RP-LC determination of oleane derivatives in Terminalia arjuna

Journal of Pharmaceutical and Biomedical Analysis 28 (2002) 447– 452 www.elsevier.com/locate/jpba RP-LC determination of oleane derivatives in Terminalia arjuna D.V. Singh, R.K. Verma, S.C. Singh, M.M. Gupta * Analytical Biophysical Chemistry Di6ision, Central Institute of Medicinal and Aromatic Plants, P.O.-CIMAP, Lucknow 226 015, India Received 3 August 2001; received in revised form 20 September 2001; accepted 30 September 2001 Abstract A rapid, sensitive and reproducible reversed phase high performance liquid chromatographic method with photo diode array detection is described for the simultaneous quantification of major oleane derivatives: arjunic acid (4), arjunolic acid (3), arjungenin (2) and arjunetin (1) in Terminalia arjuna extract. The method involves the use of a Waters Spherisorb S10 ODS2 column (250 ×4.6 mm, I.D., 10 mm) and binary gradient mobile phase profile. The various other aspects of analysis viz. Extraction efficiency, peak purity and similarity were validated using a photo diode array detector. © 2002 Elsevier Science B.V. All rights reserved. Keywords: T. arjuna; Bark; Oleane derivative; Insecticidal; Antibacterial; Triterpene; HPLC; PDA 1. Introduction The fruits and bark of different species of Terminalia trees have been used since the Vedic period (1500 – 500 BC) for the treatment of various heart diseases. The pharmacognosy, phytochemistry, pharmacology and clinical use of one of the species, Terminalia arjuna (Roxburgh) Wight and Arnott, was reviewed by Dwivedi and Udupa [1]. Recently, the plant was found useful in the treatment [2] of cancer also. The plant exhibits fungicidal [3], antimicrobial [4], antibacterial [5], antifertility [6] and antihuman immuno-deficiency virus [7] induced diseases. Clinically, the bark * Corresponding author. Fax: +91-522-342666. E-mail address: [email protected] (M.M. Gupta). stem powder/alcoholic extract was found effective in the treatment of cardiovascular diseases, including coronary artery diseases [8– 11]. Tannins and oleane type triterpenes are the major chemicals of T. arjuna [12 – 17]. Although many triterpenes are reported in the plant, no work was carried out on the biological significance of these compounds. Recently, we have found many oleane derivatives of the bark of T. arjuna possessing potent insecticidal [18,19] and antibacterial activities (unpublished data). HPLC is the most commonly used analytical technique [20 – 26] for the accurate quantification of natural products. It is important to have a suitable analytical procedure, using HPLC for the quantification of important oleane derivatives in T. arjuna, in order to evaluate different accessions of the 0731-7085/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 7 3 1 - 7 0 8 5 ( 0 1 ) 0 0 5 9 0 - 8 448 D.V. Singh et al. / J. Pharm. Biomed. Anal. 28 (2002) 447–452 plants for their genetic improvement. A normal phase HPLC method [27] is reported on other oleane derivatives of Chenopodium quinoa. In this paper, we report a reverse phase HPLC method, suitable for separating and quantifying the four major oleanes: arjunic acid (4), arjunolic acid (3), arjungenin (2) and arjunetin (1) from a single run of chromatogram using photo diode array detector. To the best of our knowledge, the HPLC method reported here is new for the simultaneous quantification of compounds 1 –4 in T. arjuna. 2. Experimental T. arjuna stem bark was collected locally and a voucher specimen of the plant material has been deposited in the Gene Bank of this Institute. 