Isolation of a deoxy lupane triterpene carboxylic acid from Finlaysonia obovata (a mangrove plant)

Fitoterapia 81 (2010) 977–981

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Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e

Isolation of a deoxy lupane triterpene carboxylic acid from Finlaysonia obovata (a mangrove plant) Pravat Manjari Mishra, A. Sree ⁎, Bandita Dash, Mallika Panigrahi, Susanta Kumar Padhan Natural Product Department, Institute of Minerals and Material Technology (Formerly RRL), Bhubaneswar-751013, Orissa, India

a r t i c l e

i n f o

Article history: Received 17 November 2009 Received in revised form 8 June 2010 Accepted 13 June 2010 Available online 25 June 2010

a b s t r a c t A deoxy lupane triterpene carboxylic acid, lup-20(29)-en-24-oic acid (1), was isolated from the active chloroform extract of Finlaysonia obovata, a latex exuding mangrove plant. Its structure was evaluated on the basis of different spectroscopic methods, including extensive 1D and 2D NMR spectroscopy. Lup-20(29)-en-24-oic acid (1) has shown moderate antimicrobial activity, against some fish pathogens. © 2010 Elsevier B.V. All rights reserved.

Keywords: Finlaysonia obovata Mangrove 3-deoxy triterpene Lup-20(29)-en-24-oic acid Antibacterial Fish pathogens

1. Introduction Literature shows the isolation of hopanoids from a number of plants [1–4], but hopanoids devoid of an oxygenated function at C-3 are the characteristic triterpenes mostly found in ferns [5–7], mosses [8,9], lichens [10], a few fungi [11,12] and also in the protozoan Tetrahymena pyriformis [13]. Some bacteria are also known to produce 3-deoxy triterpene of the hopane skeleton [14]. In contrast, triterpenes of higher plants, possess an oxygen functionality at C-3, as they are formed from a common substrate, 3(S)-2,3-oxidosqualene; by oxidosqualene cyclases (OSCs) and the epoxide oxygen is retained at C-3 in products. Absence of oxygen functionality at the C-3 of triterpenes of ferns suggests their direct derivation from cyclases (SCs). A deoxy diterpene methoxy-ent-8(14)-pimarenely-15-one of non hopane skeleton is also isolated from the mangrove plant, Ceriops tagal [15]. A lupane triterpene, 3-deoxy betulonic acid was also isolated from the active fraction of a CH2Cl2MeOH extracts of the twigs of Coussarea paniculata [16]. ⁎ Corresponding author. Tel.: +91 674 2581636x242; fax: +91 674 2581637. E-mail addresses: [email protected], [email protected] (A. Sree). 0367-326X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2010.06.016

The present paper deals with the isolation and spectral characterization of lup-20(29)-en-24-oic acid from active chloroform extract of leaves of Finlaysonia obovata. F. obovata is a mangrove plant belonging to the family Periplocaceae exuding white latex, the leaves of which are used in salad and treatment of asthma [17]. Latex bearing plants were found to show activity like anthelmintic activity [18], antiinflammatory activity [19] and other medicinal activities [20]. Earlier we have studied the antibacterial activity of extracts, lipid analysis and isolation of a rare antibacterial triterpene and a rare steroid from F. obovata [21–23]. To the best of our knowledge, this is the first report of the isolation of a 3-deoxy lupane triterpene with carboxylic acid at C-24 position from a latex bearing mangrove plant and from natural source. 2. Experimental 2.1. Plant material F. obovata is collected from Bhitarkanika mangrove forest of Orissa (during late winter season) and was identified by Mr. K.S. Murthy, I/C SMP unit, Central Research Unit (AY), Bhubaneswar.

