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Determination of parthenin in Parthenium hysterophorus L. by means of HPLC-UV: Method development and validation
Phytochemistry Letters 4 (2011) 134–137
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Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Determination of parthenin in Parthenium hysterophorus L. by means of HPLC-UV: Method development and validation Yanelis Saucedo Herna´ndez a, Luis Bravo Sa´nchez a, Mirtha Mayra Gonza´lez Bedia a, Luis Torres Go´mez a, Elisa Jorge Rodrı´guez a, Hilda M. Gonza´lez San Miguel b, Dulce Gonza´lez Mosquera a, Livier Polin Garcı´a a, Liene Dhooghe c,*, Mart Theunis c, Luc Pieters c, Sandra Apers c a
Department of Pharmaceutical Sciences, Universidad Central ‘‘Marta Abreu’’ de Las Villas, Carretera de Camajuani 5.5, Santa Clara, Cuba Institution of Pharmaceutical and Food Sciences, La Lisa, Havana, Cuba c Laboratory of Pharmacognosy and Pharmaceutical Analysis, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, Antwerp, Belgium b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 October 2010 Received in revised form 26 January 2011 Accepted 1 February 2011 Available online 16 February 2011
Parthenium hysterophorus L. or Santa Maria feverfew is a plant used in Cuba since antiquity for the treatment of several diseases. Nowadays it is still used as an antipyretic and antiparasitic agent. Parthenin, a sesquiterpene lactone, is the active secondary metabolite and the major component of the plant. In this study the development and validation of a HPLC method for the determination of parthenin in the powdered plant material are presented, making it possible to perform quality control on preparations containing P. hysterophorus. Firstly, parthenin was isolated from the plant material in order to use this as reference material. During the method development, the extraction procedure, sample preparation, and HPLC conditions were evaluated and optimized. The final method was fully validated in terms of calibration model, precision, accuracy, and specificity. Based on these results, it was concluded that the developed HPLC method is suitable for the determination of parthenin using a single-point calibration. The calibration model was linear in the concentration range from 0.05 to 0.25 mg/ml. Analysis on different days showed that the method was precise with an average concentration of 4.73% and RSD of 1.39%. A recovery experiment was performed resulting in a 95% confidence interval between 97.5% and 100.4%, meaning that the method is also accurate. Specificity was confirmed by the investigation of the peak purity. Using this newly validated method the quality of the plant material of P. hysterophorus, used as an active principle in pharmaceutical preparations, can be guaranteed. ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Parthenium hysterophorus Parthenin Isolation Quantification Validation Quality control
1. Introduction Parthenium hysterophorus L., commonly known as Santa Maria feverfew, is an annual herb native to Mexico, Central and South America. This plant is generally used by the Cuban population as an antipyretic, antiparasitic agent and for the treatment of skin infections. In spite of its common use, no commercially available preparations with determined content nor standardized products exist. The first step in the development of products from medicinal plants, is the characterization of the starting material, i.e. the powdered plant material. The main active constituent in P. hysterophorus is the sesquiterpene lactone parthenin (Fig. 1) (Dominguez and Sierra, 1970; Khare, 2007; Picman et al., 1980). This compound has antibacterial properties and it was shown to have
* Corresponding author. Tel.: +32 3 265 27 31; fax: +32 3 265 27 09. E-mail address: [email protected] (L. Dhooghe).
an anti-inflammatory effect in acute and chronic inflammations (Ramesh et al., 2003a; Recio et al., 2000). An HPLC method for the determination of parthenin in different plant parts or plant extracts of P. hysterophorus is already described in the work of research groups investigating allelochemicals (Belz et al., 2007; Reinhardt et al., 2004). However, these methods, designed for research purposes, are not suitable for routine quality control due to their complex and laborious sample preparation. Furthermore, data on calibration model, repeatability, and accuracy of the methods are lacking. The objective of this research project was to develop and validate a method suitable for controlling the quality of the plant material of P. hysterophorus, used as an active principle in pharmaceutical preparations. 2. Results and discussion Since parthenin is not only the active compound in P. hysterophorus, but also the main constituent, an HPLC method for the determination of the amount of parthenin present in the
1874-3900/$ – see front matter ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.phytol.2011.02.001
[()TD$FIG]
Y.S. Herna´ndez et al. / Phytochemistry Letters 4 (2011) 134–137
Fig. 1. The structure of parthenin.
