- Email: [email protected]
Simultaneous quantification of bergenin, catechin, and gallic acid from Bergenia ciliata and Bergenia ligulata by using thin-layer chromatography
ARTICLE IN PRESS Journal of Food Composition and Analysis 21 (2008) 496– 500
Contents lists available at ScienceDirect
Journal of Food Composition and Analysis journal homepage: www.elsevier.com/locate/jfca
Original Article
Simultaneous quantification of bergenin, catechin, and gallic acid from Bergenia ciliata and Bergenia ligulata by using thin-layer chromatography K. Dhalwal, V.M. Shinde , Y.S. Biradar, K.R. Mahadik Department of Pharmacognosy, Poona College of Pharmacy, Bharati Vidyapeeth University, Erandwane-Kothrud, Pune 411038, Maharashtra, India
a r t i c l e in f o
a b s t r a c t
Article history: Received 30 August 2007 Received in revised form 19 February 2008 Accepted 23 February 2008
Bergenia ciliata Sternb. and Bergenia ligulata Wall. (Saxifragaceae) are reputed drugs of Ayurveda, commonly known as Paashaanbhed. A simple TLC method has been developed for the simultaneous quantification of bergenin, catechin, and gallic acid from different parts of B. ciliata and B. ligulata using HPTLC plate precoated with silica gel 60 F254. The method was developed in toluene:ethyl acetate:formic acid (4:6:1, v/v) and validated in terms of precision, repeatability, and accuracy. The linearity range for bergenin, catechin and gallic acid were found to be 160–800, 160–480 and 160–560 ng/spot respectively with correlation coefficients of 0.999, 0.999 and 0.999, respectively, which were indicative of good linear dependence of peak area on concentration. The method permits reliable quantification and showed good resolution and separation from other constituents of extract. Accuracy of the method was checked by conducting recovery studies at three different levels for all the three marker compounds and the average percentage recoveries were found to 99.29%, 98.66%, and 99.23%, respectively. The rhizomes were found to contain higher concentration of bergenin, catechin, and gallic acid than other parts of the plants. The proposed method was found to be simple, precise, specific, sensitive, and accurate. It can be used for routine quality control of herbal material and formulations containing bergenia species. & 2008 Elsevier Inc. All rights reserved.
Keywords: Bergenia ligulata Bergenia ciliata Bergenin Catechin Gallic acid Thin-layer chromatography
1. Introduction Bergenia is a genus of 10 species of flowering plants in the family Saxifragaceae. It is native to central Asia and found in Afghanistan, China and in Himalaya. Bergenia ciliata Sternb. and Bergenia ligulata Wall., commonly known as Paashaanbhed in the Indian Systems of Medicine, are used as a tonic for the treatment of fevers, diarrhea, and pulmonary affections (Nadkarni, 2000). Traditionally its rhizomes are used in Ayurvedic formulations. It has a high reputation in indigenous systems of medicine for dissolving stones in the kidney (Asokar et al., 1992). Some of the major chemical constituents of this genus include bergenin (Jain and Gupta, 1962), (+)-afzelechin (Tucci et al., 1969), leucocyanidin, gallic acid, methyl gallate, catechin (Dix and Srivastava, 1989), and paashaanolactone (Chandrareddy et al., 1998). Phenolic compounds of natural origin have showed significant biological activities including bactericidal activity, inhibition of HIV replication, free radical scavenging activity, cytotoxic activity, gastric protective action and anti-inflammatory activity. The antilithic
Corresponding author. Tel.: +91 20 2543723, +91 20 25389728; fax: +91 20 25439383. E-mail address: [email protected] (V.M. Shinde).
0889-1575/$ - see front matter & 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jfca.2008.02.008
property of the crude extract has been investigated and it was concluded that B. ligulata extract had no effect in preventing stone formation in rats but was significantly beneficial in dissolving preformed stones (Garimella et al., 2001). Alcohol extract of B. ligulata exhibits significant anti-inflammatory, analgesic, and diuretic properties (Gehlot et al., 1976). The anti-inflammatory potential of B. ciliata was reported using two acute rat models (carrageenan and serotonin (5-HT)-induced rat paw oedema) and a chronic rat model (cotton pouch-induced granuloma). The methanol extract of B. ciliata exhibited significant anti-inflammatory potential by inhibition of 31.471.09% in granuloma weight at the dose level of 300 mg/kg (Sinha et al., 2001a). The methanol extract of the rhizome of B. ciliata at doses of 100, 200, and 300 mg/kg also showed significant inhibition of cough reflex by 28.7%, 33.9%, and 44.2%, respectively (Sinha et al., 2001b). Several chromatographic methods have been reported for the quantification and standardization of these potentially bioactive plants (Singh et al., 2007; Srivastava and Rawat, 2007; Umashankar et al., 1999). Quantitative analysis aims to separate and identify the marker compounds from herbs or herbal preparations and then uses them as indicators or standards to assess quality. In the last two decades, high-performance thinlayer chromatography emerged as an efficient tool for the phytochemical evaluation of herbal drugs (Dhalwal et al., 2006;
ARTICLE IN PRESS K. Dhalwal et al. / Journal of Food Composition and Analysis 21 (2008) 496–500
Ravishankara et al., 2001; Wagner and Bladt, 1996). Considering the therapeutic importance of B. ciliata and B. ligulata, we developed a simple TLC densitometric method for the simultaneous quantification of bergenin, catechin, and gallic acid in different parts of the plant.