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2.2. General experimental procedures Melting points were determined on a Buchi melting point apparatus and uncorrected. The 1H NMR spectra were recorded at 400 MHz and 13C NMR spectra were recorded at 100 MHz respectively on an AL-400 MHz FT-NMR, (JEOL, Japan). The phase-sensitive NOSEY spectra were measured with a 1024 × 256 data matrix and mixing time of 600 ms. A spectral width of 4100 Hz was used in both dimensions; 40 scans were acquired for each 256 t1 increments with a delay time of 3 s. The time domain data were zero-filled to yield a 1024 × 1024 data matrix. The ROSEY spectra were collected in a 1024 × 256 data matrix with 16 scans per t1 value. The time domain data were zero-filled to yield a 1024 × 1024 data matrix. 2.3. Extraction of the plant material The leaves of the plant (1 kg) were cut, shade dried, powdered and extraction was carried out with different solvents (1:2 vol./vol., thrice) sequentially in the increasing order of polarity like; hexane, chloroform, ethyl acetate and alcohol by soaking overnight at ambient temperature. The extracts were freed from solvent under reduced pressure. The residue thus obtained are finally dried under vacuum and used for in vitro screening of antibiotic activity. 2.4. Fractionation of chloroform extract and Isolation of pure compound The active crude chloroform extract was chromatographed on a column packed with silica gel (100–200 mesh) and the fractions were monitored by thin layer chromatography (TLC). The hexane-ethyl acetate (9:1) eluate on crystallization from CHCl3-MeOH mixture afforded a white amorphous solid, compound (1). 2.5. Antibiotic activity testing of compound 1 The antibacterial activity of compound 1 isolated from chloroform extract of F. obovata (100 μg and 50 μg/5 mm disc) was carried out against eight fresh water fish pathogenic bacteria viz., Micrococcus sp. (multidrug resistant strain), Aeromonas hydrophila (31), Aeromonas hydrophila (32), Pseudomonas aeruginosa, Vibrio alginolyticus, Staphylococcus aureus, Escherichia coli and Edwardsiella tarda by disc-assay method [24] (Table 2). Earlier the antibacterial activity of different extracts of F. obovata was studied against seven fish pathogens and the chloroform extract was found active against five pathogens [22]. The test bacterial pathogen cultures were obtained from the stock cultures maintained in the pathology Laboratory of Central Institute of Fresh water Aquaculture, ICAR, Bhubaneswar. Lup-20(29)-en-24-oic acid (1). Crystallized from CHCl3MeOH. (1 g, 0.1% Yield). mp (182–184 °C). MF: C30H48O2. o EIMS m/z: 440 [M]+. [α]29 (0.01 g/100 ml, CHCl3). D +39.60 IR (CHCl3, ν, cm−1): 3070, 1740, 885. 1H NMR and 13C NMR (See Table 1). EIMS m/z: 440[M]+, 425 [M-Me]+, 397 [M+-43], 367, 255, 221, 218, 203, 189, 177, 147, 109, 81, 55, 43.

