PHYTOCHEMICAL ANALYSIS Phytochem. Anal. 15, 241248 (2004) DETERMINATION OF FLAVONOIDS IN PASSIFLORA SPP.

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002.pca.778

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A HPTLC Densitometric Determination of Flavonoids from Passiora alata, P. edulis, P. incarnata and P. caerulea and Comparison with HPLC Method
Cntia A. M. Pereira,1 Janete H. Yariwake,1* Fernando M. Lanas,1 Jean-Nol Wauters,2 Monique Tits2 and Luc Angenot2
1 2

Universidade de So Paulo, Instituto de Qumica de So Carlos, Caixa Postal 780, 13560-970 So Carlos, SP, Brazil Universit de Lige, Institut de Pharmacie, Avenue de lHpital 1, B36, B-4000 Lige, Belgium

A high-performance thin layer chromatographic (HPTLC) method was developed in order to determine quantitatively the avonoids in leaves of Passiora alata, P. edulis, P. caerulea and P. incarnata. The content of orientin and isoorientin was determined, and the results were compared with those obtained using a quantitative HPLCUV method. The latter employed rutin as standard and was developed to analyse avonoid content from Passiora leaves for the purpose of ensuring the quality of Passiora phytomedicines. The results obtained using the two methods indicate that there are qualitative and quantitative differences in the avonoids of the reference Passiora species studied. The two methods were also employed to analyse commercial samples to illustrate their application in qualitative (ngerprint) and quantitative determination, demonstrating their feasibility in the quality control of avonoids from crude Passiora drugs and phytomedicines. The HPLC conditions used are also suitable for the quantitative analysis of aqueous extracts (Passiora infusions). Copyright 2004 John Wiley & Sons, Ltd.
Keywords: Highperformance thin layer chromatography; densitometry; high-performance liquid chromatography; avonoids; Passiora.

INTRODUCTION The unequivocal distinction among Passiora species is of utmost importance for assessing the quality of Passiora phytomedicines. Passiora incarnata L. (Passioraceae) is the main medicinal species in Europe; however, this species does not ourish in Brazils tropical climate. As an alternative to P. incarnata, the Brazilian Pharmacopoeia (Farmacopia Brasileira, 1977) recommends P. alata, which is often substituted for P. edulis (extensively cultivated in Brazil for its juice). A comparative pharmacological study of hydroethanolic extracts of P. alata and P. edulis leaves has shown that both extracts present anxiolytic activity. In addition, a correlation was reported between Passiora avonoids and the pharmacological action of these species (Petry et al., 2001). P. caerulea also merits study as a possible adulterant due to its use as a sedative in Argentina, a country that shares a common border with southern Brazil. The aforementioned Passiora species contain mostly apigenin and luteolin C-glycosylavones, which frequently occur as isomers (Pereira and Vilegas, 2000). These avonoids are important as they serve as quality markers for the species. The literature on P. incarnata
* Correspondence to: J. H. Yariwake, Universidade de So Paulo, Instituto de Qumica de So Carlos, Caixa Postal 780, 13560-970 So Carlos, SP, Brazil. Email: janete@iqsc.sc.usp.br Contract/grant sponsor: CNPq. Contract/grant sponsor: FAPESP. Contract/grant sponsor: University of So Paulos Post-graduate Deans Ofce.

also focuses on avone C-glycosides for assessment of the quality of Passiora herbal drugs or for the standardization of phytomedicines. For instance, several HPLC methods for analysing avonoids in P. incarnata have been reported (Quercia et al., 1978; Wagner et al., 1983; Pietta et al., 1986; Schmidt and Gonzlez Ortega, 1993). These methods either utilise C-glycosylavones as standards or the samples are subjected to acid hydrolysis prior to HPLC analysis. These HPLC procedures offer advantages over the spectrophotometric method (i.e., the method of the European Pharmacopoeia, 1996) because the latter is non-specic and therefore less accurate. On the other hand, HPLC-UV-photodiode array detection (PAD) has the great advantage of easily identifying avonoid peaks based on the characteristic UV prole of avonoids with two UV absorption peaks in the 240400 nm region (Mabry et al., 1970). Thus, HPLC ngerprint analysis may be a powerful tool for the quality control of raw plant material. Similarly, HPTLC (high-performance thin layer chromatography) may be an alternative technique, particularly in the analysis of crude plant extracts. An improvement over conventional TLC, HPTLC is an instrumental technique whereby special plates and instrumental resources for sampling are used and the quantitative evaluation of separations is aided by densitometry (Szepesi and Nyiredy, 1992). In comparison with HPLC, the greatest advantage of the HPTLC procedure is that it does not require extensive clean-up procedures of crude plant extracts, even for quantitative analysis. However, to the best of our knowledge, the literature contains reports only on the use of HPTLC for
Received 1 May 2003 Phytochem. Anal. 15: 241248 (2004) Revised 31 March 2004 Accepted 31 March 2004