2. Materials and methods 2.1. Plant material The plant material of B. ciliata and B. ligulata was collected from the Moussauri hills of Uttar Pradesh, India in September 2006. The samples were authenticated by our Department botanist and voucher specimens were deposited in our Department of Pharmacognosy and Phytochemistry. The collected plant material was air dried under shade. Different parts of the plant were separated as rhizomes, petiole, and leaf and powdered to 40 mesh. Estimation of the active constituents was carried out on rhizomes, petiole, and leaf. 2.2. Chemicals All the chemicals used in the experiments were of analytical grade. Reference standard bergenin (purity 98%, w/w) and catechin (purity 98%, w/w) were purchased from Natural Remedies Pvt. Ltd., Bangalore, India. Gallic acid (purity 99%, w/w) was gift sample from Tetrahedron Ltd., India. 2.3. TLC conditions Spotting device: Linomat V Automatic sample spotter; CAMAG (Muttenz, Switzerland); Syringe: 100 ml Hamilton (Bonaduz, Switzerland); TLC Chamber: Glass twin trough chamber (20 10 4 cm3); CAMAG; Densitometer: TLC Scanner 3 is controlled by WinCats software; CAMAG; HPTLC plates: 20 10 cm2, 0.2 mm thickness precoated with silica gel 60 F254; E. Merck KgaA, Cat. no. 1.05548 (Darmstadt, Germany); Experimental conditions: temperature 2572 1C, relative humidity 40% and Solvent system: toluene:ethyl acetate:formic acid (4:6:1, v/v). 2.4. Sample preparation Dried powder of rhizomes, petiole, and leaf (2.5 g) were extracted exhaustively with methanol (3 50 ml) separately under reflux for 1 h on water bath. The extracts were filtered, concentrated and their volume was made up to 25 ml in volumetric flasks with methanol. These solutions were subjected to TLC for simultaneous estimation of bergenin, catechin, and gallic acid. 2.5. Preparation of standard solution The stock solutions of bergenin, catechin and gallic acid (160 mg/ml) each were prepared in methanol. The stock solutions were quantitatively transferred into a 10 ml volumetric flask to give solution of appropriate concentration range of bergenin (16–80 mg/ml), catechin (16–48 mg/ml), and gallic acid (16–56 mg/ml) and made to volume with methanol. Standard solutions were prepared by dilution of the stock solution. 2.6. Calibration curves Standard solution (10 ml) of bergenin (16–80 ng/ml), catechin (16–48 ng/ml), and gallic acid (16–56 ng/ml) were applied in
497
triplicate on precoated silica gel 60 F254 HPTLC plates (E. Merck) of uniform thickness of 0.2 mm. The plates were developed in a solvent system of toluene:ethyl acetate:formic acid (4:6:1, v/v) in CAMAG twin trough chamber up to a distance of 8 cm. After development, the plate was dried in air and scanned at 280 nm using absorbance reflectance mode by CAMAG Scanner 3 and WINCATS software for bergenin, catechin, and gallic acid. lmax of all the three marker compounds fall in the range of 278–282 nm, hence 280 nm was chosen for scanning the three markers. The peak areas were recorded. Respective calibration curves were prepared by plotting peak area vs. concentration of bergenin, catechin, and gallic acid applied. 2.7. Simultaneous quantification of bergenin, catechin, and gallic acid in different part of B. ciliata and B. ligulata Suitably diluted sample solutions (10 ml) were applied in triplicate on a precoated HPTLC plate with the CAMAG Linomat V Automatic Sample Spotter. The band length was 6 mm and the space between two bands was 6 mm. The plate was developed and scanned at 280 nm. The peak areas and absorption spectra were recorded. To check the identity of the bands UV absorption spectrum of each standard was overlayed with the corresponding band in the sample track. Overlaying the absorption spectra at start, middle and end position of the band checked the purity of the bands in the sample extract. The amount of bergenin, catechin, and gallic acid in the sample was calculated using the respective calibration curves. 2.8. Method validation ICH guidelines were followed for the validation of the analytical procedure (CPMP/ICH/381/95; CPMP/ICH/281/95). The method was validated for precision, repeatability, and accuracy. Precision of the method was checked by repeated scanning (n ¼ 7) of the same spot of bergenin, catechin and gallic acid seven times each. The repeatability of sample application and measurement of peak area were expressed in terms of %CV. Variability of the method was studied by intra-day precision and inter-day precision. Robustness of the method was studied at three different concentration levels 320, 480, and 640 ng/band for bergenin, 240, 320, and 400 ng/band for catechin and 320, 400, and 480 ng/band for gallic acid. In order to estimate the limit of detection (LOD) and limit of quantification (LOQ), blank methanol was spotted to determine signal-to-noise ratio. LOD was considered as 3:1 and LOQ as 10:1. LOD and LOQ were experimentally verified by diluting known concentrations of bergenin catechin and gallic acid until the average responses were approximately three or ten times the standard deviation of the responses for six replicate determinations. Accuracy of the method was tested by performing recovery studies at three levels (50%, 100%, and 125% addition). For the determination of LOD and LOQ, different dilutions of the standard solutions of bergenin, catechin, and gallic acid were applied along with methanol as the blank and determined on the basis of signal-to-noise ratio.
3. Results and discussion In the present study, we quantified three marker compounds viz. bergenin, catechin and gallic acid in different parts of B. ciliata and B. ligulata by TLC densitometric method using silica gel HPTLC. The developed method was validated as per the ICH guidelines (Tables 1–3). Mobile phase composition was optimized by introducing small changes in the composition of toluene:ethyl
ARTICLE IN PRESS 498
K. Dhalwal et al. / Journal of Food Composition and Analysis 21 (2008) 496–500
acetate:formic acid. It was found that bergenin, catechin and gallic acid resolved well at RF 0.29, 0.54, and 0.60 (Figs. 1 and 2) respectively in the solvent system of toluene:ethyl acetate:formic acid (4:6:1) from other components of the sample extract. The identity of the band for bergenin, catechin, and gallic acid in the sample extract was confirmed by overlaying their UV absorption spectra with those of the respective reference standards using CAMAG TLC Scanner 3 with WINCATS software. The purity of each of these bands in the sample extract was confirmed by comparing the absorption spectra recorded at start, middle and end positions of the band (Fig. 3).
and correlation coefficients for the references were [y ¼ 5.552x+554.045] (R2 ¼ 0.9994) for bergenin, [y ¼ 11.293x142.462] (R2 ¼ 0.9994) for catechin, and [y ¼ 10.375x+203.625] (R2 ¼ 0.9996) for gallic acid. 3.2. Instrumental precision and inter-day and intra-day precision Instrumental precision was checked by repeated scanning of the same spot of bergenin (320 ng) catechin (240 ng) and gallic acid (320 ng) seven times each. The repeatability of sample application and measurement of peak area were expressed in terms of coefficient of variance (%CV) and found to be 0.74 and
3.1. Linearity A good linearity was achieved in the concentration ranges of 160–800 ng/spot for bergenin, 160–480 ng/spot for catechin, and 160–560 ng/spot for gallic acid. The regression equations
700.0 600.0
Bergenin Std.
