Isolation and structural elucidation of abietic acid as the main adulterant in an herbal drug for the treatment of psoriasis

Journal of Pharmaceutical and Biomedical Analysis 66 (2012) 345–348

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Isolation and structural elucidation of abietic acid as the main adulterant in an herbal drug for the treatment of psoriasis Yi He, Yumei Zhang, Jing Lu ∗ , Ruichao Lin National Institutes for Food and Drug Control, Tiantan Xili 2, Beijing 100050, China

a r t i c l e

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Article history: Received 16 December 2011 Received in revised form 1 March 2012 Accepted 2 March 2012 Available online 23 March 2012

a b s t r a c t An herbal drug for the treatment of psoriasis showed severe clinical adverse reactions. The main component of adulterant was isolated from the drug and its chemical structure was elucidated as abietic acid using NMR, HR–MS, and HPLC–DAD–MS. Although abietic acid had ever been used in clinical study for the treatment of psoriasis, it is not an approved drug. Its adulteration is very dangerous for the patients. © 2012 Elsevier B.V. All rights reserved.

Keywords: Abietic acid NMR HPLC–DAD–MS Herbal drug Psoriasis

1. Introduction Psoriasis is a common, chronic, immune-mediated disease that affects approximately 2% of the world population. Although this disease is rarely fatal, psoriasis has a significant adverse effect on the quality of life by its complications such as inflammatory arthropathy, disability, and myocardial infarction [1–4]. It is believed that psoriasis results from interplay between multiple genetic and environmental factors, but its pathogenesis is still incompletely understood [4,5]. Current therapies for psoriasis are far from satisfactory. Many therapies are associated with significant side effects including renal injury, liver abnormalities, vasculitis, and even cancer [4,6]. Therefore, some patients turn to herbal medicine for the treatment of psoriasis. An example of such an herbal drug, YXD capsule was composed of Scutellariae Radix, Moutan Cortex, Coptidis Rhizoma, Lonicerae japonicae Flos, Rehmanniae Radix, Zaocys, Schizonepetae Herba, Notopterygii Rhizoma et Radix, Angelicae dahuricae Radix, Saposhnikoviae Radix, Forsythiae Fructus, Cicadae Periostracum, Haematitum, Olibanum, and Glycyrrhizae Radix et Rhizoma. It was sent to our institute because several batches of the product caused severe adverse reactions including one case of death and one case of deafness.

∗ Corresponding author. E-mail address: [email protected] (J. Lu). 0731-7085/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jpba.2012.03.007

Abietic acid is an abietane diterpenoid and one of the major components of rosin (colophony), in which its concentration could reach 54% [7,8]. In vivo and in vitro studies showed that abietic acid had anti-obesity, lipoxygenase inhibition, testosterone 5␣-reductase inhibition, and anti-inflammation effect [9–12]. Oral preparations of abietic acid had been used in clinical study for the treatment of psoriasis in China [13–15], but it has not been approved as drug so far. In this paper, we describe the isolation and structure elucidation of abietic acid from YXD capsules using NMR, HR–MS, and HPLC–DAD–MS. 2. Materials and methods 2.1. Sample and chemicals YXD capsules were commercial products obtained from Ningxia Hui Autonomous Region, China. Abietic acid (purity: about 75%, GC) was purchased from Sigma–Aldrich (St. Louis, MO, USA). Silica gel was obtained from Qingdao Haiyang Chemical Co., Ltd. (Qingdao, Shandong, China). Deuterated chloroform used for NMR analysis was purchased from Cambridge Isotope Laboratories (Andover, MA, USA). All chemical reagents were either high-performance liquid chromatography (HPLC) or analytical grade obtained from Mallinckrodt Baker Inc. (Phillipsburg, NJ, USA), Fisher Scientific (Pittsburgh, PA, USA), and Beijing Chemical Works (Beijing, China). Water was purified with MilliQ system (Millipore, Billerica, MA, USA).

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2.2. Extraction and isolation The contents of two capsules (0.8 g, pale-brown powder) were suspended in water, followed by partition with chloroform. The chloroform extract was applied to flash column chromatography (silica gel) and eluted with the mixture of petroleum ether (b.p. 60–90 ◦ C) – ethyl acetate (98:2). Further purification was performed on a Delta Prep 4000 preparative chromatography system (Waters Corporation, Milford, MA, USA) using Prep Nova-Pak HR C18 (Waters Corporation, 6 ␮m, 40 mm × 100 mm) as stationary phase and 70% methanol (5 mL/min) as mobile phase to afford compound 1 (100 mg). 2.3. NMR and MS analysis 1 H-, 13 C-

