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Three new carbazole alkaloids and biological activities of Murraya koenigii a

a

a

Shaista Naz , Sumayya Saied , Afifa Ahmed & Syed Muhammad b

Shahid a

Department of Chemistry, University of Karachi, Karachi 75270, Pakistan b

The Karachi Institute of Biotechnology & Genetics Engineering (KIBGE), University of Karachi, Karachi 75270, Pakistan Published online: 07 Oct 2014.

Click for updates To cite this article: Shaista Naz, Sumayya Saied, Afifa Ahmed & Syed Muhammad Shahid (2015) Three new carbazole alkaloids and biological activities of Murraya koenigii, Journal of Asian Natural Products Research, 17:1, 7-13, DOI: 10.1080/10286020.2014.959940 To link to this article: http://dx.doi.org/10.1080/10286020.2014.959940

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Journal of Asian Natural Products Research, 2015 Vol. 17, No. 1, 7–13, http://dx.doi.org/10.1080/10286020.2014.959940

Three new carbazole alkaloids and biological activities of Murraya koenigii Shaista Naza, Sumayya Saieda*, Afifa Ahmeda and Syed Muhammad Shahidb a

Department of Chemistry, University of Karachi, Karachi 75270, Pakistan; bThe Karachi Institute of Biotechnology & Genetics Engineering (KIBGE), University of Karachi, Karachi 75270, Pakistan

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(Received 9 June 2014; final version received 26 August 2014) Three new carbazole alkaloids, mukoenigatin (1), bikoeniquinonine (2) and murrayadinal (3), were isolated from the aerial parts of Murraya koenigii, along with mukeonine-B (4). Their molecular structures were determined on the basis of spectral analysis including UV, IR, MS, and 2D NMR spectroscopy. The antimicrobial activity of different fractions of plant extract was also determined. Keywords: Rutaceae; Murraya koenigii; carbazole alkaloids; mukoenigatin; bikoeniquinonine; murrayadinal

1. Introduction Murraya koenigii (L.) Spreng. (Rutaceae) is a tree whose leaves are commonly used in India for flavoring food stuffs [1]. Commonly it occurs in the foothills of the Himalaya [2]. Its leaves contain alkaloids and volatile oils [3]. This plant is a major source of carbazole alkaloids [4]. Traditionally, it is used as a stimulant, febrifuge, and as an analgesic agent [5,6]. It showed antioxidant, hypoglycemic, antibacterial activities [7–9], antidysentery [10] property, and also acts as a hepatoprotective material [11]. This paper describes the isolation and structure elucidation of three new carbazoles named 1 – mukoenigatin, 2 – bikoeniquinonine, and 3 – murrayadinal derived from the aerial parts of M. koenigii. 2.

Results and discussion

Compound 1 was obtained as pale yellow powder, and its molecular formula was suggested as C35H51NO7 by HR-FAB-MS (pos), showing the pseudo-molecular ion peak at m/z 598.3758 [M þ H]þ. The EIMS produced fragment ions at m/z 311

[M 2 C16H16NO4]þ, 286 [M 2 C19H35O3]þ and 227 [M 2 C21H24NO5]þ, indicating the cleavage of side chain. A typical ultra violet (UV) absorption maxima at 238, 261, 308, 346, 397 nm and infrared (IR) absorption bands at 3478, 3478, 1648, 1606 cm21 suggested the presence of a carbazole nucleus [12]. The 1H NMR spectrum revealed the presence of two methoxyl singlets at d 3.62 and 3.42. The presence of ethoxy group was confirmed by a quartet at d 4.13 and a triplet at d 1.55. The stereochemistry of C-10 and C-20 is established as threo on the basis of NOESY correlations between H-10 and H-20 . The 1H NMR and 13C NMR signals of side chain of compound 1 are approximately similar to the murrangatin ester which was previously isolated from Murraya paniculata [13]. The presence of long chain fatty acid ester was shown by a two-proton triplet at d 2.33 (H2//), a broad singlet at d 1.28 and a 3H triplet at d 0.86 (H-14//), corroborating the presence of 11 methylene groups and 1 methyl group, respectively. This long chain ester group at C2/ was substantiated by HMBC cross peaks of H-20 , H-200 and H-300 to C-100 at d 175.6. In HMBC spectrum, the long range 1H– 13C

*Corresponding author. Email: [email protected] q 2014 Taylor & Francis

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S. Naz et al.

