maintenance and disease treatment. However, the use of TCM has been greatly restricted in clinical practice because of the lack of scientific and technological approaches in application. The most important reasons for this restriction are its high complexity, and the lack of knowledge of targets in the body, chemical composition, and mechanisms of action. Furthermore, the holistic and dynamic nature of disease, and the interaction among various biological components could increase the complexity of TCM studies. Therefore, we used the hPDLC/CMC–online-HPLC/MS method to raise the possibility of elucidating the active ingredients that specifically bind to the receptors of the cell membrane.
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Fig. 3. Retention times on the hPDLC/CMC column and fitting curves constructed by plotting 1/k′ versus [L]m for SIM (1), BAI (2), and WOG (3). The six concentrations of SIM were 0, 1, 2, 4, 8, and 16 × 10−7 mol/L. The retention times of BAI and WOG decreased with increasing SIM concentration in the mobile phase. From the fitting lines, the equilibrium dissociation constant for SIM was found to be KL = 1.2098 × 10−6, and those for BAI and WOG were found to be KA = 1.11 × 10−6 and 0.77 × 10−6, respectively.
The dried root of S. baicalensis Georgi (Chinese name Huang-qin) is a long-known and multipurpose medicine in oriental countries. The observed biological activities of S. baicalensis Georgi in health promotion and disease prevention [27] are credited to its abundant flavonoids [28,29]. More than 40 flavonoid derivatives from the plant, such as baicalin, baicalein (BAI) and wogonin (WOG) have been identified. Over the years, studies have mainly focused on the pharmacological effects of baicalin in the treatment of periodontitis [6], but few have reported on the effects of BAI and WOG. As a mature and creditable approach, online combined CMC-HPLC/MS method, which was used for the research of drugs acting on specific receptor, could preliminarily identify
Please cite this article as: Liu J, et al, Screening of osteoanagenesis-active compounds from Scutellaria baicalensis Georgi by hPDLC/ CMC–online-HPLC/MS..., Fitoterapia (2013), http://dx.doi.org/10.1016/j.fitote.2013.12.020
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the drugs with mass-spectrometer [7,14–16]. In this study, we used a hPDLC/CMC–online-HPLC/MS system to qualitatively screen and identify the active components from the complex samples of S. baicalensis Georgi. The SIM standard solution was used to demonstrate that the system established by using the primary cultured hPDLC was suitable for recognition, analysis, and identification of the active components acting on the hPDLC membrane receptor. Fig. 2-2A was the chromatogram of SBGEE on hPDLC-CMC, which had two retained fractions (R0 and R1). It was supposed that R0 could not interact with hPDLC. Each fraction was assessed by capturing and switching on the online HPLC/MS system for further separation and identification (showed in Fig. 2-2C and D). The eluted fraction from the CMC system was preserved in the first-dimension enrichment column due to the better retentive ability of the C18 VP-ODS than the CMC column. The retention fraction could be easily eluted into the analytical column for analysis in the second dimension. The HPLC
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Fig. 4. Effect of BAI, WOG, and SIM on hPDLC proliferation, von Kossa staining, and matrix calcification. (1). The proliferation of hPDLC by BAI and SIM in the entire period (1, 3, 5, 7, and 9 days) was determined using the MTT assay. hPDLC proliferation was increased by BAI (0.15–0.6 mg/L) and SIM (0.1 mg/L) at the fifth day; (2). Enhanced proliferation of hPDLC by WOG and SIM. The cell activity was increased by WOG (0.015–0.6 mg/L, especially 0.15 mg/L); (3). The effect of BAI 24 and WOG on the formation of calcified nodules was measured by von Kossa staining. There were some mineralization tubercles formed after 21 day incubation with 0.15 mg/L BAI or WOG or 0.1 mg/L SIM. (4). The calcium content of the matrix was determined as described in the text, and normalized to the protein content. The normalized value obtained from the cells incubated under the control conditions was set at 100%. The matrix calcification was markedly increased by 2.5-fold after 21 day treatment with 0.15 to 0.6 mg/L BAI or WOG or 0.1 mg/L SIM, compared with the control group. Data represent mean ± SEM. Statistical comparisons were made using one-way ANOVA followed by Tukey's test as a post hoc comparison. *P b 0.05, **P b 0.01, and ***P b 0.001, compared with the control group.
