Quality Control of Hypericum perforatum L. Analytical challenges and recent progress Anastasia Agapouda,1 Anthony Booker,1,2 Tivadar Kiss,3,4 Judit Hohmann,3,4 Michael Heinrich1,* and Dezső Csupor3,4,* 1 Research Cluster “Biodiversity and Medicines”, Research Group “Pharmacognosy and Phytotherapy”, UCL School of Pharmacy, University of London, 23-39 Brunswick Sq, London WC1N 1AX, UK 2 Division of Herbal and East Asian Medicine, Department of Life Sciences, University of Westminster, 115 New Cavendish St, London W1W 6UW, UK 3 Department of Pharmacognosy, University of Szeged, Eötvös u. 6., H-6720 Szeged, Hungary 4 Interdisciplinary Centre of Natural Compounds, University of Szeged, Eötvös u. 6., H-6720 Szeged, Hungary * Corresponding authors M. Heinrich: Research Cluster “Biodiversity and Medicines”, Research Group “Pharmacognosy and Phytotherapy”, UCL School of Pharmacy, University of London, 23-39 Brunswick Sq, London WC1N 1AX, UK E-mail: [email protected] D. Csupor: Department of Pharmacognosy, University of Szeged, Eötvös u. 6., H-6720 Szeged, Hungary E-mail: [email protected] Abstract Objectives The most widely applied qualitative and quantitative analytical methods in the quality control of Hypericum perforatum extracts will be reviewed, including routine analytical tools and most modern approaches. Key findings Biologically active components of H. perforatum are chemically diverse, therefore different chromatographic and detection methods are required for the comprehensive analysis of St. John’s wort extracts. Naphthodianthrones, phloroglucinols and flavonoids are the most widely analysed metabolites of this plant. For routine quality control, detection of major compounds belonging to these groups seem to be sufficient, however closer characterisation requires the detection of minor compounds as well. Conclusions TLC and HPTLC are basic methods in the routine analysis, whereas HPLC-DAD is the most widely applied method for quantitative analysis due to its versatility. LC-MS is gaining importance in pharmacokinetic studies due to its sensitivity. Modern approaches, such as DNA barcoding, NIRS and NMR metabolomics may offer new possibilities for the more detailed characterization of secondary metabolite profile of Hypericum perforatum extracts. Keywords Hypericum perforatum, St. John’s wort, HPLC, TLC, NMR metabolomics, DNA barcoding Introduction Hypericum perforatum L. (St. John’s wort - SJW) is one of the most important medicinal plants, being the active component of several products. In the modern medicine the aerial parts (Hyperici herba) are applied, usually as extracts. The efficacy of St. John’s wort has been studied in several clinical trials and according to the most recent Cochrane review, Hypericum products were superior to placebo in patients with major depression and similarly effective as standard antidepressants.[1] The European Medicines Agency granted a community herbal monograph for Hyperici herba extracts,[2] and there are several Hypericum-containing medicines on the market with well-established indications as antidepressants. Hypericum is marketed as food supplement in different countries of the world, typically with the intention to act on the central nervous system. The majority of products for internal application contain dry extracts; some preparations contain the oily extract of the herb, however these are intended for external use. Many analytical techniques have been established for the quality control of St. John’s wort products. The objective of this paper is to review the existing literature on phytochemical analysis of Hyperici herba and dry Hypericum extracts and to assess the validity of these methods for everyday use in the relevant industries. This extensive review gives an overview of all the methods that are used in the analysis of St. John’s wort-based products. Pharmacopoeias are the cornerstones of the quality control of medicinal products, since these determine the compounds to be analysed and also the methods to be applied in case of raw materials. The European Pharmacopoeia specifies a minimal (total) hypericin content of 0.08% for Hyperici herba,[3] however for the dry extract (Hyperici herbae extractum siccum quantificatum), the ranges of total hypericin (0.1-0.3%, expressed as hypericin), flavonoids (minimum 6%, expressed as rutoside), and hyperforin (maximum 6%) are defined.