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Secondary metabolites from static cultures of the fungus Rosellinia arcuata.

Introduction

Rosellinia species are found worldwide. They are common in both temperate and tropical regions [1]. Many species occur as saprobes, some live endophytically, whereas others are root pathogens. Amongst the most well-known root pathogens are R. necatrix Prill., R. pepo Pat., R. bunodes (Berk. et Br.) Sacc. and R. arcuata Petch.

R. necatrix causes white root rot in temperate fruit crops, particularly grapevine (Vitris vinefera), apple (Males pamila) and mulberry (Mores spp.). Also it infects coffee, lueceme, mandarin and tea. The roots, (young ones of tea being attacked first) are infested with a white mycelium, which in older roots turn brown and almost black. Wilt and death of the tree may be slow or fairly rapid. Infection is generally confined to the roots [2].

R bunodes causes black root rot mainly in tropical woody hosts. The most commonly infected hosts are cocoa (Theobroma cacao), quinine (Cinchona spp.), coffee (Coffee spp.), rubber (Heavea brasiliensis) and tea (Camellia sinensis) [3].

R. pepo is apparently at present restricted to Central America, the West Indies and West Africa. It is a soil inhabitant. Together with R. bunodes, they caused considerable loss, particularly on woody crops in the West Indies and other parts of tropical America. These losses occurred on land cleared from forest and immediately used for cultivation. The fungi rapidly spread to the planted crops from organic debris left after clearing [4].

The fungus R. arcuata, which is the subject of the study, shows apparent infrequent record of distribution. It has been reported from Central African Republic, Hong Kong, India, Indonesia, Kenya, Papua New Guinea, Sri Lanka and Zaire Republic. The sample of R. arcuata used for this investigation was collected from Thailand. In the wild, R. arcuata grows with brownish-black perithecias. Morphologically, this fungus is similar to R. necatrix and the two fungi can easily be confused. It differs from the latter however in the absence of pyriform swellings on the hyphae.

R. arcuata like most Rosellinia species causes black root rot disease, mainly in tropical and subtropical woody hosts. Its infection of tea is of economic significance. The advancing edge of the mycelium is white shading to black. On the root surface, the black network of strands give a woolly appearance and beneath the bark white mycelium spreads on the wood. On tea the fungus may spread up the stem and the crop often dies suddenly. The leaves of the dead crop may however remain attached to the plant for some time. Destroying surrounding bushes controls the infection. In Ceylon, tea root disease is controlled with methyl bromide (6).

The examination of R. arcuata follows the isolation of a novel metabolite, rosnecatrone, from R. necatrix by Edwards et al (7).

Results and discussion

Ethyl acetate extract of the culture medium of R. arcuata yielded a gummy yellowish liquid (10.1 g). The liquid (8.0 g) was chromatogra-phed on silica gel to give seven fractions. The column was eluted with 1.5 [dm.sup.3] chloroform-methanol (95:5) and the eluent collected in volumes of 5.0 ml. The tubes were grouped into seven fractions based on TLC studies.

Characterisation of pestalotin

Tubes (41-110), fraction 3, gave yellowish oil (1.21 g), which was triturated with acetone-hexane (1:3) mixture. This afforded a white solid (110 mg), which on crystallisation gave a white crystalline solid (96 mg), mp 71-72[degrees]C, [[[alpha]].sup.23.sub.D] + 49.2 (c = 0.89, McOH) ([lit..sup.8] 51.8 c = 0.65, CH[Cl.sub.3]) [M.sup.+] 214, [v.sub.max] (KBr) 3410, 3096, 2959 1715 and 1620 [cm.sup.-1]. TLC studies of the compound gave a yellowish colour with anisaldehyde spray reagent after heating for 3 min. at 112[degrees]C. It however did not give a positive test with p-nitroaniline spray reagent.

The HMQC, shows a correlation between the methine proton signal at [[delta.sub.H] 5.12 and [[delta].sub.c] 90.05. There is also a strong correlation between the methylene carbon at [[delta].sub.c] 29.65 with the protons at [[delta].sub.H] 2.24 and 2.77. The presence of this geminal coupling ([sup.2]J 17.0 Hz) is confirmed by the [sup.1]H-[sup.1]H COSY. A methine proton at [[delta].sub.H] 4.26 is most likely on a carbon attached to oxygen. In the HMBC, the protons of the methoxy group correlate with the [sp.sup.2] hybridised carbon at [[delta].sub.c] 173.28. This gives subunit (A).

