Journal of Pharmaceutical and Biomedical Analysis xxx (2006) xxx–xxx Short communication Analysis of neolignans compounds of Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck by HPLC 3 4 Daniele Fernanda Felipe, Benedito Prado Dias Filho, Celso Vataru Nakamura, Selma Lucy Franco, Diógenes Aparı́cio Garcia Cortez ∗ OF 5 6 Programa de Pós-Graduação em Ciências Farmacêuticas, Departamento de Farmácia e Farmacologia, Universidade Estadual deMaringá, Avenida Colombo 5790, Maringá PR 87020-900, Brazil 7 8 CT ED PR O Received 30 August 2005; received in revised form 14 February 2006; accepted 15 February 2006 9 10 Abstract 19 A high performance liquid chromatographic (HPLC) method was developed and validated for quantitative determination of neolignans in extracts of Piper regnellii var. pallescens. The analysis were carried out on a Metasil ODS column (150 mm × 4.6 mm, 5 ␮m) at 30 ◦ C, using as mobile phase acetonitrile–water (60:40, v/v) containing 2% acetic acid. The flow rate was 1.0 ml/min and the detection was at 280 nm. The validation using conocarpan as standard demonstrated that the method presents linearity (linear correlation coefficient = 0.9991), precision (relative standard deviation <5%) and accuracy (mean recovery = 104.55%) in the concentration range 31.25–500 ␮g/ml. The limit of detection (LOD) was 1.68 ␮g/ml and the limit of quantitation was 5.60 ␮g/ml. This method allowed the identification and quantification of conocarpan, eupomatenoid-5 and eupomatenoid-6 in the hydroethanolic extracts obtained from the leaves, stems and roots by maceration process. All the extracts showed the same chromatographic profile, being that the extract of the roots presented the highest concentration of neolignans. © 2006 Published by Elsevier B.V. 20 Keywords: Piper regnellii; Neolignans; HPLC; Validation 11 12 13 14 15 16 17 18 21 25 26 27 28 29 30 31 32 33 34 35 36 37 RE 24 Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck, popularly known as “pariparoba”, is a species belonging to Piperaceae family used in folk medicine, being the leaves and roots used in form of crude extracts, infusions or poultices in the treatment of wounds, swellings and skin irritations [1]. Benevides et al. [2] related the first phytochemical investigation carried out on the specie P. regnellii on the chemistry of lignans/neolignans. Among the isolated compounds from the ethyl acetate extracts of the roots of P. regnellii, are the neolignans conocarpan, eupomatenoid-3, eupomatenoid-5 and eupomatenoid-6 (Fig. 1). Benzofuran neolignans represent a sub-class with a variety of biological activities including anti-PAF, antifungal and insecticidal activity. Several compounds of this class have been isolated from Piperaceae species and in case of P. regnellii, phytochem- CO R 23 1. Introduction ∗ 1 2 UN 22 Corresponding author. Tel.: +55 44 3261 4876; fax: +55 44 3261 4999. E-mail address: dagcortez@uem.br (D.A.G. Cortez). ical studies of its roots showed the accumulation of several phenylpropanoids and benzofuran neolignans including conocarpan as the major compound [3]. Pessini et al. [1] isolated and identified the neolignans conocarpan, eupomatenoid-3, eupomatenoid-5 and eupomatenoid-6 from the hydroethanolic extract of the leaves of P. regnellii. Moreover, the antimicrobial activity of the compounds that demonstrated potential activity was evaluated, with exception of the compound eupomatenoid-3, that was inactive against bacteria and yeasts. The compounds eupomatenoid-5 and eupomatenoid-6 were active only against bacteria. The analysis of neolignans of P. regnellii by HPLC is not much explored. There is a report of Benevides et al. [2] that uses the HPLC method to demonstrate that the profile of neolignans is similar in roots, stems and leaves of P. regnellii. Moreover, Sartorelli et al. [3] carried out analysis by HPLC to verify the separation of benzofuran neolignans from the ethyl acetate extract of the leaves of P. regnellii. HPLC method is gaining increasing importance for qualitative and quantitative analysis of plant extracts, being useful for quality control of phytochemicals. 