plant material is indispensable for the quality control of any preparation containing this plant extract as active ingredient. In order to develop and validate such an HPLC method, a certain amount of reference material of parthenin is required. Therefore, parthenin was first isolated from the plant material as described in Section 3.3. Previous reports on the isolation of parthenin involved preparative HPLC (Reinhardt et al., 2004). However, in this research the isolation procedure contains a liquid–liquid extraction, separation on a silica column and finally crystallization. Overall this procedure can be considered faster, less complex and less expensive without decreasing the purity of the final compound, i.e. 98%. The structure of parthenin was confirmed by NMR and mass spectroscopy. The NMR data, shown in Table 1, were in good agreement with the literature (Ramesh et al., 2003b). The development of the HPLC method was based on the monograph on feverfew (Tanaceti parthenii herba) in the European Pharmacopoeia (EDQM, 2009). Using the determination of parthenolide in Tanacetum parthenium as starting point, the method was optimized for the determination of parthenin in P. hysterophorus. By performing different experiments for the extraction procedure, it could be concluded that for P. hysterophorus the use of the ultrasonic bath gave higher yields of parthenin than refluxing. Through investigation of the extraction solvent and volume, it was shown that the amount of plant material had to be reduced in order to obtain a more complete extraction. Usage of other solvents than methanol could, however, not improve the results. The chromatographic conditions described in the European Pharmacopoeia, could also be used for the determination of parthenin. Nevertheless, a rinsing step was added for the preservation of the HPLC column.
Table 1 NMR data of the isolated parthenin. 1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
H NMR
– 7.60, d (5.9) 6.11, d (5.9) – – 4.96, d (7.9) 3.49, m 2.32–2.19, m 1.89, m 1.67, m 2.32–2.19, m – – 6.21, d (2.6) 5.70, d (2.6) 1.11, d (7.7) 1.25, s
135
This is how the final method, as described in Section 3.4, was obtained. The resulting chromatograms of the reference solution and test solution are shown in Fig. 2(a) and (b), respectively. The peak of parthenin has a retention time of 5.3 min and shows an acceptable symmetry. In the test solution a good separation between parthenin and the other constituents is observed. Validation of the method is required to assure the suitability for its intended use. Different characteristics were investigated according to reported guidelines (ICH, 2006): calibration model, precision, accuracy, and specificity. The results on the evaluation of the calibration model are shown in Table 2. The least square line was determined by following equation: y = 1412x 74 and the correlation coefficient was at least 0.99. The intercept was not statistically different from (0.0) and its 95% confidence interval included 0. On the contrary, the regression coefficient was statistically different from 0 and therefore it could be concluded that a single-point calibration is allowed. Also graphical inspection showed a linear correlation and the highest residual of 4.5% was still lower than the limit of 5%. It could be concluded that a linear model is applicable in the concentration range from 0.05 to 0.25 mg/ml. The precision experiment which was performed included the investigation of the repeatability and the intermediate precision. The data are shown in Table 3. The mean of the days were 4.74%, 4.72%, and 4.74% with a RSD of 1.42%, 1.40%, and 1.54%, respectively. The Cochran’s test was performed in order to check whether it is allowed to compare the results obtained on the different days. Since the calculated value of 0.37 was lower than the critical value of 0.71, it is allowed to perform an ANOVA. This resulted in a calculated F of 0.29, which was lower than the critical value of 3.68, meaning that the results obtained on different days were not statistically different. Since the within-days RSD (1.45%) and the between-days RSD (1.36%) are in the same order of magnitude, the method can be considered to be precise. In addition, the between-days RSD was smaller than the limit (2.11%), calculated by the Horwitz formula: RSDmax = 2/3RSDHorwitz in which RSDHorwitz = 2(1 0.5logC) (Ermer, 2005). The overall average amount of parthenin found in the plant material was 4.71% with an RSD of 1.39%. The results of the recovery experiment are shown in Table 4. The average of the recovery was 99.0% with an RSD of 1.88%. The 95% confidence interval was situated between 97.5% and 100.4%. Student’s t-test resulted in a calculated value of 1.66 compared to the theoretical value of 2.31. Thus, it showed that the recovery was not statistically different from 100% meaning that the method is considered to be accurate. Concerning the specificity, no deviations were observed during the comparison of the UV spectra of the peaks of parthenin in the reference material and in the plant extract. In addition, the peaks showed equal symmetry factors and width at half height, indicating that no interference occurred by other constituents in the solution.
13
C NMR
84.9 165.9 131.7 213.4 60.4 80.9 45.7 29.5 31.0 41.7 172.9 142.3 122.3 17.7 18.9
3. Materials and methods 3.1. Chemicals and instrumentation HPLC grade acetonitrile and methanol were purchased at Merck. HPLC grade water was obtained from a deionized water treatment system from Millipore. The chromatographic separations were carried out on a Agilent 1100 HPLC with DAD detector, using a LiChrospher 100 ODS RP-18 5 m column (250 mm 4.6 mm) from Merck. 3.2. Plant material The plant material of P. hysterophorus was collected at the Las Villas Central University in Cuba between September and
[()TD$FIG]
Y.S. Herna´ndez et al. / Phytochemistry Letters 4 (2011) 134–137
136
Fig. 2. HPLC chromatogram of the parthenin reference material (a) and the test solution (b), using a LiChrospher 100 RP-18 5 m (250 mm 4.6 mm) with following gradient with (A) water and (B) acetonitrile at a flow of 1.0 ml/min and detection at 210 nm: 0 min, 60:40 (A:B); 10 min, 60:40; 13 min, 0:100; 15 min, 0:100; 18 min, 60:40; 20 min, 60:40.
