Introduction
Ophidic accidents are a concerning health problem, especially in tropical countries.
In Brazil, most such accidents involve snakes of the Bothrops genus, the venom of
which induces extensive local damage followed by marked edema, pain, erythema, ecchymosis,
tissue necrosis, and extensive hemorrhage through alterations of platelet function
[1]
[2]. Several studies have shown that inflammatory mediators, such as histamine, bradykinin,
and prostaglandin, may contribute to the amplification of local damage subsequent
to an intraplantar injection of Bothrops venom, and commercial anti-venom does not effectively neutralize the inflammatory
response [3]
[4]
[5]
[6]
[7]
[8].
Nonsteroidal anti-inflammatory drugs (NSAIDs) can be used to treat inflammatory processes,
including those that are elicited by snakebites [9]. However, the utility of NSAIDs is limited because of their side effects, such as
gastric and duodenal ulceration and renal failure that occurs through the inhibition
of prostaglandin synthesis [10]. Natural products with less toxic effects have been tested as alternative and complementary
treatments for inflammatory responses that are related to ophidic accidents [11]
[12].
Plant extracts have been used in folk medicine to treat or attenuate several inflammatory
conditions, including snakebites [12]
[13]. The aqueous crude extract of husk fiber from Cocos nucifera L. (Arecaceae) is widely used in northeastern Brazilian folk medicine to treat diarrhea
and arthritis, and several parts of the fruit and plant have been used by people in
different countries for the treatment of several ailments [14]
[15]. The crude extract and virgin coconut oil (VCO) from C. nucifera have been reported to have anti-inflammatory, analgesic, antimicrobial, and antiulcerogenic
properties in experimental models in rodents [16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24].
In the present study, we evaluated the possible anti-inflammatory and analgesic effects
of VCO (C. nucifera) on paw injury that was induced by Bothrops jararacussu snake venom (BjcuV) and investigated the possible pharmacological mechanisms that
underlie the effects of VCO.
Results and Discussion
The mixtures and composition of saturated and unsaturated fatty acids in the natural
oils were identified by 13C- and 1H-NMR spectra [25]. The 13C-NMR spectral data ([Fig. 1]) indicated the presence of saturated fatty acids in VCO, with signals in the range
of 14.00–34.00 ppm. In this region, there were many overlapping signals, and similar
chemical shifts were observed for different fatty acids. The presence of unsaturated
fatty acids was revealed by signals at 129.65 ppm (C9) and 129.67 ppm (C10), characteristic of the presence of oleic acid. Signals at 172.8 and 173.2 ppm were
assigned to carbonyl groups of fatty acids [25]. The presence of olefinic protons of unsaturated fatty acids was confirmed by signals
around 5.2 ppm in the 1H-NMR spectrum ([Fig. 2]).
Fig. 1 13C-NMR spectra of C. nucifera oil (CDCl3, 100 MHz).
Fig. 2 1H-NMR spectra of C. nucifera oil (CDCl3, 400 MHz).
This chemical composition of the VCO that we observed was consistent with the product
packaging, in which the main fatty acids were caproic, caprilic, capric, lauric, meristic,
palmitic, stearic, oleic, and linoleic acids. Some of these components were identified
by gas chromatography of VCO that was collected from Chiang Rai province in Northern
Thailand [18]. The presence of unsaturated acids (e. g., palmitic and stearic acids) and monounsaturated
fatty acid (oleic acid) influences the membrane permeability of many organelles and
suppresses enzyme activity (e. g., phospholipase A2 [PLA2]), leading to the suppression of inflammatory processes [18]
[26].
These fatty acids from VCO may attenuate the inflammatory response that is elicited
by BjcuV ([Fig. 3]). The choice a bothropic venom as a phlogistic agent was based on a previous study
that demonstrated that these snake venoms induce paw edema earlier than carrageenan,
which has been widely used as a pharmacological agent to induce edema [2]
[5]
[7]
[27]. Notably, neither mortality nor lethal toxicity occurred after VCO treatment or
the BjcuV injection.
