A New Cytotoxic Carbazole Alkaloid Isolated from the Stem Bark of ...

Canadian Chemical Transactions Ca

ISSN 2291-6458 (Print), ISSN 2291-6466 (Online) Year 2013 | Volume 1 | Issue 3 | Page 165-172

Research Article

DOI:10.13179/canchemtrans.2013.01.03.0027

A New Cytotoxic Carbazole Alkaloid Isolated from the Stem Bark of Malaysian Clausena excavata Tian Hai Peh1, Gin Keat Lim2**, Yun Hin Taufiq-Yap1*, Gwendoline Cheng Lian Ee1, Marwardi Rahman1, and Mohd Aspollah Sukari1 1

Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 2 School of Chemical Sciences, Universiti Sains Malaysia, 11800 Minden, Pulau Pinang, Malaysia * Corresponding author, Email: [email protected] Phone: +603 8946 6809 ** Co-author, Email: [email protected] Phone: +604 653 4028 Fax: +604 657 4854 Received: July 22, 2013 Revised: August 25, 2013 Accepted: August 30, 2013 Published: September 2, 2013

Abstract: Malaysia is one of the richest in its biodiversity in the world. There are not less than 12,000 plants species in its rain forest. The aim of this study is a continuous investigation for medicinal plants in Malaysia, especially the chemical constituents that are poses significant activities, i.e. anti-cancer. Clausena excavata (Rutaceae) has been known as a very rich in carbazole alkaloids, coumarins and limonoids species. In the present study, one new carbazole alkaloid, 1,8-dihydroxy-3-formyl-4prenylcarbazole (Clausine–TH), and two other known compounds, Clausenarin (coumarin) and Clausine–K (carbazole alkaloid), were isolated from the methanol extract of the stem bark of Clausena excavata, collected from Kedah, Malaysia. Structures of these compounds were confirmed by various spectroscopic analyses including GC-MS, NMRs, and FTIR. All pure compounds isolated were tested for their cytotoxicity against CEM-SS cell lines, with the Clausine–TH and Clausine–K gave a very strong activity with an IC50 value of 2.1 µg/mL and 5.1 µg/mL, respectively. Keywords: Clausena excavata; carbazole alkaloid; Clausine–TH; Clausine–K; cells line

Clausenarin; CEM–SS

1. INTRODUCTION Malaysia has around 12,000 species of flowering plants, of which around 1300 are said to be medicinal [1] and only about few hundreds have been investigated for their potential. Clausena excavata from the Rutaceae family is a wild shrub that grows up to 1.5 meters high is extensively distributed throughout Southeast Asian, India and China. Clausena excavata, locally known as “pokok kemantu”

Borderless Science Publishing

165

Canadian Chemical Transactions


Ca 3'

2'

5 6 7

A 8

OH

1'