2.1. Extraction Compounds 1– 4 from the stem bark of T. arjuna were isolated in the laboratory. A total of 4.5 kg of T. arjuna powdered bark was defatted using hexane. The defatted plant material was extracted using ethanol, concentrated in vacuo and reextracted by diethyl ether. Diethylether extract (100 g) was column chromatographed over silica gel (60–120 mesh; Spectrochem, India) using varying concentrations of ethyl acetate in hexane as eluent. Fractions (100 ml) were collected and monitored by TLC. Fraction Nos. 325– 442 afforded compound 4, identified as arjunic acid, m.p. \280 °C, on the basis of spectral analysis [13,14,28], by using hexane – ethyl acetate as eluent in the ratio (50:50/v:v) and crystallized by using methanol. Fraction Nos. 447– 527 afforded compound 3, identified as arjunolic acid, m.p. 296– 297 °C, by spectral analysis [13,29], by using hexane–ethyl acetate as eluent in the ratio (50:50/v:v) and crystallized by using methanol. Fraction Nos. 538– 800 afforded compound 2, identified as arjungenin, m.p. 293– 294 °C, by spectral analysis [13,15], by using hexane–ethyl acetate as eluent in the ratio (50:50/v:v) and crystallized by using methanol. Fraction Nos. 949– 1377 afforded compound 1, identified as arjunetin, m.p. 232– 234 °C (dec.), by spectral analysis [12,13], by using hexane– ethyl acetate as eluent in the ratio (20:80/v:v) and crystallized by using methanol. Solvents used were HPLC grade (JT Baker, USA). 2.2. Chromatographic instrument and conditions HPLC analysis was carried out on a Shimadzu (Japan) LC-10A gradient high-performance liquid chromatographic instrument, equipped with two LC-10AD pumps controlled by a CBM-10 interface module, a model SIL-10ADvp auto injector, an in-line degasser DGU-14A and a multidimensional UV-VIS detector SPD-10A. Photo diode array detector SPD-M10Avp (Shimadzu) was used for the peak purity and similarity test of all four terpenoids (1 – 4). Solvents were prefiltered by using a Millipore system and analysis was performed on a Waters Spherisorb S10 ODS2 reversed-phase column, 10 mm (250 ×4.6 mm, ID). The analytical parameters were selected after screening a number of solvent systems and gradient profiles. Separation was achieved with a twopump gradient program for pump A (acetonitrile:water, 30:70) and pump B (acetonitrile:water, 70:30) as follows: initially 30% B, flow rate 0.8 ml/min; then increased gradually to 50% B until 10.0 min, flow rate 0.8 ml/min; again increased gradually 50– 70% B up to 30 min, flow rate 1.2 ml/min; washed the column for 20 min, 30% B, flow rate 0.8 ml/min. The detection wavelength was 220 nm, the absorption maxima close to all the compounds. Injection size for standard and sample was 20 ml. Column temperature was 26 °C. 2.3. Sample preparation Air dried bark of T. arjuna (1 g) was extracted in ethanol for 10 h (3× 10 ml), filtered, evaporated and reextracted using diethyl ether (3×10 ml), dried and again redissolved in 10 ml of methanol for HPLC analysis. Stock solutions of compounds 1– 4 were prepared in methanol (1 mg/ml) separately and dif- D.V. Singh et al. / J. Pharm. Biomed. Anal. 28 (2002) 447–452 449 area count of each peak (X) and corresponding concentration (Y). Percent content of oleane derivatives (1 – 4) were calculated by using calibration graphs. 3. Results and discussion 3.1. Selection of mobile phase Fig. 1. Substances studied. ferent amounts of these were injected in HPLC for the preparation of calibration graphs and LC analysis. Calibration graphs were plotted by using To optimize the mobile phase for binary gradient profile, different compositions of acetonitrile in water were used. Fig. 1 and Fig. 2 illustrates the structure and separation of oleane derivatives (1 –4) in a standard mixture (A) and a plant sample extract (B) respectively. Peaks corresponding to compounds (1– 4) were base line separated and symmetrical. Retention times were 5.40, 9.03, 16.02 and 18.96 for compounds 1 –4, respectively. Recoveries of compounds 1–4 were 98, 97, 97, 96%, respectively. For the examination of recovery, known amounts of stock solution of pure