Fig. 3 Effect of virgin coconut oil on BjcuV-induced paw edema in mice. The animals were
orally pretreated with VCO (100, 200, or 400 mg), 0.15 M NaCl (0.2 ml), or dexamethasone
(Dexa; 1 mg/kg, i.p.). Each point represents the mean±SEM from 5 animals. *P<0.05,
significant difference from control group.
[Fig. 3] shows significant paw edema 30 min after the BjcuV injection, which was significantly
decreased (p<0.05) by 100, 200, and 400 mg VCO. The positive control dexamethasone
decreased (p<0.05) paw edema only 60 min after the BjcuV injection. These anti-inflammatory
effects of VCO and dexamethasone were sustained for 5 h post-venom inoculation. The
reduction of edema formation by pretreatment with the corticosteroid dexamethasone
supports previous findings that the inflammatory response amplifies local tissue damage
after bothropic inoculation [4]
[7]
[28].
The present results reinforce the potential therapeutic use of coconut products, which
have been used as nutraceutical products in folk medicine for the treatment of various
metabolic and inflammatory diseases [14]
[15]. The use of C. nucifera in folk medicine has been supported by studies of the anti-inflammatory effects of
a crude extract of C. nucifera in a rat model of carrageenan-induced paw edema [17]
[20]. Similar anti-inflammatory activity of VCO (C. nucifera) has been observed, which may be associated with its polyphenol and fatty acid content
[18]
[19].
The significant suppressive effects of VCO on the first phase of carrageenan-induced
paw edema formation in rats likely occur through an inhibitory action on the release
or synthesis of early mediators of inflammation, such as histamine, serotonin (5-hydroxytryptamine
[5-HT]), and bradykinin [18]. These mediators participate in the development of the early local inflammatory
response to carrageenan. They act in vessels by inducing vasodilation and increasing
vascular permeability, which are important triggers of edema formation [29]
[30].
The 200-mg dose of VCO was used to investigate the possible mechanism of its anti-inflammatory
effects. Rinald et al. [17] reported that a crude extract from husk fiber of C. nucifera decreased paw edema 1 h after a histamine injection in rats. Oral VCO administration
decreased (p<0.05) acute inflammation that was induced by histamine 30 min after induction,
possibly through H1 receptor blockade ([Fig. 4a]). This mechanism of action of VCO is different from dexamethasone, which modulates
histamine by attenuating mast cell degranulation [31]. Previous studies showed that histamine and mast cells do not play a primary role
in BjcuV-induced paw edema. The H1 receptor antagonist loratadine and the compound 48/80 (a mast cell degranulator/depletor)
did not prevent damage that was caused by BjcuV [7]. However, Landucci et al. [3] reported the significant participation of histamine, dependent from mast cell degranulation,
in paw edema that was induced by 2 myotoxins (i. e., bothropstoxin-I and -II) that
were isolated from BjcuV. These myotoxins have structures and activity that are similar
to PLA2. The present findings regarding the anti-histaminergic effect of VCO and the possible
applicability of VCO in other experimental models of diseases (e. g., allergies and
asthma) require further investigation.
Fig. 4 Effects of virgin coconut oil on paw inflammation induced by different phlogistic
agents. Edema was induced by histamine a, serotonin b, bradykinin c, substance P d, and prostaglandin E2 (PGE2) e. The animals were orally pretreated with VCO (200 mg) or 0.2 ml of 0.15 M NaCl. Each
point represents the mean±SEM from 5 animals. *P<0.05, significant difference from
control group.
Similar to histamine, serotonin is preformed in cytoplasmic granules of mast cells
and platelets and has proinflammatory effects. Serotonin contributes to the sensitization
of nerve fibers that is observed in inflammatory processes that are induced by Bothrops snakebites [5]. Furthermore, serotonin modulates the signal that is responsible for the chemotaxis
of neutrophil migration during the innate immune response [32] and by modify the vascular permeability induce paw edema by 120 min later [33].
[Fig. 4b] shows that VCO significantly decreased (p<0.05) edema that was induced by serotonin. A similar result was reported for a crude
extract from husk fiber of C. nucifera in rats [17]
. The possible mechanisms of action of VCO may involve the modulation of 5-HT receptors.