500.0 Table 1 Method validation parameters for the quantification of bergenin, catechin, and gallic acid
Catechin Std.
Gallic acid Std.
400.0 300.0
Parameters
Bergenin
Catechin
Gallic acid
200.0 Precision (%CV) Repeatability of application (n ¼ 7) Repeatability of measurement (n ¼ 7) Limit of detection (ng) Limit of quantification (ng) Specificity Linearity (correlation coefficient) Range (ng/spot) Robustness
0.74 0.59 120 160 Specific 0.999 160–800 Robust
0.90 0.68 80 120 Specific 0.999 160–480 Robust
0.88 0.98 40 80 Specific 0.999 160–560 Robust
100.0
B.ciliata Extract
0.0 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 [Rf ] Fig. 1. TLC densitometric chromatograms of Bergenia ciliata rhizome extract, bergenin standard, catechin standard, and gallic acid standard.
Table 2 Intra- and inter-day precision of HPTLC method (n ¼ 6) Marker compound
Amount (ng/spot)
Intra-day precision
Inter-day precision
S.D. of areas
R.S.D. (%)
S.E.
S.D. of areas
R.S.D. (%)
S.E.
Bergenin
320 480 640
1.54 2.06 1.80
0.02 0.07 0.04
0.91 0.46 0.17
2.30 3.10 1.15
0.06 0.04 0.08
0.83 0.98 0.29
Catechin
240 320 400
1.90 1.71 1.32
0.08 0.06 0.04
0.24 0.45 0.34
2.09 3.00 2.31
0.02 0.07 0.11
0.98 0.74 1.03
Gallic acid
320 400 480
2.60 1.92 2.40
0.02 0.11 0.09
0.39 0.94 0.83
2.04 1.75 2.01
0.05 0.02 0.09
0.81 1.05 0.75
Table 3 Recovery study of bergenin, catechin, and gallic acid from Bergenia ciliata rhizome extract by HPTLC method Marker compound
Amount present in the sample (mg)
Amount added (mg)
Amount founda (mg)
Recoverya (%)
Average recovery (%)
Bergenin
720
360
1076.3378.08
99.7970.99
99.29
Catechin
720 720 68
720 900 34
1430.6675.13 1604.3376.23 100.1171.60
99.0870.75 99.0070.53 98.0370.91
98.66
68 68 24 24 24
68 85 12 24 30
133.6971.52 152.1672.71 35.3071.25 47.3571.52 53.4671.50
98.5270.52 99.4471.60 99.2371.82 98.9871.78 99.4872.04
Gallic acid
a
Mean7standard deviation (S.D., n ¼ 3).
99.23
ARTICLE IN PRESS K. Dhalwal et al. / Journal of Food Composition and Analysis 21 (2008) 496–500
0.59 for bergenin, 0.90 and 0.68 for catechin, 0.88 and 0.98 for gallic acid, respectively (Table 1). Standards of bergenin (320, 480 and 640 ng/spot), catechin (240, 320 and 400 ng/spot) and gallic acid (320, 400 and 480 ng/spot) were spotted both at intra-day (spotting each concentration five times within 24 h) and inter-day (spotting each concentration four times during 5 days interval separated by at least 24 h) intervals to check the precision. The results were shown in Table 2. The results were expressed as % relative standard deviation (%R.S.D.) and standard error (S.E.) that indicated high precision.
700.0 600.0
Bergenin Std.
500.0
Catechin Std. Gallic acid Std.
400.0 300.0 200.0 100.0
B. ligulata Extract
3.3. Recovery
0.0 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 [Rf ]
The recovery was used to evaluate the accuracy of the method. The percent recovery as well as average percent recovery was calculated. Recovery studies at three different levels were done on B. ciliata rhizomes extract by accurately spiked with various
Fig. 2. TLC densitometric chromatograms of Bergenia ligulata rhizome extract, bergenin standard, catechin standard, and gallic acid standard.
100.0
100.0
90.0
90.0
80.0
80.0
70.0
70.0
60.0
60.0 [AU]
[AU]
499
50.0
50.0
40.0
40.0
30.0
30.0
20.0
20.0
10.0
10.0
0.0 200.0
250.0
300.0 [nm]
350.0
400.0
0.0 200.0
250.0
300.0 [nm]
350.0
400.0
100.0 90.0 80.0 70.0
[AU]
60.0 50.0 40.0 30.0 20.0 10.0
250.0
350.0
300.0
400.0
[nm] Fig. 3. Overlay of UV absorption spectra of the marker compounds in the sample track with respective standards: (A) bergenin, (B) catechin, and (C) gallic acid.
ARTICLE IN PRESS 500
K. Dhalwal et al. / Journal of Food Composition and Analysis 21 (2008) 496–500
Table 4 Marker compounds quantified from different parts of Bergenia ciliata and Bergenia ligulata using HPTLC method Sample tested
Content of marker compounda (% w/w) Bergenin
Catechin
Gallic acid
B. ciliate Rhizomes Petiole Leaf
0.72070.021 0.12570.019 0.14170.015
0.06870.041 0.02570.009 0.01570.010
0.02470.011 0.00870.008 0.01470.016
B. ligulata Rhizomes Petiole Leaf
0.79170.014 0.09070.022 0.11570.010
0.07070.017 0.02170.012 0.00970.014
0.03070.017 0.00770.032 0.01070.012
a
Mean7S.D., n ¼ 3.
