Determination of Tenacissoside G, Tenacissoside H, and Tenacissoside I in Rat Plasma by UPLC-MS/MS and Their Pharmacokinetics

Chen, Fan; Ma, Yizhe; Cui, Ying; Wang, Wanhang; Mei, Chenchen; Nie, Jingjing; Wen, Congcong; Shen, Xiuwei; Zhou, Xuzhao

doi:https://doi.org/10.1155/2023/4747771

International Journal of Analytical Chemistry

On this page

Abstract Introduction Experimental Discussion Conclusion Data Availability Conflicts of Interest Authors’ Contributions Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2023 | Article ID 4747771 | https://doi.org/10.1155/2023/4747771

Determination of Tenacissoside G, Tenacissoside H, and Tenacissoside I in Rat Plasma by UPLC-MS/MS and Their Pharmacokinetics

Fan Chen,¹Yizhe Ma,²Ying Cui,²Wanhang Wang,²Chenchen Mei,²Jingjing Nie,¹Congcong Wen,²Xiuwei Shen,¹and Xuzhao Zhou³

Academic Editor: Waleed Alahmad

Received29 Jun 2023

Revised18 Sept 2023

Accepted23 Sept 2023

Published28 Sept 2023

Abstract

An ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination of tenacissoside G, tenacissoside H, and tenacissoside I in rat plasma. The rat plasma was treated with liquid-liquid extraction using ethyl acetate. The determination was performed on the UPLC HSS T3 column (50 mm × 2.1 mm, 1.8 μm) with a mobile phase consisting of acetonitrile-water (containing 0.1% formic acid) and gradient elution at a flow rate of 0.4 mL/min. Electrospray (ESI) positive ion mode detection and multireaction monitoring (MRM) quantitative analysis were performed. A total of 36 rats were given tenacissoside G, tenacissoside H, and tenacissoside I, respectively, orally (5 mg/kg) and intravenously (1 mg/kg), with 6 rats in each group, to evaluate the pharmacokinetic difference of tenacissoside G, tenacissoside H, and tenacissoside I in rats. The calibration curves showed good linearity in the range of 5–2000 ng/mL, where r was greater than 0.99. The results of precision, accuracy, recovery, matrix effect, and stability met the requirements of biological sample detection methods. The established UPLC-MS/MS method was successfully applied to pharmacokinetic studies of tenacissoside G, tenacissoside H, and tenacissoside I, and the bioavailability was 22.9%, 89.8%, and 9.4%, respectively.

1. Introduction

Marsdenia tenacissima, also known as glaucescent fissistigma root, Rosa banksiae f. lutea (Lindl.) Rehd, etc., is the dry stem and rattan of Marsdenia tenacissima (Roxb) Wight et Am., a plant belonging to the family Asclepiadaceae [1–3], which was originally published in the Herbal Medicines of Southern Yunnan and is now published in the Chinese Pharmacopoeia 2010 edition [4, 5]. It is mainly distributed in Guizhou, Yunnan, Sichuan, Guangxi, and other places. Marsdenia tenacissima tastes bitter and slightly cold. It has the effects of clearing heat and detoxifying, relieving cough, and asthma, dispersing knots and relieving pain, and fighting cancer. The series of preparations made from its single medicinal material are widely used in clinics [6–8].

The roots, stems, and leaves of Marsdenia tenacissima can be used as medicine. Its effective components are steroidal glycosides, alkaloids, and polysaccharides [9–11]. Its active antitumor components are mainly steroidal components. It is reported that Marsdenia tenacissima polysaccharides and some fat-soluble components also have antitumor effects [12]. At present, dozens of steroids have been isolated from this plant. The C21 steroidal glycosides are mostly white crystals or powder, which mainly exist in the plants of Asclepiadaceae. The main antitumor active component of Marsdenia tenacissima is C21 steroidal glycosides [13]. Polysaccharide and some fat-soluble components also have antitumor effects. At present, more than 40 kinds of C21 steroidal glycosides have been isolated from the plant, which are a class of compounds formed by a class of steroidal derivatives of glycosides and 2-deoxysugars, and the sugar chain contains up to 6 sugars. These compounds contain a variety of aglycones with different structures [8, 9], of which there are 6 main configuration aglycones. In vivo pharmacokinetics of tenacissoside H and tenacissoside I have been reported in literature [14–16], but bioavailability has not been reported.