December, 2006. The aerial parts were authenticated at the botanical garden by Dr. Cristobal Rios Albuerne from the Faculty of Agriculture (Las Villas Central University), where a voucher specimen is kept (number 08133). The plant material was dried in a MLW heater HST-1512 at 38 8C during 72 h, until constant mass. The dried plant material was then reduced to a powder with particle size up to 0.315 mm using a Restsch 5657 SR.2 mill. Table 2 Overview of the results about the calibration model of parthenin. Parthenin Range (mg/ml) Number of standards Correlation coefficient Intercept standard error Confidence interval (95%) Slope standard error
0.05–0.25 5 0.997 74 46 174 to 25 1412 278
Table 3 Data on the precision of the method.
Repeatability Number of replicates Mean content (%) Standard deviation RSD (%) Intermediate precision Number of groups Overall mean (%) RSDwithin (%) RSDbetween (RSDmax) (%) Ccalc (Ccrit) Fcalc (Fcrit)
Day 1
Day 2
Day 3
6 4.74 0.067 1.42
6 4.72 0.066 1.40
6 4.74 0.073 1.54
3 4.73 1.45 1.36 0.37 0.29
(2.11) (0.71) (3.68)
3.3. Isolation of parthenin 100 g of powdered plant material was macerated with 600 ml of methanol during 24 h. The obtained extract was concentrated under reduced pressure until about half of the original volume. An equal volume of water was added. After filtration, the obtained solution was extracted three times with 60 ml of chloroform. The chloroform extract was evaporated under reduced pressure resulting in a yellowish oily substance (5.1 g). The concentrated chloroform fraction was applied on a silica gel column (50 cm 2.5 cm) and eluted with a dichloromethane:methanol (3:1) mixture. Fractions of 3 ml were collected and all were analyzed using TLC on silica gel GF 60 R plates of Merck using chloroform:acetone (3:1) as mobile phase. The spots were detected with UV at 254 nm and after spraying with vanillin reagent (5 g/l in sulphuric acid:anhydrous ethanol (4:1)). The fractions containing parthenin, recognized by the violet zone with an Rf value of about 0.6, were collected. After evaporation under reduced pressure, an oily substance was obtained (1.3 g) which was dissolved in a Table 4 Results of the recovery experiment. Solution
Determined amount
Theoretical amount
Recovery (%)
1a 1b 1c 2a 2b 2c 3a 3b 3c
4.87 4.86 4.88 10.15 10.12 10.15 14.68 14.77 14.70
5.00 5.00 5.00 10.00 10.00 10.00 15.00 15.00 15.00
97.4 97.2 97.6 101.5 101.2 101.5 97.9 98.5 98.0
Y.S. Herna´ndez et al. / Phytochemistry Letters 4 (2011) 134–137
minimal quantity of ethanol (about 1 ml). After addition of 5 ml of ether:n-hexane (1:4) mixture, the resulting solution was kept in a refrigerator for 24 h. The obtained solid was recrystallized using a minimal quantity of acetone and by adding n-hexane drop by drop until turbidity appeared. Transparent needles were obtained. The identity of the isolated compound was confirmed by means of NMR spectroscopy and comparison to literature data (Ramesh et al., 2003b). 1H NMR, 13C NMR, DEPT-135 and DEPT-90 spectra were recorded in deuterated chloroform on a Bruker DRX-400 instrument, operating at 400.15 and using standard software packages. Library search based on the 13C NMR data using the NMRPredict software (version 3.8.22) confirmed the structure as parthenin (Fig. 1). In addition, the structure assignment was confirmed by mass spectroscopy using a linear ion trap type LXQ of Thermofinnigan. Ionization was performed with electron spray ionization in positive and negative mode. An [M H] ion at m/z 261 was observed in the negative mode, and [M H]+ and [M Na]+ ions at m/z 263 and 285, respectively, in the positive mode, both indicating a relative molecular mass of 262. The purity of this reference material was determined by HPLC analysis according to the normalization method, and was calculated to be 98%. 3.4. HPLC analysis 3.4.1. Reference solution About 5.0 mg of parthenin reference material was accurately weighed in a measuring flask of 2.0 ml and dissolved in methanol. 120 ml of this stock solution was transferred into a measuring flask of 2.0 ml and diluted with acetonitrile:water (2:3). 3.4.2. Test solution 0.5 g of the powdered plant material was weighed in a flask, 40 ml methanol was added and the solution was placed in an ultrasonic bath during 10 min. The resulting extract was filtered into a round bottomed flask and the filter was rinsed with about 3 ml of methanol. This extraction procedure was then repeated, the extract was joined together in the round bottomed flask and was dried using the rotavapor. The residue was dissolved in methanol and transferred into a measuring flask of 20.0 ml and brought to volume. 1.0 ml was diluted to 10.0 ml with the mobile phase, i.e. water:acetonitrile (3:2). This solution was then filtered through a 0.45 mm membrane filter before injection into the HPLC. 3.4.3. Chromatographic conditions 10 ml of the samples and standard solutions were injected in triplicate and eluted using the following gradient at a flow of 1.0 ml/min with water (A) and acetonitrile (B): 0 min, 60:40 (A:B); 10 min, 60:40; 13 min, 0:100; 15 min, 0:100; 18 min, 60:40; 20 min, 60:40. The peaks were detected at 210 nm. 3.5. Validation of the method 3.5.1. Calibration model A series of reference solutions were prepared in a concentration ranging from 0.05 to 0.25 mg/ml. By transferring different amounts from the stock solution into measuring flasks of 2.0 ml and diluting with water:acetonitrile (3:2), five concentration levels were obtained: 0.05 mg/ml; 0.10 mg/ml; 0.15 mg/ml; 0.20 mg/ml; 0.25 mg/ml. These solutions were analyzed in triplicate using the conditions described in Section 3.4. A calibration line was made and the least square line and correlation coefficient were calculated. Using Student’s t-test, the intercept and the regression coefficient were investigated. Both the calibration line and the residuals were graphically inspected and evaluated.