correlations between the methoxyl at d 3.42 and C-1 (d 142.2), C-9a (d 136.5) and the other methoxyl at d 3.62 with C-7 (d 138.4) signified the presence of two methoxyl groups at positions C-1 and C-6, respectively. Thus, the structure of 1 was determined as 7-ethoxy-8-hydroxy-1,6dimethoxy-4-[10 -hydroxy-30 -methyl-20 -tridecanoyl-butenyl]-9H-carrbazole alkaloid, named as mukoenigatin (Figure 1). Compound 2 was obtained as green powder, and its molecular formula was suggested as C40H40N2O5 by HR-FABMS-(pos) which showed an [M þ 1]þ ion

at m/z 629.3013. In the EIMS, the peaks at m/z 254 [M 2 C25H28NO2]þ and 374 [M 2 C15H12NO2]þ indicated the cleavage of two monomeric units of carbazole alkaloids. The IR absorption bands at 1645, 3455, and 3435 cm21 as well as UV absorption maxima at 227, 240, 245, 285, and 351 nm indicated the presence of a carbazole-1,4-quinone nucleus [14,15]. The 1H NMR spectrum is approximately similar to that of bikoeniquinone-A [12]. This was supported by 13C NMR spectrum with two signals of carbonyl at d 172.8 and 172.4. In 1H NMR spectrum, a signal for a

Figure 1. Important HMBC and COSY correlations.

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Journal of Asian Natural Products Research deshielded proton appearing as doublet at d 7.99 could be assigned to H-5/ due to deshielding effect of the C-40 carbonyl moiety. The appearance of double doublet at d 4.15 and a triplet at d 1.19 indicated the presence of an ethoxy group at C-7/. The signal at d 9.96 revealed an aldehydic proton attached to C-3 on the carbazole nucleus [16]. A singlet of aryl methyl at d 3.13 (C-6) was observed along with signals assignable to the geranyl moiety at d 6.32 (H-200 ), 4.94 (H-600 ), 3.62 (H-100 ), 2.29 –2.37 (H-400 and 500 ), 1.67 and 1.71 (3H, s, C-700 ) and 1.90 (3H, s, C-300 ). The HMBC spectrum showed long-range 1 H – 13C correlations between H2-100 to C-1 (d 138.6), C-9a (d 145.3) and C-2 (d 127.6), revealing that the geranyl moiety was attached at C-1. An aryl methyl at d 2.29 also showed long-range correlations with C-5 and C-40 , which confirmed that the linkage between the two carbazole nucleus is between C-20 and C-5. In view of all these evidences, the structure of compound 2 can be represented as 1-[(20 E)-300 ,700 -dimethyl-200 ,600 octadienyl]-5-(70 -ethoxy-30 -methyl-10 ,40 dioxo-40 ,90 -dihydro-90 H-carbazol-20 -yl)-7methoxy-6-methyl-9H-carbazole-3-carbaldehyde, and has been accorded the trivial name bikoeniquinonine to reveal dimer, its species, quinone ring, and its alkaloidal nature. Compound 3 was obtained as a colorless amorphous solid, and the molecular formula was established as C19H17NO3 by HR-FAB-MS (pos), showing a pseudomolecular ion peak at m/z 308.12576 [M þ H]þ. EIMS analysis produced fragment ion at m/z 238, indicating the loss of a prenyl moiety. The IR absorption bands at 3450, 3400, and 1620 cm21 indicated the presence of a carbazole nucleus in the molecule. It was further supported by the UV spectrum showing absorption maxima at 224, 259, 303, and 380 nm which were similar to those of murrayalin-B and murrayalin-C [16]. The 1H NMR spectrum also presented a similar signal pattern to

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that of murrayalin-C except the presence of prenyl moiety which was confirmed by the presence of trans double bond indicated by the signals at d 6.33 (H-10 ) and 6.21 (H-20 ) and an isopropyl group exhibited by two multiplets at d 2.18– 2.24 (1H) and 0.61 –0.75 (6H). The 1H NMR spectrum exhibited two characteristic singlets at d 10.41 and 9.82, indicating the presence of two formyl groups attached to C-8 and C-3, respectively. A signal at d 11.25 was assignable to a strongly hydrogen bonded hydroxyl proton located ortho to the aldehyde group of C-8. The positions of formyl groups at C-3 and C-8 were also confirmed by HMBC spectrum showing long-range correlations between the formyl at d 9.82 with C-2 at d 125.9 and C-4 at d 122.1 and the other formyl group at d 10.41 correlated with C-7 at d 159.1 and C-8a at d 146.4. On the basis of all these evidences, the structure of 3 was assigned as murrayadinal (7hydroxy-1-[(E)-3-methyl-1-butenyl]-9Hcabazole-3,8-dicarbaldehyde). 3. 3.1

