Lepidosperma

King, Douglas I., Hamid, Kaiser, Tran, Van H., Duke, Rujee K. & Duke, Colin C., 2021, Kangaroo Island propolis types originating from two Lepidosperma species and Dodonaea humilis, Phytochemistry (112800) 188, pp. 1-11 : 2-4

2.1. Two propolis types sourced from resins of Lepidosperma species

The sedge, Lepidosperma sp. Flinders Chase (G. T. Plunkett 75) ( Cyperaceae ), is common and widespread on KI, and produces copious amounts of resinous exudate from the entire above ground parts of the plant; leaves, flower stem and seed head. This was observed to protect the entire plant from grazing animals such as kangaroos and wallabies. Despite the plant being widespread, common and readily accessible, propolis from this plant is relatively uncommon. Propolis from Lepidosperma sp. Flinders Chase was identified after observation of bees collecting resin from plants and matching of 1 H NMR spectral and TLC profiles of resin from the plant and bee legs, and freshly deposited propolis from bee hives.

From the two Lepidosperma species 5 previously undescribed compounds were isolated and characterised ( Fig. 1 ). Lepidosperma sp. Flinders Chase gave methyl 3-phenyl-2-(E -cinnamoyloxy)propanoate (1), 3- (E -8-methoxy-8-oxo-3,7-dimethyloct-2-enyl)-4-hydroxy- E -cinnamic acid (2) and 3-(E -6,7-dihydroxy-3,7-dimethyloct-2-enyl)-4-hydroxy- E - cinnamic acid (3). Lepidosperma viscidum R.Br. (Cyperaceae) gave 5 ′ -(E - 4-hydroxy-3-methylbut-2-enyl)-4,2 ′,4 ′ -trihydroxydihydrochalcone (4) and 5 ′ -(E -4-hydroxy-3-methylbut-2-enyl)-4 ′ -methoxy-4,2 ′ -dihydroxydihydrochalcone (5).

2.1.1. Methyl 3-phenyl-2-(E-cinnamoyloxy)propanoate (1)

Molecular formula was determined as C 19 H 18 O 4 by high-resolution mass spectrometry. 1 H NMR spectra ( Table 1 ) clearly showed two strongly coupled doublets (16 Hz) characteristic of E -cinnamate olefinic hydrogens. An ABX coupling pattern was observed for a CH 2 at 3.19 and 3.25 ppm and a CH at 5.37 ppm. The 10H multiplet, 7.24–7.54 ppm, is consistent with the molecule having two unsubstituted phenyl groups, one belonging to the cinnamate group and the other attached to the saturated CH 2 group at 3.19 and 3.25 ppm. 13 C NMR spectra ( Table 2 ) showed signals indicating two carbonyl groups, one belonging to the cinnamate group (166.1 ppm) and the other belonging to a methyl carboxylate group (170.2 ppm gHMBC correlated to the methoxy H, 3.74 ppm) ( Fig. 2 ). The hydrogens of the CH 2 (3.19 and 3.25 ppm) showed gHMBC correlation with C-1, C-2 and the phenyl C-1 ′, C-2 ′, C-6 ′, consistent with a 3-phenylpropanoate moiety. The cinnamoyloxy group was shown to be linked through C-2 by gHMBC correlation between H-2 and the cinnamate carbonyl (C-1 ′′, 166.1 ppm), other correlations are consistent with the structure 1 shown ( Fig. 1 ). The chiral structure is supported by the optical rotation measured.

3

2.1.2. 3-(E-8-methoxy-8-oxo-3,7-dimethyloct-2-enyl)-4-hydroxy-E-cinnamic acid (2)

Molecular formula was determined as C 20 H 26 O 5 by high-resolution mass spectrometry. 1 H NMR clearly showed two strongly coupled doublets (15.9 Hz) characteristic of E -cinnamate alkene hydrogens. The three hydrogens signals attributed to aromatic hydrogens showed chemical shifts and coupling characteristic of a 3-substituted p -coumaric acid. The 4-OH group was shown by the shielded aromatic H-5 (6.82 ppm) that was coupled (J = 8.8 Hz) with the adjacent H-6 (6.32 ppm). gHMBC correlation of the H-1 ′′ of the 3-position substituent showed the attachment of the 8-substituted 6,7-dihydrogeranyl group through C-1" ( Fig. 2 ). The eight-carbon chain, double bond in between C-2 ′′ and C-3 ′′ and methyl substitutions on C-3 ′′ and C-7 ′′ were determined from 1 H and 13 C NMR chemical shifts and couplings, aided by gCOSY, HSQC and gHMBC 2D correlation experiments. 13 C NMR showed two signals characteristic of carbonyl groups, one attributed to the cinnamic acid carbonyl (C-1 ′, 172.2 ppm) and the other to terminal position of the 6,7- dihydrogeranyl group (C-8 ′′, 177.6 ppm). The gHMBC correlation of the methoxy hydrogens to C-8 ′′ showed the position of the methyl carboxy ester group. The relatively shielded signal for C-10" (16.1 ppm) supports the E stereochemistry for the C-2 ′′ to C-3 ′′ olefinic bond as Z stereochemistry is predicted to show a chemical shift for the C-10 ′′ methyl group of approximately 25 ppm ( Abu-Mellal et al., 2012). The presence of a chiral centre at C-7 ′′ is supported by the optical rotation measured.