CHO

4 5a

3

4a 1a

8a

1

2

N H OH

Figure 1: Structure of Clausine–TH (1) (ghostly tree) or “pokok cerek” (diarrhea tree) is one of the Malaysian species of “ulam” (salad) that is known to have high antioxidant properties. Various parts of the plant have been used traditionally, throughout the Southeast Asia countries for the treatment of cold, malaria, AIDS, dermatopathy, abdominal pain, and snake-bite, and also as a detoxification agent [2-3]. Clausena excavata is very rich in carbazole alkaloids, coumarins and limonoids [4]. Up to date, there are approximately sixty carbazole alkaloidal compounds and fifty coumarin compounds isolated from the leaves, bark or roots of Clausena excavata [2], with the recent (years 2010 and 2012) isolations of binorponcitrin [2] (coumarin dimer), dictamine [2] (furoquinoline alkaloid), murrayacine [2] and sansokamine [5] (carbazole alkaloids). Additional to that, a new carbazole alkaloid (excavatine–A) and two new alkaloids (excavatine–B and –C) were isolated from the Clausena excavata collected from Yunnan, P. R. China reported by Peng et al. in 2013 [6]. Pharmaceutical and biological agents are derived from two major sources, natural and synthetic. The later including the biotechnological adaptation of nature compounds. These natural sources have yielded a wealth of biologically active compounds, many of these have entered the market place, either as pharmaceutical agents or biological tools for the world [7]. As for the history of Clausena excavata, as early as 1994, Clausine–D [8] and clausenaquinone–A [3] have been reported showed potent inhibitory activity of the rabbit platelet aggregation. Clausenaquinone–A [3] also gave a potent inhibitory activity of cytotoxic against the HCT-8, RPMI-7951 and TE 671 tumor cells. Several biological activities have also been on the genus of Clausena. From clausena anisata, carbazole alkaloids [9], clausenol and clausenine isolated demonstrated high antimicrobial and anti-fungi acticities against the microorganism tested. While from the extract of clausena harmandiana [10], three known compounds isolated, heptaphylline, dentatin and clausarin exhibited antiplasmodial activity against Plasmodium falciparum. 2. MATERIAL AND METHODS 2.1 Plant Material The stem bark of Clausena excavata used in this study was obtained from Jabi, Kedah, Malaysia. The sample was identified by Dr. Rusea Go of Biology Department, Universiti Putra Malaysia. Voucher specimens have been deposited in the Herbaria of this Department. 2.2 Extraction of Clausena excavata (Stem Bark) The stem bark of Clausena excavata was air dried and ground into fine powder to give 800 g sample. Extraction was performed by continuously soaking the dried ground plant material with a range of solvents such as hexane, ethyl acetate (denoted as EA thereafter) and methanol. Hexane was used first to remove non-polar organic compounds, waxes and fats. The sample was drained out after three days and


166



Ca

+

CHO

CHO

– CH3 N H

N H OH

OH

OH

m/e 295

OH

m/e 280

– CH2CH=C(CH3)2

– CH=C(CH3)2

+

+ CHO

N H

N H OH

CHO

OH

OH

m/e 240

OH

m/e 226 Figure 2: Fragmentation Patterns of (1) in the MS spectra

H3C

CH3

H H

H H

H

CHO

H

N H OH

H OH

Figure 3: 13C–1H Coupling Pattern Observed in HMBC Spectra of (1). Borderless Science Publishing

