Quantitative Determination of the Dual COX-2/5-LOX Inhibitor Tryptanthrin in Isatis tinctoria by ESI-LC-MS

• Abstract
• Introduction
• Materials and Methods
• Plant material
• Solvents and chemicals
• Equipment
• Extraction
• LC-MS
• Identification and peak purity
• Calibration curve
• Reproducibility of the integration and limit of quantitative analysis
• Quantitative analysis of woad extracts
• Validation procedures
• Results and Discussion
• Acknowledgements
• References

Abstract

Isatis tinctoria L. is an old European and Chinese dye plant and anti-inflammatory herb from which the potent cyclooxygenase-2 and 5-lipoxygenase inhibitor tryptanthrin (1) (indolo-[2,1-b]-quinazoline-6,12-dione) was recently isolated as one of the active principles. An HPLC method for the quantitative analysis of the compound in plant material was developed. Reproducible extraction was achieved by accelerated solvent extraction (ASE). Detection by UV at 254 and 387 nm and by electrospray-MS were compared. The low tryptanthrin content in the herb and possible interferences required isocratic high-performance liquid chromatography coupled with electrospray-MS in single ion mode. More than 70 Isatis samples of different origin were analyzed. The tryptanthrin content in leaf samples varied from 0.56 to 16.74 × 10^-3 %.

Introduction

Isatis tinctoria L. (woad, family Brassicaceae) is a biennial herbaceous plant distributed in Europe, Asia and Northern Africa. The plant has an extensive and well documented history in European and Chinese Medicine as an anti-inflammatory [1], [2]. We recently reported on the in vitro pharmacological profile against 16 inflammation related targets [3] [4] [5]. Among others, a pronounced COX-2 inhibitory activity in cell-based assays was observed. Subsequently, the minor indoloquinazoline alkaloid tryptanthrin (1) was identified as the COX-2 inhibitory principle in woad. The compound strongly inhibited COX-2 in cellular assays (IC₅₀ 64 nM in Mono Mac 6 cells) [4], [5]. In recent in vitro-pharmacological investigations tryptanthrin (1) was characterized as a dual COX-2/5-LOX inhibitor of an unprecedented structural class [6] and as an inhibitor of nitric oxide synthase expression [7].

Quantitative determination of active principles is a prerequisite for plant selection, breeding, optimization of post-harvest handling and extraction, in-process and stability controls of intermediate and finished products [8] [9] [10]. In view of a possible development of woad towards a rational phytopharmaceutical, a method for quantitative analysis of tryptanthrin as one of the active principles in plant material was thus required.[]

Materials and Methods

Plant material

Dried leaves of cultured Isatis tinctoria L. used for method development were obtained from Dr. A. Plescher, Pharmaplant GmbH, Artern (Germany) in July 1997 (sample ISAR97), and from Dr. B. Weinreich, Adalbert-Raps-Zentrum, Technical University München-Weihenstephan (Germany) (sample ISW). Voucher specimens (no. IT-1 and IT-2) are preserved at the Lehrstuhl für Pharmazeutische Biologie, Friedrich-Schiller-University Jena.

Plant materials for the screening were provided by the Adalbert-Raps-Zentrum TU München-Weihenstephan, Pharmaplant GmbH, Artern, Thüringer Landesanstalt für Landwirtschaft, Dornburg (Germany), and Vitaplant, Witterswil (Switzerland). All samples were dried immediately after harvest at ambient temperature or by forced air drying at 30 - 40 °C. Additional samples were collected from uncultivated sites in Thuringia and from Morocco. Samples of Isatidis folium (Daqingye) and radix (Banlangen), and Indigo naturalis (Qingdai) were purchased in Beijing from a Herbal Pharmacy store. Identity of these drugs was confirmed by macroscopic and microscopic analysis according to Hurry [1] and the Chinese Pharmacopoeia [2].

The plant material was stored in brown glass jars. Prior to extraction, the material was powdered with a M20 mill (IKA Labortechnik, Staufen, Germany) and passed through a sieve (200 μm).

Solvents and chemicals

HPLC grade acetonitrile and methanol, and analytical grade glacial acetic acid were purchased from Roth, Karlsruhe (Germany). HPLC grade water was obtained by a Sersdest SD 2800 (Seral, Ransbach-Baumbach, Germany) water purification system. Tryptanthrin (purity ≥ 99 %) was purchased from Sigma-Aldrich (Deisenhofen, Germany). Identity and purity of the compound was confirmed by HPLC, ESI-MS, ¹H- and ¹³C-NMR, in comparison with literature data [11].

