It has been presented qualitative and quantitative chromatographic analysis of hypericin containing  extracts of St. John's Wort which could be used for producing drugs for treating of oncological diseases  by the method of photodynamic therapy. The optimal conditions of carrying out of TLC and HPLC  analysis of ethanol St. John's Wort extracts have been selected. The chromatographic separation of St.  John's Wort extract has been carried out assisted by Diaion HP-20 sorbent. This separation gave the opportunity to enrich the initial extract with hypericins more than in 4 times

Kovalenko, N. A.

Leontiev, V. N.

Stasevich, O. V.

Supichenko, G. N.

It has been presented qualitative and quantitative chromatographic analysis of hypericin containing extracts of St. John's Wort which could be used for producing drugs for treating of oncological diseases by the method of photodynamic therapy. The optimal conditions of carrying out of TLC and HPLC analysis of ethanol St. John's Wort extracts have been selected. The chromatographic separation of St. John's Wort extract has been carried out assisted by Diaion HP-20 sorbent. This separation gave the opportunity to enrich the initial extract with hypericins more than in 4 times

Belarusian State Technological University

ISSN 1683-0377. Proceedings of BSTU. 2012. Issue 4. Chemistry, Organic Substances Technology and Biotechnology 140УДК 543.544 O. V. Stasevich, PhD (Chemistry), senior lecturer (BSTU);  V. N. Leontiev, PhD (Chemistry), assistant professor, head of department (BSTU) N. A. Kovalenko, PhD (Chemistry), assistant professor (BSTU);  G. N. Supichenko, PhD (Chemistry), assistant lecturer (BSTU) CHROMATOGRAPHIC ANALYSIS AND SEPARATION OF HYPERICIN CONTAINING EXTRACT OF ST. JOHN'S WORT  It has been presented qualitative and quantitative chromatographic analysis of hypericin containing extracts of St. John's Wort which could be used for producing drugs for treating of oncological diseases by the method of photodynamic therapy. The optimal conditions of carrying out of TLC and HPLC analysis of ethanol St. John's Wort extracts have been selected. The chromatographic separation of St. John's Wort extract has been carried out assisted by Diaion HP-20 sorbent. This separation gave the op-portunity to enrich the initial extract with hypericins more than in 4 times.   Introduction. At present, the important issue in the Republic of Belarus is separation, identifica-tion and determination of biologically active com-pounds from plants of various agro-climatic zones of Belarus. Based on obtained substances it be-came possible to create medicines, dietary supple-ments, herbal remedies. According to the influence on the organism the drugs of vegetable origin have several advantages over synthetic analogs, as they exhibit a mildly expressed therapeutic effect. Photodynamic therapy is a relatively new and promising method of cancer treatment based on the fact that tumor cells are destroyed by the ac-tion of reactive oxygen species, which are formed by a chemical reaction that is activated by light energy. For this reaction the photosensitive sub-stance (photo sensitizer) and also a source of light with a wave length corresponding to the maxi-mum absorption of the substance must be present in the target tissue [1]. As photo sensitizer can act only compounds having in their structure chromophore group of atoms that can absorb light in the visible or near-ultraviolet region of the spectrum. These com-pounds include hypericin. This substance is one of the main bioactive components of St. John's Wort and represents a condensed derivative of an-thraquinone. In addition to hypericin in St. John's Wort has been discovered the second potential photo sensitizer – pseudohypericin. Main part. The aim of this work is a qualita-tive and quantitative analysis of hypericins in St. John's Wort using chromatographic methods of analysis. Hypericins from St. John's Wort were isolated by extraction method using Soxhlet and ethanol as a solvent. The qualitative composition of the re-sulting extract was determined by TLC. For selec-tion of the optimal conditions for the TLC analysis the ethanol extract was separated in elution solvent systems: I) propanol-2 – hexane (7 : 3); II) 2-pro-panol – water (9 : 1); III) acetate – n-butanol – formic acid – water (5 : 3 : 1 : 1); IV) chloroform – ethyl alcohol (8 : 2); V) ethyl acetate – formic acid (50 : 6).  The substances were detected by viewing the chromatogram under UV lamp with optical filters with a wave length of 254 and 365 nm. Plates were also developed in iodine vapor. In the course of analysis of the chromatograms obtained in the five proposed eluting systems, it was found that the clearest separation of the components of ethanol extract was observed when using the eluting sys-tem V. Typical thin-layer chromatogram of the test extract obtained by using eluting system V, is pre-sented in Fig. 1.  Hypericin (Fig. 1, position 7) in the chroma-togram was identified by bright red fluorescence and the value Rf = 0.87, which coincides with the value of Rf standard hypericin sample (Carl Roth GmbH, Germany). When using this eluent system pseudohypericin (Fig. 1, position 6) was identified in the chromatogram by bright red fluorescence (in UV light with a wavelength of 365 nm) and by index Rf = 0.83, the value of which corresponds with the Rf, given in the references [2].   1 2 3 4 5 6 7 8  Fig. 1. Typical thin-layer  chromatogram of the St. John's Wort  extract in eluting system V  Biotechnology 141Thus, the analysis of hypericin containing ex-tracts by TLC method revealed that the tested ex-tracts consist of eight major components. Rf indica-tors for all components of ethanol extract are shown in Table 1. The results are presented as arithmetic average of three concurrent experiments with taking into account standard deviation.  