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A sharp peak in the IR at 3460 [cm.sup.-1] indicates the presence of an --OH group in the compound. The mass spectrum shows loss of [H.sub.2]O i.e. [[M - [H.sub.2]O].sup.+] at m/z 196. The disappearance from the [sup.1]H NMR spectrum of the peak at [[delta].sub.H] 2.32 when a sample of the compound was shaken with [D.sub.2]O confirms the presence of a hydroxyl group. In the HMQC, the proton at [[delta].sub.H] 3.59 correlates with the carbon at [[delta].sub.c] 72.47. A possible subunit is (B).

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The resonance position and multiplicity of the methyl group at [[delta].sub.H] 0.88 suggest that it is attached to a methylene group. Further evidence of this is shown in the [sup.1]H-[sup.1]H COSY, where there is correlation between the methyl protons at [[delta].sub.H] 1.34 and the methylene protons. The COSY spectrum, together with the HMBC data, supports the presence of a subunit (C).

In the HMBC, the geminal proton at [[delta].sub.H] 2.77 correlates with the carbon at [[delta].sub.H] 72.47, suggesting that fragments A, B and C are linked as shown in (D).

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In the HMBC, the geminal proton at [[delta].sub.H] 2.77 correlates with the carbon at [[delta].sub.H] 72.47, suggesting that fragments A, B and C are linked as shown in (D).

In the [sup.13]C NMR, there is a carbonyl resonance position at 166.90, suggesting an ester. Considering the molecular formula [C.sub.11][H.sub.18][O.sub.3], a CO group has not been accounted for, indicating that the compound is possibly a cyclic ester. The proof of this is found in the IR where the band at 1715 [cm.sup.-1] must be due to a six-membered [alpha], [beta] unsaturated lactone [(lit. 1720 [cm.sup.-1]).sup.9], giving 6-(1-hydroxypent-1-yl) -4-methoxy-5,6-dihydro-2H-pyran-2-one (Pestalotin) (I).

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Characterisation of didehydropestalotin

Tubes (111-130) fraction 4 gave yellowish oil (300 mg), which was treated with an acetone-hexane mixture (1:2) to give a white solid (32 mg). Recrystallisation from the same solvent system gave pure white crystals (26 mg), mp 82-84[degrees]C. The IR spectrum obtained from KBr had peaks at 3055, 2958, 1725 and 1620 [cm.sup.-1].

The [sup.13]C NMR spectrum of this compound is similar to that of Pestalotin. This suggested a compound of closely related structure.

The presence of an extra C=O signal in the [sup.13]C NMR spectrum at [[delta].sub.c] 207.52 and the absence of a hydroxyl group in the IR spectrum of the compound, supported an oxidised form of pestalotin, namely didehydropestalotin (11).

This is confirmed by a shift in [sup.13]C resonance position from [[delta].sub.c] 72.47 (C-OH) in pestalotin to [[delta].sub.c] 207.52 (C=O) in didehydropestalotin as shown in the subunits (A1) and (A2) in the two compounds respectively. The introduction of the more deshielding group in the latter compound shifted the adjacent protons downfield, the chiral proton from [[delta].sub.H] 4.26 to 4.74 and the methyl protons from [[delta].sub.H] 1.55 to 1.61. These data indicated the structure, Dide-hydropestalotin (II)

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Characterisation of Allenic epoxycyclo-hexane

Tubes (131-175), fraction 5, yielded yellowish oil (1.52 g) when the eluent was evaporated. The oil was dissolved in 1.5 ml acetone-hexane mixture (3:1) and set aside for 24 h. An impure creamy solid was obtained and filtered off (187 mg). It was purified by recrystallisation from the same solvent system to give a creamy crystalline solid (140 mg), mp 118-120[degrees]C, and m/z 194, found [C.sub.11][H.sub.14][O.sub.3]. The IR spectrum (KBr) gave signals at 3499, 2950, 2800, 1960, 1690 and 1620 [cm.sup.-1]. The strong absorption at 1960 [cm.sup.-1] is diagnostic for the allene functional group.