compounds (1 –4) were added in the T. arjuna bark extract and the quantification repeated three times. Selected wavelength (220 nm) was closed to absorption maxima of all four compounds (1 –4). Column performance report for T. arjuna plant extract is presented in Table 1. As a measure of column performance, the number of theoretical plate counts (N) for compounds 1 –4 were 5723, 11208, 16683 and 16071, respectively. Concentra- Table 1 C18 column performance in the separation of oleane derivatives (1–4) from the extract of T. arjuna bark Oleane derivatives Rt No. of theoretical plate counts (N) Capacity factor (k) Recovery (%) Separation factor Linear regression equation [Y= A9S.D. X9 C9 S.D.] Y= 4.99 0.1×10−6 X−0.139 0.02 (r= 0.9990) Y= 4.2 9 0.2×10−6 X−0.219 0.03 (r= 0.9992) Y= 6.8 9 0.1×10−6 X+0.069 0.01 (r= 0.9993) Y= 4.9 9 0.1×10−6 X−0.079 0.01 (r= 0.9992) Arjunetin (1) 5.40 9 0.02 5723 1.64 98 9 2 1.77 Arjungenin (2) 9.03 9 0.02 11208 3.40 97 9 1 2.08 Arjunolic acid (3) 16.02 9 0.03 16683 6.81 97 9 2 1.42 Arjunic acid (4) 18.96 9 0.03 16071 8.25 96 9 2 1.21 450 D.V. Singh et al. / J. Pharm. Biomed. Anal. 28 (2002) 447–452 Fig. 2. RPLC separation of oleane derivatives (1 – 4) in an artificial mixture of pure compounds (A); 1 mg/ml and a T. arjuna bark extract (B). (1) Arjunetin; (2) arjungenin; (3) arjunolic acid and (4) arjunic acid. tion of analytes was estimated at different intervals and no change was observed for the studied time, i.e. 24 h. 3.2. E6aluation of peak purity Peak purity test of compounds 1 –4 was performed using a photo diode array detector. All peaks were found pure, both up and down the peaks (Table 2). A similarity test of compounds 1– 4 in a sample extract was performed by comparing the similarity of peaks in a sample track to that of a library maintained for oleane derivatives 1– 4. Similarity of all the compounds were \ 0.99 (Table 2). The peak homogeneity was tested by examining the UV spectrum for different points of the resolving peaks. 3.3. Linearity To determine the linearity, five different concentrations of each compound (1 – 4) were used in a working range of 1– 20 mg. Linear regression Table 2 Peak purity test results of oleane derivatives (1–4) using photo diode array detector Oleane derivatives Arjunetin (1) Arjungenin (2) Arjunolic acid (3) Arjunic acid (4) Peak purity Up Down 1.00 1.00 0.99 1.00 1.00 0.99 1.00 0.99 Similarity 1.00 1.00 0.99 1.00 D.V. Singh et al. / J. Pharm. Biomed. Anal. 28 (2002) 447–452 451 Table 3 Effect of solvent in the extraction of oleane derivatives (1–4) from T. arjuna bark Solvent used for extraction % Content9 S.D. of oleane derivatives (1–4) Compound Hexane Chloroform Ethyl acetate Acetone Methanol Ethanol 1 2 3 4 ND 0.002 9 0.0002 0.008 9 0.0003 0.160 9 0.0040 0.188 9 0.0040 0.216 9 0.0030 ND 0.016 9 0.004 0.031 9 0.002 0.074 9 0.002 0.056 9 0.003 0.084 9 0.004 ND 0.012 9 0.002 0.017 9 0.003 0.020 9 0.004 0.014 9 0.003 0.022 9 0.002 ND 0.045 9 0.003 0.062 9 0.003 0.086 9 0.004 0.075 9 0.003 0.099 9 0.004 ND, not detectable. equations and correlation coefficient (r) for compounds 1 –4 have been given in Table 1. Calibration plots of peak area versus concentration are linear with r values in between 0.9990 and 0.9993. The values show good linearity in the examined concentration range. 3.4. Detection limits Detection limits, a measure of minimal mass of compounds 1– 4 that can be quantified were 0.04, 0.04, 0.08, 0.06 mg per injection, respectively. 