167



this procedure was repeated twice. This process was followed by the treatment with EA and lastly methanol was used to remove more polar compounds. Each extract obtained was filtered and evaporated to dryness. After removal of the solvent, the crude methanol extract (39.2 g) was obtained. A sample (10.0 g) of the crude methanol extract was directly chromatographed on a silica gel column and eluted with mixtures of chloroform (denoted CH3Cl thereafter), EA and acetone to afford 48 fractions. Fractions 5 and 6 were combined (1.2 g) and re-chromatographed in a silica gel mini column to give 15 fractions. Fractions 2 and 3 were eluted with 100 % chloroform, the solvent was removed and the solid obtained was recrystallized from EA to give yellow needles crystal (12 mg). This solid gave a single spot on thin-layer chromatograph (denoted as TLC hereafter) with an Rf = 0.65 (CHCl3 : EA = 9 : 1) 2.3 Cytotoxicity Assay The CEM–SS cell line (T-Lymphoblastic Leukemia) was obtained from the National Institutes Cancer, Frederick, Maryland, USA. The cells were cultured and maintained in growth medium as described [11]. Cytotoxicity was determined by performing the micro titration assay [12]. The tests were performed in 96-well micro- titer plates. Each well was added with 100 µL of varying concentrations of isolated pure compounds prepared from the stock solutions by serial dilution in RPM I–1640 medium. Subsequently, each well was filled with 100 µL of the cell suspension in complete growth medium at 1–2  105 cells/mL. The controls for each sample contain only untreated cells. The assay for each concentration of isolated compounds was performed in triplicate and the culture plates were incubated at 37 oC with 5 % (v/v) CO2 for three days. The cytotoxicity index used was IC50, the concentration that yielded 50 % cell inhibition when compared with the untreated control. Isolated compounds gave cytotoxic index (IC50)  10 µg/mL. 3. RESULTS AND DISCUSSION 3.1 Isolation of 1,8-dihydroxy-3-formyl-4-prenylcarbazole (Clausine–TH) Clausine–TH (1) (see in figure 1) was obtained as yellowish needles (12 mg) with a melting point of 194 – 196 oC. The mass spectrum indicated a molecular ion peak at m/z 295, which is consistent with the molecular formula C18H17NO3. The peak at m/z 280 was due to the loss of a methyl group from the molecular ion. One characteristic mass fragment at m/z 226 is due to the loss of a prenyl group [– CH2CH=C(CH3)2]. Meanwhile, the loss of two methyl groups gave a fragment ion at m/z 265 (refer to Figure 2 for fragmentation patterns). The IR spectrum indicated the presence of a broad band at 3438 cm-1 due to NH and OH groups. The absorption observed at 2924 cm-1 was assigned to the C–H stretching for CH3 and CH2. The spectrum also showed a strong carbonyl band at 1655 cm-1. This was further confirmed by the 13C–NMR which indicated the presence of a C=O peak at 197.0 ppm. The 1H-NMR spectrum of (1) showed a similar signal pattern to that of Clausine-D [13], except for the absence of a hydroxyl signal at  8.89. The presence of a 3,3-dimethylallyl group in the compound was suggested by the 1H-NMR signal: two singlet methyl signals at  1.68 and 1.86; a benzylic methylene doublet at  3.74 (J = 6.8 Hz); a multiplet for vinylic proton at  5.40 and the mass fragmentation ion at m/e 226 coupled with the appearance of the carbon signals at 25.9, 18.1, 23.4, 122.6 and 133.0 ppm in the 13 C-NMR spectrum which were assigned to 3’–CH3, 3’–CH3, C–1’, C–2’ and C–3’. The lack of substituent in ring A was suggested by the three mutually coupling aromatic protons at  6.91 (dd, J = 7.8, 1.0 Hz), 7.08 (t, J = 8.0, 7.8 Hz) and 7.59 (dd, J = 7.8, 0.8 Hz) which were assigned to H–7, H–6 and H– 5, respectively. The lower field signal at  7.59 is characteristic of H-5 of carbazoles [2-5, 13-17]. All


168



Ca

Table 1: 1H, 13C–NMR Chemical Shift () and Coupling Patterns of the Protons in HETCOR, COSY, DEPT and HMBC Techniques of Clausine–TH (1) H/C

 1H

 13C

1 2 3 4 5

8.29 (s) 7.59 (dd, 7.8, 0.8 Hz)

158.5 126.7 116.4 110.6 111.9

H–13C HETCOR H–2 H–5

6

7.08 (t, 8.0, 7.8 Hz)

122.2

H–6

7

6.91 (dd, 7.8, 1.0 Hz)

111.9

H–7

8 1a 4a 5a 8a 1’ 2’

3.74 (d, 6.8 Hz, 2H) 5.40 (m)

131.0 145.5 118.9 126.4 143.9 23.4 122.6

3’

-

3’–CH3 3’–CH3 1–OH 8–OH 3–CHO NH

1.68 (d, 1.2 Hz) 1.86 (s) 11.73 (s) 8.89 (br s) 9.97 (s) 10.23 (br s)

1

1

DEPT

H–1’ H–2’

H–1H COSY 7.08 (H-6), 6.91 (H-7) 7.59 (H-5), 6.91 (H-7) 7.08 (H-6), 7.59 (H-5) 5.40 (H–2’) 3.74 (H–1’)

133.0

-

-

C

25.9 18.1 197.0 -

3’–CH3 3’–CH3 -

-

CH3 CH3 CH -

C CH C C CH

HMBC (Figure 3) CHO, H–2 CHO CHO, H–1’ H–1’, CHO H–7

CH

H–5

CH

H–5

C C C C C CH2 CH

H–5 H–1’, H–2 H–5 H–6, H–5 H–7 H–2’ H–1’, 3’–CH3, 3’–CH3 H–1’, 3’–CH3, 3’–CH3 3’–CH3, H–2’ 3’–CH3, H–2’ H–2 -

Table 2: Cytotoxicity Activity of Pure Compounds against CEM–SS Cells Line (T-Lymphoblastic Leukemia) Pure Compounds