concentrations of reference solutions just prior to the extraction. The percentage recovery at three different levels for bergenin was found to be 99.79%, 99.08%, and 99.00% with an average of 99.29%, catechin was found to be 98.03%, 98.52%, and 99.44% with an average of 98.66% and gallic acid was found to be 99.23%, 98.98%, and 99.48% with an average of 99.23%. The results were shown in Table 3. 3.4. Limits of detection (LOD) and limits of quantification (LOQ) Serial dilutions of bergenin, catechin, and gallic acid were analyzed by TLC method. The LOD and LOQ were obtained with the signal-to-noise ratio of 3 and 10. LOD represents the lowest concentrations of bergenin, catechin, and gallic acid that can be detected, whereas the LOQ represents the lowest concentrations of bergenin, catechin and gallic acid that can be determined with acceptable precision and accuracy. The LOD and LOQ were found to be 120 and 160 ng/spot for bergenin, 80 and 120 ng/spot for catechin, 40 and 80 ng/spot for gallic acid, respectively. This indicated that the new method exhibited a good sensitivity for the quantification of bergenin, catechin and gallic acid from B. ciliata and B. ligulata. In order to obtain more accurate regression, the lower limit of linearity was adjusted to be higher than LOQ. The concentrations of bergenin, catechin, and gallic acid in sample solutions were within the range of linearity. 3.5. Quantitative determination All the samples were extracted, as described above and analyzed by TLC. The content of each compound was determined by the corresponding regression equation and results are summarized in Table 4. The results indicated that all three compounds were detected in six true samples, in which bergenin was obviously dominant. The densitograms of standards along with rhizome extract of B. ciliata and B. ligulata are represented in Figs. 1 and 2. B. ciliata and B. ligulata can be differentiated with there densitograms. Densitogram of B. ciliata showed two extra peaks as compared to B. ligulata whereas B. ligulata showed a peak at RF 0.21, which was not present in B. ciliata densitogram. Traditionally, its rhizomes are used in Ayurvedic formulations. The percentage amounts of bioactive constituent present in rhizome corroborate its use as compare to leaf and petiole (Table 4).
For the first time, a simple, accurate, and rapid TLC method was developed for the simultaneous determination of three major compounds from rhizome, petiole and leaf of B. ciliata and B. ligulata. The assay is proved to be accurate, reproducible, and sensitive. Furthermore, it has been successfully employed for the determination of these three compounds in different commercial samples. It was found that rhizome, petiole, and leaf samples of B. ciliata and B. ligulata show a similar but unique TLC chromatogram, thus making this method useful for the quality control of the crude drugs. 4. Conclusion We established TLC densitometric method for the simultaneous quantification of three bioactive compounds viz. bergenin, catechin, and gallic acid from different parts of B. ciliata and B. ligulata using TLC. The method was found to be simple, precise, specific, sensitive, and accurate and can be used for their quantification in the plant materials. It can be also used in routine quality control of herbal materials as well as formulations containing any or all of these compounds. References Asokar, L.V., Kakkar, K.K., Chakra, O.J., 1992. Glossary of Indian Medicinal Plants with Active Principles. Publication and Information Directorate, New Delhi, p. 122. Chandrareddy, U.D., Chawla, A.S., Mundkinajeddu, D., Maurya, R., Handa, S.S., 1998. Paashanolactone from Bergenia ligulata. Phytochemistry 47, 907–909. Dhalwal, K., Biradar, Y.S., Rajani, M., 2006. TLC densitometric method for simultaneous quantification of phyllanthin, hypophyllanthin, gallic acid and ellagic acid in Phyllanthus amarus using HPTLC. Journal of AOAC International 89, 619–623. Dix, B.S., Srivastava, S.N., 1989. Tannin constituents of Bergenia ligulata roots. Indian Journal of Natural Products 5, 24–25. Garimella, T.S., Jolly, C.I., Narayanan, S., 2001. In vitro studies on antilithiatic activity of seeds of Dolichos biflorus Linn. and rhizomes of Bergenia ligulata Wall. Phytotherapy Research 15, 351–355. Gehlot, N.K., Sharma, V.N., Vyas, D.S., 1976. Some pharmacological studies on ethanolic extract of Bergenia ligulata. Indian Journal of Pharmacology 8, 92. Jain, M.K., Gupta, K., 1962. Isolation of bergenin from Saxifraga ligulata Wall. Journal of Indian Chemical Society 39, 559–560. Nadkarni, K.M., 2000. The Indian Materia Medica. Popular Prakashan Pvt. Ltd., Mumbai, pp. 1198–1202. Ravishankara, M.N., Shrivastava, N., Padh, H., Rajani, M., 2001. HPTLC method for the estimation of alkaloids of Cinchona officinalis stem bark and its marketed formulations. Planta Medica 67, 294–296. Singh, D.P., Srivastava, S.K., Govindarajan, R., Rawat, A.K.S., 2007. High-performance liquid chromatographic determination of bergenin in different bergenia species. Acta Chromatographica 19, 246–252. Sinha, S., Murugesan, T., Maiti, K., Gayen, J.R., Pal, M., Saha, B.P., 2001a. Evaluation of anti-inflammatory potential of Bergenia ciliata Sternb. rhizome extract in rats. Journal of Pharmacy and Pharmacology 53, 193–196. Sinha, S., Murugesan, T., Pal, M., Saha, B.P., 2001b. Evaluation of anti-tussive activity of Bergenia ciliata Sternb. rhizome extract in mice. Phytomedicine 8, 298–301. Srivastava, S., Rawat, A.K.S., 2007. Simultaneous determination of bergenin and gallic acid in different Bergenia species. Journal of Planar Chromatography— Modern TLC 20 (4), 275–277. Tucci, P.A., Delle, M.F., Marini, B., Holo, B.G., 1969. Occurrence of (+)-afzelchin in Saxifraga ligulata. Annual 1st Super Sanita 5, 555–556. Umashankar, D.C.R., Chawla, A.S., Mundkinajeddu, D., Singh, D., Handa, S.S., 1999. High pressure liquid chromatographic determination of bergenin and (+)afzelechin from different parts of Paashaanbhed (Bergenia ligulata Yeo). Phytochemical Analysis 10, 44–47. Wagner, H., Bladt, S., 1996. Plant Drug Analysis: A Thin Layer Chromatography Atlas, second ed. Springer, Berlin, Germany, p. 359.