137
3.5.2. Precision Six independent samples were analyzed on three different days by the same analyst and using the same equipment. For each day and thus for every set of six samples, a fresh reference solution was prepared. From the results, the mean, the standard deviation, and the relative standard deviation (RSD) were calculated for each day. A Cochran’s test and ANOVA single factor were performed. Withinand between-days RSD were calculated. 3.5.3. Accuracy The accuracy was investigated by performing a recovery experiment. Samples were prepared at three concentration levels in triplicate by adding 2.0, 4.0, and 6.0 ml of the reference stock solution to 0.250 g of the powdered plant material. Extraction and analysis were then performed according to the described procedure. The recovery was calculated by comparing what was actually found by analysis and what theoretically calculated should be found, expressed in percentage. Student’s t-test was performed in order to investigate the results. 3.5.4. Specificity The specificity of the method was investigated by checking the peak purity of parthenin. This was performed by comparing the UV spectrum of the peak in the reference material and the peak in the plant extract.
Acknowledgement This research was financially supported by the VLIR Project (Belgium) ‘‘Strengthening undergraduate and graduate education in Pharmaceutical Sciences’’.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.phytol.2011.02.001. References Belz, R.G., Reinhardt, C.F., Foxcroft, L.C., Hurle, K., 2007. Residue allelopathy in Parthenium hysterophorus L. – does parthenin play a leading role? Crop Prot. 26, 237–245. Dominguez, X.A., Sierra, A., 1970. Isolation of a new diterpene alcohol and parthenin from Parthenium hysterophorus. Planta Med. 18, 275–277. EDQM, Council of Europe, 2009. European Pharmacopoeia, sixth ed. European Directorate for the Quality of Medicines (EDQM), Strasbourg. Ermer, J., 2005. Performance parameters, calculations and tests – precision. In: Ermer, J., Miller, J.H.Mc.B. (Eds.), Method Validation in Pharmaceutical Analysis. Wiley, Weinheim, pp. 21–51. ICH (International Conference on Harmonization of Technical Requirements for the Registration of Pharmaceuticals for Human Use), 2006. Validation of Analytical Procedures: Methodology (Q2A and Q2B). Khare, C.P., 2007. Indian Medicinal Plants: An Illustrated Dictionary. Springer, New Delhi. Picman, A.K., Ranieri, R.L., Towers, G.H.N., Lam, J., 1980. Visualization reagents for sesquiterpene lactones and polyacetylenes on thin-layer chromatograms. J. Chromatogr. 189, 187–198. Ramesh, C., Harakishore, K., Murty, U.S.N., Das, B., 2003a. Analogues of parthenin and their antibacterial activity. ARKIVOC ix, 126–132. Ramesh, C., Ravindranath, N., Das, B., Prabhakar, A., Bharatam, J., Ravikumar, K., Kashinatham, A., McMorris, T.C., 2003b. Pseudoguaianolides from the flowers of Parthenium hysterophorus. Phytochemistry 64, 841–844. ˜ ez, S., Cerda, M., De la Fuente, J.R., Rios, J.L., Recio, M.C., Giner, R.M., Uriburu, L., Man 2000. In vivo activity of pseudoguaianolide sesquiterpene lactones in acute and chronic inflammation. Life Sci. 66, 2509–2518. Reinhardt, C., Kraus, S., Walker, F., Foxcroft, L., Robbertse, P., Hurle, K., 2004. The allelochemical parthenin is sequestered at high level in capitate-sessile trichomes on leaf surfaces of Parthenium hysterophorus. J. Plant Dis. Prot. 19, 253–261.
