INTRODUCTION:

Barringtonia asiatica L. Kurz is a mangrove plant that grows in tropical Asia and the Pacific, including Northern Australia, the seeds of which are used as a fish poison.^1,2 In a previous paper,¹ we reported the isolation and structural elucidation of two major saponins, 1 and 2 putative antifeedant toward Epilachna sp larvae from seeds of B. asiatica. As part of our continuing program to explore the saponins from B. asiatica,^1,2 we now report the isolation and structural elucidation of a new antifeedant saponin, 3 from the seeds of this species.

RESULTS AND DISCUSSION:

A methanolic extract of the seeds of B. asiatica was partitioned between n-BuOH and water. Using several steps of reversed-phase HPLC afforded a saponin 3 that was obtained as a white amorphous solid purified the n-BuOH-soluble fraction.

The positive and negative FAB mass spectra of 3 showed an [M+Na+H]⁺ ion at m/z 1081 and an [M-H]^– ion at m/z 1057 suggesting an M_r of 1058 amu (C₅₃H₈₆O₂₁). The IR spectrum showed absorptions at 3416 cm^-1 (-OH), 1708 cm^-1, (ester carbonyl), 1613 cm^-1 (C=C), and 1100 – 1000 cm^-1 (glycosidic linkages).

The ¹H and ¹³C NMR spectra of 3 which are presented in Table 1, showed seven signals for methyl groups at d 0.77, 0.84, 1.02, 1.07, 1.22, 1.23, 1.85 ppm, and at d 15.6, 16.6, 16.8, 25.2, 27.5, 27.8, 33.4 ppm; one olefinic proton at d 5.33 ppm, two olefinic carbons at d 143.6 and 124.8 ppm; and methylene protons at d 3.49 and 3.66 ppm (AB d, 10.5 Hz) attached to a carbon at d 63.3 ppm, indicating a hydroxymethylene. An olean-12-ene skeleton therefore appeared likely.

The protons attached to C-16 and C-22, were identified as a broad signal at d 4.58 ppm and a deshielded doublet of doublets at d 6.13 ppm (dd, 5.5; 11.5 Hz), respectively. The deshielding of H-22 indicated an esterified position. This information was also supported by the downfield shifts in their ¹³C NMR resonances and the analysis of HMBC spectra. The low-field shift of protons attached to the 27-methyl (d 1.85 ppm) indicated a 1,3 diaxial relationship to hydroxyl, so the 16-hydroxyl should have the a configuration. Meanwhile, the b configuration for H-22 was deduced from its coupling constants. By comparison of these data with those in the literature^3-5 it could be concluded that the triterpene moiety of 3 was camelliagenin A (4).

The appearance of five signals at d 11.8 (q), 16.9 (q), 27.2 (t), 40.8 (d), and 176.2 ppm (s) in ¹³C NMR spectrum and two methyl groups at d 0.88 (t, 7.5 Hz) and 1.14 ppm (d, 7.0 Hz) in ¹H NMR spectrum which were correlated in DQCOSY spectrum with an AX₅ system signal of a methine proton at d 2.39 ppm (sxt, 7.0 Hz) indicates the presence of a 2-methylbutyroyl residue in the molecule. Long range correlation between H-22 at d 6.13 ppm and a carbonyl (C-1’’’’) at d 176.2 ppm in HMBC spectrum was also observed, indicating the 2-methylbutyroyl residue was attached to the C-22 position. The ¹H and ¹³C NMR data of the acid residue of 3 are presented in Table 2.

The identity of the sugars present in 3 was established by treatment of the latter with anhydrous methanolic HCl followed by per-trimethylsilylation. The GC profile of the products was compared with reference sugars treated under the same conditions and this indicated that D-galactose, D-glucose and D-glucuronic acid were present in approximately equal amounts. The D- configuration has been assumed for these sugars in keeping with Massiot and Lavaud’s assertion,⁶ “The enantiomers of these sugars are not found in plants, a fact used as a clue in the determination of these sugars”.