Contents lists available at ScienceDirect
Journal of Food Composition and Analysis journal homepage: www.elsevier.com/locate/jfca
Original Article
Simultaneous quantification of bergenin, catechin, and gallic acid from Bergenia ciliata and Bergenia ligulata by using thin-layer chromatography K. Dhalwal, V.M. Shinde , Y.S. Biradar, K.R. Mahadik Department of Pharmacognosy, Poona College of Pharmacy, Bharati Vidyapeeth University, Erandwane-Kothrud, Pune 411038, Maharashtra, India
a r t i c l e in f o
a b s t r a c t
Article history: Received 30 August 2007 Received in revised form 19 February 2008 Accepted 23 February 2008
Bergenia ciliata Sternb. and Bergenia ligulata Wall. (Saxifragaceae) are reputed drugs of Ayurveda, commonly known as Paashaanbhed. A simple TLC method has been developed for the simultaneous quantification of bergenin, catechin, and gallic acid from different parts of B. ciliata and B. ligulata using HPTLC plate precoated with silica gel 60 F254. The method was developed in toluene:ethyl acetate:formic acid (4:6:1, v/v) and validated in terms of precision, repeatability, and accuracy. The linearity range for bergenin, catechin and gallic acid were found to be 160–800, 160–480 and 160–560 ng/spot respectively with correlation coefficients of 0.999, 0.999 and 0.999, respectively, which were indicative of good linear dependence of peak area on concentration. The method permits reliable quantification and showed good resolution and separation from other constituents of extract. Accuracy of the method was checked by conducting recovery studies at three different levels for all the three marker compounds and the average percentage recoveries were found to 99.29%, 98.66%, and 99.23%, respectively. The rhizomes were found to contain higher concentration of bergenin, catechin, and gallic acid than other parts of the plants. The proposed method was found to be simple, precise, specific, sensitive, and accurate. It can be used for routine quality control of herbal material and formulations containing bergenia species. & 2008 Elsevier Inc. All rights reserved.
Keywords: Bergenia ligulata Bergenia ciliata Bergenin Catechin Gallic acid Thin-layer chromatography
1. Introduction Bergenia is a genus of 10 species of flowering plants in the family Saxifragaceae. It is native to central Asia and found in Afghanistan, China and in Himalaya. Bergenia ciliata Sternb. and Bergenia ligulata Wall., commonly known as Paashaanbhed in the Indian Systems of Medicine, are used as a tonic for the treatment of fevers, diarrhea, and pulmonary affections (Nadkarni, 2000). Traditionally its rhizomes are used in Ayurvedic formulations. It has a high reputation in indigenous systems of medicine for dissolving stones in the kidney (Asokar et al., 1992). Some of the major chemical constituents of this genus include bergenin (Jain and Gupta, 1962), (+)-afzelechin (Tucci et al., 1969), leucocyanidin, gallic acid, methyl gallate, catechin (Dix and Srivastava, 1989), and paashaanolactone (Chandrareddy et al., 1998). Phenolic compounds of natural origin have showed significant biological activities including bactericidal activity, inhibition of HIV replication, free radical scavenging activity, cytotoxic activity, gastric protective action and anti-inflammatory activity. The antilithic
Corresponding author. Tel.: +91 20 2543723, +91 20 25389728; fax: +91 20 25439383. E-mail address: [email protected] (V.M. Shinde).
0889-1575/$ - see front matter & 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jfca.2008.02.008
property of the crude extract has been investigated and it was concluded that B. ligulata extract had no effect in preventing stone formation in rats but was significantly beneficial in dissolving preformed stones (Garimella et al., 2001). Alcohol extract of B. ligulata exhibits significant anti-inflammatory, analgesic, and diuretic properties (Gehlot et al., 1976). The anti-inflammatory potential of B. ciliata was reported using two acute rat models (carrageenan and serotonin (5-HT)-induced rat paw oedema) and a chronic rat model (cotton pouch-induced granuloma). The methanol extract of B. ciliata exhibited significant anti-inflammatory potential by inhibition of 31.471.09% in granuloma weight at the dose level of 300 mg/kg (Sinha et al., 2001a). The methanol extract of the rhizome of B. ciliata at doses of 100, 200, and 300 mg/kg also showed significant inhibition of cough reflex by 28.7%, 33.9%, and 44.2%, respectively (Sinha et al., 2001b). Several chromatographic methods have been reported for the quantification and standardization of these potentially bioactive plants (Singh et al., 2007; Srivastava and Rawat, 2007; Umashankar et al., 1999). Quantitative analysis aims to separate and identify the marker compounds from herbs or herbal preparations and then uses them as indicators or standards to assess quality. In the last two decades, high-performance thinlayer chromatography emerged as an efficient tool for the phytochemical evaluation of herbal drugs (Dhalwal et al., 2006;
ARTICLE IN PRESS K. Dhalwal et al. / Journal of Food Composition and Analysis 21 (2008) 496–500
Ravishankara et al., 2001; Wagner and Bladt, 1996). Considering the therapeutic importance of B. ciliata and B. ligulata, we developed a simple TLC densitometric method for the simultaneous quantification of bergenin, catechin, and gallic acid in different parts of the plant.
