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].
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 (%) |
Saline |
4.53 ± 0.34 |
|
11.30 ± 1.66 |
|
Indomethacin |
1.58 ± 1.39 ** |
62.12 ** |
1.00 ± 1.43 *** |
91.15 *** |
TAF-Ap 10 mg kg−1 |
3.14 ± 1.09 |
30.68 |
6.90 ± 1.5 |
38.93 |
TAF-Ap 20 mg kg−1 |
2.98 ± 1.00 |
34.21 |
3.4 ± 2.0 ** |
69.91 ** |
TAF-Ap 30 mg kg−1 |
1.90 ± 1.09 ** |
58.05 ** |
1.63 ± 1.79 *** |
85.57 *** |
The results related to the decrease in the number of phase II licks may contribute positively to the anti-inflammatory profile observed in the decrease of carrageenan paw edema, so that both the positive control and the TAF-Ap may have caused the inhibition of mediators responsible for increasing vascular permeability after 30 min, reducing edema caused by the inflammatory agent. The results presented suggest that TAF-Ap exerts a central and peripheral effect against pain and inflammation, which corroborates data from another study [
6], in which the 100 mg kg
−1 dose of the aqueous extract of the seeds from
A. pyrifolium also showed a decrease in late phase licking time similar to that of positive controls (morphine 5 mg kg
−1 and indomethacin 10 mg kg
−1).
It can be inferred that by raising the pain threshold induced by intraperitoneal acetic acid injection and intra-implant formalin injection, and inhibiting the carrageenan-induced edematogenic effect and leukocyte migration, TAF-Ap is endowed with anti-nociceptive and anti-inflammatory activity, both of peripheral origin and associated with central mechanisms of pain and inflammation inhibition. These activities are related to the indolic alkaloids found in the extracts of the species of genus Aspidosperma, which could be confirmed from the isolation of the compounds 15-methoxyaspidospermine and 15-methoxypyrifolidine. This was the first pharmacological study conducted with the isolated fraction of alkaloids of the species A. pyrifolium.
3. Materials and Methods
3.1. Vegetable Material Collection and Identification
The stem barks from A. pyrifolium Mart. were collected at Capim Grande site, São José
da Mata district, Campina Grande, Paraíba, in August 2014 (coordinates: S 713027.5600–W
3600053.3700). After collection, the material was identified by Prof. Dr. Leonardo Pessoa
Felix and the exsiccata deposited in the Herbarium Jayme Coelho de Moraes at the Federal
University of Paraíba, under the number 20104.
3.2. Isolation and Characterization of Compounds
The stem barks from A. pyrifolium Mart. were dried in an air circulation oven (40 C)
and powdered in a 10 mesh knife mill. The powder was exhaustively extracted with
96% (v/v) ethanol and rotary evaporated (Tecnal TE-21) to yield 552 g of crude ethanolic
extract (CEE). Next, 112 g of CEE was subjected to a liquid-liquid partition, providing
the following phases: hexane (57.46%/64.36 g), chloroform (18.03%/20.2 g), ethyl acetate
(1.25%/1.4 g) and methanol: H2O—7:3 (v/v) (3.83%/4.28 g).
3.3. Obtaining of the Total Alkaloids Fraction from A. pyrifolium (TAF-Ap)
The CEE (110 g) was submitted for extraction of the total alkaloids fraction. The extract
was initially treated with 2 L of 3% hydrochloric acid solution and subsequently filtered on
filter paper. The residue was discarded and the filtrate was subjected to several extractions
with chloroform. The acidic aqueous phase was basified with NH4OH, stirred vigorously
to pH 10, and then extracted with chloroform [27]. Subsequently, the chloroform fraction
was evaporated under reduced pressure in a rotary evaporator at 40 C, yielding 2.06 g of
the TAF-Ap.
Isolation of Alkaloids from TAF-Ap
The TAF-Ap (1.90 g) was subjected to medium pressure chromatography (BUCHI
Pump Manager, model C-615, Flawil, Switzerland) using aluminum oxide 90 (70-270 mesh
ASTM, MN) and binary mixtures of hexane and methanol in an increasing polarity gradient,
providing 48 fractions. The chromatographic profile of sample 8 (126 mg) was analyzed in
an analytical HPLC (Infinity Series More Confident 1260 system, Agilent, Santa Clara, CA,
United States). The fraction was diluted in 0.2% trifluoroacetic acid (TFA):acetonitrile (ACN)
(1:1—v/v) aqueous solution at 1 mg mL?1 and the volume injected into the apparatus was
50 L. A reverse phase C-18 column was maintained at 40 C and 0.2% TFA:ACN aqueous
solution (77:23 to 65:35 v/v) was used as a mobile phase with a flow of 10 mL min?1, a
time of 15 min, and in gradient mode. After analysis in analytical HPLC, the sample was
injected into semi-preparative HPLC (2767 Sample Manager System,Waters, with a UV-Vis
photodiode detector, Milford, Massachusetts) using the same conditions as previously
mentioned. At the end of the separation, a total of 10 fractions were obtained (Ap-1 to
Ap-10). Fraction Ap-3 was directed to structural analysis, which allowed the identification
of compounds 1 and 2 (Figure 2).