[sup.1]H-[sup.1]H COSY spectrum indicated correlation between the methine protons at [[delta].sub.H] 3.27 and [[delta].sub.H] 3.32. (J 14.3 Hz). Also the proton at [[delta].sub.H] 4.30 correlates with the methylene protons at [[delta].sub.H] 2.34 and 2.46. The resonance positions of the methine protons at [[delta].sub.H] 3.27, 3.32, 4.30 and 4.55 suggest they are attached to C-O groups. The COSY spectrum shows that the protons at [[delta].sub.H] 3.27 and 3.32 are vicinally coupled and the HMQC show similar carbon resonance positions. Protons at [[delta].sub.H] 4.36 and 4.55 are not coupled but the HMQC shows that they are in a similar chemical environment. Since the molecule contains only three oxygen atoms, one of these pairs must be attached to carbon atoms attached to one oxygen atom. In the HMBC, the proton at [[delta].sub.H] 3.32 correlates with carbon atoms at [[delta].sub.c] 30.66 and 65.66. A possible fragment in this compound is (A).

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In the HMBC spectrum, the methylene protons at high field show correlations with carbon atoms at [[delta].sub.c] 65.66 and 66.07 as well as the quaternary carbon at [[delta].sub.c] 101.61. Another possible subunit is fragment (B)

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A strong band in the IR spectrum at 1960 [cm.sup.-1] indicated the presence of an allene functional group in this compound. The sp hybridised carbon atom occurs at a resonance position of [[delta].sub.c] 202.0 Two possible structures could be written for the substituent to the hexacyclic ring, (C1) and (C2).

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There is no coupling between the methine proton at [[delta].sub.H] 6.14 and the ethylene germinal protons at [[delta].sub.H] 4.89 and 4.97, rather, one of the latter protons couples with the methyl protons. This suggests that C1 is the most likely fragment in this compound. Supporting evidence for this arrangement is the fact that in the HMBC spectrum, the methyl protons correlate with the olefinic carbon at [[delta].sub.c] 115.39. A long range coupling between the olefinic proton at [[delta].sub.H] 6.14 and the methylene proton at [[delta].sub.H] 2.46 in the COSY spectrum indicates that fragment (B) is linked to (C) as shown in fragment (D). The proof of this can be found in the HMBC where there is correlation between the protons at [[delta].sub.H] 2.34 and 2.46 with the allenic carbon at [[delta].sub.c] 202.0.

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ACD Labs (Advanced Chemical Development Laboratory Software) carbon-13 predictor was used to calculate the chemical shifts of the compound. The calculated chemical shifts compared favourably with the experimental chemical shifts.

X-ray analysis results were used to confirm the structure of the compound and the relative stereochemistry of the substituents.

[FIGURE 1 OMITTED]

This confirms the structure of the compound as 3-(3-methylbuta-1,3-dien-lylidene)-7-oxabicyclo[4.1.0]heptan-2,5-diol (III).

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Identification of this compound posed a challenge. Though the IR spectrum indicated an allene peak at 1960 cm 1, peaks at 1690 and 1620 [cm.sup.-1] suggested the presence of a carbonyl group. The compound gave a positive test with 2, 4-DNP spray reagent supporting the presence of a carbonyl. It could be only one of the two functional groups, since there was only one resonance carbon position between 150-220. X-ray analysis was used to solve the problem. Possibly the two hydroxyl groups in the compound were oxidised slowly to the ketone, given a positive test for carbonyls.

Characterisation of carboxymellein

Tubes (176-190), fraction 6, gave a yellowish oil (320 mg) which was dissolved in warm alcohol (2.0 ml) and set aside for 72 h. White crystals (12.0 mg) were obtained, mp. 246[degrees]C, [[[alpha]].sup.23.sub.D] + 198.1[degrees] (c = 0.75, McOH) ([lit..sup.10] [[[alpha]].sup.23.sub.D] + 203.03[degrees]c = 0.66 EtOH), m/z 222 (100 %), [v.sub.max] (KBr) 3200, 2950, 1695, 1650, 1590 [cm.sup.-1]. Table 3, gives a summary of the NMR data.