High-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) technology has the advantages of high sensitivity, low detection limit, and small sample consumption and is widely used in drug analysis of chemical composition, drug metabolism, and impurity identification [17, 18]. UPLC, which columns with small particle sizes and under ultra-high pressure, maintains the basic principles of a traditional HPLC system but demonstrates improved separation efficiency and speed [19, 20].

Therefore, this study was to establish an UPLC-MS/MS for the determination of tenacissoside G, tenacissoside H, and tenacissoside I in rat plasma and study the pharmacokinetics and bioavailability to provide a scientific experimental basis for the basic research of clinical pharmacy.

2. Experimental

2.1. Reagents and Animals

Tenacissoside G, tenacissoside H, tenacissoside I, and astragaloside IV (internal standard) (purity ≥98%, Figure 1) were all purchased from Chengdu Master Pharmaceutical Co., Ltd. Acetonitrile and methanol in chromatographic purity were purchased from Merck. Ultra-pure water (resistance >18 mΩ) was prepared by the Milli-Q purification system in the United States. Sprague–Dawley (SD) rats (220–250 g) were from the Animal Experimental Center of Wenzhou Medical University.

(a)

(b)

(c)

(d)

2.2. Instrument Condition

A Waters XEVO TQ-S microtriple quadrupole series mass spectrometer was used for the detection of tenacissoside G, tenacissoside H, and tenacissoside I.

Chromatographic conditions were as follows: UPLC HSS T3 column (50 mm × 2.1 mm, 1.8 μm) and column temperature set at 40°C. The mobile phase was acetonitrile-water (containing 0.1% formic acid) with gradient elution at a flow rate of 0.4 mL/min and elution time of 6 min. 0–0.2 min, acetonitrile 10%; 0.2–2.4 min, acetonitrile 10%–75%; 2.4–5.0 min, acetonitrile 75%–90%; 5.0–5.1 min, acetonitrile 90%–10%; and 5.1–6.0 min, acetonitrile 10%.

Mass spectrometry conditions: nitrogen as conical gas (50 L/h) and desolvated gas (900 L/h); capillary voltage set at 2.5 kV; ion source temperature at 150°C; and desolvent temperature at 450°C. ESI positive ion mode detection and MRM were used for quantitative analysis: m/z 815.5 ⟶ 755.5 for tenacissoside G (cone voltage 96 v, collision voltage 26 v), m/z 817.4 ⟶ 757.5 for tenacissoside H (cone voltage 96 v, collision voltage 40 v), m/z 837.4 ⟶ 777.5 for tenacissoside I (cone voltage 86 v, collision voltage 30 v), and m/z 785.4 ⟶ 143.0 for astragaloside IV (cone voltage 6 v, collision voltage 46 v).

2.3. Standard Curve

Tenacissoside G, tenacissoside H, tenacissoside I, and astragaloside IV reserve solution (500 μg/mL) were prepared with methanol, respectively. Tenacissoside G, tenacissoside H, and tenacissoside I working solution was obtained by diluting the reserve solution with methanol. Both the reserve solution and working solution were stored at 4°C. Appropriate amount of tenacissoside G, tenacissoside H, and tenacissoside I working solution was added to the blank rat plasma, and then the tenacissoside G, tenacissoside H, and tenacissoside I in rat plasma were 5, 10, 20, 50, 100, 200, 500, 1000, and 2000 ng/mL. Three quality control (QC) samples with plasma concentrations (8, 180, and 1800 ng/mL) were prepared by the same method.

2.4. Sample Handling

A plasma sample of 100 μL was added to a 1.5 mL microcentrifuge tube, then 10 μL of astragaloside IV (1.0 μg/mL) and 1.0 mL ethyl acetate were added, vortex was mixed for 1.0 min, and it was centrifuged (13,000 rpm, 4°C, 5 min). The organic phase was transferred into another tube and evaporated to dryness at 40°C under a gentle stream of nitrogen. The residue was reconstituted in 100 μL methanol and centrifuged at 13,000 rpm for 5 min. The supernatant was pipetted to an auto sampler vial, and 2 μL was injected into the UPLC-MS/MS for analysis.

