Compounds isolated from the hexane fraction of P. aurantiacum
Three known compounds were identified and labelled 1, 2 and 3, (Fig. 1). These three isolated compounds belong to different classes of natural products. Compound 3 was obtained as white needles from the Hex:EtOAc (19:1) soluble fraction, mp 215°C, [α]D 25 + 27 (c 0.022, CHCl3). The HREIMS showed the [M]+ peak at m/z 426.3859 unit, which is consistent with the formula C30H50O and implying six degrees of unsaturations. The 1H-NMR showed two broad signals at δH 4.67 and δH 4.55 for the olefinic protons and a double-doublet at δH 3.19 for the oxymethine proton. Another peak at δH 3.18 could be assigned to methine proton adjacent to the olefinic bond. The protons of seven methyls showed singlets at δH 1.66, δH 1.05, δH 0.98, δH 0.95, δH 0.92, δH 0.89 and δH 0.77. The 13C NMR spectrum in association with DEPT spectrum showed thirty signals comprising, seven methyl, eleven methylene, six methine and six quaternary carbons. The olefinic carbons showed signals at δC 149.9 and δC 110.1, and the oxymethine carbon resonated at δC 77.8. The seven methyl carbons showed signals at δC 28.1, δC 19.5, δC 18.3, δC 16.3, δC 16.0, δC 15.8 and δC 14.7.
From the above spectroscopic evidence and literature data, the structure of compound 3 was assigned to be lupeol (Fig. 1). It is a known compound which was previously isolated from Zanthoxylum monogynum (Dos Santos et al., 2021).
Compound 1 was isolated as yellow powder in the mixture Hex:EtOAc (9:1). It reacted positively with ferric chloride, indicating its phenolic nature. Its “high resolution electrospray ionization-time-of-flight (HR-ESI-TOF)” MS showed a protonated molecule [M + H]+ at m/z 393.1200 (Calcd for C25H28O4, m/z 393.2067) corresponding to the molecular formula C25H28O4 with 12 degrees of unsaturation. Its broad band decoupled 13C NMR spectrum brings out twenty-five signals corresponding to the twenty-five carbon atoms appearing in the molecular formula. The analysis of these signals by the DEPT 135 technique makes it possible to highlight the presence within this compound of: four secondary carbons, all sp3 hybridized, one of which is linked to an oxygen atom at δC 66.2; six tertiary carbons all sp2 hybridized; four primary carbons. The remaining eleven other carbons are therefore quaternary, including a ketone carbonyl δC 221.8; three oxygenated sp2 carbons at δC 165.4; 165.2; 162.7. On the 1H NMR spectrum of compound 1, the presence of two signals at δH 12.58 and at 12.30 exchangeable with heavy water and of a singlet integrating two protons at δH 4.16/δC 32.8 suggests that this compound contains in its structure an anthronic type skeleton.
Also observed on the 1H NMR spectrum: four doublets of one proton each at δH 6.34 (1H; d; 1.87), 6.38 (1H; d; 1.87), 6.61 (1H; d; J = 3.0 Hz) and 6.65 (1H; d; J = 3.0 Hz), attributable to two pairs of aromatic protons. The first pair at δH 6.65/δC 119.6 and δH 6.61/δC 115.8 is attributable to protons H-5 and H-7 with a coupling constant Jmeta= 1.9 Hz while the second pair at δH 6.34/δC 100.1 and δH 6.38/δC 106, is characteristic of H-2 and H-4 protons with a coupling constant Jmeta= 3.0 Hz. A set of signals consisting of a doublet at δH 4.55 (2H; d; J = 6.0 Hz)/ δC 65.2, two triplets of one proton each at δH 5.45 (1H; t; J = 6.0 Hz)/ δC 118.5 and δH 5.08 (1H; m)/ δC 123.7; a multiplet integrating four protons at δH 2.10 and three singlets of three protons each at δH 1.60/ δC 17.7; δH 1.67/ δC 25.7 and δH 1.73/ δC 16.7, all characteristic of an O-geranyl group. Taken together, all of these data allow attribution to 1, such as 3-geranyloxyemodinanthrone (Fig. 2). It is a known anthrone that was first isolated by (Botta et al., 1983). from Psorospermum febrifugum.