and 2D NMR spectra were measured on a Bruker DRX-500 spectrometer (Bruker BioSpin, Rheinstetten, Germany). Chemical shifts were reported in ppm using the solvent peak as an internal standard. MS spectra were determined on Autospec-Ultima ETOF MS spectrometer (EI-MS, Micromass Ltd., Wythenshawe, Manchester, UK) and ZAB-HS MS spectrometer (HR–EI-MS, VG Analytical, Wythenshawe, Manchester, UK). 2.4. HPLC–DAD–MS analysis The sample solution was prepared by shaking 0.1 g of the capsule’s contents with 20 mL methanol, and the reference solution was prepared by dissolving 5 mg of abietic acid in 10 mL of methanol. HPLC–DAD–MS analysis was performed using the Agilent 1200 Series HPLC system and 6410B Triple Quadrupole LC/MS system (Agilent Technologies, Palo Alto, CA, USA). The sample solution was separated by using a Phenomenex Kinetex C18 column (2.6 ␮m, 2.10 mm × 100 mm, Phenomenex Inc., Torrance, CA, USA) with injection volume of 2 ␮L. The mobile phase was the mixture of acetonitrile and 0.1% formic acid with a flow rate of 0.2 mL/min, and the detection wavelength was set at 241 nm. MS detection was performed in positive electrospray ionization (ESI+) mode. The LC effluent was directly introduced into the ESI source without split. Nitrogen was used as desolvation gas. The optimized interface and MS conditions were as follows: gas temperature 300 ◦ C, gas flow 10 L/min, nebulizer pressure 35 psi, source voltage 4 kV, fragmentor voltage 100 V, and collision energy 10 eV (for MS/MS). 3. Results and discussion Compound 1 was obtained as a colourless amorphous powder. The molecular formula was determined to be C20 H30 O2 by HR–EIMS (m/z 302.2246 [M]+ ; calculated for C20 H30 O2 , 302.2249). Its degree of unsaturation can thus be calculated as 6. Furthermore, the characteristic fragment ion peaks at m/z 287 [M−CH3 ]+ , 259 [M−C3 H7 ]+ , and 257 [M−COOH]+ of EI-MS suggested the existence of a methyl group, a propyl group, and a carboxyl group, which were also supported by the HR–EI-MS (m/z 287.2014 [M−CH3 ]+ , calculated for C19 H27 O2 , 287.2011; m/z 259.1685 [M−C3 H7 ]+ , calculated for C17 H23 O2 , 259.1698; m/z 257.2254 [M−COOH]+ , calculated for C19 H29 , 257.2269). The 1 H NMR signals (Table 1) at ı 5.38 (1H, m, H-7) and 5.77 (1H, m, H-14) suggested the presence of ethylenic bond; whereas the 13 C NMR spectrum of 1 (Table 1) showed a carboxylic carbon signal at ı 183.7 (C-8) and four olefinic carbon signals at ı 145.2, 135.6, 122.3 and 120.4. Based on the calculation of unsaturation degree, 1 could be estimated as a tricyclic compound. The heteronuclear multiple quantum coherence (HMQC) spectrum showed that the protons at ıH 5.38 (H-7) and 5.77 (H-14) were connected to the

Table 1 NMR spectral data of compounds 1 (ı in ppm, J in Hz)a . Position

ıH

ıC

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

1.14 (m) 1.59 (m) 1.79 (m) – 2.07 (m) 1.87 (m) 5.38 (m) – 1.94 (m) – 1.79 (m) 2.07 (m) – 5.77 (s) 2.21 (m) 1.01 (d, J = 4.5) 1.00 (d, J = 4.5) – 1.26 (s) 0.83 (s)

38.3 18.0 37.2 46.3 44.9 25.6 120.4 135.6 50.9 34.5 22.5 27.4 145.2 122.3 34.9 20.9 21.4 183.7 16.8 14.0

a b

HMBCb

C-5, C-6, C-9, C-14

C-7, C-8, C-9, C-12, C-15 C-13, C-17 C-13, C-16 C-3, C-5, C-18 C-1, C-5, C-9

Data were recorded in CDCl3 at 500 MHz (1 H) and 125 MHz (13 C). HMBC correlations are from proton(s) stated to the indicated carbon.

carbons at ıC 120.5 (C-7) and 122.3 (C-14), respectively. In the heteronuclear multiple bond correlations (HMBC, Table 1) spectrum, the long range correlations between H-7 and C-14, and between H14 and C-7 suggested that those ethylenic carbons were adjacent to each other. By comparison of UV and NMR data with those reported in the literature [9,16], compound 1 was identified to be abietic acid (Fig. 1). As shown in Fig. 2, the HPLC–DAD chromatography, UV spectrum, and MS spectrum obtained from HPLC–DAD–MS confirmed the presence of abietic acid in the capsules. The presence of abietic acid could not be explained by the literature search on the chemical composition of labeled ingredients. Therefore, it is adulterated. The range of the concentration of abietic acids in rosin is very large (varying from 20 to 54%) [7,8]. Little literature reported about the chemical difference among crude abietic acid, processed (purified) rosin, and natural rosin. So it is also very hard to determine which adulterant was actually added in this herbal drug. Oral preparations of abietic acid had side-effects including nausea, discomfort in the stomach, loss of appetite, loose or solid stool, dizziness and fatigue, drug eruptions, and sleepiness [13–15]. Rosin has been regarded as a common allergen [7,17]. In an ancient book

Fig. 1. Structure of abietic acid.

