Steroids and Flavonoids of Porcelia macrocarpa

Três misturas de esteróides (1a+1b, 2a+2b e 3a+3b) e dois flavonóides glicosilados (4 e 5) foram isolados do extrato etanólico dos galhos da Porcelia macrocarpa (Warm.) R. E. Fries (Annonaceae). Os esteróides Estigmasta-4,25-dien-3-ona (2a) e (22E)-Estigmasta-4,22,25-trien-3ona (2b) são substâncias novas. As estruturas foram determinadas com base na análise de dados espectrais de RMN e de massas e por comparação com dados de substâncias da literatura usadas como modelos.


Introduction
][4][5][6] This paper describes the isolation and identification of three steroid mixtures and two flavonoid glycosides from the ethanol extract of the branches of Porcelia macrocarpa.

Results and Discussion
The ether soluble part from the ethanol extract of the branches of Porcelia macrocarpa was submitted to a partition between hexane and aqueous methanol.Chromatographic separation of the hexane phase afforded two steroid mixtures (M1 and M2) and the hydroalcoholic phase gave, after the same procedure, one mixture of steroid glycosides (M3).An insoluble material precipitated in the interface between diethyl ether and water, during the partition from the ethanol extract, afforded two flavonoid glycosides 4 and 5.
A GCMS analysis of the M1 mixture showed two compounds 1a and 1b in a 5:2 relationship, where the major compound presents a molecular peak [M] + at m/z 412 and the minor one at m/z 410.The 1 H NMR spectrum (Table 1) of the mixture M1 showed signals at δ 3.51 (m), 5.34 (m) and between δ 0.6 and 1.6 attributed to H-3, H-6 and methyl hydrogens, respectively, from a ∆ 5 -3β-hydroxy sterol with an allylic methyl group. 7Signals at δ 5.23-5.17and 4.72-4.62suggested additional double bonds in the steroidal nucleus.The 1 H and 13 C NMR data agree with the structure of ∆ 5 sterols [δ C 71.8 (oxygenated CH-3), 140.7 (C-5) and 121.7 (CH-6)] containing additionally one (1a, [M] + at m/z 412) and two double bonds (1b, [M] + at m/z 410) located at the side chain.The side chain of both steroids sustain an ethyl group at C-24, as in sitosterol.The presence in the mixture of two methylidene groups (C=CH 2 ) was deduced by 13 C signals corresponding to sp 2 non-hydrogenated and methylene carbon atoms [δ C 147.5 and 111.4 (1a); 148.6 and 109.5 (1b)].Thus, the second double bond of 1a was placed at C-25 to justify the 13 C signals and the allylic methyl group in the 1 H NMR (Table 1 and 2).The peak at m/z 84 (19%) attributed to fragment 1a-F originated by a McLafferty rearrangement (Table 3 and Figure 1) is in accordance with this suggestion.The compound 1a has been isolated before from the marine green alga Codium iyengarii. 8The low relative intensity of the peak at m/z 84 (3%) observed in the mass spectrum of 1b is justified by the presence of another peak at m/z 138 (43%) attributed to the fragment 1b-D formed also by a McLafferty rearrangement involving the additional double bond localized between the carbon atoms C-22 and C-23 (Table 3 and Figure 1).This third double bond is present only in 1b, as indicated by the 1 H NMR multiplet integration at δ 5.23-5.17and by the molecular ion.The CH signals at δ 137.2 and 130.0 observed in the 13 C NMR spectrum (Table 2) are in accordance with the proposed structure.Compound 1b is also described in the literature.