2. Materials and methods 2.1. Plant material The plant material of B. ciliata and B. ligulata was collected from the Moussauri hills of Uttar Pradesh, India in September 2006. The samples were authenticated by our Department botanist and voucher specimens were deposited in our Department of Pharmacognosy and Phytochemistry. The collected plant material was air dried under shade. Different parts of the plant were separated as rhizomes, petiole, and leaf and powdered to 40 mesh. Estimation of the active constituents was carried out on rhizomes, petiole, and leaf. 2.2. Chemicals All the chemicals used in the experiments were of analytical grade. Reference standard bergenin (purity 98%, w/w) and catechin (purity 98%, w/w) were purchased from Natural Remedies Pvt. Ltd., Bangalore, India. Gallic acid (purity 99%, w/w) was gift sample from Tetrahedron Ltd., India. 2.3. TLC conditions Spotting device: Linomat V Automatic sample spotter; CAMAG (Muttenz, Switzerland); Syringe: 100 ml Hamilton (Bonaduz, Switzerland); TLC Chamber: Glass twin trough chamber (20 10 4 cm3); CAMAG; Densitometer: TLC Scanner 3 is controlled by WinCats software; CAMAG; HPTLC plates: 20 10 cm2, 0.2 mm thickness precoated with silica gel 60 F254; E. Merck KgaA, Cat. no. 1.05548 (Darmstadt, Germany); Experimental conditions: temperature 2572 1C, relative humidity 40% and Solvent system: toluene:ethyl acetate:formic acid (4:6:1, v/v). 2.4. Sample preparation Dried powder of rhizomes, petiole, and leaf (2.5 g) were extracted exhaustively with methanol (3 50 ml) separately under reflux for 1 h on water bath. The extracts were filtered, concentrated and their volume was made up to 25 ml in volumetric flasks with methanol. These solutions were subjected to TLC for simultaneous estimation of bergenin, catechin, and gallic acid. 2.5. Preparation of standard solution The stock solutions of bergenin, catechin and gallic acid (160 mg/ml) each were prepared in methanol. The stock solutions were quantitatively transferred into a 10 ml volumetric flask to give solution of appropriate concentration range of bergenin (16–80 mg/ml), catechin (16–48 mg/ml), and gallic acid (16–56 mg/ml) and made to volume with methanol. Standard solutions were prepared by dilution of the stock solution. 2.6. Calibration curves Standard solution (10 ml) of bergenin (16–80 ng/ml), catechin (16–48 ng/ml), and gallic acid (16–56 ng/ml) were applied in
497
triplicate on precoated silica gel 60 F254 HPTLC plates (E. Merck) of uniform thickness of 0.2 mm. The plates were developed in a solvent system of toluene:ethyl acetate:formic acid (4:6:1, v/v) in CAMAG twin trough chamber up to a distance of 8 cm. After development, the plate was dried in air and scanned at 280 nm using absorbance reflectance mode by CAMAG Scanner 3 and WINCATS software for bergenin, catechin, and gallic acid. lmax of all the three marker compounds fall in the range of 278–282 nm, hence 280 nm was chosen for scanning the three markers. The peak areas were recorded. Respective calibration curves were prepared by plotting peak area vs. concentration of bergenin, catechin, and gallic acid applied. 2.7. Simultaneous quantification of bergenin, catechin, and gallic acid in different part of B. ciliata and B. ligulata Suitably diluted sample solutions (10 ml) were applied in triplicate on a precoated HPTLC plate with the CAMAG Linomat V Automatic Sample Spotter. The band length was 6 mm and the space between two bands was 6 mm. The plate was developed and scanned at 280 nm. The peak areas and absorption spectra were recorded. To check the identity of the bands UV absorption spectrum of each standard was overlayed with the corresponding band in the sample track. Overlaying the absorption spectra at start, middle and end position of the band checked the purity of the bands in the sample extract. The amount of bergenin, catechin, and gallic acid in the sample was calculated using the respective calibration curves. 2.8. Method validation ICH guidelines were followed for the validation of the analytical procedure (CPMP/ICH/381/95; CPMP/ICH/281/95). The method was validated for precision, repeatability, and accuracy. Precision of the method was checked by repeated scanning (n ¼ 7) of the same spot of bergenin, catechin and gallic acid seven times each. The repeatability of sample application and measurement of peak area were expressed in terms of %CV. Variability of the method was studied by intra-day precision and inter-day precision. Robustness of the method was studied at three different concentration levels 320, 480, and 640 ng/band for bergenin, 240, 320, and 400 ng/band for catechin and 320, 400, and 480 ng/band for gallic acid. In order to estimate the limit of detection (LOD) and limit of quantification (LOQ), blank methanol was spotted to determine signal-to-noise ratio. LOD was considered as 3:1 and LOQ as 10:1. LOD and LOQ were experimentally verified by diluting known concentrations of bergenin catechin and gallic acid until the average responses were approximately three or ten times the standard deviation of the responses for six replicate determinations. Accuracy of the method was tested by performing recovery studies at three levels (50%, 100%, and 125% addition). For the determination of LOD and LOQ, different dilutions of the standard solutions of bergenin, catechin, and gallic acid were applied along with methanol as the blank and determined on the basis of signal-to-noise ratio.
3. Results and discussion In the present study, we quantified three marker compounds viz. bergenin, catechin and gallic acid in different parts of B. ciliata and B. ligulata by TLC densitometric method using silica gel HPTLC. The developed method was validated as per the ICH guidelines (Tables 1–3). Mobile phase composition was optimized by introducing small changes in the composition of toluene:ethyl
ARTICLE IN PRESS 498
K. Dhalwal et al. / Journal of Food Composition and Analysis 21 (2008) 496–500
acetate:formic acid. It was found that bergenin, catechin and gallic acid resolved well at RF 0.29, 0.54, and 0.60 (Figs. 1 and 2) respectively in the solvent system of toluene:ethyl acetate:formic acid (4:6:1) from other components of the sample extract. The identity of the band for bergenin, catechin, and gallic acid in the sample extract was confirmed by overlaying their UV absorption spectra with those of the respective reference standards using CAMAG TLC Scanner 3 with WINCATS software. The purity of each of these bands in the sample extract was confirmed by comparing the absorption spectra recorded at start, middle and end positions of the band (Fig. 3).
and correlation coefficients for the references were [y ¼ 5.552x+554.045] (R2 ¼ 0.9994) for bergenin, [y ¼ 11.293x142.462] (R2 ¼ 0.9994) for catechin, and [y ¼ 10.375x+203.625] (R2 ¼ 0.9996) for gallic acid. 3.2. Instrumental precision and inter-day and intra-day precision Instrumental precision was checked by repeated scanning of the same spot of bergenin (320 ng) catechin (240 ng) and gallic acid (320 ng) seven times each. The repeatability of sample application and measurement of peak area were expressed in terms of coefficient of variance (%CV) and found to be 0.74 and
3.1. Linearity A good linearity was achieved in the concentration ranges of 160–800 ng/spot for bergenin, 160–480 ng/spot for catechin, and 160–560 ng/spot for gallic acid. The regression equations
700.0 600.0
Bergenin Std.