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quantitative analyses of P. caerulea, using isoorientin as the marker (Pastene et al., 1997). The fact that only a few of the C-glycosylavones occurring in Passiora are commercially available as analytical standards (owing to the high costs involved, few suppliers and the commercial availability of only very small amounts) may hinder the development of the analytical method. The HPLC method presented herein proposes to solve this problem by analysing avonoids using rutin as the standard, since it is a stable and widely available commercial reference standard. A similar approach which, for the sake of simplicity, also employs rutin as a standard, was previously reported for HPLC quantitative analyses of Hypericum perforatum extracts, which contain a broad range of avonoids and other compounds (Brolis et al., 1998). However, this strategy is usually not reported in HPTLC methods. We describe here an analytical methodology for the HPTLC analysis (ngerprint and densitometry) of the avonoids in P. alata, P. edulis, P. caerulea and P. incarnata leaves. The results of our HPTLC analysis were compared with HPLC, and 11 commercial samples were also analysed in order to illustrate the advantages and disadvantages of the HPTLC and HPLC methods in the quality control of Passiora phytomedicines.

Preparation of standards. Stock solutions of rutin (Sigma-Aldrich, Steinheim, Germany) and isoorientin (Carl Roth, Karlsruhe, Germany) were prepared in methanol (analytical grade; Merck; Darmstadt, Germany) at 500 g/mL and orientin (Carl Roth, Karlsruhe, Germany) was prepared in methanol at 400 g/mL. HPTLC analysis of Passiora (reference samples). Chromatographic analyses of the extracts were performed on silica gel 60 F254 HPTLC plates (10 10 cm; Merck, Darmstadt, Germany). Aliquots (3 L) of each extract were applied on the plates as bands using an AS30 TLC applicator (Desaga, Heildelberg, Germany). Each 10 mm-wide band was separated from its neighbouring bands by a distance of 10 mm. The step volume was 5 L, applied at a rate of 10 s/L, with 10 s intervals between applications. Plates were developed in a TLC chamber previously saturated (1 h) using ethyl acetate:formic acid:water (82:9:9 v/v) as the mobile phase. The development length was 70 mm (development time approximately 30 min). After development, plates were dried and derivatisation was carried out by immersion of the plates (one stroke) in a solution of diphenylboric acid-2aminoethylester (100 mg) and PEG 400 (500 mg) in methanol (10 mL, Brasseur and Angenot, 1986), followed by drying in a cold draft. Densitometric evaluation of HPTLC plates. Plates containing methanol extracts and standards were scanned 1 h after derivatisation, using a CD60 densitometer (Desaga, Heidelberg, Germany) operated by software running on a personal computer under the following conditions: scanning mode, re-emission-uorescence (mercury lamp); measurement wavelength, 300 nm (emission cut-off lter 550 nm); positive signal; slit width, 0.04 mm; slit height, 6.0 mm; band optimisation mode; resolution 0.025 mm; number of measurements per position, 32; signal factor, 15. Peak area measurement was utilised. Calibration and response factor studies. Calibration curves were determined using four plots (1.5, 3.0, 4.5 and 6.0 g) for rutin; four plots (1.5, 3.0, 4.5 and 6.0 g) for isoorientin and four plots (1.2, 2.4, 3.6 and 4.8 g) for orientin; each plot applied in duplicate. The general HPTLC conditions were the same as those described for the analysis of Passiora samples. Aliquots (3, 6, 9, 12 L) of standard solutions (rutin, isoorientin and orientin) were applied to the plates. The bands were 5 mm wide and the distance between the middle of the bands was 9 mm, the step volume was 3 L, applied at a rate of 10 s/L at 10 s intervals. After HPTLC development, derivatisation was carried out as described for the analysis of Passiora samples. HPTLC analysis of commercial samples. Eleven samples purchased from Brazilian pharmacies were analysed. The general HPTLC analysis and derivatisation conditions were the same as those described for the analysis of Passiora reference samples. Aliquots (5 L) of each methanol extract from the commercial samples were applied to the plates. The bands were 7 mm wide, separated by a distance of 11 mm, and the step volume was 5 L, applied at a rate of 10 s/L at 10 s intervals. Photographic record. The plates were photographed 3 h after derivatisation, using a VD40 VideoDocumentation
Phytochem. Anal. 15: 241248 (2004)

