Aspidosperma pyrifolium Mart., popularly known as “pereiro-preto”, is a small tree that is widely distributed in Northeastern Brazil, more precisely in the Caatinga. Its wood is used in the manufacture of furniture, due to its excellent quality; in traditional medicine, the extract of its leaves and bark is used for its anti-inflammatory and analgesic properties. The study made it possible to isolate 15-methoxyaspidospermine and 15-methoxypyrifolidine, corroborating the results of pharmacological assays, which showed anti-inflammatory and analgesic potential, especially at 30 mg kg−1 (p < 0.001). Thus, the species was shown to be a promising source of active substances, with special attention paid to its toxicological potential.
1. Introduction
Inflammation is an immune defense mechanism that the body uses to fight bacteria, viruses and other pathogens
[1,2][1][2]. In these processes, a variety of chemical mediators are released from damaged tissue, including excitatory amino acids, hydrogen ions, peptides, lipids, and cytokines, all of which underlie inflammation and pain
[3].
Non-steroidal anti-inflammatory drugs, such as aspirin, as well as steroidal anti-inflammatory drugs, such as dexamethasone, have been widely used to fight inflammation, but there is clinical evidence that these drugs are capable of causing adverse effects, including gastrointestinal disorders. Alternatively, natural products are growing targets in research for new drug discovery
[1,4][1][4].
Aspidosperma pyrifolium Mart., popularly known as “pereiro-preto”, is a small tree that is widely distributed in northeastern Brazil, more precisely in the Caatinga. Its wood is used in the manufacture of furniture, due to its excellent quality
[5,6,7][5][6][7]; in traditional medicine, the extract of its leaves and bark is used for its anti-inflammatory and analgesic properties
[7,8,9][7][8][9].
Previous studies have shown a hypotensive effect attributed to the alkaloids present in the bark and leaves of this species, as well as anti-plasmodic activity related to the presence of aspidosperman alkaloids, such as the alkaloid aspidospermin
[9,10][9][10].
2. Chemical Identification of Isolated Compounds
The qualitative analysis by HPLC-DAD of TAF-Ap indicated the presence of two alkaloids. The acquisition of the 3D chromatogram with scanning from 200 to 400 nm (
Figure 1) pointed the presence of the alkaloids with absorption greater than 200 mAU and retention times of 7.68 and 7.78 min, respectively.
Figure 1. 3D chromatogram of an exploratory analysis of TAF-Ap.
The mass spectra of
1 and
2 showed molecular ion peaks at
m/
z = 385.44 (calcd
m/
z 384.241) and
m/
z = 415.45 (calcd
m/
z 414.252), which allowed us to manage the following structural formulas: C
23H
32N
2O
3 and C
24H
34N
2O
4, respectively. The UV spectrum showed three absorption bands at λ
max. 222, 256 and 285 nm (
Figure S1), suggestive of indoline chromophore
[11].
The
1H NMR data (500 MHz, acetone-d
6) (
Table 1) (
Figures S2–S5) showed four signals aromatic hydrogen at δ
H 7.16 (t,
J = 7.8, 1H), δ
H 7.06 (d,
J = 8.2 Hz, 1H), δ
H 7.00 (d,
J = 8.2 Hz, 1H) and δ
H 6.82 (dd,
J = 8.3 and 1.9 Hz, 2H). In addition to these signals, others were visualized between δ
H 3.86 and 0.68, suggestive of aliphatic hydrogens
[9,11][9][11].
Table 1. 1H and 13C NMR data of 15-methoxyaspidospermine (1) and 15-methoxypyrifolidine (2) (δ, acetone-d6, 500 and 125 MHz).
5.2. Formalin-Induced Nociception Test
The injection of intraplantar formalin into the hind paw of an animal induces severe pain by the direct stimulation of nociceptors, characterized by vigorous licking, bites and bumps on the paw injected with the irritant. This test allows the verification of signals present in the modulation phase of nerve impulses, and also to observe the participation of endogenous systems, such as that of opioids
[25,26][25][26].
