Zhou and Qu,. Afr J Tradit Complement Altern Med. (2011) 8(S):33-45 33 ANALYSIS OF THE EXTRACTS OF ISATIS TINCTORIA BY NEW ANALYTICAL APPROACHES OF HPLC, MS AND NMR

The methods of extraction, separation and analysis of alkaloids and indole glucosinolates (GLs) of Isatis tinctoria were reviewed. Different analytical approaches such as High-pressure Liquid Chromatography (HPLC), Liquid Chromatography with Electrospray Ionization Mass Spectrometry (LC/ESI/MS), Electrospray Ionization Time-Of-Flight Mass Spectrometry (ESI-TOF-MS), and Nuclear Magnetic Resonance (NMR) were used to validate and identity of these constituents. These methods provide rapid separation, identification and quantitative measurements of alkaloids and GLs of Isatis tinctoria. By connection with different detectors to HPLC such as PDA, ELSD, ESIand APCI-MS in positive and negative ion modes, complicated compounds could be detected with at least two independent detection modes. The molecular formula can be derived in a second step of ESI-TOF-MS data. But for some constituents, UV and MS cannot provide sufficient structure identification. After peak purification, NMR by semi-preparative HPLC can be used as a complementary method.


Introduction
Isatis tinctoria is widely used as anti-inflammatory and dye medicinal plant in Europe and China.It has been used and cultivated in Europe and China since antiquity.Over the last decade, more than 65 compounds of Isatis tinctoria including glycosides, alkaloids (Wu et al, 1997;Liu et al, 2001a,b;Liu et al, 2003), organic acids (Wang et al, 2009), and sucrose (Liu et al, 2002a,b) were identified.Isatis tinctoria contains indolic compounds.Among these compounds, glucobrassicin and its derivatives have antitumoral effect, especially against mammary cancer (Hoessel et al, 1999;Xu et al, 1991;Lin et al, 2002;Thornalley 2002;Galletti et al, 2006).
Different analytical approaches such as High-Pressure Liquid Chromatography (HPLC) (Qin et

Alkaloids
Indigo is one of the oldest natural blue dye (Uzal et al, 2010).Indole is a product of tryptophan catabolism by gut bacteria and is absorbed into the human body in substantial amounts (Gillam et al, 2000).Indole is absorbed and metabolized within the liver to indican (indol-3-ylsulfate) (Fordtran et al, 1964;Levy 1995).Indigo and indirubin have been found in human urine.Indigo and indirubin are structural isomers, which have physiological effects on liver microsomes in mice (Sugihara et al, 2004).Tryptanthrin, is an indoloquinazoline alkaloid that strongly inhibits cycloxygenase-2 (COX-2)

Extraction and separation
Freshly harvested materials of Isatis indigotica were cut into small pieces of 2-3 cm length and immediately shock frozen with liquid nitrogen.Prior to extraction, the leaves were dried at 40 °C.Temperature, relative humidity and weight loss was monitored during the drying process.Constant weight was achieved after 3-4 days.The dried leaf samples were stored at room temperature for a few days in brown glass bottles in the dark.Immediately before extraction, dried leaf material was grounded.1.0 g frozen and powdered samples was extracted by Pressurised Liquid Extraction (PLE) with an ASE 200 instrument (Liau et al, 2007).Bagci and Özçelik (2009) investigated fatty acid in the seeds of Isatis tinctoria using GC and HPLC.

Sample preparation of Mass Spectrometry
Standard solutions (0.1 mg/ml) of indigo, isoindigo, indirubin, isatin, indican, indoxyl acetate and 2-indolinone were prepared by dissolving 5 mg of each powder in 50 ml of DMSO.The solutions were filtered over a 0.45 μm syringe filter.The solution of the mixture of active agents was prepared daily by dilution of appropriate volumes of the standard solutions with acetonitrile.

New analytical approaches
The methods used to identify indigos and its isomers include spectrophotometer in the UV, visible and IR regions (Cordy and Yeh, 1984;Miliani et al, 1998   The dichloromethane extracts of Isatis tinctoria are lipophilic with active pharmacological effect (Oberthür, 2003).Mohn   tools of this herbal medicine.HPLC separates complicated compounds.By connection with different detectors to HPLC such as PDA, ELSD, ESI-and APCI-MS in positive and negative ion modes, complicated compounds could be detected with at least two independent detection modes.The molecular formula can be derived in a second step of ESI-TOF-MS data.
But for some constituents, UV and MS cannot provide sufficient structure identification.After peak purification, NMR by semi-preparative HPLC can be used as a complementary method.Selected parameters of these analytical methods are summarized in this article.This review may be useful to guide the analysis of the extracts of Isatis tinctoria in the future.
al, 2001; Tu et al, 2009), Liquid Chromatography with Electrospray Ionization Mass Spectrometry (LC/ESI/MS), Electrospray Ionization Time-Of-Flight Mass Spectrometry (ESI-TOF-MS), and Nuclear Magnetic Resonance (NMR) were used for analysis of the compounds in Isatis tinctoria.These methods provide rapid separation, identification and quantitative measurements of glucosinolates and alkaloids from Isatis tinctoria.The methods of extraction, preparation and analysis of qlucosinolates and doi: 10.4314/ajtcam.v8i5S.13Zhou and Qu,.Afr J Tradit Complement Altern Med.(2011) 8(S):33-45 34 alkaloids of Isatis tinctoria were reviewed in this paper.