0731-7085/$ – see front matter © 2006 Published by Elsevier B.V. doi:10.1016/j.jpba.2006.02.029 PBA 5764 1–5 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 2 D.F. Felipe et al. / Journal of Pharmaceutical and Biomedical Analysis xxx (2006) xxx–xxx 2.3. HPLC analysis 88 OF 2.3.1. Reagents and chemicals Acetonitrile (HPLC grade from OmniSolv EM Science, Gibbstown, NJ), ultrapure water (Milli-Q system, Millipore, Bedford, USA) and acetic acid (analytical grade, Merck, Darmstadt, Germany) were used for the mobile phase preparation. Methanol (HPLC grade from OmniSolv EM Science, Gibbstown, NJ) was used for samples preparation. The neolignans conocarpan, eupomatenoid-5 and eupomatenoid-6 were isolated from the specie P. regnellii [1]. The conocarpan was used as external standard. Eupomatenoid-5 and eupomatenoid-6 were only used as reference to the corresponding peak in the sample extracts. CT ED PR O Fig. 1. Structures of the neolignans isolated from P. regnellii. 60 61 62 63 64 65 66 67 68 69 70 However, validated quality control methods need to be developed since the validation of analytical procedures is an important part of the registration application for a new drug. Besides the regulatory requirements, the performance and reliability of the control test procedure are essential to the quality control of drugs. Therefore, validation should be regarded as part of an integrated concept to ensure the quality, safety and efficacy of pharmaceuticals [4,5]. In this work, extraction, determination of the chromatographic conditions by HPLC method for analysis of neolignans in extracts of P. regnellii, validation of the method evaluation was developed. 71 2. Experimental 72 2.1. Plant material RE 59 84 2.2. Extract preparation 76 77 78 79 80 81 82 85 86 87 UN 74 75 CO R 83 The leaves, stems and roots of P. regnellii var. pallescens, were collected in September 2004 in the Medicinal Plants Garden “Profa . Irenice Silva” of the State University of Maringá Campus, Maringá, PR, Brazil. The plant material was identified by Marı́lia Borgo of the Botanical Departament of the Federal University of the Paraná. A voucher specimen (number HUM 11411) is deposited at the Herbarium of the State University of Maringá. The samples of leaves, stems and roots of P. regnellii were dried at 35 ◦ C in an air oven and were ground in a knife mill before extraction. 73 2.3.2. Sample preparation Stock solutions of conocarpan, eupomatenoid-5 and eupomatenoid-6 and extracts of the leaves, stems and roots from P. regnellii were prepared in methanol at a concentration of 1000 ␮g/ml. The solutions were filtered through 0.45 ␮m membrane filter (Millipore, São Paulo, Brazil). Dried leaves, stems and roots of P. regnellii (10 g) were extracted with ethanol:water (9:1, v/v, 100 ml) by maceration method at room temperature for 5 days at dark room. The extracts were filtered, evaporated under vaccum at 40 ◦ C and lyophilized. 2.3.3. Instrumentation and chromatographic conditions The analyses were carried out using a Shimadzu LC-10 liquid chromatograph equipped with quaternary pump (LC-10 AD), manual injection valve (Rheodyne) with loop of 20 ␮l, degasser (DEU-14), thermostatted column compartment (CTO-10Avp) and a UV–vis detector (SPD-10A), controlled by CLASS LC10 Software. In the chromatographic analysis was used Metasil ODS column, 5 ␮m, 150 mm × 4.6 mm, maintained at 30 ◦ C. The separation was carried out in isocratic system, using as mobile phase a mixture of acetonitrile–water (60:40, v/v) containing 2% acetic acid, with flow rate of 1.0 ml/min. The detection was carried out at 280 nm and the running time was 25 min. The sample injection volume was 20 ␮l. Three determinations were carried out for each sample. The statistical analysis of the data were performed by Statistica 6.0 software (Statsoft Inc., Tulsa, OK, USA). 