IC50 (µg/mL)

Clausine–TH (1)

2.1

Clausine–K (2)

5.1

Clausenarin (3)

 

Doxorubicin

0.1

Tamoxifen

36

Colchicine

0.02 Reference Compounds [18]


169



Ca

these assignments were further substantiated by 1H–1H COSY spectrum. The 13C–NMR spectrum showed the presence of 18 carbons atoms of which nine were quaternary carbons at 110.6, 116.4, 118.9, 126.7, 131.0, 133.0, 143.9, 145.5 and 158.5 ppm and assigned to C–4, C– 3, C–4a, C–5a, C–8, C–3’, C–8a, C–1a and C–1, respectively. Six methine carbons which were observed at 111.9, 112.0, 122.2, 122.6, 126.7 and 197.0 ppm were assigned to C–7, C–5, C–6, C–2’, C–2 and CHO, respectively. The methylene carbon at 25.9 ppm is due to C-1’ and two methyl groups resonate at 18.1 and 25.9 ppm. The 13C–NMR signals of (1) were assigned using the DEPT experiment. 1H–13C connectivity obtained from 1H–13C HETCOR and other correlations are summarized in Table 1. These spectral data suggested the structure of (1). For clarity, the proposed gross structure was unambiguously confirmed by the HMBC. 3.2 Cytotoxicity Assay The pure organic compounds were tested on the viability of CEM–SS cells line (T-Lymphoblastic Leukemia). Table 2 shows the IC50 value of each compound. Clausine–TH (1) gave an IC50 value of 2.1 µg/mL, which is considered chemotherapeutically significant (IC50  4 µg/mL). However, Clausine-K (2) is considered active against cytotoxic activity with an IC50 value 5.1 µg/mL, whereas Clausenarin (3) is considered not cytotoxic with an IC50 value  30 µg/mL. O 23 21 22

COOH

20

HO MeO

N H

OMe

17

12 11

OH

13 14

1

9

2

2 O

8

10

3

16 15

O

O

5

7 6

O

O

4

O

3

Figure 4: Structure of Clausine-K (2) and Clausenarin (3) Clausine-K (2). Yellow prisms (hot acetone), m.p. 254-256 oC. MS: C15H13NO4, m/z 271 [M]+. UV max nm (log ): 244 (2.29), 257 (0.80), 282 (1.91), 303 (0.65), 320 (0.65). IR max cm-1: 3412, 3317, 2930, 1665, 1618, 1579, 1443, 1346, 1322, 1287, 1239, 1201, 1163, 1116, 1083, 1036, 823, 679, 552. EIMS m/z (rel. int.): 271 ([M]+, 100), 256 (28), 240 (16), 212 (15), 196 (15), 183 (11), 169 (12), 153 (17), 140 (15), 127(14). 1H–NMR (DMSO–d6):  3.81 (3H, s, 7–OMe), 3.87 (3H, s, 2–OMe), 6.77 (1H, dd, J = 8.5, 2.2 Hz, H–6), 6.97 (1H, d, J = 2.2 Hz, H–8), 7.03 (1H, s, H–1), 7.94 (1H, d, J = 8.5, 2.2 Hz, H–5), 8.39 (1H, s, H–4), 11.28 (1H, br. s, NH). 13C-NMR (DMSO–d6):  55.3 (2–OMe), 56.0 (7–OMe), 94 (C–1), 95.1 (C–8), 108.1 (C–6), 112.3 (C–3), 115.8 (C–4a), 116.3 (C–5a), 120.5 (C–5), 123.1 (C–4), 141.6 (C–8a), 143.4 (C–1a), 157.4 (C–2), 158.1 (C–7) and 167.5 (C=O). Clausenarin (3). White needles (hot acetone), m.p. 292-294 oC. Lit.. R UV max nm (log ): 269 (0.05), 243 (0.03), 208 (0.91). IR max cm-1: 3483, 3432, 2950, 1719, 1638, 1398, 1301, 1162, 1117, 1065, 1028, 920, 875, 804, 601, 474. 1H–NMR (DMSO–d6):  1.05 (3H, s, CH3), 1.37 (3H, s, CH3), 1.52 (3H, s, CH3), 1.58 (3H, s, CH3), 1.65 (3H, s, CH3), 1.70 (1H, d, J =6.3 Hz, H–2), 1.86 (1H, d, J =14.9 Hz, H– 2), 2.48 (1H, dd, J =3.9, 3.9 Hz, H–12), 2.60 (1H, dd, J =3.9, 4.2 Hz, H–5), 2.63 (1H, s, H–9), 2.86 (1H,