In this model, both the indomethacin group and the 30 mg kg
−1 TAF-Ap group demonstrated a higher inhibition level than 50%, with less time to stop licking at the end of phase I (neurogenic phase). In phase II (inflammatory phase), all doses of TAF-Ap significantly decreased the time spent licking, with the 30 mg kg
−1 dose being the most effective (85.57% inhibition) (
Table 4).
Table 4. Antinociceptive effect of the total alkaloid fraction from A. pyrifolium (TAF-Ap) in phases I (0–5 min) and II (15–30 min) after 1% formalin-induced nociception.
Treatments |
Time Animals Are Licking Paw in Phase I (0–5 min) |
Inhibition (%) |
Time Animals Remain Licking Paw in Phase II (15–30 min) |
Inhibition (%) |
δ | C |
δH |
δC |
δH |
2 |
67.67 |
4.93 (dd, J = 6.2 and 10.7 Hz, 1H) |
67.90 |
4.85 (dd, |
Saline | J = | 6.1 and 10.6 Hz, 1H) |
4.53 ± 0.34 |
|
11.30 ± 1.66 |
|
3 |
52.83 |
3.26 (brd, J = 8.2 Hz, 1H) and 2.17 (m, 1H) |
Indomethacin | 52.70 |
3.26 (brd, | J | = 8.2 Hz, 1H) and 2.17 (m, 1H) |
1.58 ± 1.39 ** |
62.12 ** |
1.00 ± 1.43 *** |
91.15 *** |
5 |
52.80 |
3.37 (d, J = 6.8 Hz, 1H) and 2.36 (m, 1H) |
52.70 |
3.37 (d, J = 6.8 Hz, 1H) and 2.36 (m, 1H) |
TAF-Ap 10 mg kg−1 |
3.14 ± 1.09 |
30.68 |
6.90 ± 1.5 |
38.93 |
6 |
37.71 |
2.06 (brt, J = 2.2 Hz, 1H) and 1.92 (dd, J = 3.8 and 15.2 Hz, 1H) |
37.35 |
(brt, J = 2.2 Hz, 1H) and 1.92 (dd, J = 3.8 and 15.2 Hz, 1H) |
7 |
52.80 |
- |
52.83 |
- |
TAF-Ap 20 mg kg−1 |
2.98 ± 1.00 |
34.21 |
3.4 ± 2.0 ** |
69.91 ** |
8 |
141.65 |
- |
141.65 |
- |
9 |
110.38 |
6.82 (dd, J = 8.3 and 1.9 Hz, 2H) |
117.39 |
7.06 (d, J = 8.2 Hz, 1H) |
10 |
127.42 |
7.16 (t, J = 7.8, 1H) |
113.45 |
7.00 (d, J = 8.2 Hz, 1H) |
11 |
110.09 |
6.82 (dd, J = 8.3 and 1.9 Hz, 2H) |
154.66 |
- |
12 |
141.68 |
- |
150.10 |
- |
13 |
133.50 |
- |
134.33 |
- |
14 |
24.44 |
2.03 (m, 1H) and 1.59 (brd, J = 3.8 Hz, 1H) |
24.33 |
2.03 (m, 1H) and 1.59 (brd, J = 4.5 Hz, 1H) |
15 |
74.02 |
3.26 (brd, J = 9.0 Hz, 1H) |
74.50 |
3.17 (brd, J = 14.3 Hz, 1H) |
16 |
24.77 |
2.00 (m, 1H) and 1.35 (m, 1H) |
24.60 |
2.00 (m, 1H) and 1.35 (m, 1H) |
17 |
24.33 |
2.03 (m, 1H) and 1.37 (m, 1H) |
24.44 |
2.03 (m, 1H) and 1.37 (m, 1H) |
18 |
6.77 |
0.69 (t, J = 7.5 Hz, 3H) |
6.84 |
0.68 (t, J = 7.5 Hz, 3H) |
19 |
30.45 |
1.05 (q, J = 7.3 Hz, 2H) |
29.98 |
1.00 (q, J = 7.3 Hz, 2H) |
20 |
36.59 |
- |
36.57 |
- |
21 |
71.17 |
3.80 (s, 1H) |
71.15 |
3.81 (s, 1H) |
11-OCH3 |
- |
- |
56.53 |
3.83 (s, 3H) |
12-OCH3 |
56.01 |
3.84 (s, 3H) |
56.53 |
3.86 (s, 3H) |
15-OCH3 |
56.74 |
3.31 (s, 3H) |
56.74 |
3.31 (s, 3H) |
NCOCH3 |
169.59 |
|
170.26 |
|
NCOCH3 |
22.93 |
2.18 (s, 3H) |
22.98 |
2.19 (s, 3H) |
The