In the IR spectrum, two strong absorption bands at [v.sub.max] (KBr) 1695 and 1650 [cm.sup.-1] suggested the presence of two carbonyl groups in this compound. The resonance position of the methine proton at [[delta].sub.H] 4.55, indicates that it is attached to a C-O group. This proton must have at least five neighbours due to its multiplicity. In the [sup.1]H-[sup.1]H COSY, the methine proton correlates with the three methyl protons as well as the two non-equivalent methylene protons. A most likely subunit in this compound is (A).

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The [sup.1]H-[sup.1]H COSY spectrum shows coupling between the aromatic protons at [[delta].sub.H] 7.02 and 8.43 with a J value of 8.8 Hz, indicating an ortho coupling. A strong absorption in the IR at 1590 [cm.sup.-1] supports the presence of an aromatic ring. The compound gives a yellow colouration with diazotised p-nitroaniline and bromocresol green spray reagents (TLC). The former indicates that the compound is a phenol or an enol and the latter shows that it is an acid. In the [sup.1]H NMR, there is a broad peak at [[delta].sub.H] 6.10 which supports the presence of an acid group. The HMBC spectrum shows correlations between the aromatic proton at [[delta].sub.H] 8.43 and 7.02 with carbons at [[delta].sub.c] 143.65 and 109.30 respectively. The other portion of the compound is likely subunit (B).

[FORMULA OMITTED]

Subunits A and B may be linked in two possible ways (C1) and (C2)

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When the COOH functional group occurs at the C-5 position on mellein, it results in a split of the adjacent C[H.sub.2] protons [11]. This is the case with this compound, suggesting the structure as 5-carboxymellein (IV).

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Experimental

The fungus R. arcuata was surface cultured in 3% aqueous malt with 6% added glucose. The fungus was sub-cultured in conical flask (250 ml) for 14 days. It grew with a white upper surface and a yellow lower mycelium. It started fruiting after 10 days in the culture medium.

The cylindrical fruiting bodies grew to about 2 cm high and 1 mm in diameter. They were transferred into 28 Thompson bottles (2 [dm.sup.3]), each containing about 1.0 [dm.sup.3] of culture medium. The mycelium developed a more intense yellow colour as it matured. Filtration through a muslin cloth was used to remove the mycelium. The brownish filtrate 21 [dm.sup.3] was extracted thrice with ethyl acetate in a 5 [dm.sup.3] separating funnel and the combined extract dried over anhydrous sodium sulphate for 90 minutes.

A gummy brownish solid (27.92 g) was obtained after removal of the ethyl acetate on a rotary evaporator. TLC studies of this crude extract in different solvent systems indicated that it was a mixture of several compounds.

The gum was triturated with warm ethyl acetate and left overnight yielding 6.35 g of a white solid which was removed by filtration. Removal of the solvent from the filtrate afforded a dark brown solid (21.51 g).

The crude solid (10 g) was chromatographed over silica gel (300 g) in a column (4 x 80 cm), which was eluted with chloroform-methanol (93:7). The eluent was collected on a fraction collector in volumes of 3.0 ml. One out of every five tubes was spotted in the order in which the tubes were collected. The tubes were grouped into 6 fractions based on the TLC study.

Isolation of Pestalotin from culture medium

Tubes (41-110), fraction 3 yielded a gummy yellowish liquid (1.21 g). Triturating with acetone-hexane (1:3) mixture gave pestalotin a white solid (110 mg) which was recrystallised from the same solvent system to give white crystalline solid (96 mg), mp 71-72[degrees]C ([lit..sup.12], 71[degrees]C), [[[alpha]].sup.23.sub.D] + 49.2 (c = 0.89, McOH) (lit. 51.8 c = 0.65, CH[Cl.sub.3]), m/z 214 ([M.sup.+]), [v.sub.max] (KBr) [cm.sup.-1] 3410, 3096, 2959, 1695 and 1620. [[delta].sub.H] (CD[Cl.sub.3]) 0.88 (3H, t, J 7.6 Hz, C[H.sub.3]) 1.34 (3H, m, C[H.sub.2], 1.47 (1H, m, C[H.sub.2]), 1.55 (2H, m, CHA 2.24 (1H, dd, J 4.0, 17.0 Hz, C[H.sub.2]) 2.32 (1H, s(br), OH), 2.77 (1H, ddd, J 2.0, 17.0, 12.7 Hz, C[H.sub.2]), 3.73 (3H, s, OC[H.sub.3]), 4.26 (1H td, J 13.1, 8.3 Hz, CH) and 5.12 (1H, s, CH). [[delta].sub.c] (CD[Cl.sub.3]) 14.06 (5'-C[H.sub.3]), 22.67 (4'-C[H.sub.2]), 27.67 (3'-C[H.sub.2]), 29.65 (5-C[H.sub.2]), 32.43 (2'-C[H.sub.2]), 56.27 (7-C[H.sub.3]O), 72.47 (1'-CH), 78.50 (6-CH), 90.05 (3-CH), 166.90 (2-C=O) and 173.28 (4-C).