Figure 1 :
Figure 1: Relationship of tryptophan, indigo, indirubin and indican Mohn et al (2007a) isolated GLs from Isatis tinctoria seeds by Soxhlet-extraction for 8 h with 400mL petrol ether (boiling range 40-60 °C).After evaporation to dryness, the residue was extracted three times with water (room temperature, 3×150 mL), centrifuged (5 min, 1600×g, room temperature) and filtered.The aqueous solutions were concentrated to 45mL in vacuo, and 5mL acetonitrile were added.After centrifugation, the supernatant was introduced into a column packed with DEAE-Sephadex A-25 (50 g).The column was eluted with water/acetonitrile 80:20 until the eluate was colorless.GLs were eluted with a mixture of 0.1M K 2 SO 4 /acetonitrile 80:20 at 3.2 mL/min and monitored with UV detection at 229 nm.Fractions were analyzed by HPLC-MS.Finally, isolated GLs were freeze dried.Purity and structures of isolated compounds were confirmed by NMR and LC-MS experiments.New analytical approachesIn the recent years, new analytical approaches such as LC-based on-line spectroscopy, MS, NMR opened new avenues for increasingly comprehensive analysis of plant extracts and secondary metabolites.Researches by LC-based on-line spectroscopy has been widely taken on metabolite profiling studies(Burns et  al, 2003; Yamazaki et al, 2003; Le Gall et al, 2005; Long et al, 2006; Dan et al, 2008; Ding et al, 2008; Iijima et al, 2008; Qiao et al, 2008; Schliemann et al, 2008).Previous reports on the indigo precursors and GLs showed harvest regimen affected the chemical composition of Isatis tinctoria leaves (Oberthür et al, 2004b; Mohn and Hamburger, 2008).Mohn et al (2008) carried out a quantitative assay for the direct analysis of intact GLs in Isatis tinctoria leaves.In their research, pressurized liquid extraction (PLE) was used.Detection was carried out by ESI/MS in the negative ion mode.Mohn and Hamburger (2008) used atmospheric pressure chemical ionization (APCI) and ESI/MS, and ESI-TOF-MS detectors to detect the extracts of Isatis tinctoria.

Data processing was with Topspin 2 \
Isatis tinctoria has been cultivated throughout Europe, especially in Western and southern Europe, since ancient times.In China, Isatis tinctoria has been used thousands of years as an important and popular herbal medicine in Traditional Chinese Medicine (TCM) for the treatment of inflammatory diseases.This paper reviewed the extraction methods of hydrophilic compounds (alkaloids) and lipophilic compounds (indole glucosinolates) of Isatis tinctoria.Multiple analytical approaches including HPLC, LC/ESI/MS, ESI-TOF-MS, and NMR have been reported as validation and quality control doi: 10.4314/ajtcam.v8i5S.13

Table 2 :
The parameters of tryptanthrin, indigo, and indirubin by HPLC Previous reports reveal that LC/MS does not require sample pretreatment and allows for the detection of tryptanthrin, indigo, and indirubin at the same time.ESI/MS with high sensitivity, offers detection limits in the range 0.03-5.00μg/ml for these compounds examined.Some selected parameters of these methods used to analyze alkaloid in Isatis tinctoria are presented in 2 Aobchey et al, 2007 Reversed-phase LC/ESI/MS and UV/visible spectrophotometic methods were elaborated for the identification of indigoid (indigo, indirubin, isoindigo, isoindirubin) color components of natural dye stuffs and their natural or synthetic precursors (indican, isatin, indoxyl, 2-indolinone) (Puchalska et al, 2004).Gillam et al (2000) reported the definitive identification of indigo and indirubin as products of human cytochrome P450 (P450)-catalyzed metabolism of indole by visible, 1 H NMR and MS.Mohn et al (2009) discovered a new indolic alkaloid from Isatis tinctoria by combined several detectors such as photodiode array detector (PDA), ESI/MS, and APCI-MS (positive and negative ion modes).Structural information was obtained by unspecific evaporative light scattering detector (ELSD)/MS/MS (Xiao et al, 2007) experiments and by high-resolution mass spectra recorded by ESI-TOF-MS.Different analytic methods have been used to identity and determine the compounds in Isatis tinctoria.

Table 3 :
The parameters of tryptanthrin, indigo, and indirubin analyzed by MS

Table 4 :
The results of indigoids by HPLC/ESI/MS 60echard et al, 2001).Some reports demonstrated that qlucosinolates is very sensitive to temperature.60-70°C have been used in validated PLE methods for many classes of natural products such as isatan A and B (Benthin et al., 1999; Oberth¨ur et al, 2003; Oberth¨ur et al, 2004a, b; Basalo et al, 2006).For the extraction and isolation of GLs of Isatis tinctoria, existing methods (Kushad et al, 1999; Kiddle et al, 2001; Verkerk et al, 2001; Bennett et al, 2007) has a problem with the extent of thermal degradation at temperature above 50 °C with.Extractions were typically carried out at temperatures between 70 °C and 100 °C.Thermal degradation of GLs during cooking has been reported (Slominski and Campbell, 1989; Oerlemans et al, 2005; Bones and Rossiter,, 2006).

Table 5 :
The parameters of the dichloromethane extracts by HPLC In previous reports, trifluoroacetic acid and formic acid are often used as mobile phase for chromatography of intact GLs (Mellon et al., 2002; Bringmann et al., 2005; Song et al., 2005).The parameters in Table 6 present GLs extracts analysed by HPLC-PDA-MS and HPLC-TOF-MS.The parameters of LC/TOF/ MS methods for GLs are presented in

Table 7 :
The parameters of GLs analyzed by LC/TOF/ MS

Table 8 :
The parameters of GLs analyzed by NMR