13C-NMR spectrum (125 MHz, acetone-d
6) (
Table 1) (
Figures S6 and S7) showed 31 signals: 9 from unhydrogenated carbons, 7 from CH carbons, 7 from CH
2 carbons and 8 from CH
3 carbons. The signals at δ
C 110.38, 110.09, 127.42, 117.39 and 113.45 are characteristic of aromatic methinic carbons. In addition, the signals at δ
C 141.65, 141.68, 133.50, 154.66, 150.10 and134.33 are consistent with non-hydrogenated aromatic carbons
[9,12][9][12].
The heteronuclear correlation map-HSQC (
Figures S8 and S9) showed the following correlations: δ
H 6.82/δ
C 110.38 and 110.09 (C-9 and C-11) and δ
H 7.16/δ
C 127.42 (C-10), suggestive of a tri-substituted aromatic nucleus. The correlations of δ
H 7.06/δ
C 117.39 (C-9) and δ
H 7.00/δ
C 113.45 (C-10) are suggestive of another tetra-substituted aromatic ring
[9]. The correlations between δ
H 3.84/δ
C 141.68 e δ
H 3.83/δ
C 154.66 e δ
H 3.86/δ
C 150.10, seen in the heteronuclear correlation map-HMBC (
Figures S10–S12), confirm the presence of methoxyl groups in C-11, C-11 and C-12 in the two aromatic nuclei, respectively. According to
[9], the signals at δ
C 169.59 and δ
C 170.26 suggested the presence of
N-acethyl carbonyl groups, in addition to a signal at δ
C 52.80 and 52.83, characteristic of C-7 indolic-ring quaternary carbon. These positions were confirmed by long-range heteronuclear correlation at δ
H 2.18/δ
C 169.59; δ
H 2.19/δ
C 170.26 and δ
H 6.82/δ
C 52.80; and δ
H 7.06/δ
C 52.83.
All the methylene carbons were displayed by
13C NMR and experiment APT and the hydrogens attached to them were revealed by chemical shifts, coupling constants and comparison with data from previous research. The signals at δ
C 71.17 and 71.15 were attributed to the methinic carbons of the position 21, respectively.
A signal at δ
H 3.31 (s, 6H) showed a correlation in the HSQC with δ
C 56.74 and suggested the presence of other methoxyl groups in the compounds. Methoxyl insertion at C-15 was reinforced by
α deprotection in this carbon,
β deprotection at C-14 and C-20 and
γ protection at C-19 and C-3, when compared to a structure without this substituent and confirmed by correlations of δ
H 3.31/δ
C 74.02 and 74.50, seen in the HMBC. In addition, the coupling constants for the two broad doublets at δ
H 3.26 (
J = 9.0 Hz) and 3.17 (
J = 14.3 Hz) were in accordance with the expected values when the hydrogens H-15 were located in the
α position in relation to ring
D. Therefore, it can be deduced that OCH
3-15 features pseudo-equatorial stereochemistry
[13]. The correlation between δ
H 2.03/δ
H 3.17 and 3.26, observed in the NOESY spectrum (
Figure S13), reinforce this argument.