Equipment

For accelerated solvent extraction (ASE), a Dionex ASE 200 instrument (Dionex Sunnyvale, CA, USA) coupled with a solvent controller for ASE 200 was used. Extraction was carried out in 11 ml steel cartridges. The HPLC instrumentation consisted of a HP 1100 system (Hewlett Packard, Waldbronn, Germany) with degasser, binary high-pressure mixing pump, autosampler, column oven, diode array detector (DAD) and a HP Chem Station. The HPLC was coupled on-line with an API 165 single quadrupole mass spectrometer equipped with a turbo-ionspray interface (PE Sciex, Toronto, Kanada). PE Sciex Analyst software was used for system control and data evaluation.

Extraction

Extraction under reflux was carried out as follows : the powdered drug (0.5 g) was extracted 3 times with 150 ml methanol (70 % v/v) for 1 h. After filtration, the extracts were evaporated to dryness in vacuo, and the dry residues kept in a freezer at -32 °C until analysis. ASE was carried out with powdered drug (0.5 g) under the following conditions: solvent : methanol, temperature: 60 °C, pressure: 120 bar, extraction cycles of 5 min, followed by a rinsing step. The solvent consumption of an extraction/flush cycle was 23 ml. The combined extracts were evaporated to dryness and kept in a freezer at -32 °C under argon until analysis. Stability : The dry extracts were stable for up to 14 days.

LC-MS

The HPLC separation was carried out under the following conditions: LiChrospher 100 RP18 cartridge (5 μm, 150 × 4 mm I.D.) (Merck, Darmstadt, Germany), 40 % MeCN (containing 2 % acetic acid) over 10 min, flow rate: 1 ml min^-1, temperature: 25 °C, DAD detection: 254 and 387 nm, injection volume: 20 μl. For analysis of crude extracts, a rinsing step (40 % to 95 % MeCN over 6 min, 95 % MeCN for 4 min) was added at the end of the isocratic run of 9 min.

ESI-MS conditions for quantitative analysis were optimized for maximum sensitivity and were as follows: split: 1 : 4, positive ion mode, temperature: 350 °C, ion spray voltage: 4.8 kV, declustering potential: 50 V, focussing potential: 220 V, entrance potential: -10 V, detection for quantitative analysis in single ion mode (SIM): m/z 248.5 - 249.5. Full scan ESI-MS for identification of tryptanthrin peak and purity check were obtained under the following conditions: ion spray voltage: 5 kV, declustering potential: 20 V, focussing potential: 230 V, entrance potential: -10 V, scan range: 150 - 1000 amu. Other parameters were as for SIM measurements.

Identification and peak purity

Tryptanthrin (1) was identified in the extract by LC-DAD-MS and by comparison of peak retention time (7.5 min) in the extracts and in the reference. The DAD and ESI-MS spectral data of 1 recorded in the extract were comparable to those of the reference compound and in accord with literature [11]. Peak purity check was by DAD with the aid of the HP Chem Station routine, and by comparison of the ESI-MS recorded on-line at the slopes and in the apex.

Calibration curve

A stock solution of 2.50 mg tryptanthrin in 250 ml methanol was used. This solution was found to be stable at room temperature for at least four weeks. Geometric dilutions of 0.05 to 10 μg ml^-1, corresponding to 1 - 200 ng per injection, were prepared for recording the calibration curve. For each data point in the calibration curves, 6 injections were carried out and separately integrated. For the range of 1 - 50 ng, a linear function y = ax + b could be applied to the calibration curve (r ≥ 0.999). Extending the range up to 200 ng, the curve fitted a quadratic function of the type y = ax² + bx + c (r ≥ 0.999).

Reproducibility of the integration and limit of quantitative analysis

The reproducibility of the integration procedure was determined with the references used for the calibration curve by calculation of the relative standard deviation (R.S.D.) for MS and DAD at 254 and 387 nm (n = 6). The data are summarized in Table [1].

The limit for quantitative analysis (signal/noise ratio ≥ 10 : 1) was determined as 1 ng per injection (see Fig. [3] B).

Quantitative analysis of woad extracts

The dry extracts were redissolved in 5 ml methanol and transferred to a 10 ml volumetric flask. Quantitative recovery was achieved by rinsing of the storage vessel with chloroform (1.0 ml) and twice with methanol (1.5 ml each), followed by adjustment of the volume to 10.0 ml. An aliquot (1 ml) of the solution was passed through a Minisart SRP 4 filter (0.45 μm pore size) (Sartorius, Göttingen, Germany) into an autosample vial and immediately analysed. The tryptanthrin content was determined by the external standard method, via integration of the peak area and the calibration curve equation calculated for the tryptanthrin reference. The recovery of tryptanthrin from the plant material was checked by repeated extraction (see Extraction) and subsequent analysis of the extracts obtained. With the ASE procedure, which was ultimately used for all plant samples listed in Table [2], the first extraction cycle yielded > 93 % of total tryptanthrin, the second to fourth cycles > 4 %, < 2 %, and < 1 %, respectively (see Fig. [4]).