Compound 2 was isolated as yellow needles from the mixture Hex:EtOAc (17:3). Soluble in DMSO, it melted between 120–121°C and reacted positively to both the ferric chloride test and the Bornträger test suggesting its phenolic and quinonic nature. Moreover, the fact that 2 gave a positive reaction to magnesium acetate in methanolic medium permitted to conclude that it possessed a 1,8-dihydroxyanthraquinone skeleton (Bruneton, 2009). Its high-resolution mass spectrum under electrospray ionization showed, in positive mode, the peak of the molecular ion [M + H]+ at m/z 406.2507 (Cald for C25H27O5 at m/z 406.2851). This makes it possible to deduce the peak of the molecular ion [M] + at m/z 406.1773, compatible with the molecular formula C25H26O5, containing thirteen degrees of unsaturation. Its broad band decoupled 13C NMR spectrum, revealed the presence of twenty-five carbon signals. The analysis of these signals by DEPT techniques revealed the presence two carbonyl signals of conjugated ketones at δC 182.0 and at δC 190.7; three sp2-hybridized oxygenated carbon signals and one sp3-hybridized oxygenated carbon signal (δC 65.8). The 1H NMR spectrum of 2 showed many similarities to that of emodin previously isolated by (Cohen and Neil Towers, 1995) from Nephroma laevigatum, especially with regard to the aromatic proton region. The difference between these two spectra lies in the presence on the 2 spectrum, in the region of the olifenic and aliphatic protons, of a set of signals consisting of a doublet of two protons at δH 4.66 (2H ; d ; J = 8.0 Hz)/δC 65.8, two triples of one proton each at δH 5.48 (1H ; t ; J = 8.0 Hz)/δC 118.3 and at δH 5.05 (1H ; t ; J = 8.0 Hz)/δC 123.5 a multiplet of four protons at δH 2.11 and three singlets of three protons each at δH 1.75/ δC 16.7 ; δH 1.56/ δC 25.6 and δH 1.52/ δC 17.6, characteristic of an O-geranyl group that we located in position 3 thanks to the comparison of the spectral data of compounds 2 with those of the literature. This is how we attributed to this compound the structure of 3-geranyloxyemodin (Fig. 1) isolated for the first time from the fruits of Psorospermum febrifugum by (Botta et al., 1983).
Molecular docking
Conformational Modes in the elastase Binding-Site area
The visualization of the interactions between the amino acid residues present at the active site of elastase and isolated compounds (Fig. 2) revealed that 3-geranyloxyemodinanthrone is stabilized by hydrophobic interactions formed by the amino acid residues His 200, Ser 29, Pro 28, Trp 27, Gln 119, Leu 114, Tyr 207, Val 122 and Leu120 (Fig. 2A). 3-geranyloxyemodin forms four hydrogen bonds, between its C13-OH group and the amine group of His 57, the carboxylic group of Ser 195, the oxygen of Ser 214 and the amine group of Val 216. The molecule also stabilized by hydrophobic interactions formed by the amino acid residues Cys191A, Gln 192A, The 215A, Val 199, Trp 172, Arg 217A (Fig. 2B). Lupeol is stabilized by the hydrophobic interactions of the residues: Asp 60, Val 99, Phe 215, His 67, Arg 61, Tyr 96, Asn 214 in the active site of elastase, and by two hydrogen bonds between the C9-OH group of lupeol and the carboxylic and amino group of Val 216 of (Fig. 2C).
Conformational Modes in the lipoxygenase Binding-Site area
The interactions between the amino acid residues of lipoxygenase and compounds (Fig. 3) shown the hydrophobic interactions of amino acid residues Ile325, Leu 266, Ile 267, Val 322, Ser 269, ILE 323, Gln 704 with 3-Geranyloxyemodinanthrone (Fig. 3A); the compound 3-geranyloxyemodin form a hydrogen bond between its C17-OH group and the amine group of His, the molecule is also stabilized by hydrophobic interactions of residues Val 112 A, Leu 173A, Val 212, Phe 113, Gln 71A, Met 154A of lipoxygenase (Fig. 3B); lupeol is stabilized by hydrophobic interactions of amino acid residues Gln 264, Phe 112, Asp 150, Leu 15, Tyr 181, Tyr 183 of the lipoxygenase active site (Fig. 3C).
Conformational Modes with Activator Protein-1(AP-1)
The interactions between the amino acid residues of the transcription factor Activator protein-1 (AP-1) and the isolated compounds were visualized as shown in Fig. 4. two hydrogen bonds were observed between the oxygen of carbons 16 and 17 of 3-geranyloxyemodinanthrone and the amine groups of amino acid residues Ala 133 and Ala131and, the hydrophobic interactions of acid residues Ser 154, Val 150, Met 15, Ser 132, Leu 127, Ser 129, Thr 131, Gly 152 (Fig. 4A). Figure 4B reveals a hydrogen bond between the C-OH of carbon 26 of 3-geranyloxyemodin and the carboxylic group of Gln 204. The molecule is also stabilized by the hydrophobic interactions of acid residues, Phe 211, Gln 213, Phe 194, Gln 207, Phe 219, Phe 218 of factor AP-1. Lupeol is stabilized by hydrophobic interactions formed by amino acid residues Ser 194, Gln203, Gln 21, Phe 112, Phe 114, His 211, Ile 210 of AP-1(Fig. 4C).