Isolation of Didehydropestalotin

Tubes (111-130), fraction 4 afforded a yellowish liquid (300 mg). The liquid was dissolved in limited amount of acetone and hexane mixture (1:2) to give a white solid (32 mg), which was recrystallised from the same solvent mixture to give didehydropestalotin as pure white crystals (26 mg) mp 82-84[degrees]C ([lit..sup.13] 82[degrees]C). [v.sub.max] (KBr) [cm.sup.-1] 3055, 2958, 1725, and 1620, [[delta].sub.H] (CD[Cl.sub.3]) 0.89 (3H, t, J 7.4 Hz, C[H.sub.3]) 1.32 (2H, m, C[H.sub.2]), 1.61 (4H, m, 2C[H.sub.2]), 2.71 (2H, m, C[H.sub.2]), 3.74 (3H, s, OC[H.sub.3]) 4.74 (1H, dd, J 7.5, 8.0 Hz, CH) and 5.14 (1H, s, CH). [sup.13]C-NMR [[delta].sub.c] (CD[Cl.sub.3]) 13.92 (5'-C[H.sub.3]) 22.25 (4'-C[H.sub.2]), 25.05 3'-C[H.sub.2]), 28.77 (5-C[H.sub.2]), 38.69 (2'-[H.sub.2]), 56.36 (7-C[H.sub.3 O), 78.88 (6-CH), 90.50 (3-CH), 165.47 (2-C=O), 172.37 (4-C=C) and 207.52 (1'-C=O).

Purification of Allenic epoxyclohexane

Tubes (131-175), fraction 5 yielded a yellowish oil (1.52 g) when the eluent was evaporated. The oil was dissolved in 1.5 ml acetone-hexane mixture (3:1) and set aside for 24 h. An impure creamy solid was obtained and filtered off (187 mg). It was purified by recrystallisation in the same solvent system to give allenic epoxycyclohexane, a creamy crystalline solid (140 mg) mp 118-120[degrees]C. ([lit..sup.14], 118 [degrees]C). The solid gave a bright pink spot when sprayed with anisaldehyde spray reagent. It did not give a positive test with p-nitroaniline spray reagent. However when sprayed with 2, 4-DNP, it slowly developed a yellow spot. (Found, C, 68.0: H, 7.2, [C.sub.11][H.sub.14][O.sub.3], required C, 68.1, H 7.2 %, m/z 194 ([M.sup.+]). vmax (KBr) [cm.sup.-1] 3499, 2950, 2800, 1960 and 1620. [[delta].sub.H] (CD[Cl.sub.3]), 1.73 (3H, s), 2.34 (1H, dd, J 14.3, 4.12 Hz), 2.46 (1H, td, J 3.61, 14.26 Hz), 3.27 (1H, d, J 3.0 Hz), 3.32 (1H, d, J 2.9 Hz), 4.30 (1H, s), 4.55 (1H, s), 4.89 (1H, s), 4.97 (1H, s) and 6.14 (1H, s). [[delta].sub.c] (CD[Cl.sub.3]), 19.70 (5'-C[H.sub.3]) 30.66 (4-C[H.sub.2]), 54.53 (6-CH), 55.84 (1-CH), 65.66 (5-CH), 66.08 (2-CH), 101.00 (3-C), 101.61 (2-CH), 115.39 (4'-C[H.sub.2]) 138.66 (3'-C) 202.00 (1'-C).