Table 1 Rf values for components of hypericin  containing extract Substance Rf 8 0.97 ± 0.01 7 (hypericin) 0.87 ± 0.02 6 (pseudohypericin) 0.83 ± 0.02 5 0.65 ± 0.02 4 0.42 ± 0.02 3 0.34 ± 0.02 2 0.12 ± 0.02 1 0.020 ± 0.006  The quantitative content of hypericin in the ex-tract was determined by HPLC method when using GC-MS “Waters Micro-mass ZQ 2000”, and the column BDS HYPERSIL C18 250×4.6 mm. De-tection was performed by diode matrix detector with the wave length of 590 nm and mass detector with electrosprayionization. Elution was performed in a linear gradient us-ing a system consisting of acetonitrile (solution A) and 0,01 M water ammonium acetate solution (so-lution B) (A : B: 0 min – 15 : 85, 0–5 min – 30 : 70; 5–10 min – 45 : 55, 10–15 min – 60 : 40, 15– 20 min – 75 : 25, 20–40 min – 90 : 10) with a flow rate of 1 ml/min. Hypericin in the extracts was identified in the chromatogram (Fig. 2, a) according to the retention time 35.95 min, which coincided with the retention time of the standard hypericin sample. In the mass spectra of this compound signals were observed corresponding to the molecular hy-pericin ions with m / z = 503,66 for negative ions and with m / z = 505.64 for positive ions (Fig. 2, b). Pseudohypericin in the extracts was identified by the mass spectrum of negative ions, where there was a signal with m / z = 519.63, corresponding to the ion [M–H]– for connection with the molecular formula C30H16O9. In the field of positive ions for pseudohypericin was also observed a molecular ion [M + H]+ with m / z = 521.61. It was found that, under given conditions of the HPLC analysis pseu-dohypericin had a shorter retention time than hy-pericin – 28.7 min. Electronic pseudohypericin spectrum was simi-lar to hypericin spectrum, where there was a band with an absorption maximum of 590 nm, which is typical of condensed derivatives of antrohinon. Quantitative determination of hypericin in St. John's Wort was performed by the calibration curve constructed according to solutions of the standard hypericin sample of (equation of line  y = 328 775x – 278 5034, R2 = 0.962068). To get hypericin enriched fraction was con-ducted preparative separation of St John's Wort extract by chromatographic method. The separa-tion of the extract was carried out on the sorbent Diaion HP-20, elution was performed with applica-tion of aqueous ethanol in increasing concentra-tions (0, 20, 40, 60, 80 and 100%) and a mixture of ethanol and chloroform (1 : 1).    а   b Fig. 2. HPLC chromatogram of St John's Wort extract (a), the mass spectrum of hypericin in the field of positive ions (b)        5.00      10.00      15.00      20.00     25.00     30.00     35.0    0 40.00    45.00 m / z100  %   –1 Pseudohypericin Hypericin 22.68 202.4 15.11 202.4 5.18 202.4 4.85 202.4 3.50 201.4 43.86 200.4 35.95 200.4 28.70 205.4 100   %   0   440   460   480   500    520   540   560   580   600  620  640   660   680  700  720  m / z 505.64 448.63 468.23 475.95 506.96 505.04 508.41 551.96680.35 681.27 728.71722.18662.14 654.55618.07611.40578.08592.20529.65542.98ISSN 1683-0377. Proceedings of BSTU. 2012. Issue 4. Chemistry, Organic Substances Technology and Biotechnology 142Fractions obtained after separation were ana-lyzed on a spectrophotometer resulting in the elu-tion profile as shown in Fig. 3.   Fig. 3. Elution profile on the sorbent Diaion HP-20   As a result of the TLC analysis of fractions corresponding to peaks in the profile, it was found out that hypericin containing fraction eluted from the column with 80% aqueous ethanol in volume of 304–356 ml (fr. 1), and assisted with a mixture of ethanol and chloroform eluted in volume of 540–564 ml (fr. 2). The results of the HPLC analy-sis of obtained fractions are shown in Table 2. As shown in table 2, the separation of St John's Wort on the sorbent Diaion HP-20 provides an op-portunity to enrich the original extract by hy-pericins more than 4 times. This confirms the fea-sibility of separation of hypericin containing ex-tract on this sorbent.  Table 2 Results of separation of the hypericin  containing extract on the sorbent Diaion HP-20 Fraction Hypericin content in original extract, %  Fraction out-put towards the inserted extract, % Hypericin content in the frac-tion, %  1 9.98 0.34 2 0.081 14.20 0.13  However, the purity of the fractions obtained by chromatographic separation of the ethanol hy-pericin extract is not fully satisfactory. Therefore, for further use of hypericins as photosensitizer in photodynamic therapy, more research is needed regarding the cleaning and enriching of hypericin containing extracts. Conclusion. Optimal conditions for HPLC and TLC analyses have been selected. It has been con-ducted chromatographic separation of St John's Wort on the sorbent Diaion HP-20, which made it possible to enrich the original extract by hypericins in more than 4 times. References 1. Karioti, A. Hypericins as potential leads for new therapeutics / A. Karioti, A. R. Bilia // Int. J. Mol. Sci. – 2010. – Vol. 11. – P. 562–594. 2. Китанов, Г. М. Фитохимическое изучение и анализ видов Hypericum L., произрастающих в Болгарии / Г. М. Китанов // Растительные ре-сурсы. – 1988. – Т. 241. – С. 114–121. Recieved 19.03.2012fr. 1 fr. 2 0      100     200    300     400    500     600    700Volume, ml Optical density under 590 nm 0,50,40,30,20,10,0Hypericin containing fractions

Chromatographic analysis and separation of hypericin containing extract of St. john's wort

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Chromatographic analysis and separation of hypericin containing extract of St. john's wort

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