Phytoconstituents of Adenanthera pavonina Linn from the bark extracts

Adenanthera pavonina L. is an important medicinal plant and its barks are used in traditional medicine for treating different diseases. Therefore, a phytochemical investigation was carried out to isolate and identify secondary metabolites from its barks. Seven compounds namely ethyl 3,3-dimethyl-13-hydroxytridecanoate (1), stigmasta-5,22-dien-3β-ol (2), tert.butyl tridecanoate (3), 6-α-hydroxy stigmast-20(21)-en-3-one (4) of dichloromethane extract and 18-(2′, 3′-dihydroxyphenyl)nonadec-17-en-2-ol (5), 1-(N-propyl amino)-2-henecosanone (6), and stigmast–5(6), 20(21)-diene-3-one (7) were isolated from the barks of Adenanthera pavonina Linn. Of these compounds, 1, 4, 5, 6, and 7 appear new. The structures of these compounds were elucidated by spectroscopic techniques, mainly by NMR. Five new and two known compounds have been isolated and characterized from the bark of A. pavonina. The isolated compounds could be a potential template for the synthesis and development of new lead compounds with interesting pharmacological properties.


Background
Adenanthera pavonina L (Bengali: Rakta kambal) is an erect medium-sized tree (6-15 m tall and up to 45-cm diameter) with dark brown to grayish bark belongs to the family Leguminosae. The plant is native to the Asian continent and mostly found in Africa, Pacific and Caribbean regions [1]. It is also indigenous to India and Bangladesh particularly in the South-eastern region [2]. Different parts of this plant have been used in traditional medicine for the treatment of various diseases. The bark and leaves are used as a remedy for diarrhea, gout, hematuria, hematemesis, and chronic rheumatism [1][2][3][4][5]. The anti-inflammatory, analgesic, antioxidant, cytotoxic, anti-diarrheal, acute toxicity, antibacterial, antifungal, and blood pressure-reducing activities of the bark, leaf, and seed extracts and its isolated compounds have been reported [6][7][8][9][10][11][12][13]. Previous phytochemical investigation reported the presence of many bioactive compounds like robinetin, chalcone, butin and flavanol ampelopsin, stigmasterol glucosides, oleanolic acid, echinocystic acid, and sapogenins from the leaves and seeds of the plant [6,[14][15][16][17][18][19]. Very few compounds like as stigmasterol glucosides, oleanolic acid, and echinocystic acid have been reported from the bark part [15,20]. Our previous in vitro studies have shown that the bark extracts of the plant possessed significant pharmacological activities [11][12][13]. Hence, our aim is to isolate bioactive compounds from this plant part. spectra were recorded using Nicolet iS10 FT-IR spectrometer by potassium bromide (KBr) pellets. 1 H NMR, 13 C NMR, DEPT 135 spectra, and attached proton test (APT) spectra were recorded in CDCl 3 , CD 3 OD, and mixture of CDCl 3 and CD 3 OD with a 300-MHz NMR Spectrometer (Varian MERCURY-VX-300). Chemical shifts are presented in δ (ppm) using tetramethylsilane (TMS) as an internal standard, and coupling constants (J) are expressed in Hertz (Hz). Mass spectra were recorded by infusion into the ESI source using CH 3 OH/CH 3 OD as a solvent with a LC-ESI-MS/MS-System TSQ Quantum Ultra AM Finnigan and Triple Quadrupole MS with Mikro-HPLC (Surveyor plus). Pre-coated glass plates of silica gel (Keiselgel 60, F254, Merck KGaA, Darmstadt, Germany) were used for TLC analysis. The TLC spots were observed under long and short wavelength UV light (Fisher Scientific LCF-445) at 366 and 254 nm and the plates were sprayed with vanillin-sulfuric acid solution.

Plant material
The barks of Adenanthera pavonina were collected from the capital city Dhaka of Bangladesh. The plant was authenticated by Dr. Sardar Nasir Uddin of Bangladesh National Herbarium, Dhaka, and a voucher specimen (accession number-34196) was deposited in the Herbarium.

Compound 3
Compound 3 was identified as tert.butyl tridecanoate by comparing its spectral data with those reported for this compound (http://www.nmrdb.org) (Additional file 1).