Experimental General experimental procedures

UV spectra were recorded on UV-1601 (Shimadzu, Tokyo, Japan), UV – visible spectrophotometer in methanol. IR spectra were recorded on FTIR-8900 (Shimadzu) spectrophotometer. 1H NMR spectra were recorded on Bruker Aspect AM-400, and AM-500 nuclear magnetic resonance spectrometers used SiMe4 as an internal standard. EI-MS was recorded with MAT312 (ThermoFischer Scientific, Bermen, Germany). High-resolution mass measurements and fast atom bombardment (FAB) mass measurements were carried out on Jeol – JMS-HX 110 mass spectrometer (JEOL Ltd, Tokyo, Japan). FAB source used glycerol as the matrix and cesium iodide (CsI) as internal standards. Vacuum liquid chromatography (VLC) and column chromatography (CC) were performed using silica gel (Si 60, 70– 230 mesh,

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S. Naz et al.

E. Merck, Darmstadt, Germany). Precoated silica gel60 F254 preparative plates (20 £ 20, 0.5 mm thick; E. Merck) were used for thick-layer chromatography.

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3.2

Plant material

The plant was collected from Baluchistan, Pakistan in July 2008 and identified by the taxonomist, Dr Rubina Dawar, Department of Botany, University of Karachi, Pakistan. A voucher specimen (No. 86445) has been deposited in the Herbarium of Botany Department, University of Karachi. 3.3

Extraction and isolation

The shade-dried aerial parts of M. koenigii (10 kg) were divided in two portions; one part was soaked in ethanol and the other in acetone. The extractions were repeated thrice at room temperature. The ethanolic extract was concentrated under reduced pressure to yield a gum (502 g). The gum was suspended in water and partitioned with n-hexane (1 L) and ethyl acetate (1 L), respectively, to yield n-hexane soluble fraction (240 g) and EtOAc soluble fraction (22.65 g). The EtOAc fraction was subjected to VLC (n-hexane – EtOAc; v/v 8:2, 6:4, 1:1, 2:8 and EtOAc –MeOH; v/v 8:2, 6:4, 1:1, 2:8, respectively). Totally eight fractions (A – H) were collected; fraction B (4.5 g) was subjected to CC over silica gel. As a result, 55 sub-fractions were collected, which were combined on the basis of TLC profile to get 7 fractions (B1 –B7). The fraction B4 (n-hexane – EtOAc; 7:3 eluted) was purified through preparative TLC (CHCl3 –MeOH; 9.5:0.5) to give 25 mg of mukoenigatin (1). Fraction C (6.1 g) was subjected to CC yielding 65 fractions which were combined on the basis of TLC to get 20 subfractions (C1 – C20). The fraction C8 (n-hexane –EtOAc; 1:1) was rechromatographed over silica gel. In total, 40 fractions were collected and combined to

get 10 fractions (CI –CX). Of these fractions, CVI (n-hexane – EtOAc; 8:4) was purified through preparative TLC (CHCl3 – MeOH; 9:1) to give 20 mg of bikoeniquinonine (2). The acetone extract was concentrated under reduced pressure. The crude extract obtained (85 g) was subjected to VLC. Five fractions (M – Q) were obtained (as n-hexane, n-hexane –EtOAc 1:1, EtOAc, EtOAc – MeOH 1:1). Fraction N (35 g, n-hexane – EtOAc 1:1) was subjected to CC over silica gel. The column was eluted with n-hexane/EtOAc and EtOAc/MeOH gradient system in increasing order of polarity. As a result, 146 fractions were collected, which were combined on the basis of TLC (25 fractions, N1 – N25). The fraction N15 (EtOAc– MeOH; 8:2) exhibited two major spots and purified through preparative TLC (CHCl3 –MeOH; 9:1, two developments). The faster moving band yielded 20 mg of mukeonine-B (4) and the slower moving band gave 30 mg of murrayadinal (3). 3.3.1 Mukoenigatin (1) Pale yellow powder (yield 25 mg). UV (CHCl3): lmax 238, 261, 308, 346, 397 nm; IR (KBr): nmax 3478, 1648, 1606 cm21; 1H and 13C NMR spectral data: see Table 1. EIMS m/z (rel. int. %): 311 (6.4), 286 (4.9), 255 (8.7), 227 (15.1), 224 (5.8), 179 (9.8), 162 (11.9), 84 (20.7), 57 (100). HRFAB-MS (pos): m/z 598.3758 [M þ H]þ (calcd for C35H52NO7, 598.3745). 3.3.2 Bikoeniquinonine (2) Green powder (yield 20 mg). UV (CHCl3): lmax 227, 245, 285, 351 nm. IR (KBr): nmax 3455, 3435, 1645, 1610 cm21. 1H and 13 C NMR spectral data: see Table 1. EIMS m/z (rel. int. %): 374 [M 2 C15H12NO3] (7.8), 254 (5.4), 305 (19.2), 239 (10.9), 195 (9.6), 137 (45.3). HR-FAB-MS (pos): m/z 629.3013 [M þ H] þ (calcd for C40H41N2O5, 629.3017).