2.4. Validation parameters 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 2.4.1. Linearity The linearity of the calibration curve for the conocarpan was determined by the external standard method. Stock standard solution at a concentration of 1000 ␮g/ml was diluted in methanol yielding concentrations of 31.25 ␮g/ml, 62.5 ␮g/ml, 125 ␮g/ml, 250 ␮g/ml and 500 ␮g/ml. Three determinations were carried out for each solution. The calibration curves were obtained by plotting the peak area of the conocarpan versus the concentration of the standard solutions. The statistical parameters of the calibration curve as slope, intercept and correlation coefficient were calculated by linear regression analysis. 2.4.2. Precision The repetibility of the method was evaluated on the same day while the intermediate precision was determined for 2 nonPBA 5764 1–5 125 126 127 128 129 130 131 132 133 134 135 136 137 138 3 D.F. Felipe et al. / Journal of Pharmaceutical and Biomedical Analysis xxx (2006) xxx–xxx 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 2.4.3. Limit of detection and quantification The limit of detection (LOD) and the limit of quantification (LOQ) were determined from the calibration curve of the standard conocarpan. LOD was calculated according to the expression 3σ/S, where σ is the standard deviation of the response and S is the slope of the calibration curve. LOQ was established by using the expression l0σ/S. 2.4.4. Accuracy The accuracy was evaluated with the recovery test by analysing the mixture prepared by adding of the conocarpan solution at the three concentration levels (31.25 ␮g/ml, 125 ␮g/ml and 500 ␮g/ml) to extract of the leaves of P. regnellii, containing known amount of the analyte. Three determinations were carried out for each solution. The percentage recovery was calculated by subtracting the values obtained for the control matrix preparation from those samples that were prepared with the added standards, divided by the amount added and then multiplied by 100. 166 3. Results and discussion 167 3.1. Optimization of the chromatographic conditions 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 To develop a HPLC method for the analysis of neolignans in P. regnellii extracts, several parameters were optimized to select the proper conditions. An isocratic system was chosen since allowed a good separation of neolignans within a short analysis time. To optimize the mobile phase different compositions of acetonitrile in water containing 2% of acetic acid were tested. The acetonitrile–water 60:40 (v/v) ratio showed to be adequated. The addition of acetic acid decreased the peak tailing of the neolignan eupomatenoid-5, being essencial to improve the resolution of the chromatogram. Flow rates between 0.6 ml/min and 1.0 ml/min were studied. The value of 1.0 ml/min allowed good separation with a analysis time of 25 min. The separation was further improved when column temperature was 30 ◦ C. The maximum absorption of the neolignans was found to be 280 nm, and this wavelength was chosen for the analysis. The Fig. 2 shows the chromatogram of standard mixture of neolignans. The chromatographic profile showed three well-resolved peaks (Rs1–2 = 15.93; Rs2–1 = 15.93; Rs2–3 = 3.54) when the chromatographic conditions described were employed. Peak 1 with a retention time of 11.23 min was identified as conocarpan. Peak 2 with a retention time of 16.63 min can be CO R 164 UN 163 RE 165 2.4.5. Stability of the analyte during analysis The stability was evalueted with standard solutions and sample solutions that were stored at 4 ◦ C and at room temperature during 72 h. The solutions were analyzed every 24 h. 162 OF 141 consecutive days. The standard solution was analysed at three concentration levels (31.25 ␮g/ml, 125 ␮g/ml and 500 ␮g/ml). Three determinations were carried out for each solution. The precision was expressed as relative standard deviation (R.S.D.