chromatograms and mass-spectrograms were obtained and used to identify R1–1 as BAI and R1–2 as WOG. The chromatograms of BAI and WOG standard solutions in the online system are shown in Fig. 2-3 and -4. Fig. 2-2B was the chromatogram of SBGEE analyzed by being injected directly into HPLC/MS. The comparison of Fig. 2-2B and C could prove that BAI and WOG were the major retained components in SBGEE on hPDLC/CMC column. Through our system, we screened the SBGEE as the active extracted part. The active ingredients were BAI and WOG, instead of the typically identified compound baicalin. This finding indicates that S. baicalensis Georgi could interact with the hPDLC membrane receptor via two main flavonoids BAI and WOG. Do other types of flavonoids would turn out positive results in our assay? In order to solve this problem, we chose Baicalin as the testing object for analyzing. The chromatogram result of baicalin standard solution which was analyzed by being injected into hPDLC/CMC directly shows
Please cite this article as: Liu J, et al, Screening of osteoanagenesis-active compounds from Scutellaria baicalensis Georgi by hPDLC/ CMC–online-HPLC/MS..., Fitoterapia (2013), http://dx.doi.org/10.1016/j.fitote.2013.12.020
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This work was supported by the National Natural Science Foundation of China (no. 81173311) and Shaanxi Province “13115” Science and Technology Innovation Engineering Major Projects (no. 2009ZDKG-77).
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compounds from medicinal herbs. The beneficial influence of the active ingredients BAI and WOG on bone metabolism might contribute to their use in new and effective therapeutic drugs for treating periodontitis.
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no retained peak (the Chromatograms have not shown). In other words, baicalin does not show to be positive in this system because it could not be combined with the receptor of hPDLC. However, another experiment has already screened baicalin as the active compounds acting on the epidermal growth factor receptor from S. baicalensis Georgi [7]. So it is conjectured that baicalin may have a good effect in the treatment of periodontitis through other approaches, such as direct stimulation of intracellular structure of hPDLC. Therefore, we can explain that the advanced technique is specific for screening the active components from Chinese herbal medicines. Competitive displacement experiments were employed in investigating whether BAI and WOG share a common binding receptor site with SIM on the hPDLC membrane. The retention times of BAI and WOG continuously decreased with increasing concentration of SIM. The degree of shift of the retention times is relevant to the affinity. The ligands interact with the membrane receptor and the effective concentration of the binding sites that both the markers and the ligands occupy. Therefore, the trend suggests that the two molecules acted on the same site as that of SIM on the hPDLC receptor. The KA values of the various drugs indicate that BAI and WOG had low affinity to hPDLC compared with SIM. Our study confirmed that the active ingredients of S. baicalensis Georgi, BAI, WOG, and SIM, combined with the cell membrane receptor on hPDLC, and that they bound to the same receptor to exert their pharmacodynamic effect. Therefore, our next research direction is finding the membrane protein receptors on hPDLC. Although the ingredients screened from S. baicalensis Georgi interact with hPDLC, it remains to be determined whether this interaction is positive regulation. Skeletal tissues, which consist of cells and matrixes, undergo uninterrupted bone remodeling, including old bone absorption and new bone formation. Bone mineralization is a process that requires inorganic minerals such as calcium and phosphorus deposited in the bone matrix in the form of crystals. Thus, cells secrete calcium salt crystals into the extracellular matrix to promote matrix mineralization, which begins bone reconstruction. Our study illustrates this process by the detection of the concentration of Ca2+ ion in the matrix, and staining of the mineralized nodules. The retention times of BAI and WOG in the hPDLC/CMC system were shorter than that of SIM because the interactions of BAI and WOG with hPDLC were weaker. The in vitro activity experiments clearly showed that BAI (0.15–0.6 mg/L) and WOG (0.015–0.6 mg/L) could increase the proliferative activity of hPDLC, promote cell matrix Ca2+ ion secretion, and enhance bone mineral nodule formation. According to this, we could confirm that the screened active substances BAI and WOG induced hPDLC to undergo osteogenic conversion and further osteoanagenesis. The Department of Orthopedics and Traumatology confirmed that BAI could influence bone metabolism by promoting osteoblast differentiation [30], inhibiting osteoclast formation, and increasing osteoclast apoptosis [31] through different pathways. All these reports and our findings are in good agreement with the concept that the active substances may promote bone formation. In summary, we directly and rapidly screened osteoanagenesis-active compounds from the flavonoids of S. baicalensis Georgi that bind to hPDLC membrane receptors by the hPDLC/CMC–online-HPLC/MS method. This could efficiently drive the screening process in the discovery of lead drug
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Please cite this article as: Liu J, et al, Screening of osteoanagenesis-active compounds from Scutellaria baicalensis Georgi by hPDLC/ CMC–online-HPLC/MS..., Fitoterapia (2013), http://dx.doi.org/10.1016/j.fitote.2013.12.020
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