2.5. Pharmacokinetic Study

Tenacissoside G, tenacissoside H, and tenacissoside I were given sublingual intravenous administration (iv) of 1 mg/kg and oral administration (po) 5 mg/kg, respectively, with 6 rats in each group, for a total of 36 rats. All experimental procedures and protocols were approved by the Animal Care Committee of Wenzhou Medical University (xmsq 2023-0689). The 0.4 mL blood was collected from the caudal vein at 0.083 3, 0.5, 1, 2, 3, 4, 6, and 8 h for tenacissoside G; 0.083 3, 0.5, 1, 2, 3, 4, 6, 8 and 12 h for tenacissoside H; and 0.083 3, 0.5, 1, 2, 3, 4, and 6 h for tenacissoside I, collected in heparinized test tubes and centrifuged at 13000 r/min for 10 min. 100 μL of the plasma was then transferred to a new 1.5 mL microcentrifuge tube and held at −80°C prior to analysis. Pharmacokinetic parameters were statistically calculated using the pharmacokinetic software (DAS 2.0 version).

3. Result

3.1. Selectivity

The retention times of tenacissoside G, tenacissoside H, tenacissoside I, and astragaloside IV were 3.32, 3.42, 3.41, and 2.63 min, as shown in Figure 2, respectively. The optimized gradient elution procedure was used to isolate tenacissoside G, tenacissoside H, and tenacissoside I, effectively, and no interference of endogenous components was observed in the retention time of tenacissoside G, tenacissoside H, and tenacissoside I. This method has good selectivity.

(a)

(b)

3.2. Standard Curve

The calibration curves of tenacissoside G, tenacissoside H, and tenacissoside I in rat plasma showed good linearity in the range of 5–2000 ng/mL, with r greater than 0.99. The typical regression equation of the tenacissoside G in rat plasma was as follows: y1 = 0.0045x1 + 0.0102 (r = 0.9976), x1 was the concentration of tenacissoside G in plasma, and y1 was the ratio of tenacissoside G peak area to the internal standard. The typical regression equation for tenacissoside H in rat plasma was y2 = 0.0046x2 + 0.0022 (r = 0.9986), where x2 was the concentration of tenacissoside H in plasma, and y2 was the ratio of the tenacissoside H peak area to the internal standard. The typical regression equation for tenacissoside I in rat plasma was y3 = 0.0020x3 + 0.0097 (r = 0.9977), x3 was the concentration of tenacissoside I in plasma, and y3 was the ratio of tenacissoside I peak area to internal standard. The lower limit of quantitation of tenacissoside G, tenacissoside H, and tenacissoside I in rat plasma was 5 ng/mL, and the detection limit was 1.5 ng/mL.

3.3. Precision, Accuracy, Recovery, and Matrix Effect

The intraday and interday precision of tenacissoside G was within 10%, the accuracy was 90% to 111%, the recovery was over 92%, and the matrix effect was in the range of 94% to 109% (Table 1).

The intraday and interday precision of te nacissoside H was within 13%, the accuracy was 88% to 115%, the recovery was above 88%, and the matrix effect was in the range of 101% to 108% (Table 1).

The intraday and interday precision of tenacissoside I was within 15%, the accuracy was 88% to 110%, the recovery was above 80%, and the matrix effect range was 91% to 99% (Table 1).

3.4. Stability

The plasma samples of rats were stored in an automatic injector for 2 h, pretreated, and placed at room temperature for 24 h. The plasma samples underwent three freeze-thawing cycles, and the stability test was conducted at −20°C for 30 days. The accuracy of tenacissoside G was 88%–112%; RSD was within 13%. The accuracy of tenacissoside H was 88%–111%; RSD was within 15%. The accuracy of tenacissoside I was 88%–111%; RSD was within 15% (Table 2). The results indicated that tenacissoside G, tenacissoside H, and tenacissoside I were stable.

3.5. Pharmacokinetic Study

The concentration-time curves of tenacissoside G, tenacissoside H, and tenacissoside I in rat plasma are shown in Figure 3. The main pharmacokinetic parameters are listed in Table 3, and oral bioavailability was 22.9%, 89.8%, and 9.4%, respectively.