5-HT receptors have been reported to participate in paw edema that is elicited by
toxins from the venom of Bothrops jararacussu, Bothrops jararaca, and Bothrops lanceolatus [3]
[4]
[5]. The peripheral activity of serotonin that acts at 5-HT1 and 5-HT2 receptors modifies vascular permeability and indirectly allows the flow of other
pronociceptive and proinflammatory factors, such as bradykinin and eicosanoids [34].
Bradykinins participate in the genesis of local edema and act as important pain mediators
in the pathophysiological process of Bothrops snakebite accidents [35]. In the present study, to reproduce the effects of bradykinin, the animals were
first intraperitoneally pretreated with 5 mg/kg captopril 30 min before the injection
of bradykinin to inhibit the activity of the angiotensin-converting enzyme, which
is involved in the biological degradation of bradykinin and blocked by Bothrops toxins [35]. Thus, the bradykinin injection resulted in edema formation, which peaked at 10 min
and was attenuated (p<0.05) by VCO treatment ([Fig. 4c]).
The participation of bradykinin in the process of BjcuV-induced edema formation is
still uncertain. Wanderley et al. [7] reported that pretreatment with the bradykinin receptor antagonist HOE140 did not
prevent edema that was induced by BjcuV. Rioli et al. [6] reported the presence of a bradykinin-potentiating peptide in B. jararacussu toxin. Such compounds increase the recruitment and adherence of leukocytes and vasodilation.
In the present study, substance P induced paw edema, and this effect was not prevented
by VCO pretreatment (p>0.05; [Fig. 4d]). The desensitization of afferent C-fibers by capsaicin did not affect the formation
of edema or migration of neutrophils to the site of injury that was caused by BjcuV
[7]. Thus, we speculate that substance P does not participate in the inflammatory response
that is induced by BjcuV. Our results indicate that VCO does not modulate substance
P in this experimental mouse model of inflammation.
According to Zakaria et al. [19], VCO inhibits phlogistic mediators, such as autacoids. Prostaglandins, unlike many
autacoids, are not stored in vesicles or other organic compartments, and they require
synthesis that depends on the enzymatic activity of PLA2 that is present in phospholipids within cell membranes. The product of this reaction
is arachidonic acid, which is then acted upon by cyclooxygenase (COX), producing the
final synthesis of prostaglandins. BjcuV increases the activity of COX2, and prostaglandins are major inflammatory mediators that are involved in paw edema
that is induced by BjcuV in mice [7]. We further investigated the effect of VCO on inflammation that was elicited by
exogenous prostaglandin E2. Pretreatment with VCO did not prevent (p>0.05) edema that was triggered by prostaglandin
E2 ([Fig. 4e]). We cannot exclude the possibility that the anti-inflammatory effect of VCO occurs
through the attenuation of COX2 activation that is elicited by BjcuV and not through the direct blockade of prostaglandin
receptors.
Notably, BjcuV stimulates the migration of neutrophils to the paw in mice [7]. In a previous study, an aqueous crude extract of C. nucifera inhibited the inflammatory process that was induced by a subcutaneous injection of
carrageenan by reducing leukocytes and protein extravasation [21]. In the present study, carrageenan increased the number of leukocytes in the peritoneal
exudate (7.74±0.23×106 cells/ml vs. 14.23±1.85×106 cells/ml), which was prevented by 200 mg VCO (6.57±0.5×106 cells/ml) and dexamethasone (6.20±0.68×106 cells/ml). These results suggest that VCO exerts anti-inflammatory effects at least
partially through interactions with several of these pathways that are involved in
leukocyte migration. Including the VCO to neutralize reactive oxygen species (ROS)
that are produced by neutrophils in the inflammatory process [21]
[26].
