Effectiveness of red propolis and Mikania glomerata in bitches ́ s surgical analgesia

The aim of this research was to evaluate the analgesia provided by red propolis and Mikania glomerata oral administration in 40 female dogs submitted to ovariohysterectomy (OH) compared to standard treatment with no steroidal anti-inflammatory ketoprofen. Through of a doble-blind and randomized study, the animals were divided in four different groups with 10 animals of treatments: control treatment with ketofen (CT), propolis treatment (PT), Mikania glomerata treatment (MT), and propolis-Mikania glomerata treatment (PGT). All pacients received one of these treatments two hours before OH and the pain evaluation was performed 1 (T1), 6 (T6), 12 (T12), and 24 hours (T24) after OH using University of Melbourne Pain Scale and the Glasgow Composite Measure Pain Scale. Glucose levels were also measured at the same times, except at 6 hours after OH (T6). All treatments achieved similar and satisfactory analgesia. During the experiment, only three animals were rescued, two belonging to the PT and another of the MT. Considering the lower rate of complications and no rescue necessity of in PGT and CT treatments, they were considered the most effective and safer. The combined treatment with propolis and Mikania glomerata could be a promising alternative method for OH surgery analgesia in bitches.


Introduction
In the last decade, the pain measurement and control in pets has been the subject of increasing concern and more researches due the improvement of pain pathophysiology scientific understanding and the relationship between pets and their owners.
Ovariohysterectomy (OH) is among the most frequently performed and under-monitored postoperative pain, although it is presumed to cause moderate to severe abdominal pain (Slingsby et al, 2011;Morgaz et al, 2013).
As a result of it being impossible to quantify pain intensity in nonhuman animals due to their inability to rationalize and express their perceptions verbally, pain scales based on the analysis of physiological and behavioral parameters are used and extremely important in veterinary medicine (Zanuzzo, 2014). In dogs, the pain scale of the University of Melbourne (Muir and Gaynor, 2009) and the Glasgow composite measure pain scale (Murrell et al, 2008) are the most commonly used.
The efficacy of analgesic drugs is frequently investigated for use in the pet veterinary market, and many of non-steroidal anti-inflammatory drugs have been indicated for use in bitches submitted to OH, including ketoprofen, vedaprofen, meloxicam, and dipyrone (Selmi et al, 2009;Morgaz et al, 2013;Zanuzzo et al, 2015). However, the use of natural products as analgesics is still relatively little exploredr. This is especially surprising considering that many potential natural products are of national and regional importance to Brazil, such as Mikania glomerata and red propolis from Alagoas, a protected and endemic species of this region designated recently by the National Institute of Industrial Property (INPI) (under registration number IG201101) (INPI -Brazil, 2012).
Ketoprofen is a non-steroidal anti-inflammatory drug with anti-inflammatory, antipyretic, and strong analgesic properties, which is known to be an effective treatment for pain due OH (Alves et al, 2001;Fantoni et al, 2015) that minimizes hyperglycemia and cortisol release in response to surgical pain in the 24-hour period containing and following OH surgery (Lacerda and Nunes, 2008).
The leaves of Mikania glomerata, popularly known as guaco, have been used for centuries in traditional and popular medicine for their tonic, bronchodilatory, anti-flu, antipyretic, and appetite-stimulating actions. According to Castro et al. (2006), they are extremely beneficial in the treatment of asthma, which suggests that guaco also has antiinflammatory and antiallergic action, in addition to its known ability to block calcium channels (Czelusniak et al, 2012). Development, v. 9, n. 10, e4879108733. 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8733 5 Propolis is widely used in Brazilian medicine and is marketed in many ways. Its main components are isoflavonoids and flavonoids (Bueno-Silva et al, 2015), the latter of which interact with different physiological processes, such as the action and absorption of vitamins, healing processes, antioxidant pathways, and antimicrobial and modulating functions of the immune system (). Additionally, Batista et al. (2015) verified an antinociceptive (i.e. painrelieving) action similar to dipirone in mice.
The aim of this study was to evaluate the analgesic action of Mikania glomerata and red propolis from Alagoas (Brazil), in single and combinated applications, in bitches submitted to OH. The analgesic effects of these compounds were compared with ketoprofen, using the University of Melbourne pain scale (UMPS) and the Glasgow composite measure pain scale (GCMPS) to assess pain levels in patients.