2.1.3. 3-(E-6,7 -dihydroxy-3,7-dimethyloct-2-enyl)-4-hydroxy-E-cinnamicacid (3)

Molecular formula was determined as C 19 H 26 O 5 by high-resolution mass spectrometry. Comparison of the 1 H and 13 C NMR spectra with 2 showed that 3 had an identical carbon skeleton and that the methoxy group of 2 was absent from 3. The data also showed that, like 2, 3 was a

3-substituted p -coumaric acid. 1 H and 13 C NMR spectra showed that the main differences between 2 and 3 were for hydrogen and carbon signals for positions 5 ′′ to 9 ′′ of the dihydrogeranyl group. A vicinal diol moiety was identified from 13 C NMR chemical shifts for the tertiary C-7" (74.0 ppm) and the secondary C-6" (79.1 ppm). H-6" (3.27 ppm, dd, J =10.6, 1.6 Hz) showed coupling to the adjacent CH 2, C-5" (30.9 ppm). The carbon interconnections for positions 5 ′′ to 9 ′′ and connection through C- 4 ′′ are shown by gHMBC correlations observed ( Fig. 2 ). The relatively shielded signal for C-10" (16.4 ppm) supports the E stereochemistry for the C-2 ′′ to C-3 ′′ olefinic bond. The presence of a chiral centre at C-6 ′′ is supported by the optical rotation measured.

2.1.4. 5 ′ -(E-4-hydroxy-3-methylbut-2-enyl)-4,2 ′,4 ′ -trihydroxydihydrochalcone (4)

Molecular formula was determined as C 20 H 22 O 5 by high-resolution mass spectrometry. A 1,4-disubstituted benzene ring is shown in the 1 H NMR spectrum by two 2H doublets at 7.04 and 6.69 ppm with a 8.6 Hz coupling indicating coupling of ortho aromatic hydrogens. The chemical shifts are consistent with a p -hydroxyphenyl group attached to an alkyl group. Two linked CH 2 groups at 3.25 and 3.13 ppm appear as 2H triplets, J 7.6 Hz. The 13 C NMR spectrum shows a signal at 205.76 = ppm consistent with a ketone carbonyl group and signals at 156.82, 164.39 and 164.91 ppm consistent with three aromatic carbons with hydroxyl groups attached. The remaining aromatic H signals at 7.47 and 6.25 ppm appear as singlets indicating a 1,2,4,5-tetrasubstituted benzene ring. The deshielded signal at 7.47 ppm is consistent with this hydrogen being adjacent to the carbonyl group and the strongly shielded hydrogen at 6.25 ppm being between two hydroxyl groups 2D NMR experiments established a 4,2 ′,4 ′ -dihydrochalcone partial structure. The remaining signals are recognisable as a prenyl group attached to an aromatic ring through a CH 2 at 3.25 ppm appearing as a broad doublet, J =7.2 Hz, coupled to an olefinic CH at 5.56 ppm. A prenyl methyl group is observed at 1.74 ppm corresponding to H-5". H-4 ′′ at 3.96 ppm is a 2H singlet consistent with a CH 2 of a 4-hydroxyprenyl group. 13 C NMR chemical shifts are also consistent with a 4-hydroxyprenyl group and the chemical shift of the C-5 ′′, 14.0 ppm, indicates E stereochemistry through the C-2 ′′ to C-3 ′′ olefinic bond ( Abu-Mellal et al., 2012). Attachment of the 4-hydroxyprenyl group through C-1 ′′ and C-5 ′ is confirmed by gHMBC long range correlation from H-6 ′ to C––O and C-1 ′′, and H-1 ′′ to H-4 ′, H-5 ′ and H-6 ′. Other gHMBC correlations are in agreement with the structure shown for 4.

2.1.5. 5 ′ -(E-4-hydroxy-3-methylbut-2-enyl)-4 ′ -methoxy-4,2 ′ -dihydroxydihydrochalcone (5)

Molecular formula was determined as C 21 H 24 O 5 by high-resolution mass spectrometry. Compounds 4 and 5 showed similar 1 H and 13 C NMR chemical shifts other than a methoxy group showing at δH 3.86 ppm and δC 56.42 ppm. The location of the methylated phenolic hydroxy group was shown by gHMBC long range correlation from the methoxy hydrogens to C-4 ′. Other gHMBC correlations are in agreement with the structure shown for 5.

Figures

Fig. 1. Cinnamic acid derivatives and prenylated dihydrochalcones identified in this study.

Fig. 2. Key heteronuclear multiple bond correlation (gHMBC) of compounds 1 to 5.