500.0 Table 1 Method validation parameters for the quantification of bergenin, catechin, and gallic acid
Catechin Std.
Gallic acid Std.
400.0 300.0
Parameters
Bergenin
Catechin
Gallic acid
200.0 Precision (%CV) Repeatability of application (n ¼ 7) Repeatability of measurement (n ¼ 7) Limit of detection (ng) Limit of quantification (ng) Specificity Linearity (correlation coefficient) Range (ng/spot) Robustness
0.74 0.59 120 160 Specific 0.999 160–800 Robust
0.90 0.68 80 120 Specific 0.999 160–480 Robust
0.88 0.98 40 80 Specific 0.999 160–560 Robust
100.0
B.ciliata Extract
0.0 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 [Rf ] Fig. 1. TLC densitometric chromatograms of Bergenia ciliata rhizome extract, bergenin standard, catechin standard, and gallic acid standard.
Table 2 Intra- and inter-day precision of HPTLC method (n ¼ 6) Marker compound
Amount (ng/spot)
Intra-day precision
Inter-day precision
S.D. of areas
R.S.D. (%)
S.E.
S.D. of areas
R.S.D. (%)
S.E.
Bergenin
320 480 640
1.54 2.06 1.80
0.02 0.07 0.04
0.91 0.46 0.17
2.30 3.10 1.15
0.06 0.04 0.08
0.83 0.98 0.29
Catechin
240 320 400
1.90 1.71 1.32
0.08 0.06 0.04
0.24 0.45 0.34
2.09 3.00 2.31
0.02 0.07 0.11
0.98 0.74 1.03
Gallic acid
320 400 480
2.60 1.92 2.40
0.02 0.11 0.09
0.39 0.94 0.83
2.04 1.75 2.01
0.05 0.02 0.09
0.81 1.05 0.75
Table 3 Recovery study of bergenin, catechin, and gallic acid from Bergenia ciliata rhizome extract by HPTLC method Marker compound
Amount present in the sample (mg)
Amount added (mg)
Amount founda (mg)
Recoverya (%)
Average recovery (%)
Bergenin
720
360
1076.3378.08
99.7970.99
99.29
Catechin
720 720 68
720 900 34
1430.6675.13 1604.3376.23 100.1171.60
99.0870.75 99.0070.53 98.0370.91
98.66
68 68 24 24 24
68 85 12 24 30
133.6971.52 152.1672.71 35.3071.25 47.3571.52 53.4671.50
98.5270.52 99.4471.60 99.2371.82 98.9871.78 99.4872.04
Gallic acid
a
Mean7standard deviation (S.D., n ¼ 3).
99.23
ARTICLE IN PRESS K. Dhalwal et al. / Journal of Food Composition and Analysis 21 (2008) 496–500
0.59 for bergenin, 0.90 and 0.68 for catechin, 0.88 and 0.98 for gallic acid, respectively (Table 1). Standards of bergenin (320, 480 and 640 ng/spot), catechin (240, 320 and 400 ng/spot) and gallic acid (320, 400 and 480 ng/spot) were spotted both at intra-day (spotting each concentration five times within 24 h) and inter-day (spotting each concentration four times during 5 days interval separated by at least 24 h) intervals to check the precision. The results were shown in Table 2. The results were expressed as % relative standard deviation (%R.S.D.) and standard error (S.E.) that indicated high precision.
700.0 600.0
Bergenin Std.
500.0
Catechin Std. Gallic acid Std.
400.0 300.0 200.0 100.0
B. ligulata Extract
3.3. Recovery
0.0 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 [Rf ]
The recovery was used to evaluate the accuracy of the method. The percent recovery as well as average percent recovery was calculated. Recovery studies at three different levels were done on B. ciliata rhizomes extract by accurately spiked with various
Fig. 2. TLC densitometric chromatograms of Bergenia ligulata rhizome extract, bergenin standard, catechin standard, and gallic acid standard.
100.0
100.0
90.0
90.0
80.0
80.0
70.0
70.0
60.0
60.0 [AU]
[AU]
499
50.0
50.0
40.0
40.0
30.0
30.0
20.0
20.0
10.0
10.0
0.0 200.0
250.0
300.0 [nm]
350.0
400.0
0.0 200.0
250.0
300.0 [nm]
350.0
400.0
100.0 90.0 80.0 70.0
[AU]
60.0 50.0 40.0 30.0 20.0 10.0
250.0
350.0
300.0
400.0
[nm] Fig. 3. Overlay of UV absorption spectra of the marker compounds in the sample track with respective standards: (A) bergenin, (B) catechin, and (C) gallic acid.
ARTICLE IN PRESS 500
K. Dhalwal et al. / Journal of Food Composition and Analysis 21 (2008) 496–500
Table 4 Marker compounds quantified from different parts of Bergenia ciliata and Bergenia ligulata using HPTLC method Sample tested
Content of marker compounda (% w/w) Bergenin
Catechin
Gallic acid
B. ciliate Rhizomes Petiole Leaf
0.72070.021 0.12570.019 0.14170.015
0.06870.041 0.02570.009 0.01570.010
0.02470.011 0.00870.008 0.01470.016
B. ligulata Rhizomes Petiole Leaf
0.79170.014 0.09070.022 0.11570.010
0.07070.017 0.02170.012 0.00970.014
0.03070.017 0.00770.032 0.01070.012
a
Mean7S.D., n ¼ 3.