Plants 2021, 10, 2526 11 of 15
3.4. Techniques Used for Structural Identification
The mass spectra were obtained by injecting the samples into a high-performance liquid
chromatograph with a diode array detector coupled with a low resolution electrospray
ionization mass spectrometer (HPLC-DAD-ESI-MS; 2767 Sample Manager System, Waters,
Milford, Massachusetts) and a quadrupolar analyzer ion trap (trap ions) operating in the
positive mode. The 1H and 13C NMR spectra (500 and 125 MHz, acetone-d6) (1D and 2D)
were recorded on a Varian Mercury spectrometer using TMS as the internal standard.
3.5. Animals
The study was carried out in strict accordance with the Standard Operating Procedures
(Laboratory of Pharmacology at the State University of Paraiba, Campina Grande, Brazil)
and approved by a veterinarian, who frequently monitored the health of the animals
through physical condition assessments. All efforts were made to reduce the suffering of
the experimental animals.
The animals were obtained from the Institute for Research in Pharmaceuticals and
Medications (Iperfarm) of the Federal University of Paraíba (UFPB). Disease-free adult mice
Swiss (Mus musculus) of both sexes, weighing between 25 and 30 g, were used. The animals
were housed in standard plastic cages in an environment with a controlled temperature and
humidity, a light-dark cycle of 12 h and food and water ad libitum. All the mice underwent
a period of at least 7 days of acclimatization prior to the procedure, being socialized through
contact, including with humans. The animals were handled with care to minimize stress.
The researchers confirm that the laboratory established a protocol for the use of humane
endpoints in cases where animals became severely ill or moribund during the experiment,
but none reported death or behavioral changes in animals. The animals received standard
laboratory pellets and water ad libitum for both the adaptation period (7 days) and during
the trial, except for the period of 12 h prior to the experiments, in which the access to
food was restricted. Throughout the experiments, all of the animals received humane
care according to the “Guide for the Care and Use of Laboratory Animals” prepared by
the National Academy of Sciences [28]. At the end of the experiments, the animals were
euthanized by intraperitoneal anesthetic overdose (ketamine 50 mg kg?1 and xylazine—
3 mg kg?1), following the recommendation of resolution number 714 of 20 June 2002 of
the Federal Council of Veterinary Medicine. The experimental protocols were submitted
and approved by the Ethics Committee on Animal Use (CEUA) of the Faculty of Social and
Applied Sciences of Campina Grande (FACISA)—Project Number/Protocol: 5402042015
and CIAEP/CONCEA: 01.001.2012.
3.6. Chemicals and Reagents
The following drugs and chemicals were used: indomethacin 10 mg (Bayer, Leverkusen,
Germany), dipyrone sodium 500 mg (TEUTO, Goiás, Brazil), carrageenan (laboratory
(BDH Chemicals®, London, UK), formalin (BDH Chemicals®, London, UK), (Merck,
Kenilworth, NJ, USA), HPLC-grade acetonitrile (Merck, Germany, USA) and trifluoroacetic
acid (Merck, Kenilworth, NJ, USA). TAF-Ap was dissolved in 0.9% saline solution. All the
solutions were prepared immediately prior to the start of the experiments.
3.7. Pharmacological Tests
Acute Toxicity Assessment
The acute toxicity test was based on ANVISA Resolution No. 90/2004 and the Almeida
Experimental Protocol [29], with some adaptations according to [30], where the behavioral
alterations in the central and autonomic nervous system were evaluated along with the occurrence
of death [31]. In this trial, a total of 48 adult mice (n = 5), corresponding to 8 groups,
with 24 males and 24 females, were treated orally at doses of 50, 100 and 200 mg kg?1 of
TAF-Ap; a control group received saline solution only. Parameters such as body mass,
water and feed intake and excreta production were evaluated for 30 min, 1, 2, 4, and every
24 h for 14 days. On day 15, the animals were weighed and euthanized. After euthanasia,
Plants 2021, 10, 2526 12 of 15
weighing and macroscopic analysis of the viscera (liver, kidneys, spleen, lungs and heart)
was carried out [32].