Journal of Asian Natural Products Research Table 1.

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C and 1H NMR spectral data of compounds 1 – 3 (CDCl3, d in ppm, J in Hz). 1

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Position d (C) 1 2 3 4 4a 4b 5 6 7 8 8a 9a 10 20 30 40 4a0 4b0 50 6/ 70 80 8a0 9a0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 OCH3 OCH3 OCH2 CH3 NH OH CH3 CH3 CH3 CH3 CHO CHO

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2

d (H)

142.2 – 113.1 6.18 (d, J ¼ 7.8) 130.2 7.99 (d, J ¼ 7.8) 131.2 – 116.9 – 132.5 – 105.7 7.02 (s) 147.3 – 138.4 – 125.3 – 136.1 – 136.5 – 70.3 5.35 (d, J ¼ 8.5) 61.7 4.39 (d, J ¼ 6.9) 134.6 – 120.4 4.26 (dd, J ¼ 11.8, 4.1) – – – – 17.2 1.58 (s) – – – – – – – – – – 175.6 – 33.2 2.33 (t, J ¼ 7.5) 31.9 1.28 (brs) 29.7 1.28 (brs) 29.6 1.28 (brs) 29.4 1.28 (brs) 29.3 1.28 (brs) 29.2 1.28 (brs) 29.0 1.28 (brs) 29.1 1.28 (brs) 27.5 1.28 (brs) 24.9 1.28 (brs) 24.7 1.28 (brs) 14.1 0.86 (t, J ¼ 6.6) 56.2 3.62 (s) 55.9 3.42 (s) 65.4 4.13 (q, J ¼ 11.1, 5.9) 14.3 1.55 (t, J ¼ 6.9) – 9.50 (s) – 8.68 (s) – – – – – – – – – – – –

d (C)

3

d (H)

138.6 – 127.6 7.92 (s) 135.3 – 123.8 8.61 (s) 129.1 – 137.8 – 128.5 – 125.7 – 155.3 – 93.6 7.74 (s) 142.1 – 145.3 – 172.4 – 151.4 – 138.7 – 172.8 – 118.3 – 115.1 – 117.1 7.99 (d, J ¼ 7.7) 104.3 6.34 (d, J ¼ 7.7) 162.5 – 98.0 7.96 (s) 136.5 – 131.4 – 31.5 3.62 (dd, J ¼ 9.4, 8.3) 128.8 6.32 (t, J ¼ 10.1) 136.8 31.1 2.29 –2.37 (m) 19.5 2.29 –2.37 (m) 129.9 4.94 (d, J ¼ 7.0) 131.1 22.4 1.67 (s) – – – – – – – – – – – – – – 53.4 3.67 (s) 60.6 4.15 (dd, J ¼ 6.9, 2.4) 14.1 1.19 (t, J ¼ 7.1) – 10.05 (s) – – 17.3 1.71 (s, C-700 ) 14.7 3.13 (s, C-6) 15.9 2.29 (s, C-30 ) 22.6 1.91 (s, C-300 ) 192.7 9.96 (s, C-3) – –

d (C)

d (H)