Validation procedures

Tryptanthrin recovery from plant samples was determined by repeated ASE as described above. Repeatability was determined by one worker. Extractions were carried out on three different days, as well as the LC-MS determinations. Robustness testing: HPLC solvents from different manufacturerers, HPLC columns from different batches of same manufacturer had no influence on result. MS signal stability was checked daily with aid of a tryptanthrin reference solution.

Results and Discussion

Due to the low tryptanthrin content and the poor solubility of the compound, major attention had to focus on an extraction method of high recovery and reproducibility, and on a selective and sensitive method for quantitation. Also, some validation of the method according to pharmaceutical guidelines was required [12] [13] [14].

Whereas gradient HPLC was the method of choice for activity directed identification of 1 [4], isocratic separation was given preference for the quantitative analysis for reasons of reproducibility, simplicity and speed. The presence of highly lipophilic substances in the extract, however, required column rinsing after elution of tryptanthrin (see Materials and Methods). Typical HPLC chromatograms of a extract recorded at 254 and 387 nm, the absorption maxima of tryptanthrin, are shown in Fig. [1]. Tryptanthrin elutes at 7.50 ± 0.02 min, whereas the preceeding peak at 6.0 min, on the basis of its characteristic DAD and ESI-MS, was characterized as a flavonoid glycoside. Despite the high concentration of the extract (approx. 5 mg ml^-1), the peak intensity and area at 254 and 387 nm were too low for reliable integration. Considering the versatility, selectivity and sensitivity required in possible future applications of the method to be developed, MS detection appeared as a prefereable option over DAD, despite inherent drawbacks such as higher operating cost and lower long-term stability.

The ionization of an analyte in ESI-MS is governed by several factors, such as eluent, addition of modifiers and their concentration, and instrument parameters such as electrospray voltage, vaporizer temperature in ionspray interfaces, and orifice voltage. Depending on the conditions selected, the relative responses for the quasimolecular ion may vary by more than an order of magnitude and significant differences with respect to fragmentation may occur [15], [16]. Therefore, ESI-MS conditions were optimized with respect to maximum intensity of the quasimolecular ion. The ESI-MS of tryptanthrin recorded in the positive ion mode is shown in Fig. [2]. Besides a strong quasimolecular ion at m/z 249 [M+H]⁺, only a fragment ion at m/z 221 (elimination of CO) can be observed. Small changes of the electrospray and orifice voltages did not affect the ratio of the two ions, and hence the peak area of the [M+H]⁺ ion was selected for quantitative determination in the single ion mode (SIM). Fig. [3] A shows the LC-MS chromatogram corresponding to Fig. [1]. To minimize interface contamination for optimal stability of MS detection, the column effluent was directed to the interface only during the critical time window of 6.5 - 9 min. The limit for quantitative analysis was 1 ng. The LC-MS run of a corresponding injection is shown in Fig. [3B]. Calibration curves were subsequently recorded for the range of 1 - 200 ng tryptanthrin. In the range of 1 - 50 ng, a linear function can be applied (r = 0.9992). For the extended range 1 - 200 ng, the curve is best described by a quadratic function (r = 0.9998). Analysis of samples ISAR97 and ISW as well as other Isatis leaf and root samples (see Table [3]) showed that the tryptanthrin content was in the lower range of the calibration curve. Hence a linear function was used for calculation. The relative standard deviations (R.S.D.) for ESI-MS and DAD detection at 254 and 387 nm were calculated (Table [1]). In general, the R.S.D.s for MS detection were higher than those for DAD. In the range of 20 - 200 ng, DAD at 254 nm was clearly superior in that respect. In the range 1 - 10 ng, however, the higher sensitivity of MS provided a lower detection limit and comparable to lower R.S.D.s. Repeatability (6 injections in a row) was acceptable. For repeated tryptanthrin injections of 5, 10 and 20 ng, the R.S.D. were 1.3, 0.6 and 1.7 %, respectively (Table [2]). DAD at the absorption maximum of tryptanthrin at 387 nm was least suitable due to poor sensitivity. Given the fact that the tryptanthrin content in most extracts was towards the lower end of the calibration range and that interfering substances appeared under the tryptanthrin peak in certain Isatis extracts, MS detection was ultimately given preference over DAD.