EXPERIMENTAL Plant material Authenticated leaf samples of P. edulis Sims. and P. alata Dryander (Passioraceae) were supplied by Dr. Ana Maria Soares Pereira (Laboratory of Vegetal Biotechnology, UNAERP, Ribeiro Preto, SP, Brazil). Professor Cozimo Pizza (Salerno University, Italy) and CPQBA (UNICAMP, Campinas, Brazil) provided authenticated samples of P. incarnata L. leaves. An authenticated leaf sample of P. caerulea was supplied by Dr. Laura Meletti (Campinas Agronomical Institute, Campinas, SP, Brazil). Voucher specimens of all plant material employed in the study are archived in the Laboratory of Phytochemical Analysis (IQSC-USP, So Paulo, SP, Brazil) with reference numbers FITO-Pe-001/ 1998 (P. edulis), FITO-Pa-001/1999 (P. alata), FITOPi-001/1995 and FITO-Pi-001/1999 (P. incarnata), and FITO-Pc-001/2000 (P. caerulea), respectively. Plant material was dried at 35C for 48 h, powdered and ground; only particles between 0.5 and 1.0 mm were utilised for the extractions. The powdered plant material was stored in glass asks protected from light and humidity. Commercial samples were purchased in Brazilian drugstores and subjected to the same procedure as the reference samples of Passiora. HPTLC analysis Preparation of samples. A sample (500 mg) of leaves was reuxed with 10 mL of methanol for 10 min at 60C. The extract was ltered and concentrated under pressure until dry. Prior to the HPTLC analysis, 2.5 mL of methanol were added to the extracts, which were ltered (Millipore Durapore, Waters, Milford, MA, USA; 0.45 m).
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System (Desaga), at an excitation wavelength of 366 nm, and the data was processed by DocIt (Desaga) software running on a personal computer. HPLC analysis Preparation of samples. A sample (1.0 g) of leaves was stir-mixed with 10 mL of ethanol (Merck, Rio de Janeiro, Brazil) and water (puried using the Milli-Q system, Millipore, Milford, MA, USA, 2:1, v/v) for 24 h at room temperature. The extract was ltered and the solvent evaporated in a rotary evaporator under reduced pressure at 40C until it was reduced to approximately 2 mL (Farmacopia Brasileira, 1977). A clean-up step was used to remove interfering materials, such as pigments (chlorophylls) etc.; the avonoids were extracted using solid-phase extraction (SPE) on C18 Sep-Pak cartridges (400 mg, Waters Associates, Milford, MA, USA). An appropriate volume of the sample (2 mL of extract) was drip-percolated through the cartridge, which was preconditioned with 5 mL of methanol (HPLC-grade from Mallinckrodt, Xalostoc, Mexico) and 5 mL of water. The avonoid fraction was eluted with 10 mL of methanol 60%, with the volume adjusted to 10 mL in a volumetric ask. This solution was directly injected into the HPLC system after being ltered through 0.45 m membranes (Millipore, Milford, MA, USA). Analytical HPLC method. The HPLC analyses were carried out at 35C on a Shimadzu (Kyoto, Japan) LC10AD liquid chromatograph equipped with a Supelco (Bellefonte, PA, USA) RP18 column (250 4.0 mm i.d.; 5 m) with a Supelco LC18 Supelguard guard column (2 cm 4.0 mm i.d.; 5 m). The samples were injected using a 10 L loop (Rheodyne Rohnet Park, CA, USA). The mobile phase consisted of solvent A [2.0% formic acid (99%; Carlo Erba, Milano, Italy) in water] and solvent B [HPLC grade acetonitrile (Mallinckrodt, Xalostoc, Mexico)]. The separation was performed using gradient elution: 010 min 15% B in A, 1040 min 1530% B in A and 4050 min 3015% B in A. The ow-rate was 0.8 mL/min. A Shimadzu SPD-M10A photodiode array detector was used at 337 nm for detection. The data were processed on a Shimadzu LC WorkStation Class LC-10 System. Method validation. Samples for validation analyses were obtained by the same procedure as described for plant analyses. A rutin solution in three different concentrations was added to avonoid fractions of P. edulis to evaluate the methods performance. The method was validated for precision, accuracy, limit of detection and of quantication, linearity, range and sensitivity. Intra- and inter-day variability measurements were used to determine the methods precision and accuracy. The intra-day precision of ve individual samples was examined on one day, and inter-day precision was determined on ve independent days. The intra- and inter-day accuracy was determined by the same procedure. Linearity was determined using rutin, vitexin, isoorientin and orientin reference standards. Linearity of response was determined at ve levels of concentration (ranging from 50 to 250 mg avonoid/L), each level in triplicate.
Copyright 2004 John Wiley & Sons, Ltd.