The GC-MS of M2 showed two compounds, in the same proportion of M1, with molecular peaks at m/z 410 and 408 (Table 3).The 1 H NMR spectrum (Table 1) was very similar with that of M1, but presented a signal at δ 5.72 instead of the signals at δ 3.51 and 5.34, suggesting a ∆ 4 conjugated double bond as in sitostenone. 9The 13 C NMR spectra (Table 2) showed signals that confirmed this suggestion, when compared with literature data of model compounds. 10The MS fragmentation followed the same pattern as M1 (Figure1 and Table 3).Hydrogenation of M2 mixture, at room pressure, gave the steroid sitostenone, as proved by GCMS and 1 H NMR spectra. 9M2 is then composed by a mixture of 2a and 2b which are described here for the first time.
The 1 H NMR spectrum (Table 1) of M3 differs from that of M1 only in the carbinolic hydrogen region.It showed signals between, δ 4.05 and 5.07, from a sugar unit.The H-3 multiplet is deshielded by 0.4 ppm suggesting that the mixture is composed by, glycopyranosyl steroids, with β configuration on the anomeric carbon, evidenced by the doublet at δ 5.02 (7.6 Hz). 11The 13 C NMR (Table 2) data suggested that the compounds are steroid glucosides. 11,12All the other signals are compatible with the structure of 3a and 3b.The steroid 3a has been isolated from Clerodendron colebrookianum (Verbenaceae). 11he 1 H NMR spectrum of 4 (Table 4) showed five signals in the aromatic region with the pattern of the flavonoid quercetin. 13Besides of those a singlet at δ 3.84 (6H) and signals between δ 3.05 and δ 5.35 were assigned to methoxyl groups and sugar hydrogens respectively.A doublet at δ 0.95 suggested that rhamnose should be present in the molecule and the singlet at δ 12.5 indicated the presence of one hydroxyl at C-5 of the aglycone.The 13 C NMR spectrum (Table 5) showed 29 signals indicating that two sugar units and two methoxyl groups are present in the flavonoid.The chemical shifts of the sugar moiety are consistent with those of rutinosyl (rhamnopyranosyl-(1→6) glucopyranosyl).Comparison between the 13 C NMR data of 4 with those of 7,4'-dimethoxyquercetin (6) 13,14 showed that C-3 is shielded and C-2, C-4 and C-10 are deshielded in agreement with the glycosylation at C-3 of 4. [14][15][16] The 1 H NMR spectrum of 5 (Table 4) had the same pattern as that of 4 but the signal of C-5 OH was absent.The 13 C NMR spectrum showed 33 signals indicating the presence of 3 sugar units in the dimethoxyflavonoid, one of them should be linked to C-5.Signals of rutinosyl and glucosyl groups were present.Hydrolysis of 5 led to the isolation of 7,4'-dimethoxyquercetin (6). 14To determine which group, glicosyl or rutinosyl is bounded to C-5, a COLOC 1 H- 13 C was obtained and a long range correlation between δ 4.84, assigned to the anomeric hydrogen of glucose and δ 158.6, assigned to C-5 of the flavonoid was observed.To confirm the structure of 5 as quercetin-5-Oglucoside-3-O-rutinoside-7,4'-dimethyl ether a comparison between the 13 C NMR data of 4 and 5 and those of 5glucopyranosyl luteolin and luteolin 17 was made.The difference found in the chemical shifts of rings A and C of those flavonoids confirmed the proposed structure, as showed in Table 6.

Plant material
The branches of P. macrocarpa (Warm.)R.E.Fries were collected at Instituto of Botânica de São Paulo in June, 1991.A voucher specimen is deposited in the herbarium of the Instituto de Botânica, São Paulo, Brazil under reference SP76791.

Extraction and isolation of the compounds
Dried and powdered branches (800 g) of P. macrocarpa were extracted with EtOH.The EtOH extract, after concentration in vacuum, was partitioned between Et 2 O/H 2 O (1:2) giving a water-soluble fraction (31 g), an ether soluble (20 g) and an insoluble interface (2.0 g).The ether soluble part was then partitioned between MeOH-H 2 O (9:1) and hexane.

Table 3 .
Mass spectrometric fragments of the side-chain of steroids

Table 6 .
Differences found in the carbon chemical shifts of rings A and C between 5 and 4 and those between 5-glucopyranosyl luteolin