%) of the concentrations of conocarpan. Fig. 2. Chromatogram of the standard mixture of neolignans: (1) conocarpan , (2) eupomatenoid-6 and (3) eupomatenoid-5. Chromatographic conditions: Metasil ODS column; mobile phase: acetonitrile/water (60:40, v/v) with 2% acetic acid; flow rate: 1.0 ml/min; temperature: 30 ◦ C; detection: 280 nm. CT ED PR O 139 140 assigned to the eupomtenoid-6 and peak 3, with a retention time of 18.07 min as eupomatenoid-5. 3.2. Validation 189 190 191 For the validation of the analytical method, the guideline of the International Conference on Harmonization of Technical Requirements for the Registration of Pharmaceuticals for Human Use was followed [6]. The conocarpan was used as standard because it can be found as the major compound in P. regnellii [3]. Moreover, the conocarpan presents high antimicrobial activity [1]. 3.2.1. Linearity Excellent linearity was observed for conocarpan between peak area and concentration in the range 31.25–500 ␮g/ml, as confirmed by the correlation coefficient of 0.9991. The validating parameters of the calibration curve, obtained by linear regression analysis, are described in Table 1. 3.2.2. Precision The method precision was evaluated in terms of repeatability and intermediate precision, by performing three repetitive analyses for each concentration levels (31.25 ␮g/ml, 125 ␮g/ml and 500 ␮g/ml). The repeatability test showed R.S.D. values lower than 2.16% and the intermediate precision, evaluated on 2 nonconsecutive days, presented R.S.D. between 2.83% and 4.70% (Table 2). These results could be considered satisfactory since the majority of phytochemicals present R.S.D. values lower than 6% according to literature [7]. Table 1 Linearity parameters for the calibration curve of the conocarpan Compound Linearity range (␮g/ml) Slope (a) Intercept (b) (r2 ) Conocarpan 31.25–500 55797 −595314 09991 r2 , correlation coefficient. PBA 5764 1–5 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 4 D.F. Felipe et al. / Journal of Pharmaceutical and Biomedical Analysis xxx (2006) xxx–xxx Table 2 Repeatability and intermediate precision data for the determination of conocarpan by HPLC Compound Concentration (␮g/ml) Repeatability (R.S.D%) Intermediate precision (R.S.D%) Conocarpan 31.25 125 500 1.18 2.16 1.42 4.14 4.70 2.83 R.S.D., relative standard deviation. For each sample n = 3. 217 218 219 220 221 222 223 224 225 226 227 228 229 3.2.3. Limit of detection and quantification The limit of detection, defined as the lowest concentration of conocarpan in a sample, which can be detected but not necessary quantified under the stated experimental conditions, was 1.68 ␮g/ml. The limit of quantification, defined as the lowest concentration of conocarpan in a sample that can be determined with acceptable precision and accuracy, was 5.60 ␮g/ml. 3.2.4. Accuracy The accuracy of the method was evaluated with the recovery test. Table 3 shows the recovery data, which were obtained by relationship between the amount of added standard and the amount detected. The method produced a medium recovery of 104.55% with R.S.D. below 4% for all analysed concentrations, confirming accuracy of the method. 