Initial experiments with classical extraction methods were not satisfactory. Extraction under reflux (see Materials and Methods) yielded extracts with erratic tryptanthrin content. Ultimately, accelerated solvent extraction (ASE) [17] was found to combine high tryptanthrin recovery with satisfactory reproducibility. The conditions were optimized by determination of tryptanthrin in a series of four consecutive extraction cycles of 5 min each (Fig. [4]). Yields of > 93 %, > 4 %, < 2 % and < 1 %, respectively, of total tryptanthrin were obtained. Hence, an ASE protocol with two extraction cycles and > 97 % yield was considered sufficient. Finally, the repeatability of the method was determined with the aid of the ISAR97 leaf sample. The tryptanthrin content found was between 1.22 × 10^-3 and 1.49 × 10^-3 % (mean value 1.38 × 10^-3 %), and the between-day R.S.D. was 5.57 % (n = 18).

A screening of Isatis samples was subsequently carried out with the above method, including 67 samples of dry leaves, four samples of dried roots, and one sample each of dried fruit and Indigo naturalis (Qingdai), respectively (Table [3]). Due to the geographical origin of the material ranging from Western Europe to East Asia and the number of leaf samples included in the study, these specimens were probably quite representative of the variability of the species. The tryptanthrin content in the leaves ranged from 0.56 to 16.74 × 10^-3 %, corresponding to an almost 30-fold difference. The majority of the samples were in the range of 3 to 8 × 10^-3 %. Interestingly, the highest tryptanthrin level were found in a Daqingye sample from China (sample no. 65) and two woad samples from Thuringia (samples no. 57 and 58). Compared to the leaves, the tryptanthrin yield in roots, fruits and Indigo naturalis was significantly lower.

A method for the quantitative determination of tryptanthrin, an active principle in Isatis tinctoria with potent dual COX-2/5-LOX inhibitory properties, has been developed and validated with respect to specificity, selectivity, linearity, working range, quantitation limit, extraction yield, precision and repeatability. A major obstacle in the method development, namely the erratic results obtained with extraction under reflux, could be resolved with an ASE extraction protocol. Besides acceptable recovery and reproducibility, the ASE method offered significant advantages with respect to speed, possibility for automation, and solvent economy over conventional extraction methods. Solvent consumption was 23 ml per extraction cycle including rinsing, hence 46 ml were required per analysis. These findings confirm earlier reports by us and others [18], [19] regarding the potential of ASE in medicinal plant analysis. Dosage by ESI-LC-MS has become routine in biopharmaceutical applications such as determination of drugs and metabolites in biological fluids, but has been rarely used up till now in quantitative medicinal plant analysis. The intrinsic advantages of the MS detector such as high selectivity and sensitivity are to some extent counterbalanced by its lower signal stability. Frequent calibration is a prerequisite for satisfactory reproducibility. Unlike with UV-vis detection, the use of an internal standard is not applicable due to differential ionization behaviour of analyte and a structurally differing standard. Suitably isotope-labelled analyte references could be used in principle, but are only accessible via partial or total synthesis.

The screening of over 70 Isatis samples revealed large differences with respect to the tryptanthrin content. These findings will serve as a basis for further plant selection and breeding.

Acknowledgements

The present study was supported, in part, by Zeller AG, Romanshorn (Switzerland). The HPLC equipment used in this study was purchased with a grant from the Ministry for Science, Research and Culture of the State of Thuringia (TMWFK, grant B 311 - 97 014). Thanks are due to Dr. B. Büter, Vitaplant, Witterswil, Dr. A. Plescher, Pharmaplant, Artern, Dr. A. Vetter, Thüringer Landesanstalt für Landwirtschaft, Dornburg, Dr. B. Weinreich, Adalbert-Raps-Zentrum, TU München-Weihenstephan, for the provision of plant samples. Mr. B. Benthin is kindly acknowledged for his skillful assistance and advice in the development of the ASE method.

References

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• 15 Zhou S, Hamburger M. Effects of solvent composition on molecular ion response in electrospray mass spectrometry: Investigation of the ionization processes.  Rapid Communications in Mass Spectrometry. 1995;  9 1516-21
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Prof. Dr. Matthias Hamburger