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Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Determination of parthenin in Parthenium hysterophorus L. by means of HPLC-UV: Method development and validation Yanelis Saucedo Herna´ndez a, Luis Bravo Sa´nchez a, Mirtha Mayra Gonza´lez Bedia a, Luis Torres Go´mez a, Elisa Jorge Rodrı´guez a, Hilda M. Gonza´lez San Miguel b, Dulce Gonza´lez Mosquera a, Livier Polin Garcı´a a, Liene Dhooghe c,*, Mart Theunis c, Luc Pieters c, Sandra Apers c a
Department of Pharmaceutical Sciences, Universidad Central ‘‘Marta Abreu’’ de Las Villas, Carretera de Camajuani 5.5, Santa Clara, Cuba Institution of Pharmaceutical and Food Sciences, La Lisa, Havana, Cuba c Laboratory of Pharmacognosy and Pharmaceutical Analysis, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, Antwerp, Belgium b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 October 2010 Received in revised form 26 January 2011 Accepted 1 February 2011 Available online 16 February 2011
Parthenium hysterophorus L. or Santa Maria feverfew is a plant used in Cuba since antiquity for the treatment of several diseases. Nowadays it is still used as an antipyretic and antiparasitic agent. Parthenin, a sesquiterpene lactone, is the active secondary metabolite and the major component of the plant. In this study the development and validation of a HPLC method for the determination of parthenin in the powdered plant material are presented, making it possible to perform quality control on preparations containing P. hysterophorus. Firstly, parthenin was isolated from the plant material in order to use this as reference material. During the method development, the extraction procedure, sample preparation, and HPLC conditions were evaluated and optimized. The final method was fully validated in terms of calibration model, precision, accuracy, and specificity. Based on these results, it was concluded that the developed HPLC method is suitable for the determination of parthenin using a single-point calibration. The calibration model was linear in the concentration range from 0.05 to 0.25 mg/ml. Analysis on different days showed that the method was precise with an average concentration of 4.73% and RSD of 1.39%. A recovery experiment was performed resulting in a 95% confidence interval between 97.5% and 100.4%, meaning that the method is also accurate. Specificity was confirmed by the investigation of the peak purity. Using this newly validated method the quality of the plant material of P. hysterophorus, used as an active principle in pharmaceutical preparations, can be guaranteed. ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Parthenium hysterophorus Parthenin Isolation Quantification Validation Quality control
1. Introduction Parthenium hysterophorus L., commonly known as Santa Maria feverfew, is an annual herb native to Mexico, Central and South America. This plant is generally used by the Cuban population as an antipyretic, antiparasitic agent and for the treatment of skin infections. In spite of its common use, no commercially available preparations with determined content nor standardized products exist. The first step in the development of products from medicinal plants, is the characterization of the starting material, i.e. the powdered plant material. The main active constituent in P. hysterophorus is the sesquiterpene lactone parthenin (Fig. 1) (Dominguez and Sierra, 1970; Khare, 2007; Picman et al., 1980). This compound has antibacterial properties and it was shown to have
* Corresponding author. Tel.: +32 3 265 27 31; fax: +32 3 265 27 09. E-mail address: [email protected] (L. Dhooghe).
an anti-inflammatory effect in acute and chronic inflammations (Ramesh et al., 2003a; Recio et al., 2000). An HPLC method for the determination of parthenin in different plant parts or plant extracts of P. hysterophorus is already described in the work of research groups investigating allelochemicals (Belz et al., 2007; Reinhardt et al., 2004). However, these methods, designed for research purposes, are not suitable for routine quality control due to their complex and laborious sample preparation. Furthermore, data on calibration model, repeatability, and accuracy of the methods are lacking. The objective of this research project was to develop and validate a method suitable for controlling the quality of the plant material of P. hysterophorus, used as an active principle in pharmaceutical preparations. 2. Results and discussion Since parthenin is not only the active compound in P. hysterophorus, but also the main constituent, an HPLC method for the determination of the amount of parthenin present in the
1874-3900/$ – see front matter ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.phytol.2011.02.001
[()TD$FIG]
Y.S. Herna´ndez et al. / Phytochemistry Letters 4 (2011) 134–137
Fig. 1. The structure of parthenin.