170



s, H–6), 3.14 (1H, t, J =14.2 Hz, H–12), 3.47 (1H, d, J =15.4 Hz, H–6), 3.72 (1H, s, H–12), 4.00 (1H, d, J =2.6 Hz, 1–OH), 4.15 (1H, t, J =6.6 Hz, H–1), 4.62 (1H, d, J =4.4 Hz, 11–OH), 4.76 (1H, t, J =5.1 Hz, H–11), 5.54 (1H, s, H–17), 6.48 (1H, d, J =1.0 Hz, H–22), 7.57 (1H, t, J =1.7 Hz, H–23), 7.59 (1H, t, J =0.7 Hz, H–21). 13C-NMR (DMSO–d6):  18.4 (CH3), 19.6(CH3), 20.5(CH3), 23.9(CH3), 33.6(CH3), 36.9 (C–13), 40.0 (C–12), 40.2 (C–6), 44.6 (C–2), 46.2 (C–8), 47.1 (C–9), 52.4 (C–10), 50.7 (C–5), 54.4 (C–15), 65.5 (C–14), 66.6 (C–11), 70.5 (C–C–1), 79.0 (C–17), 84.4 (C–4), 111.0 (C–22), 121.8 (C–20), 142.3 (C–21), 144.1 (C–23), 168.0 (C–16), 170.8 (C–3), 208.7 (C–7). 4. CONCLUSION A new carbazole alkaloid (Clausine–TH) and two known compounds Clausine–K and Clausenarin were successfully isolated as pure compounds from the methanol crude extract of the stem back of Clausena excavata collected from Jabi, Kedah, Malaysia. These compounds were isolated from the conventional column chromatography method and the structure elucidations we done using the standard spectroscopic analyses (i.e. GC-MS, 1D-NMR, 2D-NMR and FTIR spectroscopy). The pure compounds isolated were also tested for their cytotoxic activity against the CEM-SS cell lines. The Clausine–TH and Clausine–K gave very significant activities, with the IC50 value of 2.1 µg/mL and 5.1 µg/mL, respectively. This study again proven that there are still lot more natural products in our nature need further investigation before it could be beneficial to us. ACKNOWLEDGEMENT Authors wish to thank Mr. Tan Boon Keat for his help in conducting the cytotoxic testing and Dr. Rusea Go for identifying the plant materials. This project was financially supported by the Malaysian Government under the IRPA programme. REFERENCES [1] [2] [3]

[4] [5] [6] [7]