In order to determine the accuracy of the method, known amounts of rutin were added in three concentrations (50, 150 and 250 mg/L) to P. edulis leaves prior to extracting the avonoids. Recovery rates were calculated by the following equation:

Average of the amount of rutin quantied in the sample R(%) = 100 Average of the amount of rutin added to the sample
Quantitative analysis of total avonoids. The total avonoids were determined by an external standard method, using rutin, vitexin, isoorientin and orientin as references. The standard solutions were prepared in methanol (50250 mg/L). The total avonoid content of each Passiora sample was found by adding up all areas (normalised) of the peaks identied as originating from avonoids (peaks with UV-PAD spectra max around 254 and 337 nm; Harborne, 1988). The response factors were calculated from the peak area (mAbs) divided by the corresponding concentration (62.5 mg/L) of each avonoid (vitexin, orientin and isoorientin), and rutin was chosen arbitrarily as the reference (response factor = 1.00).

RESULTS AND DISCUSSION HPTLC ngerprint analysis Four different mobile phases previously described for the separation of avonoids were tested, using silica gel HPTLC plates, namely ethyl acetate:formic acid:water (6:1:1, v/v; Brasseur and Angenot, 1984), ethyl acetate:formic acid:acetic acid:water (100:11:11:26, v/v; Wagner and Bladt, 1996), ethyl acetate:methyl ethyl ketone:formic acid:water (50:30:10:10, v/v; European Pharmacopoeia, 1996), and ethyl acetate:formic acid: water (82:9:9, v/v; Pharmacopoeia Helvetica, 1990). The only phase that allowed us to visualise differences among the Passiora extracts studied was the mobile phase ethyl acetate:formic acid:water (82:9:9, v/v). The use of these solvent systems provides good separation of the avonoids with Rf above rutin (Rf = 0.36; Fig. 1). This mobile phase offers an improvement over the earlier method described by Brasseur and Angenot (1984) and may replace the one described in the last edition of the European Pharmacopoeia (1996), since the mobile phase (ethyl acetate:methyl ethyl ketone:formic acid:water; 50:30:10:10, v/v) proposed in that method does not allow for very efcient separation and characterisation of Passiora avonoids. Isoorientin, orientin, isovitexin and vitexin are avonoids known to be present in Passiora species (Pereira and Vilegas, 2000). Isoorientin and orientin (Rf = 0.45 and 0.65, respectively) were found in all the species studied (Fig. 1). The vitexin (Rf = 0.73) band was not identied in P. alata or P. edulis, but appeared as a pale band in P. caerulea extract. The HPTLC analysis also showed in P. alata extract a yellow uorescent band (Rf = 0.23) followed by a green uorescent band (Rf = 0.30) identied as vitexin 2-O-rhamnoside. These two bands are characteristic of P. alata, the latter being a very
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HPTLC densitometric evaluation The best scanning conditions were chosen using P. incarnata (the Brazilian sample). The range 200400 nm (with intervals of 20 nm) was monitored, with the cutoff lter set at 370, 420, 450 and 550 nm. Because the Passiora extract ngerprint is highly complex, and due to the difculty of meeting commercial standards for Cglycosylavonoids, the method with detection at 300 nm and cut-off lter at 550 nm, positive signal, which corresponds to the maximum intensity of uorescence and a condition selective for the uorescence yellow bands corresponding to luteolin derivatives was chosen as the best condition. The densitometric scan of each extract