4. Conclusions The reported reversed phase LC method, using photo diode array detector, is suitable for the analysis of oleane derivatives 1 –4 (possessing potent insecticidal and antibacterial activities. These are new findings on these compounds and detailed results will be published elsewhere) in T. arjuna extract. The method is simple, rapid and precise. A base line separation of all four oleane derivative has been achieved and could be used for rapid screening of T. arjuna plant for genotypic quality assessment, drug analysis, etc. 3.5. Extracti6e 6alue in different organic sol6ents Experiments were performed to select the suitable solvent for the maximum extraction of oleane derivatives (1 – 4). Different solvents, viz. hexane, chloroform, ethyl acetate, acetone, methanol and ethanol, were used for the extraction of plant and the results of compounds 1 –4 content are presented in Table 3. Ethanol was found a suitable solvent for extraction of maximum oleane derivatives (1– 4). 3.6. Precision Precision of the method was measured by repeating each experiment four times. Mean and S.D. values for the retention times and recoveries for the compounds 1– 4 were 9 0.02 to 90.03 and 91.0 to 92.0, which are presented in Table 1. Acknowledgements We are grateful to Director, CIMAP, Lucknow for providing the facilities for the above work and the Department of Biotechnology (DBT), New Delhi, India for financial support. References [1] S. Dwivedi, N. Udupa, Fitoterapia 60 (1989) 413 – 420. [2] G.R. Pettit, M.S. Hoard, D.L. Doubek, J.M. Schmidt, R.K. Pettit, L.P. Tackett, J.C. Chapuis, J. Ethanopharm. 53 (1996) 57 – 63. [3] R. Kumar, R.K. Verma, Ind. Phytopathol. 40 (1987) 274. [4] P.G. Ray, S.K. Majumdar, Econ. Bot. 30 (1976) 317 – 320. [5] Y.N. Shukla, A. Srivastava, T.R. Santha Kumar, S.P.S. Khanuja, S. Kumar, Ind. Drugs 37 (2000) 60 – 61. 452 D.V. Singh et al. / J. Pharm. Biomed. Anal. 28 (2002) 447–452 [6] C. Seshadri, S.R. Pillai, Venkataraghawan, J. Sci. Res. Plants Med. 2 (1/2) (1981) 1 – 3. [7] I.T. Kusumoto, T. Nakabayashi, H. Kida, H. Miyashiro, M. Hattori, T. Namba, K. Shimotohno, Phytother. Res. 9 (1995) 180 – 184. [8] S. Dwivedi, R. Jauhari, Ind. Heart J. 49 (1997) 507 – 510. [9] A. Bharani, A. Ganguly, K.D. Bhargava, Int. J. Cardiol. 49 (1995) 191 – 199. [10] A. Ram, P. Lauria, R. Gupta, P. Kumar, V.N. Sharma, J. Ethanopharm. 55 (1997) 165 – 169. [11] N. Singh, K.K. Kapur, S.P. Singh, K. Shanker, J.N. Sinha, R.P. Kohli, Planta Med. 45 (1982) 102 – 104. [12] T. Tsuyuki, Y. Hamada, T. Honda, T. Takahashi, K. Matsushita, Bull. Chem. Soc. (Japan) 52 (1979) 3127 – 3128. [13] T. Honda, T. Murae, T. Tsuyuki, T. Takahashi, M. Sawai, Bull. Chem. Soc. (Japan) 49 (1976) 3213 – 3218. [14] A.S.R. Anjaneyulu, A.V. Rama Prasad, Phytochemistry 21 (1982) 2057 – 2060. [15] T. Honda, T. Murae, T. Tsuyuki, T. Takahashi, Chem. Pharm. Bull. 24 (1976) 178 – 180. [16] A.S.R. Anjaneyulu, A.V. Rama Prasad, Ind. J. Chem. 21B (1982) 530 –533. [17] V.K. Tripathi, V.B. Pandey, K.N. Udupa, G. Rucker, Phytochemistry 31 (1992) 349 – 351. [18] D.V. Singh, M.M. Gupta, A.K. Tripathi, V. Prajapati, S. Kumar, Phytother. Res. (2001) (communicated). [19] R.S. Bhakuni, Y.N. Shukla, A.K. Tripathi, V. Prajapati, S. Kumar, Phytother. Res. (2001) (in press). [20] M.M. Gupta, R.K. Verma, G.C. Uniyal, S.P. Jain, J. Chromatogr. A 637 (1993) 209 –212. [21] R.K. Verma, K.G. Bhartariya, M.M. Gupta, S. Kumar, Phytochem. Anal. 10 (1999) 191 – 193. [22] D.V. Singh, A. Maithy, R.K. Verma, M.M. Gupta, S. Kumar, J. Liq. Chromatogr. 23 (2000) 601 – 607. [23] S. Srivastava, R.K. Verma, M.M. Gupta, S. Kumar, J. Chromatogr. A 841 (1999) 123 – 126. [24] D.V. Singh, S. Prajapati, S. Bajpai, R.K. Verma, M.M. Gupta, S. Kumar, J. Liq. Chromatogr. 23 (2000) 1757 – 1764. [25] S. Srivastava, R.K. Verma, M.M. Gupta, S.C. Singh, S. Kumar, J. Liq. Chromatogr. 24 (2001) 13 – 19. [26] D.C. Jain, M.M. Gupta, S. Saxena, S. Kumar, J. Pharm. Biomed. Anal. 22 (2000) 705 –709. [27] M. Burnouf-Radosevich, N.E. Delfel, J. Chromatogr. 292 (1984) 403 – 409. [28] L. Ramchandra Row, P.S. Murty, G.S.R. Subba Rao, C.S.P. Sastry, K.V.J. Rao, Ind. J. Chem. 8 (1970) 716 – 721. [29] F.E. King, T.J. King, J.M. Ross, J. Chem. Soc. (1954) 3995 – 4003.