Table 1. ¹H and ¹³C NMR data of aglycone moiety of 3 in pyridine-d₅^a.

Position	d C (ppm)	DEPT	d H (ppm) multiplicity J (Hz)
1	38.7	CH₂	0.80 dm, 14.0 1.34 dm, 14.0
2	26.5	CH₂	1.80 dm, 13.0 2.10 dm, 13.0
3	89.4	CH	3.25 dd, 3.5; 11.0
4	39.6	C	-
5	55.6	CH	0.72 br, d 11.5
6	18.4	CH₂	1.47 dm, 13.0 1.56 dm, 13.0
7	33.0	CH₂	1.30 m, 1.58 m
8	40.0	C	-
9	46.8	CH	1.74 m
10	36.7	C	-
11	23.7	CH₂	1.69 m
12	124.8	CH	5.33 br, s
13	143.6	C	-
14	41.7	C	-
15	35.1	CH₂	1.59 m, 1.89 m
16	69.9	CH	4.58 br, s
17	44.8	C	-
18	41.9	CH	3.01 dd, 3.5; 14.0
19	47.4	CH₂	1.29 m, 2.87 t, 13.5
20	32.0	C	-
21	41.7	CH₂	1.90 m 2.78 t, 11.5
22	71.8	CH	6.13 dd, 5.5; 11.5
23	27.8	CH₃	1.22 s
24	16.6	CH₃	1.07 s
25	15.6	CH₃	0.77 s
26	16.8	CH₃	0.84 s
27	27.5	CH₃	1.85 s
28	63.3	CH₂	3.49 d, 10.0 3.66 d, 10.0
29	33.4	CH₃	1.02 s
30	25.2	CH₃	1.25 s

^a Assignment was also based upon DQCOSY, HMQC, HMBC, HMQC-TOCSY.

The first step in identifying the number of sugar residues present in 3 involved beginning with the anomeric proton and carbon resonances. The number of sugars can usually be determined by counting both the number of anomeric protons (d 4.5 – 6.5 ppm) and carbons (d 90 – 112 ppm) present in the 1-dimensional ¹H and ¹³C NMR spectra.⁷ In this manner, one unit each of D-galactose, D-glucose and D-glucuronic acid as a carbohydrate trisaccharide [individual sugars are indicated by bold capital letters (A – C)] were identified in 3 based on their characteristic proton and carbon signals (Table 1). Moreover, the presence of carbonyl resonance at d 170.0 ppm and two hydroxymethyl resonances at d 61.9 and 63.4 ppm (Table 1) further confirmed that the trisaccharide consists of an acid and two hexose sugars. Note that the additional proton resonance (d 5.33 ppm, br s) in the anomeric region has been previously assigned to the vinylic proton (H-12) of the aglycone part of 3.

Due to severe overlap in the d 3.5 – 5 ppm regions of the ¹H NMR spectrum of the sugar moiety of 3, only the H-1,H-2 connectivities from the anomeric protons can be unambiguously identified from a DQCOSY spectrum. Thus the full assignment of the spin systems of each individual sugar of 3 was derived from an HMQC-TOCSY experiment.⁷ The HMQC part leads to direct (one-bond) ¹H – ¹³C correlations,^8,9 and the TOCSY part was used to obtain correlations for all of the protons of an isolated spin network.¹⁰ The application of this experiment to the three monosaccharides present in 3 are discussed below. All NMR data are presented in Table 2 and the relayed correlations observed in the HMQC-TOCSY spectrum are presented in Table 3.