[4] The U.S. Pharmacopeia National Formulary contains three Hypericum monographs in order to regulate the quality of Hypericum-based food supplements. The St. John’s wort monograph specifies not less than 0.6% hyperforin content and not less than 0.04% combined hypericin and pseudohypericin content for the herb[5] and the powdered herb as well.[6] For the powdered St. John’s wort extract, only the acceptable deviations (90-110%) from the declared hypericin and hyperforin contents are prescribed, there are no upper or lower limits for the concentrations of these analytes.[7] The Chinese Pharmacopoeia defines a lower limit of hyperoside content (0.1%) in the herb.[8] SJW preparations are usually quantified to their content of hypericin-type compounds, which may be determined by spectrophotometric measurement[9] or for their content of hypericin-derivatives and hyperforin derivatives. Hypericin and pseudohypericin result in red solutions with organic solvents and have characteristic UV spectra with a maximum at 590 nm. One major limitation of spectrophotometric quantifications is that there is possible interference from other plant metabolites, eg. chlorophylls, that may have absorption overlapping directly with hypericin derivatives. Further, using this method only the total amount of hypericin-derivatives can be determined, the quantification of individual compounds is not possible. Therefore, UV spectrophotometry is not considered as the most appropriate tool for the quality control of SJW products and the plant material. Moreover, it has been shown that by adulterating SJW with food dyes, it is possible to mimic the UV-spectrum and produce substandard material that passes the analytical test.[10] However, the European Pharmacopoeia still prescribes UV spectrophotometry as quantitative assay for Hyperici herba.[11] Other methods, such as TLC and HPTLC may be applied primarily for qualitative analysis of SJW extracts. In recent studies (similarly to pharmacopoeia monographs of dry extracts),[4,7] HPLC-DAD is most widely applied for quantification, whereas for qualitative analysis, primarily LC-MS is used. DNA barcoding and NMR metabolomics belong to the most modern tools of instrumental analysis, which are under development for use also within pharmacopoeias. Sample preparation Sample preparation has a major impact on the reliability of analytical experiments. In the case of SJW, the polarity of the extracting solvents and light exposure are the most determinative factors, whereas pH and temperature have less impact on the recovery of analytes. Hypericin, hyperforin and their derivatives are unstable under certain conditions. Light catalysis the transformation of protoderivatives to their respective hypericins (hypericin and pseudohypericin as the main components). Hyperforin is unstable at higher temperatures and in the presence of air and in apolar solvents such as n-hexane, resulting in the formation of furohyperforin derivatives. It is more stable in protic solvents.[12] When exposed to light, hyperforin and adhyperforin in a MeOH extract solution degraded rapidly, particularly at pH 7, where within 12 h, complete transformation was observed. Interestingly, hyperforin was more stable in an acidic milieu. When protected from light, the solutions regardless of pH, underwent minimal transformation after 36 h.[13] A 5 min exposure of the crude extract of SJW to sunlight induced a 96% loss of hyperforins.[14] Hypericin and pseudohypericin show low stability to air and light. Rapid degradation of total naphthodianthrone content (only about 30% of the theoretical content) was detected after three months of storage, even if antioxidants were added to the extracts.[15] In order to simplify and increase the reliability of methods for the determination of hypericins, experiments have been carried out to assess the effect of light exposure on the transformation of protohypericins to hypericins. One method combines on-line, precolumn photochemical conversion followed by photodiode-array detection to allow convenient quantification of hypericins. A photochemical reactor was used in order to transform the light sensitive naphthodianthrones, protohypericin and protopseudohypericin, into hypericin and pseudohypericin, respectively.[16] Using a photo halogen lamp (1000 W), the plateau of the hypericin content expressed as the sum of areas of hypericin and pseudohypericin peaks was achieved after 10 min of light exposure in the liquid extracts of SJW-containing samples.