Conformational Modes with Nuclear Factor Kappa B (NF-KB)
Based on the visualization of the complex between target compounds and Nuclear Factor Kappa B (Fig. 5), compound 3-geranyloxyemodinanthrone is stabilized by hydrophobic interactions of amino acid residues including Gln 170, Leu 172, His 173, Gly 206, His 172, Ser 174, Gln 177, Thr 169, Leu 205(Fig. 5A); 3-geranyloxyemodin forms a hydrogen bond between its C-OH group and the carboxylic group of His and it is also stabilized by hydrophobic interactions of amino acid residues Gly 168, Asp 136, Pro 137, His 171, Leu 172, Ile 175, Ser 174 (Fig. 5B); complex with lupeol reveals two hydrogen bonds between the its C-OH group and the carboxylic group of Asp 242 and the amine group of Val 243. Lupeol is also stabilized by hydrophobic interactions of amino acid residues Ala 241, Leu 272, Ser 234, Leu 237, Lys 238, Gly240, Asn 244 (Fig. 5C).
Binding free- energy of compound
The binding free-energies of complex formed between compounds and macromolecules were calculated and data are summarized in Table 1. The results shown that the binding free- energies of the complexes by elastase with 3-geranyloxyemodinanthrone (M) and elastase with 3-geranyloxyemodine (S) are respectively − 7.42 Kcal/mol and − 7.3 Kcal/mol while the elastase-lupeol (E) complex is lower − 12 .83Kcal/mol. No difference was observed between the binding free- energies of the complexes formed by the compounds and the transcription factor AP-1. The binding free- energies lipoxygenase-3-geranyloxyemodinanthrone (M) and lipoxygenase-E complexes are respectively − 7. 92 Kcal/mol and − 7.44 Kcal/mol while that of the lipoxygenase-3-geranyloxyemodine (S) complex is lower and is -8.98 Kcal/mol. No significant difference is observed between the binding free- energies of the complexes formed by the compounds and the transcription factor NF-KB.
Table 1
Docking results (scoring functions and amino acids interactions) of the isolated compound from P. aurantiacum hexane fraction on elastase, lipoxygenase, nuclear factor Kappa B and activator protein-1.
Compounds | Binding free- energy with elastase (Kcal /mol) | Interactions with elastase | Binding free- energy with LOX (Kcal /mol) | Interaction with LOX | Interactions with AP-1 | Binding free- Energy with AP-1 (Kcal /mol) | Interaction with NF-KB | Binding free-energy (Kcal /mol) with NF-KB |
1 | -7.42 | 8 AA involved in vanderwall and coloumb interaction ; noneAA involved in H bonding | -7.92 | 7 AA involved in vanderwall and coloumb interaction ; | 8 AA involved in vanderwall and coloumb interaction ; 2AA involved in H bonding : | − 6.49 | 9 AA involved in vanderwall and coloumb interaction | -6.35 |
2 | -7.3 | 4 AA involved in vanderwall and coloumb interaction, 3 AA involved in H Bonding | -8.98 | 8 AA involved in vanderwall and coloumb interaction ; 1AA involved in H bonding 7 | 6 AA involved in vanderwall and coloumb interaction, 1 AA involved in H Bonding : | -6.94 | 8 AA involved in vanderwall and coloumb interaction ; 2AA involved in H bonding | -6.84 |
3 | -12.83 | 8 AA involved in vanderwall and coloumb interaction ; 2AA involved in H bonding | -7.44 | 6 AA involved in vanderwall and coloumb interaction | 7 AA involved in Vanderwall and coloumb interaction:, | 6.37 | 7 AA involved in vanderwall and coloumb interaction ; 2AA involved in H bonding | -6.76 |
(1): 3-geranyloxyémodinanthrone; (2): 3-geranyloxyemodine; (3): Lupeol, LOX: lipoxygenase, NF-B: Nuclear Factor Kappa B, AP-1: Activator protein-1 |
Tyrosinase and elastase inhibition
In vitro tests were performed using purified porcine elastase and mushroom tyrosinase. The anti-tyrosinase enzymatic activity was evaluated using L-tyrosine as substrate and detecting the produced chromophore (dopachrome) at 490 nm as described previously by (Manosroi et al., 2010). The obtained results are shown in Table 2. All compounds exhibited a tyrosinase inhibitory concentration-depending activity as well as vitamin C (positive control). Inhibition percentages ranged from 2 to 100%, and 3-geranyloxyemodinanthrone (1) exhibited a higher activity compared to others compounds with an IC50 value of 65.00 ± 7.60 µg/mL significantly (p < 0.05) different from that of vitamin C (48.85 ± 1.25 µg/mL).