Residue A.^___ The anomeric proton and carbon of residue-A show four cross-peaks in the HMQC-TOCSY spectrum (Table 3) indicating the correlations between four carbons at d 78.5, 77.7, 76.3, 72.4 ppm, which were later assigned to C-3, C-5, C-2, and C-4 respectively, with the anomeric proton at d 5.64 ppm (1H, d, 7.5 Hz), and the correlations between four protons at d 4.22, 4.15, 4.05, 3.80 ppm with the anomeric carbon at d 103.8 ppm. Those protons were assigned to H-3, H-4, H-2, and H-5 respectively. The four cross-peaks of the anomeric proton indicated the presence of a large vicinal coupling among ring protons due to a trans diaxial orentation, suggesting a gluco configuration. Further correlation occurred between the methylenic protons AH-6 (A stands for residue-A, H-6 for the methylenic group at position 6) at d 4.30 and 4.44 ppm with C-5, C-4, and C-3, as well as H-5, H-4, H-3 (Table 3), indicated that this residue was a hexose sugar. Meanwhile, the ³J_H-1,H-2for this residue was 7.5 Hz, indicating a b-anomeric configuration. Therefore, residue-A was assigned as b-glucopyranose.

Residue B. ^__ In the HMQC-TOCSY spectrum (Table 3), the anomeric proton and carbon of residue-B show only three cross-peaks, thus indicating the existence of small ³J_H-4,H-5 value and suggesting a galacto configuration. Moreover, the correlations occurred between the methylenic protons BH-6 at d 4.33 and 4.42 ppm with BH-5, then BH-5 with BH-4 (Table 3), indicated that this residue was a hexose sugar. Based on its ³J_H-1,H-2 of 8.0 Hz, this residue also had a b-anomeric configuration. Thus, residue-B was assigned as b-galactopyranose.

Tabel 2. ¹H and ¹³C NMR data of osidic moieties of 3 in pyridine-d₅.

	dC (ppm)	dH (ppm) multiplicity J (Hz)		dC (ppm)	dH (ppm) multiplicity J (Hz)
b-D-Glc-A			b-D-Gal
1’	105.1	4.92 (d, 7.5)	1’’’	105.1	5.31 (d, 8.0)
2’	78.9	4.42 (m)	2’’’	72.9	4.48* (m)
3’	87.7	4.34 (m)	3’’’	75.3	4.14 (m)
4’	71.8	4.46^a (m)	4’’’	70.1	4.45^a (m)
5’	77.3	4.48*(m)	5’’’	77.3	4.16 (m)
6’	172.0	-	6’’’	61.9	4.33 (m) 4.42 (m)
b-D-Glc			2-methylbutyroyl
1’’	103.8	5.64 (d, 7.5)	1’’’’	176.2	-
2’’	76.3	4.05 (m)	2’’’’	40.8	2.39 (sxt, 7.0)
3’’	78.5	4.22 (m)	3’’’’	27.2	1.44 (m) 1.74 (m)
4’’	72.4	4.15 (m)	4’’’’	11.8	0.88 (t, 7.5)
5’’	77.7	3.80 (m)	5’’’’	16.9	1.14 (d, 7.0)
6’’	63.3	4.30 (m) 4.44 (m)

* overlapping signals; ^a may be interchangeable

Table 3. Relayed correlations of the sugar moiety of 3. One-bond correlations are shown bold.

Residue-3A (glucose)		Residue-3B (galactose)		Residue-3C (glucuronic acid)
NO. C	NO. H	NO. C	NO. H	NO. C	NO. H
C1	H1,H2,H3,H4,H5	C1	H1,H2,H3,H4	C1	H1,H2,H3,H4
C2	H1,H2,H3,H4,H5	C2	H1,H2,H3	C2	H1,H2,H3,H4
C3	H1,H2,H3,H4,H5,H6	C3	H1,H2,H3,H4	C3	H1,H2,H3,H4
C4	H1,H2,H3,H4,H5,H6	C4	H1,H2,H3,H4,H5	C4	H1,H2,H3,H4
C5	H1,H2,H3,H4,H5,H6	C5	H4,H5,H6	C5	n.o.
C6	H3,H4,H5,H6	C6	H5,H6	C6	^__

n.o. : not observed

Based on the above observations, the structure of 3 was determined to be 3-O-{[b-D-galactopyranosyl(1®3)-b-D-glucopyranosyl(1®2)]-b-D-glucuronopyranosyloxy} -22-O-[2-methylbutyroyloxy]-16,28-dihydroxy-(3b,16a,22a)-olean-12-ene. To the best of our knowledge, this compound has not been characterized previously.