plant material is indispensable for the quality control of any preparation containing this plant extract as active ingredient. In order to develop and validate such an HPLC method, a certain amount of reference material of parthenin is required. Therefore, parthenin was first isolated from the plant material as described in Section 3.3. Previous reports on the isolation of parthenin involved preparative HPLC (Reinhardt et al., 2004). However, in this research the isolation procedure contains a liquid–liquid extraction, separation on a silica column and finally crystallization. Overall this procedure can be considered faster, less complex and less expensive without decreasing the purity of the final compound, i.e. 98%. The structure of parthenin was confirmed by NMR and mass spectroscopy. The NMR data, shown in Table 1, were in good agreement with the literature (Ramesh et al., 2003b). The development of the HPLC method was based on the monograph on feverfew (Tanaceti parthenii herba) in the European Pharmacopoeia (EDQM, 2009). Using the determination of parthenolide in Tanacetum parthenium as starting point, the method was optimized for the determination of parthenin in P. hysterophorus. By performing different experiments for the extraction procedure, it could be concluded that for P. hysterophorus the use of the ultrasonic bath gave higher yields of parthenin than refluxing. Through investigation of the extraction solvent and volume, it was shown that the amount of plant material had to be reduced in order to obtain a more complete extraction. Usage of other solvents than methanol could, however, not improve the results. The chromatographic conditions described in the European Pharmacopoeia, could also be used for the determination of parthenin. Nevertheless, a rinsing step was added for the preservation of the HPLC column.
Table 1 NMR data of the isolated parthenin. 1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
H NMR
– 7.60, d (5.9) 6.11, d (5.9) – – 4.96, d (7.9) 3.49, m 2.32–2.19, m 1.89, m 1.67, m 2.32–2.19, m – – 6.21, d (2.6) 5.70, d (2.6) 1.11, d (7.7) 1.25, s
135
This is how the final method, as described in Section 3.4, was obtained. The resulting chromatograms of the reference solution and test solution are shown in Fig. 2(a) and (b), respectively. The peak of parthenin has a retention time of 5.3 min and shows an acceptable symmetry. In the test solution a good separation between parthenin and the other constituents is observed. Validation of the method is required to assure the suitability for its intended use. Different characteristics were investigated according to reported guidelines (ICH, 2006): calibration model, precision, accuracy, and specificity. The results on the evaluation of the calibration model are shown in Table 2. The least square line was determined by following equation: y = 1412x 74 and the correlation coefficient was at least 0.99. The intercept was not statistically different from (0.0) and its 95% confidence interval included 0. On the contrary, the regression coefficient was statistically different from 0 and therefore it could be concluded that a single-point calibration is allowed. Also graphical inspection showed a linear correlation and the highest residual of 4.5% was still lower than the limit of 5%. It could be concluded that a linear model is applicable in the concentration range from 0.05 to 0.25 mg/ml. The precision experiment which was performed included the investigation of the repeatability and the intermediate precision. The data are shown in Table 3. The mean of the days were 4.74%, 4.72%, and 4.74% with a RSD of 1.42%, 1.40%, and 1.54%, respectively. The Cochran’s test was performed in order to check whether it is allowed to compare the results obtained on the different days. Since the calculated value of 0.37 was lower than the critical value of 0.71, it is allowed to perform an ANOVA. This resulted in a calculated F of 0.29, which was lower than the critical value of 3.68, meaning that the results obtained on different days were not statistically different. Since the within-days RSD (1.45%) and the between-days RSD (1.36%) are in the same order of magnitude, the method can be considered to be precise. In addition, the between-days RSD was smaller than the limit (2.11%), calculated by the Horwitz formula: RSDmax = 2/3RSDHorwitz in which RSDHorwitz = 2(1 0.5logC) (Ermer, 2005). The overall average amount of parthenin found in the plant material was 4.71% with an RSD of 1.39%. The results of the recovery experiment are shown in Table 4. The average of the recovery was 99.0% with an RSD of 1.88%. The 95% confidence interval was situated between 97.5% and 100.4%. Student’s t-test resulted in a calculated value of 1.66 compared to the theoretical value of 2.31. Thus, it showed that the recovery was not statistically different from 100% meaning that the method is considered to be accurate. Concerning the specificity, no deviations were observed during the comparison of the UV spectra of the peaks of parthenin in the reference material and in the plant extract. In addition, the peaks showed equal symmetry factors and width at half height, indicating that no interference occurred by other constituents in the solution.
13
C NMR
84.9 165.9 131.7 213.4 60.4 80.9 45.7 29.5 31.0 41.7 172.9 142.3 122.3 17.7 18.9
3. Materials and methods 3.1. Chemicals and instrumentation HPLC grade acetonitrile and methanol were purchased at Merck. HPLC grade water was obtained from a deionized water treatment system from Millipore. The chromatographic separations were carried out on a Agilent 1100 HPLC with DAD detector, using a LiChrospher 100 ODS RP-18 5 m column (250 mm 4.6 mm) from Merck. 3.2. Plant material The plant material of P. hysterophorus was collected at the Las Villas Central University in Cuba between September and
[()TD$FIG]
Y.S. Herna´ndez et al. / Phytochemistry Letters 4 (2011) 134–137
136
Fig. 2. HPLC chromatogram of the parthenin reference material (a) and the test solution (b), using a LiChrospher 100 RP-18 5 m (250 mm 4.6 mm) with following gradient with (A) water and (B) acetonitrile at a flow of 1.0 ml/min and detection at 210 nm: 0 min, 60:40 (A:B); 10 min, 60:40; 13 min, 0:100; 15 min, 0:100; 18 min, 60:40; 20 min, 60:40.