Methylation of compound (1). Compound 1 (10 mg) was dissolved in 5 ml of MeOH, and treated with an ether solution of CH2N2 at room temp. for 2 h. After evaporation of the solvent, 8 mg of methyl ester 1a was obtained and crystallized from MeOH. 1H NMR (400 MHz, CDCl3):δ: 0.77 (3H, s), 0.79 (3H, s), 0.96 (3H, s), 1.06 (3H, s), 1.34 (3H, s), 1.68 (3H, s), 2.38 (1H, m), 4.57 (1H, brs), 4.69 (1H, brs), 3.640 (1H, s, COOCH3), 2.38 (1H, m, H-19). 3. Results and discussion The antibacterial assay of chloroform extract of F. obovata has shown strong activity against some fish pathogens [22]. This extract was further taken up for fractionations and isolations of secondary metabolites. The column fraction (Hexane-Ethyl acetate, 9:1) eluate on crystallization from CHCl3:MeOH afforded white amorphous powder (1). The compound is optically active and its molecular weight by mass spectrometry was 440 and elemental analysis gave the formula C30H48O2. It gave a positive Liebermann– Buchard test for triterpenes. The IR spectrum showed the absorption for a carboxylic group (1690 cm−1) and a terminal double bond (3070, 1740 and 885 cm−1). The 13C NMR spectra of compound revealed 30 carbon signals which were sorted by DEPT 13C NMR as six methyls, 11 methylenes, 6 methines, five quaternary carbons, one carboxylic acid and two olefinic carbons (one = CH2 and one quaternary). The Δ20,29-functionality of a lupene skeleton was inferred for this compound from the resonance of the SP2 carbons at C-29 (secondary carbon signal deduced by DEPT pulse sequence) at δ 109.42 and C-20 (quaternary carbon) at δ 150.08. A detailed analysis of the 1H NMR and 13C NMR spectra of compound 1 confirmed the characteristic features for a triterpenic lup-20(29)-ene parent structure. It was characterized by signals for five tertiary methyl at δ 0.77, 0.79, 0.96, 1.04 and 1.34 (3H each, Me-25, Me-28, Me-27, Me-26 and Me-23, respectively). One vinylic methyl at δ 1.68 (3H, s, Me30), two protons of an isopropyl moiety at δ 4.69 and 4.57 (1H each, brs, Ha-29 and Hb-29). In 13C NMR spectrum, the signal for the carboxylic function was observed at 180.43. The formation of a methyl ester also corroborated the presence of one carboxylic group. The position of the absorption of the methoxyl moiety of a methyl ester is also partially indicative of relative position of the carbomethoxyl group in the triterpene. The carbomethoxyl group absorbed at δ 3.640, which indicates the carboxylic group may be at C-24 or at C30 [25].The mass fragmentation pattern m/z 43, 55, 81, 109, 121, 147, 177, 189, 203, 218, 221, 255, 367, 397, 425 suggests that the carboxylic group might be located at ring A and B [26]. Generally the down field chemical shift showed by Hβ19 at δ 2.98 indicates the presence of carboxylic group at C-17 [27], but in the present investigated compound Hβ-19 comes at δ 2.38 which indicates the presence of carboxylic group at C-24 [28]. Localization of the carboxylic acid function at C-24 was also realized through the observation on the HMBC spectrum of 3J C-H interaction between the carboxylic carbon signal at δ 180.43 (C-24) and H-3 (δ 1.62), H-5 (δ 1.45) and H23 (δ 1.34) shown in Fig. 1 (Table 1). In the H1 × H1-ROSEY experiment, the compound showed correlation between proton signal of δ 1.34 (H-23) and δ 1.62 (α H-3), which

P.M. Mishra et al. / Fitoterapia 81 (2010) 977–981 Table 1 1 H (400 MHz) and

13

979

C NMR (100 MHz) data for compound 1/. HMQC

HMBC

δ (C)

δ (H)

C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 C-10 C-11 C-12 C-13 C-14 C-15 C-16 C-17 C-18 C-19 C-20 C-21 C-22 C-23 C-24 C-25 C-26 C-27 C-28 C-29

32.19 t 25.40 t 43.00 t 43.19 s 47.96 d 18.20 t 32.67 t 40.76 s 48.19 d 38.08 s 21.54 t 25.11 t 39.90 d 39.96 s 27.42 t 29.69 t 35.48 s 40.57 47.21 d 150.08 s 29.78 t 24.69 t 28.25 q 180.43 s 14.40 q 15.91 q 18.84 q 18.00 q 109.42 t

1.07 (m), 1.48 (m) 1.89 (m), 1.58 (m) 1.62 (m)

C-30

19.26 q

1.45 (1H, m) 1.82 (m), 1.61 (m) 1.64 (m), 1.35 (m)

(13

C-1H)

CH2(2), Me (25)

Me(25), H-18

H-5, Me(23), C-24 Me(23), CH2-3, C-24 Me(23), C-24 CH2(7)

H-5, Me(23)

CH(9), Me(26), Me(27) Me(27)

1.38 (1H,d, J = 11.2 Hz)

H1 ×H1-ROSEY

H-3

Me(25)

1.41 (m), 1.21 (m) 1.06 (m), 1.62 (m) 1.64 (1H.ddd, J = 11.2, 11.2, 2.8 Hz) Me(26), Me(27) 1.02 (m), 1.6 (m) 1.46 (m) 1.44 2.38 (1H, ddd, J = 11.2, 11.2, 3.6 Hz) 1.38 (m), 1.98 (m) 1.17 (m), 1.37 (m) 1.34 (3H, s) 0.77 (3H, s) 1.04 (3H, s) 0.96 (3H, s) 0.79 (3H, s) 4.57 (1H, brs, J = 2.0 Hz), 469 (1H, brs, J = 2.0 Hz) 1.68 (3H, s)