4. Discussion

In order to obtain the best mass spectrum conditions, the positive and negative ion modes were used for monitoring. The responses of tenacissoside G, tenacissoside H, and tenacissoside I were higher in the positive ion mode than the negative ion modes. After optimizing various parameters, the mass spectrum parameters can meet the requirements of accurate quantification of effective substances in biological samples. Through the standard sample, the capillary voltage and the collision energy were optimized.

The commonly used biological sample pretreatment methods were liquid-liquid extraction (LLE) [21–24], solid-phase extraction (SPE) [25, 26], and protein precipitation method (PPT). The SPE method has complicated operation steps and a high extraction column price. LLE could efficiently extract target substances, especially for low-concentration samples, with higher extraction efficiency than PPT. LLE and PPT methods were tried in this work, and LLE with ethyl acetate was found to be a better extraction efficiency (around 90%) than PPT with acetonitrile (around 60%).

During quantitative analysis, it is necessary to add an internal standard substance of known concentration as a quantitative reference for the compounds to be measured in the sample. The internal standard substance should have similar physical and chemical properties to the compound to be tested, and be stable in the sample, and be easy to detect and quantify. Commonly used internal standard substances include isotope-labeled compounds and structural analogues. Astragaloside IV has similar physical and chemical properties to tenacissoside, and it was selected as the internal standard.

Zhao et al. studied the plasma concentration and pharmacokinetic process of tenacissoside A in rats by the LC-MS/MS method [27]. Medroxyprogesterone acetate was used as the internal standard. The oral bioavailability of the drug was low (2.6%), the elimination was faster, and the first-pass effect was obvious. Li established a LC-MS/MS method for simultaneous determination of garcinia extract in rat plasma samples of tenacissoside B, tenacissoside H, tenacissoside I, caffeic acid, cryptochlorogenic acid, chlorogenic acid, and neochlorogenic acid [16]. Digoxin was used as an internal standard reference. Zeng et al. have developed a LC-MS/MS method for simultaneous determination of three isomerized gestrins (17β-tenacigenin B, tenacigenine B, and tenacigenine A) and their corresponding glycosides (tenacissoside A and tenacissoside B) in rat plasma [14]. After dexamethasone acetate was added as an internal standard, a simple liquid-liquid extraction technique was used. This method was successfully applied to the pharmacokinetic study after intravenous injection of Xiao-Ai-Ping in rats. However, these methods did not study the bioavailability of tenacissoside B, tenacissoside H, and tenacissoside I.

5. Conclusion

In this study, the UPLC-MS/MS technique was established for the determination of tenacissoside G, tenacissoside H, and tenacissoside I in rat plasma in the range of 5–2000 ng/mL. The rat plasma was treated with liquid-liquid extraction using ethyl acetate, and astragaloside IV was used as internal standard. The selectivity, linearity, precision, accuracy, recovery, and stability of this method have been verified, and it has been applied to the pharmacokinetic study of tenacissoside G, tenacissoside H, and tenacissoside I in rats, and the bioavailability was calculated to be 22.9%, 89.8%, and 9.4%, respectively.

Data Availability

The data used to support the findings of this study are included within the article.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors’ Contributions

Fan Chen and Yizhe Ma contributed equally to this work.

Acknowledgments

This study was supported by grants from the Zhejiang Provincial Natural Science Foundation (LQ22H090013).