235 3.3. Analysis of leaves, stems and roots of P. regnellii 233 236 237 238 239 240 241 242 243 244 The retention times of the standards conocarpan, eupomatenoid-6 and eupomatenoid-5, were used to identify the corresponding peaks in the extracts of P. regnellii. For determination of conocarpan content in the extracts of P. regnellii was used the regression equation of y = 55797x − 595314. The concentrations of eupomatenoid-5 and eupomatenoid-6 were expressed in conocarpan. Fig. 3a–c shows the chromatograms of the extracts of P. regnellii obtained from leaves, stems and roots, respectively. CO R 232 Table 3 Results of the recovery test for conocarpan from the extract of P. regnellii Compound Conocarpan UN 231 RE 234 3.2.5. Stability of the analyte during analysis The analytes in solution did not show any appreciable change in chromatographic profile for at least 72 h. No degradation products were observed, confirming the stability of the samples under the studied conditions. 230 OF 216 CT ED PR O 215 Spiked concentration (␮g/ml) Recovery (%) (mean ± S.D.) Mean ± S.D. 31.25 125 500 100.69 ± 2.00 107.45 ± 0.09 105.49 ± 1.07 104.55 ± 3.48 3.33 R.S.D. (%) S.D., standard deviation; R.S.D., relative standard deviation. For each sample n = 3. Fig. 3. Chromatograms of the P. regnellii extracts, (a) leaves; (b) stems and (c) roots. Chromatographic conditions: Metasil ODS column; mobile phase: acetonitrile/water (60:40, v/v) with 2% acetic acid; flow rate: 1.0 ml/min; temperature: 30 ◦ C; detection: 280 nm. The extracts demonstrate the same chromatographic profile, but there were differences in the concentrations of neolignans according to Table 4, that shows the content of neolignans in the different parts of the plant. As can be seen, roots presented a higher concentration of conocarpan than the leaves and stems, but the difference was significant (p < 0.05) in relation to leaves. Leaves and roots presented higher content of eupomatenoid-5 Table 4 Quantification of neolignans in hydroethanolic extracts of the leaves, stem and root of P. regnellii by HPLC Material Conocarpan (␮g/ml) Eupomatenoid-6a (␮g/ml) Eupomatenoid-5a (␮g/ml) Leaves Stems Roots 71.88* ± 1.05 79.09 ± 1.46 84.32* ± 2.11 79.20* ± 1.10 60.28* ± 2.33 66.76 ± 3.16 81.55 ± 1.14 71.97* ± 5.41 85.97* ± 0.61 * a p < 0.05, n = 3. Calculated as conocarpan. PBA 5764 1–5 245 246 247 248 249 250 251 D.F. Felipe et al. / Journal of Pharmaceutical and Biomedical Analysis xxx (2006) xxx–xxx 257 258 259 260 261 262 263 264 [1] G.L. Pessini, B.P. Dias Filho, C.V. Nakamura, D.A.G. Cortez, Mem. Inst. Oswaldo Cruz. 98 (2003) 1115–1120. [2] P.J.C. Benevides, P. Sartorelli, M.J. Kato, Phytochemistry 52 (1999) 339–343. [3] P. Sartorelli, P.J.C. Benevides, R.M. Ellensohn, M.V.A.F. Rocha, P.R.H. Moreno, M.J. Kato, Plant Sci. 161 (2001) 1083–1088. [4] T.P.D. Souza, M.H. Holzschuh, M.I. Lionço, G.G. Ortega, P.R. Petrovick, J. Pharm. Biomed. Anal. 30 (2002) 351–356. [5] J. Ermer, J. Pharm. Biomed. Anal. 24 (2001) 755–767. [6] International Conference on Harmonization of Technical Requirements for the Registration of Pharmaceuticals for Human Use (ICH) Q2B, Validation of analytical procedures. Methodology, 1996. [7] W. Andlauer, M.J. Martena, P. Fürst, J. Chromatogr. A 849 (1999) 341–348. OF 256 The HPLC method developed allowed the detection and quantification of the neolignans conocarpan, eupomatenoid-5 and eupomatenoid-6 in the extracts of P. regnellii. The validation procedure demonstrated the method presented linearity, precision and accuracy in the range studied. This procedure confirms that developed technique affords reliable analysis of the neolignans and is appropriate for the quality control of extracts and phytopharmaceutical preparations produced with P. regnellii. Moreover, the validated method complies with regulatory requirements if the plant is to be used by the pharmaceutical industry. 265 CT ED PR O 255 4. Conclusion References RE 254 and eupomatenoid-6, respectively, and both showed significant difference (p < 0.05) in relation to stems. CO R 253 UN 252 5 PBA 5764 1–5 266 267 268 269 270 271 272 273 274 275 276 277 278 279