December, 2006. The aerial parts were authenticated at the botanical garden by Dr. Cristobal Rios Albuerne from the Faculty of Agriculture (Las Villas Central University), where a voucher specimen is kept (number 08133). The plant material was dried in a MLW heater HST-1512 at 38 8C during 72 h, until constant mass. The dried plant material was then reduced to a powder with particle size up to 0.315 mm using a Restsch 5657 SR.2 mill. Table 2 Overview of the results about the calibration model of parthenin. Parthenin Range (mg/ml) Number of standards Correlation coefficient Intercept standard error Confidence interval (95%) Slope standard error
0.05–0.25 5 0.997 74 46 174 to 25 1412 278
Table 3 Data on the precision of the method.
Repeatability Number of replicates Mean content (%) Standard deviation RSD (%) Intermediate precision Number of groups Overall mean (%) RSDwithin (%) RSDbetween (RSDmax) (%) Ccalc (Ccrit) Fcalc (Fcrit)
Day 1
Day 2
Day 3
6 4.74 0.067 1.42
6 4.72 0.066 1.40
6 4.74 0.073 1.54
3 4.73 1.45 1.36 0.37 0.29
(2.11) (0.71) (3.68)
3.3. Isolation of parthenin 100 g of powdered plant material was macerated with 600 ml of methanol during 24 h. The obtained extract was concentrated under reduced pressure until about half of the original volume. An equal volume of water was added. After filtration, the obtained solution was extracted three times with 60 ml of chloroform. The chloroform extract was evaporated under reduced pressure resulting in a yellowish oily substance (5.1 g). The concentrated chloroform fraction was applied on a silica gel column (50 cm 2.5 cm) and eluted with a dichloromethane:methanol (3:1) mixture. Fractions of 3 ml were collected and all were analyzed using TLC on silica gel GF 60 R plates of Merck using chloroform:acetone (3:1) as mobile phase. The spots were detected with UV at 254 nm and after spraying with vanillin reagent (5 g/l in sulphuric acid:anhydrous ethanol (4:1)). The fractions containing parthenin, recognized by the violet zone with an Rf value of about 0.6, were collected. After evaporation under reduced pressure, an oily substance was obtained (1.3 g) which was dissolved in a Table 4 Results of the recovery experiment. Solution
Determined amount
Theoretical amount
Recovery (%)
1a 1b 1c 2a 2b 2c 3a 3b 3c
4.87 4.86 4.88 10.15 10.12 10.15 14.68 14.77 14.70
5.00 5.00 5.00 10.00 10.00 10.00 15.00 15.00 15.00
97.4 97.2 97.6 101.5 101.2 101.5 97.9 98.5 98.0
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minimal quantity of ethanol (about 1 ml). After addition of 5 ml of ether:n-hexane (1:4) mixture, the resulting solution was kept in a refrigerator for 24 h. The obtained solid was recrystallized using a minimal quantity of acetone and by adding n-hexane drop by drop until turbidity appeared. Transparent needles were obtained. The identity of the isolated compound was confirmed by means of NMR spectroscopy and comparison to literature data (Ramesh et al., 2003b). 1H NMR, 13C NMR, DEPT-135 and DEPT-90 spectra were recorded in deuterated chloroform on a Bruker DRX-400 instrument, operating at 400.15 and using standard software packages. Library search based on the 13C NMR data using the NMRPredict software (version 3.8.22) confirmed the structure as parthenin (Fig. 1). In addition, the structure assignment was confirmed by mass spectroscopy using a linear ion trap type LXQ of Thermofinnigan. Ionization was performed with electron spray ionization in positive and negative mode. An [M H] ion at m/z 261 was observed in the negative mode, and [M H]+ and [M Na]+ ions at m/z 263 and 285, respectively, in the positive mode, both indicating a relative molecular mass of 262. The purity of this reference material was determined by HPLC analysis according to the normalization method, and was calculated to be 98%. 3.4. HPLC analysis 3.4.1. Reference solution About 5.0 mg of parthenin reference material was accurately weighed in a measuring flask of 2.0 ml and dissolved in methanol. 120 ml of this stock solution was transferred into a measuring flask of 2.0 ml and diluted with acetonitrile:water (2:3). 3.4.2. Test solution 0.5 g of the powdered plant material was weighed in a flask, 40 ml methanol was added and the solution was placed in an ultrasonic bath during 10 min. The resulting extract was filtered into a round bottomed flask and the filter was rinsed with about 3 ml of methanol. This extraction procedure was then repeated, the extract was joined together in the round bottomed flask and was dried using the rotavapor. The residue was dissolved in methanol and transferred into a measuring flask of 20.0 ml and brought to volume. 1.0 ml was diluted to 10.0 ml with the mobile phase, i.e. water:acetonitrile (3:2). This solution was then filtered through a 0.45 mm membrane filter before injection into the HPLC. 3.4.3. Chromatographic conditions 10 ml of the samples and standard solutions were injected in triplicate and eluted using the following gradient at a flow of 1.0 ml/min with water (A) and acetonitrile (B): 0 min, 60:40 (A:B); 10 min, 60:40; 13 min, 0:100; 15 min, 0:100; 18 min, 60:40; 20 min, 60:40. The peaks were detected at 210 nm. 3.5. Validation of the method 3.5.1. Calibration model A series of reference solutions were prepared in a concentration ranging from 0.05 to 0.25 mg/ml. By transferring different amounts from the stock solution into measuring flasks of 2.0 ml and diluting with water:acetonitrile (3:2), five concentration levels were obtained: 0.05 mg/ml; 0.10 mg/ml; 0.15 mg/ml; 0.20 mg/ml; 0.25 mg/ml. These solutions were analyzed in triplicate using the conditions described in Section 3.4. A calibration line was made and the least square line and correlation coefficient were calculated. Using Student’s t-test, the intercept and the regression coefficient were investigated. Both the calibration line and the residuals were graphically inspected and evaluated.