Institute of Pharmacy

Friedrich-Schiller-University Jena

Semmelweisstrasse 10

07743 Jena

Germany

Email: B7HAMA@rz.uni-jena.de

Fax: +49-3641 949842

References

• 1 Hurry J B. The woad plant and its dye. London; Oxford University Press 1930: 249-56
  Search in Google Scholar
• 2 Arzneibuch der Chinesischen M edizin. Stuttgart: Deutscher Apotheker Verlag 1999
• 3 Danz H, Stoyanova S, Hamburger M, Brattström A. Identification and isolation of the COX-2 inhibitory principle in Isatis tinctoria L. (Brassicaceae).  Archiv der Pharmazie. 2000;  333 (Suppl. 1) 11
  PubMedSearch in Google Scholar
• 4 Danz H, Stoyanova S, Wippich P, Brattström A, Hamburger M. Identification and isolation of the cyclooxygenase-2 inhibitory principle in Isatis tinctoria .  Planta Medica. 2001;  67 411-6
  Thieme ConnectPubMedSearch in Google Scholar
• 5 Danz H. Untersuchungen zur antiinflammatorischen Wirkung und zur Analytik von Tryptanthrin in Isatis tinctoria L. Ph. D. Thesis. University of Jena 2001
  Search in Google Scholar
• 6 Danz H, Stoyanova S, Thomet O AR, Simon H U, Dannhardt G, Ulbrich H, Hamburger M. Inhibitory activity of tryptanthrin on cyclooxygenases-1 and -2, and 5-lipoxygenase. European Journal of Pharmacology submitted
• 7 Ishihara T, Kohno K, Ushio S, Kanso I, Ikeda M, Kurimoto M. Tryptanthrin inhibits nitric oxide and prostaglandin E2 synthesis by murine macrophages.  European Journal of Pharmacology. 2000;  407 197-204
  CrossrefPubMedSearch in Google Scholar
• 8 Harnischfeger G. Qualitätskontrolle von Phytopharmaka. Stuttgart; Thieme 1985
  Search in Google Scholar
• 9 Hamburger M, Hostettmann K. Analytical aspects of drug of natural origin.  Journal of Pharmaceutical and Biomedical Analysis. 1989;  7 1337-49
  CrossrefPubMedSearch in Google Scholar
• 10 Franz C. Züchtung und Anbau von Arzneipflanzen. In: Rimpler H. editor Biogene Arzneistoffe. Stuttgart; Deutscher Apotheker Verlag 1999: 1-24
  Search in Google Scholar
• 11 George V, Koshy A S, Singh O V, Nayar M NS, Pushpangadan P. Tryptanthrin from Wrightia tinctoria .  Fitoterapia. 1996;  67 53-4
  PubMedSearch in Google Scholar
• 12 ICH . (International conference on harmonisation of technical requirements for registration of pharmaceuticals for human use), harmonised tripartite guideline: Validation of analytical methods: Definitions and terminology. London; 1994
  Search in Google Scholar
• 13 USP . 24 - NF 19, General information [1225] : Validation of compendial methods. United States Pharmacopeial Convention Inc Rockville; 2000
  Search in Google Scholar
• 14 Söfftge M. Statistische Datenauswertung und Validierung von Analysenverfahren. In : Adam KP, Becker H, editors Analytik biogener Arzneistoffe. Stuttgart; Wissenschaftliche Verlagsgesellschaft 2000: 293-318
  Search in Google Scholar
• 15 Zhou S, Hamburger M. Effects of solvent composition on molecular ion response in electrospray mass spectrometry: Investigation of the ionization processes.  Rapid Communications in Mass Spectrometry. 1995;  9 1516-21
  CrossrefPubMedSearch in Google Scholar
• 16 Zhou S, Hamburger M. Application of liquid chromatography - atmospheric pressure ionization mass spectrometry in natural product analysis: Evaluation and optimization of electrospray and heated nebulizer interfaces.  Journal of Chromatography A. 1996;  755 189-204
  CrossrefPubMedSearch in Google Scholar
• 17 Richter B E, Jones B A, Ezzell J L, Porter N L, Avdalovic N, Pohl C. Accelerated solvent extraction: A technique for sample preparation.  Analytical Chemistry. 1996;  68 1033-9
  CrossrefPubMedSearch in Google Scholar
• 18 Benthin B, Danz H, Hamburger M. Pressurized liquid extraction of medicinal plants.  Journal of Chromatography A. 1999;  837 211-9
  CrossrefPubMedSearch in Google Scholar
• 19 Kawamura F, Kikuchi Y, Ohira T, Yatagai M. Accelerated solvent extraction of paclitaxel and related compounds from the bark of Taxus cuspidata .  Journal of Natural Products. 1999;  62 244-7
  CrossrefPubMedSearch in Google Scholar

Prof. Dr. Matthias Hamburger

Institute of Pharmacy

Friedrich-Schiller-University Jena

Semmelweisstrasse 10

07743 Jena

Germany

Email: B7HAMA@rz.uni-jena.de

Fax: +49-3641 949842