Compound 3 was tested for antifeedant activity toward Epilachna sp.¹ and it exhibited 100 % activity at a concentration of 1000 mg mL^-1. From this assay, compound 3 could be developed for natural pesticide as well as two saponins, 1 and 2 that were previously isolated from B. asiatica.¹ However, the crude saponin from the seed of B. asiatica could be also developed as natural pesticide because it showed antifeedant activity.¹

MATERIAL AND METHODS:

General Experimental Procedures:

The IR spectrum was determined using a Perkin-Elmer 1800 FTIR spectrophotometer. Optical rotation was measured in a 1 decimeter path cell with a Perkin-Elmer 241 polarimeter. The FABMS was measured in a 3-nitrobenzyl-alcohol matrix on a VG Analytical ZAB-SEQ2 Mass Spectrometer. ¹H and ¹³C NMR spectra were recorded using a Varian INOVA instrument at 500 MHz (¹H) and 125 MHz (¹³C). All of the NMR data were measured in pyridine-d₅ at 25 ⁰C and chemical shifts are expressed in d (ppm). 2D experiments were performed using standard INOVA programs.

Two step semi-preparative RP-HPLC was performed on YMC-Pack ODS-AQ, 5mm 120Å 250 mm columns of 10 and 20 mm internal diameter, thermostatted at 40ºC. The mobile phase was generated by blending A: MeOH/THF/H₂O/HOAc (9/1/90/0.05), B: MeOH/ THF/HOAc (90/10/0.05) in 60%B of isocratic elution using a flow rate of 16 mL min^-1 (step 1); and blending A: H₂O/HOAc (100/0.05), B: CH₃CN/HOAc (100/0.05) in 45%B of isocratic elution using flow rate of 4 mL min^-1 (step 2). Instrumentation consisted of two Waters 510 and 481 pumps, a Rheodyne 7125 injector fitted with a 4.4 mL sample loop, and a Waters 481 UV/visible detector fitted with a 2.3 mm path flow cell, monitoring absorbance at 210 nm.

GC was performed on a Varian-3400 instrument with an FID and a 12 meter 0.22 mm i.d., BP-1 (100% polydimethylsiloxane), column was used. The injector temperature was 250ºC, and the detector 320ºC. The temperature program used was 50ºC (2 min hold) then heated at 10ºC min^-1 to 300ºC and held 3 min at this temperature before cooling down. The retention times were compared with those of authentic sugars, treated under identical conditions.

Plant Material:

The seeds of Barringtonia asiatica were collected on 1996 in Sangihe Talaud region, North Sulawesi province, Indonesia. This plant was identified by Mr Djuandi of the Herbarium of Department of Biology, Bandung Institute of Technology, Indonesia, where a voucher specimen has been deposited.

Extraction and Isolation:

Extraction of a brown residue containing crude saponin was described previously.^1,2 The crude saponin (4.35 g) was dissolved in methanol and then injected on to a semi-preparative RP-HPLC using mobile phase of step 1 (see general experimental) to obtain five fractions. Rechromatography of fraction 5 on the semi-preparative RP-HPLC using mobile phase of step 2 gave five fractions, where the subfraction 1 was a single pure 3 (70 mg).

Compound 3:

White amorphous solid, 70 mg; [a]²⁵_D –1.4º CH₃OH; IR (KBr) v_max 3427 (OH), 1709 (C=O), 1613 (C=C), 1100-1000 cm^-1 (glycosidic linkages); NMR (see Table 1 and 2); FABMS m/z 1081 [M+Na+H]⁺ and 1057 [M-1]^–, anal. C 57.0%, H 7.6% calcd for C₅₃H₈₆O₂₁ + 2CH₃OH + H₂O, C 57.0%, H 8.3%.