Compound 4
The IR spectrum of compound 4 showed an absorption band at 3676 cm −1 indicated a hydroxyl group (-OH) and the band at 1685 is responsible for C=O bond. The sharp absorption band at 2940 and 2869 cm −1 were demonstrative of aliphatic C-H stretching. The bands at 983 and 883 cm −1 were demonstrative for the steroidal nature [22]. 1 H-NMR data ( Table 1) of 4 showed two singlets at δ 0.74 and 0.81 (2×CH 3 , C-18, 19) of 3H proton intensity of each and two doublets found at δ 0.94 and 0.96 (2×CH 3 , C-26,27) of 3H proton. Moreover, 3H distorted triplet found at δ 0.87 (1×CH 3 , C-29) is typical steroidal signal [22]. The distorted triplet for single proton at δ 4.15 is suggested for an oxymethine proton flanked by one methylene groups of cyclohexane ring system of a steroidal compound. The oxymethine proton may be attached to C-1, C-2, C-6, or C-12. If the oxymethine proton is attached to C-1, C-2, or C-12, it will find a triplet, but oxymethine proton showed a broad multiplet at δ 4.15 due to its β-axial orientation [23]. So, the oxymethine proton is at C-6. The spectrum displayed signals at δ 4.73 and 4.65 (1H, each, s, br) attributable to an exomethylene protons [24]. The presence of the double bonds at C-20 in this structure received support from 13 C-NMR data (Table 1) at δ 150.79 for C-20 and δ 109.2 for C-21. The presence of a keto group (C-3) and a hydroxyl group (C-6) is also confirmed by the 13 C-NMR at δ 180.3 for C-3 and δ 79.1 for C-6. Moreover, it responded to the Salkowsky and Liebermann-Burchard [25] color tests to exhibit its steroidal nature. On the basis of spectral data, we can assign the structure of compound 4 as 6-α-hydroxy stigmast-20(21)-en-3one. The structure of 4 was confirmed on the basis of the comparison of their data with lupeol [24] and 12α-Hydroxystigmast-4-en-3-one [23].

Compound 5
The IR spectrum of compound 5 was assigned for the presence of hydroxyl group (-OH) at 3425 cm −1 . The band at 1506 cm −1 is responsible for double bond stretching of aromatic carbon, whereas the band at 1660 cm −1 indicated the aliphatic C=C stretching. From the 1 H-NMR data (Table 1) (Table 1) assigned for N-H proton. The 13 C-NMR also showed the peak at δ 57.6 indicated that amino-substituted carbon is there. The C-1 is flanked by a keto group as well as by NH group is indicated by the 1 H-NMR value at δ 4.04 (s, br) and 13  On the other hand, the distorted triplet at δ 5.35 is suggestive of an alkene proton of cyclohexane ring system of a steroidal compound [23]. The spectrum attributable to an exomethylene protons at δ 4.72 and 4.59 (1H, each, br.s) [24]. The 1 H-NMR for H-2 at δ 3.17 and H-4 at δ 3.65 (s) indicated that the keto group is present in C-3 position. We could not do any 13 C-NMR and mass spectra due to the isolation of a very small amount of 7.
But the 1 H-NMR spectra is almost similar to that for compound 4. Moreover, it responded to the Salkowsky and Liebermann-Burchard color tests [25] of steroidal compounds. Therefore, 1 H-NMR spectral data of compound 7 is suggested as stigmast-5(6), 20(21)-diene-3one by comparison with 4 which molecular formula is C 29 H 46 O. The structure of 7 is attained only by the removal of water from 4, that results in a C=C double bond at C-5 and C-6.

Conclusions
Adenanthera pavonina Linn. has been reported for its various pharmacological activities in the field of traditional medicines. Present investigation has unfolded its seven compounds from the bark extract especially dichloromethane and ethyl acetate extract for the first time. The compounds were isolated through chromatographic methods and their structures were established by extensive spectroscopic techniques, particularly NMR. This investigation may open up future research in the field of synthetic chemistry to synthesis new series of compounds with immense medicinal importance.
Additional file 1: Figure S1. 1 H-NMR spectrum of the compound 1. Figure S2. ESI-MS (Negative ion) spectrum of the compound 1. Figure  S3. IR spectrum of the compound 2. Figure S4. 1 H-NMR spectrum of the compound 2. Figure S5. 13 C-NMR spectrum of the compound 2. Figure  S6. ESIMS spectrum of the compound 2. Figure S7. IR spectrum of the compound 3. Figure S8. 1 H-NMR spectrum of the compound 3. Figure  S9. 13 C-NMR spectrum of the compound 3. Figure S10. IR spectrum of the compound 4. Figure S11. 1 H-NMR spectrum of the compound 4. Figure S12. 13 C-NMR spectrum of the compound 4. Figure S13. IR spectrum of the compound 5. Figure S14. 1 H-NMR spectrum of the compound 5. Figure S15. ESI-MS (positive ion) spectrum of the compound 5. Figure S16. GC-MS spectrum of the compound 5. Figure  S17. IR spectrum of the compound 6. Figure S18. 1 H-NMR spectrum of the compound 6. Figure S19. 13 C-NMR spectrum of the compound 6. Figure S20. 1 H-NMR spectrum of the compound 7. Table  S1. 1 H-NMR of compound 2 and Comparison of 13 C-NMR data of compound 2 with those of the published data [19].