3.8. Evaluation of Anti-Inflammatory Activity
3.8.1. Carrageenan-Induced Paw Edema
The test was based on the methodology used by Zayed and Hassan (2014) [33]. Five
groups of six animals each (n = 6) were treated orally with saline (0.9%; negative control),
10 mg kg?1 indomethacin (positive control) and TAF-Ap at doses of 10, 20 and 30 mg kg?1.
After 30 min, 0.1 mL of 1% carrageenan solution was injected into the right hind paw
subplantar region. Paw thickness was measured with a digital caliper before and up to the
fourth hour after edematogenic stimulation. The anti-edematogenic effect was obtained
by calculating the difference between the initial thickness of the paw that received the
phlogistic agent and the thickness measured after each hour. The inhibition percentage was
calculated using the following formula: Inhibition = (V ? X)/V 100%, where V is the
measure of vehicle group edema and X tests or positive control. To complement the results,
the paw that received the phlogistic agent was cut with surgical scissors in the tibio-tarsal
region for later comparison between weights, according to the methodology used by [34].
3.8.2. Carrageenan-Induced Peritonitis
Four groups of six animals each (n = 6) were treated orally with 10 mg kg?1 saline (negative
control), 10 mg kg?1 indomethacin and TAF-Ap at doses of 20 and 30 mg kg?1. After
30 min, a 1% carrageenan solution (0.1 mL/10 g) was injected into the intraperitoneal cavity
of the animals. Four hours after the induction of inflammation, the animals were sacrificed
and the intraperitoneal cavity washed with 2 mL of alkaline phosphate buffer (pH 7.2). The
solution containing the buffer plus the peritoneum cells was transferred to tubes containing
0.4 mL of Turk’s solution. After five minutes, the global polymorphonuclear leukocyte
count was performed in the Neubauer Chamber [35].
3.9. Evaluation of Anti-Nociceptive Activity
3.9.1. Acetic Acid-Induced AbdominalWrithing Test
Five groups of six animals each (n = 6) were treated orally with 10 mg kg?1 saline
(negative control), dipyrone 500 mg kg?1 (positive control) and TAF-Ap at doses of 10,
20 and 30 mg kg?1. After 40 min, a 1% acetic acid solution (0.1 mL 10 g?1) was injected
into the intraperitoneal cavity and the animals were transferred to clear glass funnels and
observed for 20 min to account for the number of abdominal contortions, followed by
stretching of the lower limbs [36].
3.9.2. Formalin-Induced Nociception
Five groups with six animals each (n = 6) were treated orally with 10 mg kg?1 saline
(negative control), 10 mg kg?1 indomethacin (positive control) and TAF-Ap at doses of G10,
20 and 30 mg kg?1. After 30 min, 20 L of 1% formalin was injected into the subplantar
region of the right paw of the animals. The animals’ reactivity was observed by recording
the start and end time of licking the paw that received the phlogistic agent during the initial
5 min (Phase I—neurogenic) and between 15 and 30 min (Phase II—inflammatory). The
calculation involved subtracting the final time by the initial time in each phase (Tf—To) [37].
3.10. Statistical Analysis
The results were evaluated using analysis of variance (ANOVA) followed by Tukey’s
post-test. All the results were expressed as mean standard deviation (s.d.) with a
minimum significance level of p < 0.05 and analyzed using GraphPad Prism 5.0 software.
4. Conclusions
The results obtained in this research showed that Aspidosperma pyrifolium is an important
plant species for the research of bioactive compounds. In this study, two monoterpenic
Plants 2021, 10, 2526 13 of 15
indole alkaloids were isolated from the total alkaloid fraction. Through spectroscopic techniques,
it was possible to identify 15-methoxyaspidospermine and 15-methoxypyrifolidine,
compounds previously found in the species.
In the acute toxicity test, it was demonstrated that the TAF-Ap has considerable toxicity
under the conditions evaluated and the LD50 was estimated at 160 mg kg-1. However,
a potent anti-inflammatory effect was demonstrated in the paw edema and peritonitis
tests induced by carrageenan, especially at the dose of 30 mg kg-1 (p < 0.001) of TAF-Ap,
promoted by the inhibition of the release of inflammation mediators and the modulation of
neutrophil migration, respectively. In addition, TAF-Ap also showed interesting analgesic
properties in the acetic acid-induced nociception test, in which there was an 84% decrease
in the number of abdominal contortions at the dose of 30 mg kg-1, when compared to the
negative control; and in the formalin test, with a significant decrease in the number of licks
on the paw where the phlogistic agent was applied. Thus, the species whas shown to be a
promising source of substances with pharmacological activity, with special attention paid
to the toxicological potential presented. Therefore, its use should be conditioned to the
determination of safe doses.
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