was therefore performed under the specied conditions and the quantication accomplished as isoorientin and orientin (identied in the samples at Rf 0.45 and 0.65, according to Fig. 1). All these avonoids displayed a linear response, with values of r 2 > 0.99 and the intercept of the y-axis close to zero (Table 1). Because isoorientin and orientin are commercially less accessible avonoids than rutin, the total avonoid content was also calculated, using the analytical curve for rutin and correcting the results based on the response factor for each avonoid. The results obtained for isoorientin and orientin by the two procedures are given in Table 2. Students t-test results showed that the application of the response factor can give a good indication avonoid content of the species if a laboratory does not have the analytical standards of C-glycosylavonoids. The isoorientin content in P. incarnata (Brazilian sample) was found to be three times higher than the P. incarnata (Italian sample), while the orientin content was four times higher in P. incarnata (Brazilian sample) than in P. incarnata (Italian sample). These differences suggest the inuence of the cultivation and climate on the avonoid content of the species. P. edulis displayed the highest isoorientin content, while P. caerulea and P. incarnata (Brazilian samples) showed the highest orientin content, with the latter showing statistically similar results. HPLC analysis Optimisation of chromatographic conditions. Optimisation was performed to identify a single chromatographic condition that would serve to analyse all the Passiora samples under study. To this end, the HPLC conditions were optimised using the avonoid fraction of P. edulis and monitoring the separation performance parameters (N, H, Rs, k, ). An earlier test had yielded better results (narrower peaks) with acetonitrile than with

Figure 1. Photographic record of HPTLC plate treated with diphenylboric acid-2-aminoethylester-PEG 400 reagent (excitation = 366 nm) of crude methanolic extracts and standards. For detailed chromatographic protocol see the Experimental section. Lane number, from left to right: 1, P. edulis, 2, P. alata; 3, standard mixture: rutin (Rf = 0.36), isoorientin (Rf = 0.45), orientin (Rf = 0.65), vitexin (Rf = 0.73); 4, P. incarnata (Brazilian sample); 5, P. caerulea.

important characteristic of the chromatographic prole of this species. The blue bands in P. alata and also in P. edulis extracts indicated the presence of phenolic carboxylic acids, such as caffeic acid, which occurs frequently in herbs containing avonoids (Wagner and Bladt, 1996). The sample of Brazilian P. incarnata displayed two very close bands at Rf = 0.42, unlike the Italian sample, which showed the four bands considered by Brasseur and Angenot (1984) and by the European Pharmacopoeia (1996) as a characteristic ngerprint of P. incarnata: yellow/green/yellow/green, being, respectively, isoorientin, isovitexin (Rf = 0.57), orientin and vitexin. The comparative analysis of the samples demonstrated the difculty of the choice of a single marker for Passiora. All the species revealed the presence of isoorientin and orientin, indicating that the choice of one of them as a marker may lead to a false result, since its presence does not conclusively identify the species. Among the species studied here, P. alata was the only one that contained vitexin-2-O-rhamnoside, which could be proposed as a marker for differentiating this species. A practical problem in routine analysis, however, may be the commercial availability of this avonoid.