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3.5.2. Precision Six independent samples were analyzed on three different days by the same analyst and using the same equipment. For each day and thus for every set of six samples, a fresh reference solution was prepared. From the results, the mean, the standard deviation, and the relative standard deviation (RSD) were calculated for each day. A Cochran’s test and ANOVA single factor were performed. Withinand between-days RSD were calculated. 3.5.3. Accuracy The accuracy was investigated by performing a recovery experiment. Samples were prepared at three concentration levels in triplicate by adding 2.0, 4.0, and 6.0 ml of the reference stock solution to 0.250 g of the powdered plant material. Extraction and analysis were then performed according to the described procedure. The recovery was calculated by comparing what was actually found by analysis and what theoretically calculated should be found, expressed in percentage. Student’s t-test was performed in order to investigate the results. 3.5.4. Specificity The specificity of the method was investigated by checking the peak purity of parthenin. This was performed by comparing the UV spectrum of the peak in the reference material and the peak in the plant extract.
Acknowledgement This research was financially supported by the VLIR Project (Belgium) ‘‘Strengthening undergraduate and graduate education in Pharmaceutical Sciences’’.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.phytol.2011.02.001. References Belz, R.G., Reinhardt, C.F., Foxcroft, L.C., Hurle, K., 2007. Residue allelopathy in Parthenium hysterophorus L. – does parthenin play a leading role? Crop Prot. 26, 237–245. Dominguez, X.A., Sierra, A., 1970. Isolation of a new diterpene alcohol and parthenin from Parthenium hysterophorus. Planta Med. 18, 275–277. EDQM, Council of Europe, 2009. European Pharmacopoeia, sixth ed. European Directorate for the Quality of Medicines (EDQM), Strasbourg. Ermer, J., 2005. Performance parameters, calculations and tests – precision. In: Ermer, J., Miller, J.H.Mc.B. (Eds.), Method Validation in Pharmaceutical Analysis. Wiley, Weinheim, pp. 21–51. ICH (International Conference on Harmonization of Technical Requirements for the Registration of Pharmaceuticals for Human Use), 2006. Validation of Analytical Procedures: Methodology (Q2A and Q2B). Khare, C.P., 2007. Indian Medicinal Plants: An Illustrated Dictionary. Springer, New Delhi. Picman, A.K., Ranieri, R.L., Towers, G.H.N., Lam, J., 1980. Visualization reagents for sesquiterpene lactones and polyacetylenes on thin-layer chromatograms. J. Chromatogr. 189, 187–198. Ramesh, C., Harakishore, K., Murty, U.S.N., Das, B., 2003a. Analogues of parthenin and their antibacterial activity. ARKIVOC ix, 126–132. Ramesh, C., Ravindranath, N., Das, B., Prabhakar, A., Bharatam, J., Ravikumar, K., Kashinatham, A., McMorris, T.C., 2003b. Pseudoguaianolides from the flowers of Parthenium hysterophorus. Phytochemistry 64, 841–844. ˜ ez, S., Cerda, M., De la Fuente, J.R., Rios, J.L., Recio, M.C., Giner, R.M., Uriburu, L., Man 2000. In vivo activity of pseudoguaianolide sesquiterpene lactones in acute and chronic inflammation. Life Sci. 66, 2509–2518. Reinhardt, C., Kraus, S., Walker, F., Foxcroft, L., Robbertse, P., Hurle, K., 2004. The allelochemical parthenin is sequestered at high level in capitate-sessile trichomes on leaf surfaces of Parthenium hysterophorus. J. Plant Dis. Prot. 19, 253–261.