References

S. Li, W. Pei, W. Yuan, D. Yu, H. Song, and H. Zhang, “Multi-omics joint analysis reveals the mechanism of action of the traditional Chinese medicine Marsdenia tenacissima (Roxb.) Moon in the treatment of hepatocellular carcinoma,” Journal of Ethnopharmacology, vol. 293, Article ID 115285, 2022.
View at: Publisher Site | Google Scholar
S. Lin, Q. Sheng, X. Ma et al., “Marsdenia tenacissima extract induces autophagy and apoptosis of hepatocellular cells via MIF/mToR signaling,” Evidence-based Complementary and Alternative Medicine, vol. 2022, Article ID 7354700, 10 pages, 2022.
View at: Publisher Site | Google Scholar
B. Xie, S. Q. Jiang, X. L. Shen, H. Q. Wu, and Y. J. Hu, “Pharmacokinetics, plasma protein binding, and metabolism of a potential natural chemosensitizer from Marsdenia tenacissima in rats,” Journal of Ethnopharmacology, vol. 281, Article ID 114544, 2021.
View at: Publisher Site | Google Scholar
N. Yu, Y. L. Wei, Y. Zhu et al., “Integrated approach for identifying and evaluating the quality of Marsdenia tenacissima in the medicine market,” PLoS One, vol. 13, no. 4, Article ID e0195240, 2018.
View at: Publisher Site | Google Scholar
E. Li, Z. Teng, S. Liu, S. Qin, and C. Zhang, “[HPLC-ELSD fingerprint of Marsdenia tenacissima from different habitats],” Zhongguo Zhongyao Zazhi, vol. 37, no. 11, pp. 1610–1613, 2012.
View at: Google Scholar
R. Li, Z. Zhang, X. Su, J. Yu, L. Lu, and T. Liu, “Nontargeted metabolomics study and pharmacodynamic evaluation of bidirectional fermentation for Ganoderma lucidum with Marsdenia tenacissima,” Frontiers in Pharmacology, vol. 13, Article ID 1012063, 2022.
View at: Publisher Site | Google Scholar
X. P. Jiang, S. Jin, W. Shao, L. Zhu, S. Yan, and J. Lu, “Saponins of Marsdenia Tenacissima promotes apoptosis of hepatocellular carcinoma cells through damaging mitochondria then activating cytochrome C/Caspase-9/Caspase-3 pathway,” Journal of Cancer, vol. 13, no. 9, pp. 2855–2862, 2022.
View at: Publisher Site | Google Scholar
J. L. Fu, H. F. Hao, S. Wang, Y. N. Jiao, P. P. Li, and S. Y. Han, “Marsdenia tenacissima extract disturbs the interaction between tumor-associated macrophages and non-small cell lung cancer cells by targeting HDGF,” Journal of Ethnopharmacology, vol. 298, Article ID 115607, 2022.
View at: Publisher Site | Google Scholar
P. Liu, D. W. Xu, R. T. Li et al., “A combined phytochemistry and network pharmacology approach to reveal potential anti-NSCLC effective substances and mechanisms in Marsdenia tenacissima (roxb.) moon (stem),” Frontiers in Pharmacology, vol. 12, Article ID 518406, 2021.
View at: Publisher Site | Google Scholar
B. Xie, Y. Y. Lu, Z. H. Luo et al., “Tenacigenin B ester derivatives from Marsdenia tenacissima actively inhibited CYP3A4 and enhanced in vivo antitumor activity of paclitaxel,” Journal of Ethnopharmacology, vol. 235, pp. 309–319, 2019.
View at: Publisher Site | Google Scholar
P. Wang, J. Yang, Z. Zhu, and X. Zhang, “Marsdenia tenacissima: a review of traditional uses, phytochemistry and pharmacology,” The American Journal of Chinese Medicine, vol. 46, no. 07, pp. 1449–1480, 2018.
View at: Publisher Site | Google Scholar
Y. Pan, X. Liao, L. Yang et al., “Extract of Marsdenia tenacissima (roxb.) moon [apocynaceae] suppresses hepatocellular carcinoma by inhibiting angiogenesis,” Frontiers in Pharmacology, vol. 13, Article ID 900128, 2022.
View at: Publisher Site | Google Scholar
X. Pang, L. P. Kang, X. M. Fang et al., “C(21) steroid derivatives from the Dai herbal medicine Dai-Bai-Jie, the dried roots of Marsdenia tenacissima, and their screening for anti-HIV activity,” Journal of Natural Medicines, vol. 72, no. 1, pp. 166–180, 2018.
View at: Publisher Site | Google Scholar
Q. Zeng, F. Zhang, S. Gao, L. Sun, B. Jiang, and W. Chen, “Simultaneous determination of six C21 steroids of Xiao-Ai-Ping injection in rat plasma by LC-MS/MS,” Biomedical Chromatography, vol. 28, no. 2, pp. 223–230, 2014.
View at: Publisher Site | Google Scholar