Acid hydrolysis of 3:

The acid hydrolysis was performed according to Chapman and Kennedy¹¹ using 1 mg of 3. The hydrolysate was dissolved in 100 ml pyridine, 100 ml BSTFA containing 1% TMCS added, then heated at 95 ⁰C for 1 hour before GC analysis.

ACKNOWLEDGEMENT:

This work was supported by the Research School of Chemistry (RSC), Australian National University (ANU), Canberra ACT 0200, Australia. The authors are grateful to Mr. Chris Blake of the ANU University NMR Center who ran all of the NMR spectra, to Mrs. Jenny Rothschild of the RSC Mass Spectroscopy Unit and to the RSC Microanalytical Unit for their assistance.

REFERENCES

1. Herlt AJ, Mander LN, Pongoh EJ, Rumampuk RJ, and Tarigan P. Two major saponins from seeds of Barringtonia asiatica: Putative antifeedants towards Epilachna Sp. Larvae. Journal of Natural Products. 65(2); 2002:115–120.

2. Rumampuk RJ. Structure Elucidation of Saponins from The Seeds of Barringtonia asiatica (L) Kurz. PhD Thesis, Padjadjaran University, Bandung, Indonesia, September 2001.

3. Chakravarty AK, Das B, and Pakrashi SC. Triterpenoid prosaponins from leaves of Maesa chisia var. angustifolia. Phytochemistry. 26(8); 1987:2345–2349.

4. Dimbi MZ, Warin R, Delaude C, Huls R, and Mpuza K. Triterpenoïdes de Harpullia Cupanioïdes. Bulletin des Sociétés Chimiques Belges. 92(5); 1983:473–484.

5. Voutquenne L, Lavaud C, Massiot G, and Delaude C. Saponins from Harpullia cupanioides. Phytochemistry. 49(7); 1998:2081–2085.

6. Massiot G. and Lavaud C. “Studies in Natural Products Chemistry,” Vol, 15, Elsevier Sciences, Amsterdam, 1995, pp 187 – 224.

7. Delay C, Gavin JA, Aumelas A, Bonnet PA, and Roumestand C. Isolation and structure elucidation of a highly haemolytic saponin from the Merck saponin extract using high-field gradient-enhanced NMR techniques. Carbohydrate Research. 302(1-2); 1997:67–78.

8. Bax A, and Subramanian S. Sensitivity-enhanced two-dimensional heteronuclear shift correlation NMR spectroscopy. Journal of Magnetic Resonance. 67(3); 1986:565–569.

9. Lerner L, and Bax A. Sensitivity-enhanced two-dimensional heteronuclear relayed coherence transfer NMR spectroscopy. Journal of Magnetic Resonance. 69(2); 1986:375–380.

10. Davis DG, and Bax A. Assignment of Complex 'H NMR Spectra via Two-Dimensional Homonuclear Hartmann-Hahn Spectroscopy. Journal of American Chemical Society. 107; 1985:2820–2821.

11. Chapman MF, and Kennedy JF. “Carbohydrate analysis – a practical approach,” IRL Press, 1986, pp. 25 and 78 – 79.

Received on 26.09.2011 Modified on 17.10.2011

Asian J. Research Chem. 4(12): Dec., 2011; Page 1935-1940

An Antifeedant Saponin from Seeds of Barringtonia asiatica

Emma J. Pongoh¹, Wilson A.R. Rombang¹, Rymond J. Rumampuk¹*, Ponis Tarigan².

RESULTS AND DISCUSSION:

Position

An Antifeedant Saponin from Seeds of Barringtonia asiatica

Emma J. Pongoh1, Wilson A.R. Rombang1, Rymond J. Rumampuk1*, Ponis Tarigan2.

RESULTS AND DISCUSSION:

Position

Emma J. Pongoh¹, Wilson A.R. Rombang¹, Rymond J. Rumampuk¹*, Ponis Tarigan².