[17] A HPLC method for the determination of hypericin and pseudohypericin included the use of a light reaction coil, installed between the autosampler and the analytical column to convert potentially existing protohypericin and protopseudohypericin into hypericin and pseudohypericin to make quantification more reproducible.[18] SJW contains marker compounds of different polarity, therefore sample preparation has major influence on the composition of extracts. Different extraction procedures are described in different pharmacopoeias (eg. in the European Pharmacopoeia 80% THF[11]), and quantitative data reported in the literature are obtained from experiments with samples gained by different extraction methods. Avato and Guglielmi performed a systematic study to assess the hypericin content of different SJW extracts. Soxhlet extraction was carried out with MeOH or EtOH (in the latter case, after pre-extraction with diethyl ether). Extracts with solvents of different polarity (petroleum ether, CHCl , EtOAc and 3 MeOH) were prepared by sonication. Macerate was gained with MeOH. One extract was prepared with 90% aqueous acetone under stirring and one sample was extracted with hot methanol. These experiments revealed, that extracts poor in chlorophylls and relatively rich in hypericins can be obtained by Soxhlet extraction with ethanol (after pre-extraction with diethyl ether) and with 90% aqueous acetone. Hot MeOH and Soxhlet extraction with MeOH resulted in the highest hypericin content. Soxhlet extracts contained the highest amount of hyperforin, whereas ultrasonic extracts were relatively poor in this compound. HPLC analyses of the various extracts provided useful information on the quantities of flavonoids and chlorogenic acid in the extracts. Based on these results the best extraction procedure to obtain an extract representative of all the major metabolites (hypericins, hyperforins and flavonoids) involves the use of a polar solvent such as MeOH or EtOH.[19] Milevskaya et al carried out extensive experiments to study the influence of different factors on extraction efficiency based on the quantification of 15 constituents (phenolcarboxylic acid, flavonoids, naphthodianthrones and phloroglucinols) of SJW. It was concluded that the effects of temperature and microwave radiation, as well as the combination of temperature and pressure offer the greatest degree of extraction.[20] In one experiment, extraction with hot MeOH after pre extraction with CHCl 3 (to remove chlorophyll) resulted in an extract with higher flavonoid content than that of a macerate prepared with EtOH.[21] Optimal conditions for extraction of H. perforatum samples in a water bath shaker were determined using response surface methodology. Extraction efficiency was defined by comparing either the total extractable material weight or individual component (rutin, isoquercitrin, quercitrin, quercetin and hypericin) peaks. Of the tested variables, the extraction temperature most significantly affected extraction efficiency, but high temperature also caused decomposition of hypericin. Considering all variables, optimum ranges for extraction time and extraction solvent concentration (percent ethanol in acetone) were 5.0-6.7 h and 44-74% at 23 °C, 5.4-6.9 h and 45-72% at 40 °C, and 5.3-5.9 h and 44- 69% ethanol in acetone at 55 °C, respectively.[22] In one experiment, extraction of dried plant material with MeOH in the dark, at room temperature for 2 h, led to a complete recovery of naphthodianthrones but only a partial recovery of the phloroglucinol derivatives. Extraction with water – EtOH 4:6 in a water bath shaker at 80 °C led to the total extraction of hypericins with a 90% recovery of hyperforins.[14] The optimum conditions for extraction of rutin and quercetin from H. perforatum were investigated by Biesaga et al. Aqueous methanol (40-80%) is the most efficient extracting solvent. The aglycone quercetin could be obtained from its glycosides most efficiently after 5/10 min hydrolysis wit 2.8/2.1 M HCl.[23] Pages et al used different chemometric approaches to evaluate the influence of extraction factors on the detectable amount of hypericin. An asymmetric screening design was built in order to evaluate the weight of each level for each factor – sonication duration, magnetic stirring, light exposure duration on the response, the total hypericin content. Stirring has no real impact on efficiency and there is no direct association between the sonication time and hypericin content, however, it was confirmed that light exposure catalyses the breakdown of hypericin.