The interpretation of spectral data, in addition to the comparison with the previous research data, made it possible to identify two monoterpenoid indole alkaloids of the plumeran class. Compound
1 was identified as 15-methoxyaspidospermine. The presence of a methoxyl group at position C-11 in compound
2 allowed us to identify it as being t 15-methoxypyrifolidine (
Figure 2).
Figure 2. Compounds isolated from A. pyrifolium present in TAF-Ap. 1—R=H, 2—R=OCH3.
3. Acute Toxicity
The TAF-Ap dose of 200 mg kg
−1 was 100% lethal to animals within 30 min of its administration and death was preceded by severe tremors. The 100 mg kg
−1 dose caused death in a female one hour after administration; by the fourth hour, all the animals showed poor responses to tail pinching, low auricular reflex and impaired posture, as well as tremors and signs of sedation and forced breathing. Regarding the dose of 50 mg kg
−1, no animal died; however, the animals presented signs of mild sedation and forced breathing in the first 24 h. The lethal dose capable of killing 50% of animals (LD
50) was estimated to be 160 mg kg
−1.
The mean values obtained for each group in the weight evolution of the animals showed significant differences for the dose of 100 mg kg
−1; although they gained weight, the values for body mass increase in the animals of this group were lower than those of the negative control group and of the groups receiving the 50 mg kg
−1 dose (
Table 2).
Table 2. Effect of oral administration of the total alkaloid fraction from A. pyrifolium (TAF-Ap) on weight evolution (change in body mass) by water and feed intake in 14 day-old male and female Swiss mice.
Parameter |
Sex |
Saline Solution |
TAF-Ap (50 mg kg−1) |
TAF-Ap (100 mg kg−1) |
Initial W. (g) |
M |
28.83 ± 1.32 |
27.67 ± 2.58 |
30.67 ± 0.81 |
Liver |
M |
5.11 ± 0.65 |
5.06 ± 0.55 |
4.13 ± 0.54 * |
Final W. (g) |
33.17 ± 1.31 |
34.50 ± 3.72 |
31.50 ± 3.72 |
Spleen |
0.59 ± 0.32 |
0.73 ± 0.47 |
0.80 ± 0.21 |
Gain (%) |
4.34 |
6.83 |
0.83 *** |
Heart |
0.47 ± 0.02 |
0.50 ± 0.05 |
0.52 ± 0.11 |
Initial W. (g) |
F |
27.50 ± 0.83 |
25.00 ± 1.41 |
25.33 ± 3.14 |
Kidneys |
1.23 ± 0.64 |
1.37 ± 0.11 |
1.24 ± 0.16 |
Final W. (g) |
32.00 ± 1.26 |
32.17 ± 1.54 |
26.31 ± 3.18 |
Liver |
F |
TAF-Ap 30 mg kg−1 | 4.98 ± 0.20 |
4.95 ± 0.47 |
4.03 ± 0.53 * |
Gain (%) |
4.50 |
7.17 |
0.98 *** |
1.90 ± 1.09 ** |
58.05 ** |
Spleen |
0.59 ± 0.11 |
0.45 ± 0.11 |
0.48 ± 0.25 |
Feed intake per day (g) |
M |
34.57 ± 2.92 |
37.29 ± 3.42 |
25.50 ± 3.25 *** |
Heart |
0.48 ± 0.03 |
0.45 ± 0.04 |
0.36 ± 0.18 |
|
F |
36.71 ± 2.94 |
35.86 ± 2.65 |
Kidneys | 23.07 ± 2.30 *** |
1.08 ± 0.11 |
Water consumption per day (mL) |
M |
52.14 ± 4.25 |
52.86 ± 4.68 |
56.43 ± 2.25 *** |
|
F |
46.79 ± 4.64 |
50.00 ± 3.39 *** |
60.29 ± 3.93 *** |