Table 1. HPTLC calibration curves and response factors using different analytical standards of avonoids
Standard Rutin Isoorientin Orientin
a b

Mass rangea (g) 1.56.0 1.56.0 1.24.8

Calibration curves (y = ax + b)

r2
0.9922 0.9997 0.9956

Response factorb 1.00 0.42 0.22

y = 5.3.101x + 1.9.101 y = 1.3.102x 1.1.102 y = 2.4.102x 4.7.101

Mass calculated by each spot of calibration curve. Rutin was arbitrary chosen as reference (response factor = 1.00).
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Table 2. Content of isoorientin and orientin in Passiora reference samples using HPTLC method
Isoorientin content (g SD)a Quantication standard Species Isoorientin 12.65 0.47A 4.21 0.41B 7.92 0.48C 25.13 0.19D 14.29 0.05F Rutinb 11.56 0.47A 3.16 0.41B 6.85 0.48C 23.99 0.19E 13.20 0.05G Orientin content (g SD)a Quantication standard Orientin 4.74 0.64H 0.91 0.15I 0.21 0.01J 2.38 0.09K 4.88 0.02L Rutinc 4.43 0.64H 0.63 0.15I Not detectedJ 2.08 0.09K 4.56 0.02L

P. incarnata (Brazil) P. incarnata (Italy) P. alata P. edulis P. caerulea

a

SD, standard deviation (n = 3). Mean values followed by the same letter do not differ from each other (Students t-test, p = 0.05). b Values corrected using the response factor of isoorientin (Table 1). c Values corrected using the response factor of orientin (Table 1).

Figure 2. HPLC-UV/DAD chromatograms of avonoids extracts from: (A) P. incarnata, (B) P. alata, (C) P. caerulea and (D) P. edulis. Detection at 337 nm. (For chromatographic protocol see the Experimental section.)

methanol, and because of the complexity of the extracts, only a gradient programming of acetonitrile: 2% aqueous formic acid successfully separated the avonoid peaks and suppressed tailing. The ow rate was also optimised, with the condition of 0.8 mL/min showing the highest efciency (N values), without signicant peak broadening or increase in the duration of the analysis. The 337 nm wavelength (B band characteristic for these avonoids) was chosen because it proved highly sensitive to Passiora avonoids. The chromatograms in Fig. 2 reveal differences among the Passiora species studied here; hence, this method may be useful for ngerprint quality control of phytomedicines. Of the species studied, P. caerulea and P. edulis showed fewer polar avonoids (tR > 30 min). P. incarnata and P. alata displayed peaks up to 25 min, suggesting the predominance of glycosylated avonoids, some with similar tR. P. caerulea showed the most complex avonoid mixture, with peaks eluting at very close tR. This
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nding suggests that the structures of some avonoids can be very similar (they may be isomers). Quantitative analysis of total avonoids The HPLC method was carefully evaluated according to its analytical characteristics in order to evaluate its performance, using rutin as a standard. Procedures for the assessment of the quality of phytomedicines require that analytical parameters such as linearity, accuracy, precision, limit of detection (LOD) and limit of quantitation (LOQ) must be determined. For statistical recovery data, rutin was spiked in P. edulis extract and the relative standard deviation (RSD) was calculated and used as a measurement of the methods precision (Table 3). The data obtained demonstrated that the analytical method employed provided acceptable levels of precision (<5% RSD). Recovery data were obtained at three
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Table 3. Statistical recovery data found with P. edulis extracts spiked with rutin and HPLC validation parameters
Rutin concentration (mg/L) 50 150 250
a b

Recovery % 98.5 4.5 95.6 7.2 95.4 10.6

Intraday precisiona RSD (%) 2.7 2.3 0.7

Interday precisiona RSD (%) 4.4 2.2 1.4

Interday accuracyb (%) 124.7 4.5 105.9 2.1 92.9 0.5

Intraday accuracyb (%) 124.5 2.7 108.3 2.2 91.9 0.6

n = 5; RSD: relative standard deviation. Calculated as (mean analysed concentration)/(nominal concentration) 100.

Table 4. HPLC calibration curve and sensitivity data found using different analytical standards (external standard method)
Standard Rutin Vitexin Isoorientin Orientin
a b c

Curve equation (y = ax + b) 1.9.104x 2.3.105 2.4.104x 2.9.105 2.3.104x 2.5.105 2.1.104x 1.1.106

r2
0.9988 0.9994 0.9993 0.9997

Response factora 1.00 0.53 0.44 0.63

LODb (mg/L) 0.46 0.10 0.42 0.08 0.47 0.12 0.47 0.11

LOQc (mg/L) 1.37 0.04 1.41 0.03 1.39 0.04 1.39 0.05

Concentration range (mg/L) 1.371000 1.411000 1.391000 1.391000

Rutin was arbitrary chosen as reference (response factor = 1.00). Limit of detection (signal-to-noise = 3). Limit of quantication (signal-to-noise = 10).