[24] These results point out that the light exposure, recommended in the monograph as sample pretreatment in the European Pharmacopoeia[11], does not permit reproducible quantification of the hypericin content. A comparison of sonication, Soxhlet extraction and pressurised-fluid extraction was conducted for several major constituents in SJW. It was confirmed that there is a direct link between sonication time and extraction efficiency. In case of pressurised-fluid extraction, moderate changes in pressure did not significantly affect extraction efficiency. Poor extraction efficiency was observed for the most polar analytes (e.g., chlorogenic acid and flavonoids) with acetone, methylene chloride, and hexane. Acetone was more effective for extraction of the nonpolar naphthodianthrones. The extraction efficiency, especially for non-polar components was relatively constant at 20, 60 and 100 °C, however levels for polar flavonoids were significantly reduced for extractions at 200 °C. Comparing these 3 methods, the highest recoveries of the major constituents were achieved with Soxhlet extraction.[25] Optimisation of ultrasonic-assisted extraction of H. perforatum for quercetin was carried out using the Box-Behnken design combined with response surface methodology. The effects of temperature (30- 70 °C), extraction time (20-80 min), methanol (20-80%), and HCl concentration (0.8-2.0 M) on quercetin concentration were assessed. The optimum conditions were determined as follows: 67 °C, 67 min, 77% MeOH, HCl concentration 1.2 M. The method was validated by experimental confirmation of the predicted quercetin content in the extract.[26] In case of in vivo studies, sample preparation of biological samples usually includes solvent extraction from blood plasma to enrich the analytes. For hyperforin, apolar extracting solvents, such as n-hexane- EtOAc 9:1-7:3 are used.[27] In one experiment, solid phase extraction on C8 column was carried out prior to the HPLC analysis of hypericin[28], others used Oasis HLB.[29] In a study biapigenin was extracted from biological tissues using Oasis HLB 1-cc extraction cartridges.[30] Solid phase extraction prior to HPLC is also necessary when analysing oily extracts. From SJW oil (extract prepared with fatty oil), an aminopropyl SPE cartridge may be used. Conditioning was reported sequentially with NaOH, MeOH, acetone, and heptane and rinsing with heptane, elution was carried out with 5 % oxalic acid dihydrate in acetone – MeOH 1:1.[31] As the result of miniaturisation in analytical chemistry several new liquid–liquid extraction have been developed to reduce the consumption of organic solvents and the time needed for analysis and to facilitate towards automation. In the so-called single-drop liquid-phase microextraction the organic micro droplet is placed into the aqueous sample and the analytes are extracted into the organic droplet based on passive diffusion. This method, with good extraction efficiency, was optimised for the quantification of hypericin, pseudohypericin and hyperforin from biological fluids.[32] Thin Layer Chromatography TLC is the method of preference for identification and quality control of H. perforatum (both plant and extract) by the European and the United States Pharmacopoeias. Both pharmacopoeias describe the analysis procedure of the SJW plant and extract as well as the compounds that should be seen in their fingerprint. According to the European Pharmacopoeia, both the plant material and the extract are prepared in a concentration of 50 mg/mL in methanol for TLC analysis and the standards rutin and hyperoside are prepared at concentrations of 1 mg/ml for SJW plant and 0.5 mg/ml for SJW extract. The TLC plate is developed with the mobile phase anhydrous formic acid – water – ethyl acetate (6: 9: 90 v/v/v). After the development, the plate is sprayed with solvent 1: 10 g/L diphenylboric acid aminoethyl ester in methanol and solvent 2: 50 g/L macrogol 400 in methanol and is visualised under UV light at 365 nm. The chromatogram of SJW plant should illustrate the fluorescent bands of rutin, hyperoside, hypericin and pseudohypericin while it is claimed that other bands of yellow or blue colour are visible. The chromatogram of SJW extract needs to have the yellow band of rutin, the blue zone of chlorogenic acid and the yellow band of hyperoside in the lower third of the chromatogram. In the top third of the chromatogram 2 red bands due to hypericin and pseudohypericin and one yellow band due to quercetin have to be visible, while in the middle third, three yellow bands can be seen. The pharmacopoeia states that other fluorescent bands can also be illustrated in the chromatogram of SJW extract.