Burkill, I. H.; A Dictionary of the Economic Products of the Malay Peninsula. Crown agents for the Colonies, London, The Malaya Nature Society: Kuala Lumpur 1935, Vol. 1 Sripisut, T.; Cheenpracha S.; Ritthiwigrom T.; Prawat U.; Laphookieo, S. Chemical constituents from the roots of Clausena excavata and their cytotoxicity. Records Nat. Prod. s 2012, 6, 386-389. Wu, T. S.; Huang, S. C.; Wu, P. L.; Lee, K. H. Structure and synthesis of clausenaquinone–A. A novel carbazolequinone alkaloid and bioactive principle from Clausena excavata. Bioorg. Med. Chem. Lett. 1994, 4, 2395-2398. Taufiq-Yap, Y. H.; Peh, T.H.; Ee, G. C. L.; Rahmani, M.; Sukari, M. A.; Muse, R. A new cytotoxic carbazole alkaloid from Clausena excavata, Nat. Prod. Res. 2007, 21, 310-313. Sripisut T.; Laphookhieo, S. Carbazole alkaloids from the stems of clausena excavata. J. Asian Nat. Prod. Res. 2010, 12, 614-617. Peng, W. W.; Zeng, G. Z.; Song, W. W.; Tan, N. H. A new cytotoxic alkaloid and two new other alkaloids from Clausena excavata. Chem. Biodiversity 2013, 10, 1317-1321. Cordell, G. A.; Beecher, W. W. C.; Fransworth, N. R.; Kinghorn, A. D.; Pezzuto, J. M.; Chai, H. B.; Constant H. L.; Fang, L. Q.; Likhitwitayawuid, K.; Long, L.; Ma, X. J.; Shieh, H. L.; Mahinda ,D. B.; Wikramaratne, Y. M. Recent Studies on biologically active natural products, In New Trends in Natural Products Chemistry Rahman A. and Choudhary M. I., Ed.; Harwood Academic Publisher; Australia; 1998, p 57-78.


171

Canadian Chemical Transactions Ca [8] [9] [10] [11]

[12] [13] [14] [15] [16] [17] [18]


Wu, C. C.; Ko, F. N.; Wu, T. S.; Teng, C. M. Antiplatelet effects of Clausine–D isolated from Clausena excavata. Biochimica et Biophysica Acta 1994, 1201, 1-6. Chakraborty, A.; Chowdhury, B. K. and Chattacharyya, P. Clausenol and clausenine – Two carbazole alkaloids from Clausena anisata. Phytochem. 1994, 40, 295-298. Yenjai, C.; Sripontan, P.; Kittakoop, P.; Jintassirikul, A.; Tanticharoen, M.; Thebtaranonth, Y.; Coumarins and carbazoles with antiplasmodial activity from Clausena harmandiana. Planta Medica 2000, 66, 277-278. Ali, A. M.; Mackeen, M. M.; Safinar, I.; Hamid, I.; Lajis, N. H.; El-Sharkawy, S. H.; Murakoshi, M. Antitumor promoting and antitumor activities of the crude extract from the leaves of Juniperus chinensis. J. Ethnopharmacol. 1996, 53, 165-169. Shier, W. T. An undergraduate experiment to demonstrate the use of cytotoxic drugs in cancer chemotherapy. Am. J. Pharm. Edu. 1983, 47, 216-220. Wu, T. S.; Huang, S. C. Clausine–D and –F, two new 4-prenylcarbazole alkaloids from Clausena excavata. Chem. Pharm. Bulletin 1992, 40, 1069-1071. Wu, T. S.; Huang S. C.; Wu P. L.; Teng, C. M. Carbazole Alkaloids from Clausena excavata and Their Biological activity. Phytochem. 1996, 43, 133-140. Wu, T. S.; Huang, S. C.; Wu, P. L.; Teng, C. M. Carbazole alkaloids from the stem bark of Clausena excavata. Phytochem. 1996, 43, 1427-1429. Wu, T. S.; Huang, S. C.; Wu, P. L.; Teng, C. M. Carbazole-pyranocoumarin dimer and binary carbazole alkaloid from Clausena excavata. Tetrahedron Lett. 1996, 37, 7819-7822. Wu, T. S.; Huang, S. C.; Wu, P. L. Lactonic Carbazoel Alkaloids from the Root Back of Clausena excavata. Chem. Pharm. Bulletin 1998, 46, 1451-1461. Ali, A. M.; Ismail, N. H.; Mackeen, M. M.; Yazan, L. S.; Mohamed, S. M.; Ho, A. S. H.; Lajis, N. H. Antiviral, cytotoxic and antimicrobial activities of anthraquinones isolated from the roots of Morinda elliptica. Pharm. Bio. 2000, 38, 298-301.

© 2013 By the Authors; Licensee Borderless Science Publishing, Canada. This is an open access article distributed under the terms and conditions of the Creative Commons Attribution license http://creativecommons.org/licenses/by/3.0/


172