Table 5. Content of total avonoidsa in Passiora samples using HPLC method

Quantication standardsb Species Rutin 11.25 1.54A 11.05 3.84A 15.94 2.16B 16.00 1.63B 19.81 0.11 28.80 0.10 Vitexin 11.27 1.54A 11.08 3.84A 15.96 2.16B 16.03 1.63B Isoorientin 11.15 1.54A 10.95 3.84A 15.84 2.16B 15.90 1.63B Orientin 11.19 1.54A 10.99 3.84A 15.88 2.16B 15.94 1.63B

Passiora alata Passiora edulis Passiora incarnata Passiora caerulea

Aqueous extractsc Passiora alata Passiora edulis
a

Results are given expressed as (mg avonoid/g dried plant) SD. Mean values followed by the same letter do not differ from each other (n = 10; Students t-test, p = 0.05). b A calibration curve for each standard was plotted and results were expressed as the respective compound. c Aqueous extracts were prepared according to Farmaropoea Ufciale della Republica Italiana (1998) procedure (1.0 g of plant infused in 100 mL of boiling water).

concentrations levels and are useful to evaluate the accuracy of the analytical method proposed herein. The results found with Passiora extracts were quite satisfactory, remaining within the recommended range of 70120% (ICH, 1996), except at the lower concentration level (50 mg/L rutin). However, our previous experience with Passiora extracts (which are very complex matrices) shows that this deviation at lower levels from the recommendation does not affect the general performance of the method. Quantitative data obtained using rutin as quantication standard were compared with those obtained with vitexin, isoorientin and orientin. All the analytical standards utilised in the validation showed a linear response and the curves displayed values of r 0.999 with y-intercepts close to zero (Table 4). The LOQ was used as the lower range limit, while the upper limit was considered as the concentration at which no linear response
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was observed. Vitexin showed the highest sensitivity (ability to discriminate among similar concentrations of the analyte, indicated by the greatest inclination angle of the standards calibration curves). The comparison of quantitative data obtained using a rutin quantication standard with those obtained using vitexin, isoorientin and orientin revealed that the total avonoid content for the same species showed no signicant statistical difference (Table 5). In addition, these data showed that the total avonoid content of P. alata and P. edulis was very similar and signicantly lower than in the P. incarnata and P. caerulea samples. To study the matrix effects in the quantitative analysis, two analytical curves were compared, the rst using the P. edulis extracts to which rutin (which was absent from this extract) was added in different concentrations (50250 mg/L), and the second obtained from rutin in methanol. The quantication using the external standard
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method was applied to both curves and the results compared using the Students t-test, which revealed no statistic difference, i.e. the HPLC quantication of avonoids was not inuenced by the matrix. Our previous studies on analysis of pesticide residues by Highresolution gas chromatography-electron capture detector (HRGC-ECD) in Passiora extracts showed signicant matrix effects (Zuin et al., 2003). In order to evaluate the usual intake of avonoids from Passiora phytomedicines, the HPLC method was applied to aqueous extracts (infusions) prepared according to the procedure described in the Farmaropoea Ufciale della Republica Italiana (1998) from P. alata and P. edulis leaves. The avonoid ngerprint pattern, i.e. the entire spectrum of avonoids found in hydroalcoholic extract, was present also in the infusion. Quantitative data (Table 5) indicated higher values of avonoids per gram of dry plant in the infusions than in the hydroalcoholic extracts prepared using the same plant material. This was probably a consequence of the higher temperature of extraction, solvent volume and the higher solubility of the glycosylated avonoids in water than in ethanol:water. However, in summary, the HPLC method also proved to be feasible for the analysis of Passiora aqueous extracts. Comparison of the methods (HPTLC vs. HPLC) The HPLC and HPTLC methods, which were developed using reference Passiora samples, were applied to 11 commercial samples to evaluate the methods applicability in the quality control of Passiora phytomedicines. From the qualitative standpoint, only one commercial sample resembled P. alata. The main adulterant appeared to be P. edulis (four commercial samples), while the other samples showed no resemblance to any of the species studied, indicating another kind of adulterant. Using HPTLC analysis, isoorientin was found to be present in all but two samples; hence, its presence