142.8 – 125.9 7.62 (s) 136.6 – 122.1 8.55(s) 127.6 – 111.0 – 132.6 7.96 (d, J ¼ 8.5) 103.5 6.74 (d, J ¼ 8.5) 159.1 – 105.9 – 146.4 – 149.0 – 128.5 6.33 (d, J ¼ 17.5) 139.1 6.21 (dd, J ¼ 17.5, 5.5) 33.2 2.18 –2.24 (m) 22.7 0.61 –0.75 (m) – – – – 22.7 0.61 –0.75 (m) – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – 8.54 (s) – 11.25 (s) – – – – – – – – 190.4 9.82 (s) 192.5 10.41 (s)

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3.3.3 Murrayadinal (3) Colorless amorphous solid (yield 30 mg). UV (CHCl3): lmax 224, 259, 380 nm. IR (KBr): nmax 3350, 3445, 1670, 1640, 1610 cm21. 1H and 13C NMR spectral data: see Table 1. EIMS m/z (rel. int. %): 307 (Mþ, 10), 278 (15), 251 (43), 238 (100), 209 (13), 163 (32), 69 (56). HRFAB-MS (pos): m/z 308.1258 [M þ H]þ (calcd for C19H18NO3, 308.1287).

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3.4

Antimicrobial activity

The antimicrobial activities of different extracts of M. koenigii have been analyzed against methicillin-resistant Staphylococ-

cus aureus (MRSA), vancomycin-resistant Staphylococcus aureus (VRSA) and ciprofloxacin-resistant V-MRSA (CVM-RSA). The obtained extracts, including n-hexane extract (MKAH – 35 g), acetone extract (MKA – 8.5 g), and methanol extract (MKAM – 5.2 g), were evaluated by disc diffusion method. Among all, the methanol extract (MKAM) showed most promising against VRSA, MRSA, and CVM-RSA, as it was found to be most effective against highest number of clinical isolates of VRSA (68.2%), MRSA (65.8%), and CVM-RSA (65%) with an activity zone ranging from 6 to 16 mm, as shown in Tables 2– 4.

Table 2. Antimicrobial activity against VRSA (n ¼ 44). Zone of activity (mm) Extract

Minimum

Maximum

NO activity (number of strains)

MKA MKAH MKAM

8 6 6

20 18 16

30 27 14

Activity (number of strains)

Percentage

14 17 30

31.8 38.6 68.2

Activity (number of strains)

Percentage

14 14 27

34.1 34.1 65.8

Activity (number of strains)

Percentage

14 13 26

35.0 32.5 65.0

Table 3. Antimicrobial activity against MRSA (n ¼ 41). Zone of activity (mm) Extract

Minimum

Maximum

No activity (number of strains)

MKA MKAH MKAM

8 6 6

20 16 16

27 27 14

Table 4. Antimicrobial activity against CMV-RSA (n ¼ 40). Zone of activity (mm) Extract

Minimum

Maximum

No activity (number of strains)

MKA MKAH MKAM

8 6 6

20 16 16

26 27 14

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[9] S.M. Zachariah, P. Muthumani, and K. Ramaseshu, J. Pharmacol. 6, 4 (2009). [10] A.C. Adebajo, O.F. Ayoola, E.O. Iwalewa, A.A. Akindahunsi, N.O. Omisore, C.O. Adewunmi, and T.K. Adenowo, Phytomedicine 13, 246 (2006). [11] J.E. Wilking, J.K. Mathias, K. Das, I.S. R. Nidhaya, and G. Sudhakar, Indian J. Nat. Prod. 23, 13 (2006). [12] C. Ito, Y. Thoyama, M. Omura, I. Kajiura, and H. Furukawa, Chem. Pharm. Bull. 41, 2096 (1993). [13] F. Imai, K. Itoh, N. Kishibuchi, T. Kinoshita, and U. Sankawa, Chem. Pharm. Bull. 37, 119 (1989). [14] T.S. Wu, T. Ohta, H. Furukawa, and C.S. Kouh, Heterocycles 20, 1267 (1983). [15] H. Furukawa, T.S. Wu, T. Ohta, and C.S. Kouh, Chem. Pharm. Bull. 33, 4132 (1985). [16] C. Ito, M. Nakagawa, T. Sheng Wu, and H. Furukawa, Chem. Pharm. Bull. 39, 2525 (1991).

Three new carbazole alkaloids and biological activities of Murraya koenigii.

Three new carbazole alkaloids, mukoenigatin (1), bikoeniquinonine (2) and murrayadinal (3), were isolated from the aerial parts of Murraya koenigii, a...
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