Table 2
Tyrosinase and elastase inhibitory activities of isolated compound from P. aurantiacum hexane fraction
Compounds | Tyrosinase % inhibition (at 10 µg/mL) | Tyrosinase % inhibition (at 100 µg/mL) | Elastase % Inhibition (at 10 µg/mL) | Elastase % Inhibition (at 100 µg/mL) | Tyrosinase IC50 (µg/mL) |
1 | 22.00 ± 1.05 | 81.11 ± 14.00 | 57.73 ± 3.44 | 86.41 ± 0.84 | 65.00 ± 7.60* |
2 | 10.23 ± 3.15 | 36.21 ± 13,75 | 3. 68 ± 11.01 | 83.96 ± 0.01 | ˃ 100 |
3 | 5.02 ± 3.75 | 29.28 ± 4.76 | 89.09 ± 4.05 | 100.00 ± 6.11 | ˃ 100 |
Vitamin C | 24.20 ± 2.00 | 96 .88 ± 0.01 | ND | ND | 41.85 ± 1.25 |
Epigallocatechin gallate | ND | ND | 98.00 ± 2.03 | 100.00 ± 7.90 | ND |
Results are represented as means ± standard error of mean (SEM), n = 3; Significant difference *(p˂0.05); compared to vitamin (positive control); |
IC50: Half inhibitory concentration; ND: not determined; (1): 3-geranyloxyémodinanthrone; (2): 3-geranyloxyemodine; (3): Lupeol. |
For elastase inhibition, the percentage inhibitions were calculated at 100 and 10 µg/mL (Table 2). All the compounds and epigallocatechin gallate (positive control) showed good inhibitory activity (inhibition greater than 50%) at 10 µg/mL except 3-geranyloxyemodine (2) as indicated in Table 2. Lupeol (3) (88.09%) showed the highest percentage of elastase inhibition at 10 µg/mL compared to other compounds.
Anti-inflammatory activities
The NO inhibitory activity of isolated compounds was evaluated on LPS-activated RAW 264.7 macrophage cells. Nitrite accumulation, a stable oxidized product of NO was measured in culture media. The levels of nitrites in the cells stimulated by LPS increased compared to the control cells (untreated cells), the quantities of nitrites produced in the presence of the various compounds at 100 µg/mL are represented in Table 3. The viability percentage of RAW 264.7 cells at 100 µg/mL varied considerably between 0 and 86%. Lupeol significantly (p < 0.05) inhibited NO production with an inhibitory concentration (IC50 = 28.74 µg/mL) compared to quercetin (IC50 = 15.13 µg/ mL), the results are grouped together in the following Table 3.
Table 3
Percentage of 15-lipoxygenase (15-LOX) and nitric oxide production inhibitory activities and half inhibitory concentrations (IC50) compounds from P. aurantiacum on LPS-activated RAW264.7 macrophages and their percentage of viability after 24hours of incubation.
Compounds | NO (µM) | % NO inhibition (at 100 µg/mL) | % viability (at 100 µg/mL) | NO IC50 (µg/mL) | 15-LOX % Inhibition (at 50 µg/mL) | 15-LOX % Inhibition (at 100 µg/mL) | 15-LOX IC50 (µg/mL) |
1 | ND | ND | 1.51 ± 9.35 | ND | 62.99 ± 3.75 | 65.19 ± 3.75 | 35.35 ± 0.35 |
2 | ND | ND | 0.86 ± 0,19 | ND | 0 ± 1.20 | 80.04 ± 3 .73 | NA |
3 | 0.74 ± 0.01 | 80.12 ± 1.12 | 79.47 ± 2.84 | 28.74 ± 0.05* | 54.28 ± 3.75 | 5.52 ± 3.75 | NA |
Quercetin | 0.74 ± 0.19 | 86.72 ± 3.56 | 67.24 ± 6.72 | 15.13 ± 0.25 | 63.12 ± 3.75 | 84.12 ± 3.75 | 26.79 ± 3.97 |
Results are represented as means ± standard error of mean (SEM), n = 3; Significant difference *(p˂0.05); compared to quercetin (positive control), NO: Nitric oxide; IC50: Half inhibitory concentration; 15-LOX: 15-Lypoxygenase; ND: not determined; NA: Not active |
The 15-lipoxygenase (15-LOX) inhibitory activity was performed using purified soybean lipoxygenase and results are summarized in Table 3. In general, the percentage of inhibition of 15-lipoxygenase increases with the concentration of compounds, which was not the case for lupeol (3) and 3-geranyloxyemodine (2). The percentage inhibition of all compounds ranged from 0.00 to 100% and 3-geranyloxyemodinanthrone (M) exhibited good anti 15-LOX inhibitory activity with an IC50 value of 35.35 ± 0.35µg/mL comparable to that of quercetin (26.79 ± 3.97 µg/mL).