was not considered a valid indicator of a samples authenticity. Table 6 summarises the results of analyses of commercial samples by HPLC and HPTLC and compares the results obtained from these techniques with microscopic (morphological) analyses of plant material. Microscopic analysis is a technique that can be used to validate the authenticity and quality of powdered Passiora material (Brasseur and Angenot, 1984; Pereira, 2002). Table 6 data show that HPTLC may be the best choice for ngerprint analysis, since identication of species was fully coincident with the morphological identication of authentic Passiora samples. In addition, the simplicity of HPTLC in the sample preparation step and the possibility of simultaneously analysing several samples in less time than by HPLC are important points that recommend HPTLC as the method of choice in routine ngerprint analyses. As for quantication, we have found that the selectivity of uorescence detection in HPTLC with densitometry may itself limit the application of this method in routine quantitative analyses of avonoids in complex mixtures such as Passiora extracts. As the uorescence emission of avonoids may occur in a broad colour spectrum after derivatisation (Brasseur and Angenot, 1986), their selective detection involves measuring the uorescence radiation emission of each spot and using distinct standards for calibration plots. Therefore, in the present work, HPTLC conditions for quantitative analysis were established selectively only for orientin and isoorientin (yellow uorescence spots detected at 300 nm; for details see Experimental section), because apigenin derivatives such as vitexin or isovitexin (green uorescence spots) were not available in our laboratory. In short, the availability (or unavailability) of avonoid standards may be a decisive factor for the applicability of the HPTLC method. On the other hand, the proposed HPLC method using rutin as standard for quantitative analyses has proven to be fully appropriate for the analysis of commercial

Table 6. Comparison of analysis of commercial samples of Passiorae herba using HPTLC and HPLC methods and by microscopical examination
Analytical method HPLC Total avonoids content b 9.78 0.09 10.51 0.02 10.28 0.03 b b b b 3.42 0.02 3.49 0.10 b microscopical examinationa () P. edulis P. edulis P. edulis () () () () P. edulis P. alata ()

Samples 1 2 3 4 5 6 7 8 9 10 11

HPTLC () P. edulis P. edulis P. edulis () () () () P. edulis P. alata ()

Identication

P caerulea (?) P. edulis P. edulis P. edulis () () () () P. edulis P. alata ()

() Samples not identied (not similar to Passiora). a Detailed morphological characteristics of each Passiora specie are given elsewhere (Pereira, 2002). b Only data from authenticated samples are given.
Copyright 2004 John Wiley & Sons, Ltd. Phytochem. Anal. 15: 241248 (2004)

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samples. Total avonoid content (expressed as rutin) of authentic commercial samples was found to range from 1.05 to 26.32 mg rutin/g dried plant (Table 6). Samples with higher avonoid content (samples 24) displayed values close to those of the Passiora reference samples (Table 5), but this wide range of results reveals the lack of standardisation of commercial samples. The HPTLC and HPLC methods described in this work proved mutually complementary, each offering advantages and limitations in the analysis of Passiora avonoids. The advantages of HPTLC (such as fast development of the method, good post-chromatographic visualisation and the fact that crude extracts can be analysed without requiring cleanup) indicate the potential of this method in routine ngerprint analyses for quality and authenticity validations of plant material, extracts and preparations containing Passiora in the phyto-

pharmaceutical industry. HPLC, however, plays an important role, particularly in the quantitative analysis of Passiora avonoids. The proposed HPLC method offers the advantage of using rutin (a widely available, low-cost analytical standard) with nal results statistically similar to those obtained with C-glycosylavone standards. It may therefore serve as an alternative HPLC method to the more specic ones previously reported. Acknowledgements
The authors wish to thank Dr. Ana Maria Soares Pereira (UNAERP), Professor Cozimo Pizza (Salerno University, Italy) and Dr. Laura Meletti (IAC) for supplying the plant material, CNPq and FAPESP (Brazilian research funding agencies) for their nancial support and fellowships, and the University of So Paulos Post-graduate Deans Ofce for their nancial support and fellowships granted.

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Phytochem. Anal. 15: 241248 (2004)