[33] The United States Pharmacopoeia requires that 100 mg/mL of SJW plant and 50 mg/mL of SJW extract in methanol are analysed. The development solvent proposed is ethyl acetate – glacial acetic acid – formic acid: water (10: 1.1: 1.1: 2.6 v/v/v/v) and the development distance is 18 cm. After development the plate is derivatized with 10 mg/ml solution of diphenylboric acid aminoethyl ester in methanol and 50 mg/mL solution of polyethylene glycol 400 in ethanol and visualized under UV light at 365 nm. The acceptance criteria for SJW plant is the presence of some yellowish bands on the chromatogram, one of which travels at R=0.5. The bands of hypericin (R=0.85) and pseudohypericin (R=0.8) should be f f f present while two blue bands below the yellow hyperoside band are described and correspond to chlorogenic and neochlorogenic acids. The chromatogram of SJW extract should contain the bands of rutin, hyperoside, hypericin and pseudohypericin as described above, but other bands of different colour and intensity might be present in the chromatogram. The USP Pharmacopoeia, unlike other Pharmacopoeias, describe a different solvent system for the analysis of hyperforin, hexane – ethyl acetate (4:1 v/v), while the plate is derivatized with a solution containing 0.38 g ceric ammonium sulfate and 3.8 g ammonium molybdate in 100 mL of 2N sulfuric acid and visualized under UV light (hyperforin is a blue band around R=0.54).[34] f TLC published studies have mostly focused on the identification and separation of hypericin and pseudohypericin.[35,36] However there are some TLC studies which analysed the phenolic content of Hypericum species, including the study of Jesionek et al and Males et al.[37,38] Mulinacci et al used TLC-densitometry in combination with HPLC-DAD in order to identify and quantify hypericin in SJW extracts. Hydroethanolic extracts (EtOH 80%) of SJW aerial parts were analysed and the silica gel TLC plates were developed with the solvent system toluene– ethyl acetate – formic acid (50: 40:10 v/v/v). The team used Incremental multiple development in an unsaturated horizontal chamber which means that they developed the plate twice with the same solvent in order to maximize the separation. No dipping or spraying solvents were used, while the densitometric assessment was conducted under an excitation wavelength of 313 nm. Hypericin and pseudohypericin were well separated and HPLC-DAD were used for their quantification.[35] Kitanov et al used TLC to identify, and spectrophotometry to quantify, hypericin and pseudohypericin in 36 Hypericum species.[36] The different Hypericum extracts were applied on silica gel TLC plates and the plates were developed with two mobile phases; toluene – ethyl acetate – formic acid (50: 40: 10 v/v/v), as Mulinacci et al did, and with ethyl acetate: formic acid (50:6 v/v). After development the plates were sprayed with 0.5 N KOH in ethanol and visualised under UV 366 nm. Hypericin and pseudohypericin were well separated and existed in 27 out of 36 Hypericum species. Males et al used TLC not only to separate and analyse flavonoids and phenolic acids from Croatian Hypericum species but they were also the first research team to analyse the amino acid content in those species. For the flavonoids and the phenolic acids methanolic solutions of the samples were spotted on TLC silica plates which were developed with the mobile phases ethyl acetate – formic acid – acetic acid – water (100: 11: 11: 26 v/v) and ethyl acetate – formic acid – water (8:1:1 v/v) and derivatized with NP and PEG reagents. For the separation of amino acids, aqueous solutions of the samples were spotted on cellulose TLC plates, which were developed with the mobile phases n-butanol – acetone – acetic acid – water (35: 35: 10: 20 v/v) and n-butanol – acetic acid – water (40:10 :10 v/v) and derivatized with ninhydrin reagent. UV spectrophotometry was used for quantitative analysis. Overall, 16 amino acids, 10 flavonoids and 3 phenolic acids were separated and H. perforatum subspecies were found to be the richest in these constituents. In particular, H. perforatum subsp perforatum was the richest in rutin, hyperoside and isoquercitrin as well as in tryptophan (which was not detected in the rest of the samples).