Intraplantar injections of B. jararaca venom caused hyperalgesia in rodents [36]
[37]. Crude extracts and VCO of C. nucifera have been reported to have antinociceptive effects in different rodent models of
pain [17]
[18]
[19]
[20]
[21]. We investigated the possible analgesic effects of VCO on hyperalgesia that was
induced by BjcuV. Virgin coconut oil at a dose of 200 mg significantly decreased (p<0.05) mechanical hypernociception that was induced by BjcuV 2 h after pain stimulation,
and this analgesic effect persisted throughout the study. The positive control tramadol
significantly decreased (p<0.05) mechanical hypernociception 1, 2, 4, and 5 h after the BjcuV injection ([Fig. 5]).
Fig. 5 Antinociceptive effect of virgin coconut oil on BjcuV-induced mechanical hyperalgesia
in mice. The data represent variations in mechanical hypernociception that was evaluated
for 5 h in mice that were orally treated with 0.2 ml of 0.15 M NaCl and then received
an intraplantar injection of saline (30 µl, negative control) and in mice that were
treated with 0.2 ml of 0.15 M NaCl (p.o., control), virgin coconut oil (VCO; 200 mg,
p.o.), or tramadol (40 mg/kg, i.p., positive control) and then received an intraplantar
injection of BjcuV (8 μg/paw) 60 min later. Other animals received VCO (p.o.) and
then BjcuV, followed by naloxone. The data are expressed as the mean±SEM from 5 animals
per group. *P<0.05, vs. negative control group; # p<0.05, vs. control group; Ψ p<0.05, vs. VCO group.
To elucidate the mechanism of the antinociceptive effect of VCO, the animals were
pretreated with the opioid receptor antagonist naloxone. [Fig. 5] shows that naloxone blunted the antinociceptive effect of VCO on BjcuV-induced mechanical
hypernociception 3 h after the BjcuV injection. A previous study also reported that
the analgesic effects of C. nucifera occurred through opioid receptors [17].
In summary, in the present study VCO decreased paw edema that was induced by BjcuV
and inflammatory mediators, including histamine, serotonin, and bradykinin. Virgin
coconut oil also inhibited leukocyte migration to the inflammatory focus and decreased
BjcuV-induced mechanical hypernociception. The antinociceptive effects of VCO appeared
to occur through opioid receptors. The present results support the possible therapeutic
use of coconut products, which have been used a nutraceutical products in animals
and humans for the treatment of inflammatory diseases.
Materials and Methods
Drugs and venom
Virgin coconut oil (Batch no. 91026716) was purchased from Copra Sul Natural Products.
It was obtained from the fruit endosperm of C. nucifera. Lyophilized BjcuV was obtained from the Butantan Institute, São Paulo, Brazil. The
venom was maintained at 20°C and diluted in 0.9% sterile saline. Dexamethasone (Decadron®, batch no. 1113428) was purchased from Aché Pharmaceutical Laboratories SA. Carrageenan,
histamine, serotonin, bradykinin, substance P, and prostaglandin E2 were purchased from Sigma. Tramadol (purity 99.7%, batch no. AW 022/14) was obtained
from Hipolabor. Naloxone (purity>95%, batch no. 08129231) was obtained from Cristália.
Nuclear magnetic resonance spectra
1H- and 13C-NMR spectra were acquired in CDCl3 at 301 K using a BrukerAVANCE III 400 NMR spectrometer that operated at 9.4 Tesla,
observing 1H and 13C at 400 and 100 MHz, respectively. The spectrometer was equipped with a 5-mm multinuclear
inverse detection probe with a z-gradient. All of the 1H- and 13C-NMR chemical shifts are presented in ppm and were related to the TMS signal at 0.00 ppm
as an internal reference. The coupling constants (J) are presented in Hz.
Animals
Female Swiss mice (n=125; 25–30 g body weight) were housed at a temperature of 25°C±2°C
under a 12 h/12 h light/dark cycle (lights on at 6:00 AM) with food and water available
ad libitum (Purina Lab). All experiments were performed in accordance with the Guide for the
Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD, USA)
and were approved by the Ethics Committee in Research of the Federal University of
São Francisco Valley (protocol no. 12081038).