concentrations of reference solutions just prior to the extraction. The percentage recovery at three different levels for bergenin was found to be 99.79%, 99.08%, and 99.00% with an average of 99.29%, catechin was found to be 98.03%, 98.52%, and 99.44% with an average of 98.66% and gallic acid was found to be 99.23%, 98.98%, and 99.48% with an average of 99.23%. The results were shown in Table 3. 3.4. Limits of detection (LOD) and limits of quantification (LOQ) Serial dilutions of bergenin, catechin, and gallic acid were analyzed by TLC method. The LOD and LOQ were obtained with the signal-to-noise ratio of 3 and 10. LOD represents the lowest concentrations of bergenin, catechin, and gallic acid that can be detected, whereas the LOQ represents the lowest concentrations of bergenin, catechin and gallic acid that can be determined with acceptable precision and accuracy. The LOD and LOQ were found to be 120 and 160 ng/spot for bergenin, 80 and 120 ng/spot for catechin, 40 and 80 ng/spot for gallic acid, respectively. This indicated that the new method exhibited a good sensitivity for the quantification of bergenin, catechin and gallic acid from B. ciliata and B. ligulata. In order to obtain more accurate regression, the lower limit of linearity was adjusted to be higher than LOQ. The concentrations of bergenin, catechin, and gallic acid in sample solutions were within the range of linearity. 3.5. Quantitative determination All the samples were extracted, as described above and analyzed by TLC. The content of each compound was determined by the corresponding regression equation and results are summarized in Table 4. The results indicated that all three compounds were detected in six true samples, in which bergenin was obviously dominant. The densitograms of standards along with rhizome extract of B. ciliata and B. ligulata are represented in Figs. 1 and 2. B. ciliata and B. ligulata can be differentiated with there densitograms. Densitogram of B. ciliata showed two extra peaks as compared to B. ligulata whereas B. ligulata showed a peak at RF 0.21, which was not present in B. ciliata densitogram. Traditionally, its rhizomes are used in Ayurvedic formulations. The percentage amounts of bioactive constituent present in rhizome corroborate its use as compare to leaf and petiole (Table 4).
For the first time, a simple, accurate, and rapid TLC method was developed for the simultaneous determination of three major compounds from rhizome, petiole and leaf of B. ciliata and B. ligulata. The assay is proved to be accurate, reproducible, and sensitive. Furthermore, it has been successfully employed for the determination of these three compounds in different commercial samples. It was found that rhizome, petiole, and leaf samples of B. ciliata and B. ligulata show a similar but unique TLC chromatogram, thus making this method useful for the quality control of the crude drugs. 4. Conclusion We established TLC densitometric method for the simultaneous quantification of three bioactive compounds viz. bergenin, catechin, and gallic acid from different parts of B. ciliata and B. ligulata using TLC. The method was found to be simple, precise, specific, sensitive, and accurate and can be used for their quantification in the plant materials. It can be also used in routine quality control of herbal materials as well as formulations containing any or all of these compounds. References Asokar, L.V., Kakkar, K.K., Chakra, O.J., 1992. Glossary of Indian Medicinal Plants with Active Principles. Publication and Information Directorate, New Delhi, p. 122. Chandrareddy, U.D., Chawla, A.S., Mundkinajeddu, D., Maurya, R., Handa, S.S., 1998. Paashanolactone from Bergenia ligulata. Phytochemistry 47, 907–909. Dhalwal, K., Biradar, Y.S., Rajani, M., 2006. TLC densitometric method for simultaneous quantification of phyllanthin, hypophyllanthin, gallic acid and ellagic acid in Phyllanthus amarus using HPTLC. Journal of AOAC International 89, 619–623. Dix, B.S., Srivastava, S.N., 1989. Tannin constituents of Bergenia ligulata roots. Indian Journal of Natural Products 5, 24–25. Garimella, T.S., Jolly, C.I., Narayanan, S., 2001. In vitro studies on antilithiatic activity of seeds of Dolichos biflorus Linn. and rhizomes of Bergenia ligulata Wall. Phytotherapy Research 15, 351–355. Gehlot, N.K., Sharma, V.N., Vyas, D.S., 1976. Some pharmacological studies on ethanolic extract of Bergenia ligulata. Indian Journal of Pharmacology 8, 92. Jain, M.K., Gupta, K., 1962. Isolation of bergenin from Saxifraga ligulata Wall. Journal of Indian Chemical Society 39, 559–560. Nadkarni, K.M., 2000. The Indian Materia Medica. Popular Prakashan Pvt. Ltd., Mumbai, pp. 1198–1202. Ravishankara, M.N., Shrivastava, N., Padh, H., Rajani, M., 2001. HPTLC method for the estimation of alkaloids of Cinchona officinalis stem bark and its marketed formulations. Planta Medica 67, 294–296. Singh, D.P., Srivastava, S.K., Govindarajan, R., Rawat, A.K.S., 2007. High-performance liquid chromatographic determination of bergenin in different bergenia species. Acta Chromatographica 19, 246–252. Sinha, S., Murugesan, T., Maiti, K., Gayen, J.R., Pal, M., Saha, B.P., 2001a. Evaluation of anti-inflammatory potential of Bergenia ciliata Sternb. rhizome extract in rats. Journal of Pharmacy and Pharmacology 53, 193–196. Sinha, S., Murugesan, T., Pal, M., Saha, B.P., 2001b. Evaluation of anti-tussive activity of Bergenia ciliata Sternb. rhizome extract in mice. Phytomedicine 8, 298–301. Srivastava, S., Rawat, A.K.S., 2007. Simultaneous determination of bergenin and gallic acid in different Bergenia species. Journal of Planar Chromatography— Modern TLC 20 (4), 275–277. Tucci, P.A., Delle, M.F., Marini, B., Holo, B.G., 1969. Occurrence of (+)-afzelchin in Saxifraga ligulata. Annual 1st Super Sanita 5, 555–556. Umashankar, D.C.R., Chawla, A.S., Mundkinajeddu, D., Singh, D., Handa, S.S., 1999. High pressure liquid chromatographic determination of bergenin and (+)afzelechin from different parts of Paashaanbhed (Bergenia ligulata Yeo). Phytochemical Analysis 10, 44–47. Wagner, H., Bladt, S., 1996. Plant Drug Analysis: A Thin Layer Chromatography Atlas, second ed. Springer, Berlin, Germany, p. 359.