5-Carboxymellein

Tubes (176-190), fraction 6 were yellow liquid (320 mg). The liquid was dissolved in warm alcohol (1.5 ml) and set aside for 72 h, yielding white needles of 5-carboxymellein (12.0 mg) mp 246[degrees]C ([lit..sup.15], 247[degrees]C), m/z 222 ([M.sup.+]), [v.sub.max] (KBr) [cm.sup.-1] 3200, 2950, 1695, 1650 and 1590, [[delta].sub.H] ([C.sub.5][D.sub.5]N) 1.27 (3H, d, J 6.2 Hz), 3.02 (1H, m), 4.10 (1H, m), 4.55 (1H, m), 7.02 (1H, d, J 8.8 Hz) and 8.43 (1H, d, J 8.8). [[delta].sub.c] ([C.sub.5][D.sub.5]N) 20.43 (11-C[H.sub.3]), 32.93 (4-C[H.sub.2]), 75.68 (3-CH), 109.30 (9-C), 115.85 (7-CH), 139.11 (6-CH), 143.65 (10-C), 165.15 (8-C), 168.47 (12-C=O) and 170.35 (1-C=O).

Conclusion

The Xylariaceae are amongst the best investigated filamentous fungi with regards to the production of secondary metabolites. In spite of this, only a few of the large number of species (over 1000 species), have been extensively studied for their secondary metabolites. Numerous biologically active compounds with potential for application in human and vetenary medicine or as crop protection agents have been reported. Most of them through various culture techniques. The structural resemblance within these secondary metabolites appears useful for the taxonomic classification of their producers.

This is the first report of pestalotin, didehydropestalotin and allenic epoxycyclo-hexane from a member of the family Xylariaceae.

Reference

[1] L. E. Petrini, Rosellinia species of the temperate zones, Sydowia, 1993, 44, 169-281.

[2] A. Sivanesan and P. Holliday, Description of Pathogenic Fungi and Bacteria, CMI, Commonwealth Agic. Bur., 1972, No. 352.

[3] A. Sivanesan and P. Holliday: Description of Pathogenic Fungi and Bacteria, CMI, Commonwealth Agic. Bur., 1972, No. 351.

[4] C. Booth and P. Holliday: Description of Pathogenic Fungi and Bacteria, CMI, Commonwealth Agic. Bur., 1972, No. 354.

[5] A. Sivanesan and P. Holliday: Description of Pathogenic Fungi and Bacteria, CMI, Commonwealth Agic. Bur., 1972, No. 353.

[6] D. Petch: The Disease of The Tea Bush, 1923. 13.

[7] R. L. Edwards, D. J. Maitland, I. J. Scowen, and A. J. S. Whalley, J. Chem.

Soc. Perkin Trans, 1, 2001, 537-542.

[8] S. F. Meyer, A. Steinreiber, M. Goriup, R. Saf and K. Faber, Tetrahedron, 2002, 13(5), 523-526.

[9] R. M. Silverstein, G. C. Bassler and T. C. Morrill, Spectroscopic identification of organic compounds, 4th ed. 1981, 122.

[10] Tanticharoen and Y. Thebtaranonth, J.Chem. Soc. Perkin Trans. 1, 2002, 2173-2478.

[11] M. A. Alvarenga and R. Braz-Fo, et al., Phytochemistry, 1978, 17, 511.

[12] D. Seebach and H. Meyer, Angew.Chem. Int. Ed., 1974, 13.

[13] D. Seebach and H. Meyer, Angew.Chem. Int. Ed., 1974, 13, 77.

[14] J. M. Renaud, G. Tsoupras, H. Stoeckli-Evans, R. Tabacchi, Helv. Chim. Acta 1989, 72, 1262.

[15] N. Claydon, F. Grove and M. Pople, Phytochemistry, 1985, 24, 937.

E. K. Oppong (1), R. L. Edwards (2), D. J. Maitland (3), A. J. S. Whalley (4) and Y. Ameyaw (5)

(1,5) Department of Science Education, University of Education, P. O. Box 25, Winneba, Ghana

(2,3) Department of Chemistry and Forensic Science, University of Bradford, Richmond Road, West Yorkshire, BD7 1DP 'United Kingdom

(4) Department of Biology, Liverpool polytechnique, Liverpool, L3 3AF United Kingdom
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Author:Oppong, E.K.; Edwards, R.L.; Maitland, D.J.; Whalley, A.J.S.; Ameyaw, Y.
Publication:International Journal of Applied Chemistry
Article Type:Report
Geographic Code:1USA
Date:Sep 1, 2009
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