Effect of virgin coconut oil on paw edema induced by B. jararacussu venom
To investigate the local effects of BjcuV, the mice were divided into groups of 5
animals each. They received injections of the following in the subplantar region of
the right hindpaw: 30 µl of 0.15 M NaCl (control group) or BjcuV (8 µg/paw) plus oral
treatment with 100, 200, or 400 mg VCO in oil solution or 0.2 ml of 0.15 M NaCl. The
VCO was weighed, and respective volumes were administered by gavage 1 h before the
injection of BjcuV. In other experimental groups, dexamethasone (1 mg/kg, positive
control) was administered intraperitoneally 1 h before the injection of BjcuV.
Paw edema was measured by plethysmography (PanLab 7500 water plethysmometer) immediately
before (basal volume) and then hourly for 5 h after the BjcuV or saline injection.
The results are expressed as the difference between the final and basal paw volumes
(% variation of paw volume) [38].
Effect of virgin coconut oil on paw edema induced by different phlogistic agents
To investigate the possible mechanisms that are involved in the anti-inflammatory
activity of VCO, additional groups of 5 animals each were orally pretreated with 0.2 ml
of 0.15 M NaCl (control) or 200 mg VCO. One hour after pretreatment, paw edema was
induced by an intraplantar injection of serotonin (300 ng/paw), histamine (100 μg/paw),
prostaglandin E2 (30 ng/paw), substance P (800 pg/paw), or bradykinin (320 pg/paw) in the right hindpaw
[34]. Paw volume was measured immediately before (basal volume) and 30, 60, 90, and 120 min
after the histamine, serotonin, prostaglandin E2, and substance P injections. For bradykinin, paw edema was evaluated 10, 20, 30,
60, 90, and 120 min after the injection. The edema response was measure as described
previously [38].
Effect of virgin coconut oil on carrageenan-induced peritonitis
To determine neutrophil migration to the peritoneal cavity, the mice were treated
with 0.2 ml of 0.15 M NaCl solution (p.o.), 200 mg VCO (p.o.), or 1 mg/kg dexamethasone
(i.p.). Each group consisted of 5 mice each. One hour later, 250 μl of carrageenan
was administered (500 μg/cavity, i.p.). The mice were euthanized 4 h later, and the
peritoneal cavity was washed with 1.5 ml of heparinized phosphate-buffered saline
to harvest peritoneal cells. The recovered volumes were similar in all experimental
groups and equivalent to ~95% of the injected volume. Total cell counts were performed
in a Neubauer chamber. The results are presented as the total number of leucocytes
per milliliter of peritoneal exudate as previously described [39].
Effect of virgin coconut oil on B. jararacussu venom-induced mechanical hypernociception
The animals were first fasted for 18 h and then orally treated with 0.2 ml of 0.15 M
NaCl (p.o., negative control), 200 mg VCO (p.o.), or 40 mg/kg tramadol (i.p., positive
control). One hour later, they received an intraplantar injection of 30 µl of 0.15 M
NaCl or BjcuV (8 µg/paw) in the right hindpaw. Mechanical hypernociception was then
assessed for 5 h. The mechanical nociceptive threshold was assessed by stimulating
the hindpaws with a pressure meter that consisted of a handheld force transducer that
was fitted with a 0.5 mm2 polypropylene tip (electronic von Frey Digital Analgesymeter; Insight Instruments).
In a quiet room, the mice were placed in acrylic cages (12 cm×20 cm×17 cm) with wire
grid floors 1 h before the test. A tilted mirror was placed under the grid to provide
a clear view of the hindpaw. The investigator was trained to apply the tip perpendicularly
to the central area of the hindpaw using a gradual increase in pressure. The stimulus
was discontinued and its intensity recorded when the paw was withdrawn. The end-point
was characterized by removal of the paw in a clear flinch response after paw withdrawal.
The difference in mechanical nociceptive thresholds before and after the BjcuV injection
was calculated [40]. To assess the possible analgesic mechanism of action of VCO, the animals were treated
with 1 mg/kg naloxone (i.p). 15 min later, they received 200 mg VCO (p.o.) to evaluate
mechanical hypernociception that was induced by BjcuV.
Statistical analysis
The data are expressed as mean±standard error of the mean (SEM). The data were analyzed
using 2-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls test,
as appropriate. Values of p<0.05 were considered statistically significant.