Y. Li, H. Xu, L. Chen, and L. Tan, “A simple and sensitive UHPLC-MS/MS method for quantification of buddlejasaponin IV in rat plasma and its application to a pharmacokinetic study,” Journal of Pharmaceutical and Biomedical Analysis, vol. 120, pp. 374–382, 2016.
View at: Publisher Site | Google Scholar
S. Li, W. H. Pei, and H. Zhang, “Simultaneous determination of eight bioactive components in Marsdenia tenacissima extract in rat plasma by LC-MS/MS and its application in a pharmacokinetic study,” Biomedical Chromatography: Biomedical Chromatography, vol. 34, no. 11, Article ID e4946, 2020.
View at: Publisher Site | Google Scholar
T. Bach and G. An, “Importance of utilizing natural isotopologue transitions in expanding the linear dynamic range of LC-MS/MS assay for small-molecule pharmacokinetic sample analysis A mini-review,” Journal of Pharmaceutical Sciences, vol. 111, no. 5, pp. 1245–1249, 2022.
View at: Publisher Site | Google Scholar
Y. Wang, L. Zhang, S. Gu et al., “The current application of LC-MS/MS in pharmacokinetics of traditional Chinese Medicines (recent three years): a systematic review,” Current Drug Metabolism, vol. 21, no. 12, pp. 969–978, 2020.
View at: Publisher Site | Google Scholar
L. Nahar, A. Onder, and S. D. Sarker, “A review on the recent advances in HPLC, UHPLC and UPLC analyses of naturally occurring cannabinoids (2010-2019),” Phytochemical Analysis, vol. 31, no. 4, pp. 413–457, 2020.
View at: Publisher Site | Google Scholar
F. Blum, “High performance liquid chromatography,” British Journal of Hospital Medicine, vol. 75, no. 2, pp. C18–C21, 2014.
View at: Publisher Site | Google Scholar
D. Cibotaru, M. N. Celestin, M. P. Kane, and F. M. Musteata, “Comparison of liquid-liquid extraction, microextraction and ultrafiltration for measuring free concentrations of testosterone and phenytoin,” Bioanalysis, vol. 14, no. 4, pp. 195–204, 2022.
View at: Publisher Site | Google Scholar
J. Karasiński, A. Tupys, L. Halicz, and E. Bulska, “A novel approach for the determination of the Ge isotope ratio using liquid-liquid extraction and hydride generation by multicollector inductively coupled plasma mass spectrometry,” Analytical Chemistry, vol. 93, no. 40, pp. 13548–13554, 2021.
View at: Publisher Site | Google Scholar
M. Pavan, P. Yamamoto, R. Moreira da Silva et al., “Chemometric optimization of salting-out assisted liquid-liquid extraction (SALLE) combined with LC-MS/MS for the analysis of carvedilol enantiomers in human plasma: application to clinical pharmacokinetics,” Journal of Chromatography B, vol. 1205, Article ID 123338, 2022.
View at: Publisher Site | Google Scholar
J. Zhang, Y. Zhang, X. Liu, X. Xu, Y. Li, and T. Zhang, “Supercritical fluid chromatography tandem mass spectrometry employed with evaporation-free liquid-liquid extraction for the rapid analysis of cinnarizine in rat plasma,” Journal of Separation Science, vol. 45, no. 4, pp. 968–975, 2022.
View at: Publisher Site | Google Scholar
B. Qin, X. Yu, M. Gao et al., “Magnetic molecularly-imprinted microspheres with a core-shell structure for the extraction of catalpol from both Rehmannia glutinosa Libosch and biological samples,” Journal of Chromatography A, vol. 1705, Article ID 464183, 2023.
View at: Publisher Site | Google Scholar
N. Tryon-Tasson, D. Ryoo, P. Eor, and J. L. Anderson, “Silver-mediated separations: a comprehensive review on advancements of argentation chromatography, facilitated transport membranes, and solid-phase extraction techniques and their applications,” Journal of Chromatography A, vol. 1705, Article ID 464133, 2023.
View at: Publisher Site | Google Scholar
L. Zhao, B. Xiang, J. Chen, X. Tan, D. Wang, and D. Chen, “Determination of Tenacissoside A in rat plasma by liquid chromatography-tandem mass spectrometry method and its application to pharmacokinetic study,” Journal of Chromatography B, vol. 877, no. 20-21, pp. 1799–1804, 2009.
View at: Publisher Site | Google Scholar

Copyright

Copyright © 2023 Fan Chen et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

PDF Download Citation

Download other formats

Order printed copies

Views

131

Downloads

209

Citations