[38] Jesionek et al separated and identified phenolic compounds in hydroethanolic (70% EtOH) extracts of five plants including aerial parts of SJW and they optimized the TLC conditions for better separation of those phenolic compounds. In addition TLC was hyphenated to the (in silico) DPPH assay to evaluate the antioxidant potential of the compounds. The silica gel TLC plates were developed with 7 different mobile phases and then derivatized with NP reagent and PEG reagent. The research team found that flavonoid aglycons like quercetin were better separated with the system toluene – diethyl ether – acetic acid (60:40:10 v/v/v), the flavonoid glycosides like rutin and hyperoside with the system ethyl acetate – acetic acid – formic acid – water (100: 11: 11: 26 v/v/v/v) and the phenolic acids like chlorogenic acid with the system chloroform – ethyl acetate – acetone – formic acid (40: 30: 20: 10 v/v/v/v).[37] High performance thin layer chromatography HPTLC is an improved form of thin layer chromatography, more automated and reproducible, and which provides better separation of compounds and better detection. The European Pharmacopoeia is currently updating the identification method from TLC to HPTLC on the monograph of SJW.[33] In addition, the HPTLC association recommends a well-established method for the identification of compounds in SJW while several studies have been published analysing SJW with HPTLC. The HPTLC association proposes a method for the analysis of SJW for both crude material and extract. 100 mg/mL and 50 mg/mL methanolic solutions for crude material and extract respectively are prepared as well as the standards rutin and hyperoside at a concentration of 1 mg/mL in methanol. The HPTLC silica gel plates are developed with the solvent system ethyl acetate – dichloromethane – water – formic acid – acetic acid (100: 25: 11: 10: 10 v/v/v/v/v) in a saturated chamber with the humidity set at 33%. After development, the plates are derivatized with Natural Product reagent (NP) and Polyethylene glycol 400 reagent (PEG) for detection of phenolic compounds. The yellow bands of rutin and hyperoside should be seen at R=0.1 and R=0.25 respectively, as well as the red bands of f f hypericin and pseudohypericin at R= 0.57 and R=0.63 respectively. Other yellow bands can be seen f f between hyperoside and hypericin.[39] Two HPTLC studies of SJW adulteration have been published.[10,40] Huck-Pezzei et al used a combination of analytical techniques, including TLC, HPLC, MS, NIR (near-infrared) spectroscopy and imaging methods coupled to multivariate data analysis, in an attempt to identify adulteration in 32 SJW samples (both plant material and finished products) and to differentiate between Hypericum of European and Chinese origin. HPTLC was used to identify some unusual ingredients present in Chinese samples. Methanolic SJW extracts were applied on HPTLC plates and developed in a saturated chamber with the mobile phase ethyl acetate – water – formic acid (42.5: 2.5: 5 v/v/v). The plates were sprayed with 1% methanolic diphenylboryloxyethylamine and 5% methanolic PEG 400 and were visualized under UV light at 365 nm. They found that SJW of Chinese origin contained a yellow-orange band under hypericin in the chromatogram which they suggested that it might belong to the compounds Kushenol G and H (present in H. hirsutum L.) after MS analysis. They also identified different concentrations of phenolic compounds between European and Chinese SJW with European SJW containing higher concentrations of rutin, hyperoside and isoquercitrin. Frommenwiler et al used HPTLC to investigate adulteration on crude SJW herbs, commercial finished SJW products and dry SJW extract.[10] The team analysed the samples using the HPTLC association method described above and they detected an extra yellow band at R=0.4-0.5 as Huck-Pezzei et al did f but additionally they observed the absence of a yellow band at R=0.18 for the samples with the extra f