Me(28) Me(25) C(20), Me(30) Me(30)

CH(5) CH2(3), CH(5), Me(23) CH2(1) CH(9), CH2(7) CH2(15) CH2(16), CH2(22) Me(30), CH(19) CH-19, CH2-29

H-3

H-5 Me(26) Me(30)

suggest the presence of carboxylic group at C-24 position. The position of carboxylic group at C-24, is also clearly proved from NOSEY spectrum. In the NOSEY spectrum, the compound showed correlation between the proton signals δ 1.34 (H-23) and δ 1.45 (H-5), which confirms that the presence of carboxylic group at C-24 (Fig. 2). 13 C NMR assignment of compound (1) based on DEPT experiments and in comparison with the data of related compounds viz., α-boswellic acid, β-boswellic acid and 3αhydroxy-lup-20(29)-en-24-oic acid, its structure was eluci-

dated as lup-20(29)-en-24-oic acid [28]. The MS-data further confirmed the identity of compound (1) as lup-20(29)-en24-oic acid (Fig. 3) [26,28]. The all trans nature of A/B, B/C, C/D and D/E ring junctions and the absolute configuration of the ten asymmetric centres in the compound are determined to be 4S, 5R, 8R, 9R, 10R, 13R, 14R, 17R, 18R and 19R by NOSEY spectrum, 2D NMR and comparison of data with other lupane triterpenes. The antibacterial screening of compound 1 (Table 2) has shown broad spectrum activity against four fish pathogens viz. Micrococcus sp., A. hydrophila (31), P. aeroginosa and E. tarda and trace activity against two pathogens namely,

Fig. 1. HMBC correlations of compound 1.

Fig. 2. Important NOE correlations of compound 1.

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Institute (AY), Bhubaneswar for identification of the plant. The authors thank DOD/MoES for funding the project. References

Fig. 3. Lup-20(29)-en-24-oic acid.

Table 2 Antibacterial activity screening of lup-20(29)-en-24-oic acid against fish pathogens (zone of inhibition in mm including 5 mm disc). Compound 1 Fish pathogens

100 μg

50 μg

Micrococcus sp. Aeromonas hydrophila (31) Aeromonas hydrophila (32) Escherichia coli Pseudomonas aeroginosa Vibrio alginolyticus Staphylococcus aureus Edwardsiella tarda

7.0 7.0 Trace Trace 6.0 –ve –ve 6.5

6.0 Trace –ve –ve Trace – – 6.0

(–) = no zone, Trace = Trace inhibition (although observable, the zone of inhibition could not be measured).

A. hydrophila (31) and E. coli at 100 μg. At 50 μg, it has also shown activity against Micrococcus sp. and E. tarda and trace activity against A. hydrophila (31) and P. aeroginosa. Pentacyclic triterpenes have recently attracted much attention due to their great potential as multitarget therapeutics. Literature shows the isolation of a number of rare and novel triterpenoids from plants viz. deoxybetulonic acid [16], oxo-azukisapogenol [29], divergioic acid [30], 2βhydroxy-3-oxo-D:A-friedooleanan-29-oic acid [31], resinone (16-hydroxylup-20(29)-en-3-one) etc [32]. Structural modifications based on natural triterpenes have been extensively explored to find more potential pentacyclic triterpenes as preventive and therapeutic agents [33–37]. Studies of Structure-Activity relationships (SAR) of pentacyclic triterpenes showed that A-ring functions had a significant impact of biological activities [34–39]. The utilities of 3-deoxy pentacyclic triterpenes as therapeutic agents against metabolic diseases, tumors and HIV infections is also studied by Hao et al. [40]. Hence, the activity of chloroform extract may be due to the presence of the deoxy compound lup-20(29)en-24-oic acid. The isolation of biologically active 3-deoxy compound i.e. lup-20(29)-en-24-oic acid, can establish a scientific base for the use of this latex bearing mangrove plant F. obovata in modern medicine. Acknowledgments The authors thank Director, IMMT, Bhubaneswar for the facilities, Mr. K.S. Murthy, I/C SMP Unit, Central Research

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