Y. He et al. / Journal of Pharmaceutical and Biomedical Analysis 66 (2012) 345–348

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Fig. 2. HPLC–DAD chromatograms of the YXD capsule sample (a) and abietic acid reference (b); the UV spectra of compound 1 (c) and abietic acid reference (d); mass spectra of compound 1 (e) and abietic acid reference (f); MS/MS spectra of the m/z 303 ion of compound 1 (g) and abietic acid reference (h).

of traditional Chinese medicine “Xu Yi Shuo” (AD 1522), it was recorded that someone died after ingesting rosin without careful purification [15]. The severe adverse reactions of YXD capsule may be caused by the adulterant. 4. Conclusion In this study, the main component of adulterant, abietic acid, was isolated from an herbal drug by flash column chromatography and preparative HPLC, and its chemical structure was elucidated using NMR, HR–MS, HPLC–DAD–MS. In view of the fact that abietic acid is not an approved drug till present, its adulteration is very dangerous for the patients, and the severe adverse reactions of YXD capsule may be caused by the adulterant. References [1] J.M. Gelfand, A.L. Neimann, D.B. Shin, X. Wang, D.J. Margolis, A.B. Troxel, Risk of myocardial infarction in patients with psoriasis, JAMA 296 (2006) 1735–1741.

[2] S.R. Rapp, S.R. Feldman, M.L. Exum, A.B. Fleischer, D.M. Reboussin, Psoriasis causes as much disability as other major medical diseases, J. Am. Acad. Dermatol. 41 (1999) 401–407. [3] C.L. Leonardi, J.L. Powers, R.T. Matheson, B.S. Goffe, R. Zitnik, A. Wang, A.B. Gottlieb, Etanercept as monotherapy in patients with psoriasis, N. Engl. J. Med. 349 (2003) 2014–2022. [4] B.J. Nickoloff, F.O. Nestle, Recent insights into the immunopathogenesis of psoriasis provide new therapeutic opportunities, J. Clin. Invest. 113 (2004) 1664–1675. [5] P. Rahman, J.T. Elder, Genetic epidemiology of psoriasis and psoriatic arthritis, Ann. Rheum. Dis. 64 (2005) ii37–ii39. [6] W. Weger, Current status and new developments in the treatment of psoriasis and psoriatic arthritis with biological agents, BJP 160 (2010) 810–820. [7] S. Sadhra, I.S. Foulds, C.N. Gray, Oxidation of resin acids in colophony (rosin) and its implications for patch testing, Contact Dermatitis 39 (1998) 58–63. [8] J. Zhang, X. Bi, X. An, C. Wu, Study on rosin decarboxylation under microwave irradiation, J. Southwest Forestry Coll. 24 (2004) 55–57. [9] S. Roh, M. Park, Y. Kim, Abietic acid from resina pini of Pinus species as a testosterone 5˛-reductase inhibitor, J. Health Sci. 56 (2010) 451–455. [10] K. Hwang, J. Ahn, S. Kim, J. Park, T. Ha, Abietic acid has an anti-obesity effect in mice fed a high-fat diet, J. Med. Food 14 (2011) 1052–1056. [11] N.N. Ulusu, D. Ercil, M.K. Sakar, E.F. Tezcan, Abietic acid inhibits lipoxygenase activity, Phytother. Res. 16 (2002) 88–90.

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[12] M.A. Fernández, M.P. Tornos, M.D. García, B. De las Heras, A.M. Villar, M.T. Sáenz1, Anti-inflammatory activity of abietic acid, a diterpene isolated from Pimenta racemosa var. grissea, J. Pharm. Pharmacol. 53 (2001) 867–872. [13] C. Mi, H., Shi, F. Li, A botanical drug for treating psoriasis and preparation process of its crude drug abietic acid, Chinese patent CN 1883519A (27 December 2006). [14] Y. Dong, C., Wang, Q., Xu, C., Tang, X., Xia, S. Han, Medicine for treating psoriasis, Chinese patent CN 1397341A (19 February 2003).

[15] Editorial board of China herbal, State administration of traditional Chinese medicine, China Herbal, book two, Shanghai Science and Technology Publishers, Shanghai, 1999, pp. 303–305. [16] S. Budavari, M.J. O’Neil, A. Smith, P.E. Heckelman, J.F. Kinneary, The Merck Index, 12th ed., Merck Research Laboratories, Division of Merck & Co., Inc., New Jersey, 1996, pp. 1–2. [17] A.M.R. Downs, J.E. Sansom, Colophony allergy: a review, Contact Dermatitis 41 (1999) 305–310.