Materials and Methods
The experiment has a quali-quantitative nature and was approved by Animals Ethics Committee on the use of animals/Federal University of Alagoas (CEUA/UFAL), under approval number 039/2016. Forty healthy bitches were studied, with a mean ± standard deviation (SD) age of 2.77 ± 1.38 years old and mean body mass of 13.15 ± 4.88 kg. All were subjected to clinical examinations to evaluate their heart rates (HR), respiratory rates (RF), rectal temperatures (T, in ºC), detailed cardiac and pulmonary auscultations, mucosa, appetite disturbance, weight, and complete blood cell counts. All animals used in this study had normal values for the species of all of these measured parameters (Muir et al, 2013), and were thus considered healthy.
Adaptation of the animals to recovery conditions occurred over a minimum period of 72 hours, during which they were kept in individual cages 2 m 2 in floor area and fed with commercial dog food and provided with water ad libitum. Walks were conducted twice daily for 15 minutes each throughout the experiment. Complete blood cell counts and blood glucose tests were performed 24 hours prior to the experiment.
After blood sample through the cephalic vein, the animals were submitted to trichotomy in the surgical area. The experimental procedures were performed at the Laboratory of Innovation in Surgery, Hemotherapy, and Veterinary Cellular Therapies of the Federal University of Alagoas (LABINOVET-UFAL). After 12 and 4 hours of food and water fast, respectively, before surgery. Research, Society and Development, v. 9, n. 10, e4879108733. 2020 (CC BY 4 All procedures were performed by the same surgeon, using the same surgical technique.
Throughout the surgical procedure, heart rate, respiratory rate, and rectal temperature were monitored. At the end of the procedure, all animals received a single dose of 30000 IU/kg benzathine penicillin intramuscularly.
Pain evaluation was performed 1 (T1), 6 (T6), 12 (T12), and 24 hours (T24) after OH using both the University of Melbourne Pain Scale (UMPS) (Muir and Gaynor, 2009) and the Glasgow Composite Measure Pain Scale (GCMPS) (Murrell et al, 2008). Glucose levels were also measured at the same times, except at 6 hours after OH (T6). After collection, blood samples for glucose measurement were processed within less than 20 minutes.
In the cases of pain positivity, analgesic rescues of bitches with tramadol were performed intramuscularly at the dose of 2 mg/kg, as this has been shown to be an effective analgesic for use after OH (Mastrocinque and Fantoni, 2003;Morgaz et al, 2013), in addition to oral administration of ketoprofen at a dose of 2 mg/kg for another 4 days. The animals were evaluated for 24 hours because this was the period over which ketoprofen has been previously shown to have effective analgesic action (Selmi et al, 2009); the ketoprofen-treated CT animals were thus used as the control group for pain evaluation.
Research, Society and Development, v. 9, n. 10, e4879108733. 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8733 7 Statistical analyses were performed using repeated measures ANOVAs in time and a significance level of 5% (p ≤ 0.05) of the clinical parameters used in the pain scales (heart rate, respiratory rate, and temperature) and glucose measurements, which were then followed by multiple comparisons using LSD test. The non-parametric Kruskal-wallis test was used for analyses of the pain scale scores. All statistical analyses were performed using SPSS® software 17.0. A minimum confidence level of 95% (p < 0.05) was considered in all statistical analyses.

Results and Discussions
The surgical data collected shows that the mean ± SD length of the incision was 4 ± 1.2 cm, and the mean surgery time was 20 ± 4.62 minutes, requiring no additional anesthetic doses in all experimental dogs.
In terms of the clinical parameters evaluated (heart rate, respiratory rate, and temperature) and glucose, there were no statistically significant differences among treatments.
The most significant differences (p < 0.05) were only found among times in all treatments (Tab.  Table 1 shows the data before and after the surgery where was a visible increase in HR between T0 (where the animals had their normal parameters) and T1. Between T6 and T12, the Development, v. 9, n. 10, e4879108733. 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8733 8 averages still differed significantly from baseline values, and baseline values were not restored until the T24 evaluation. It is also notable that regarding RR, it was noticed that there was no difference between T0 and T1, but there was a decrease at T6. There was a decrease in T°C by T1 At T0, glucose levels in all bitches were within the normal range for the species, which is from 77 mg/dL to 126 mg/dL (Muir et al, 2013). However, at T1 a significant increase was observed (p = 0.042).
For pain scores evaluated using the UMPS, a intense decrease in the score in all 4 treatments between T1 and the remaining times (T6, T12, and T24) was observed (Figure 1).  Research, Society and Development, v. 9, n. 10, e4879108733. 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8733 9 There was a significant difference in the magnitude of the decrease in the UMPS score at T6 between the GT and PGT (p = 0.001). The decrease in the UMPS score at T6 was also significantly less for the GT than for either CT (p = 0.007) or PT (p = 0.051) in T6.
During the experiment, only three animals were rescued, two belonging to the PT and one to the GT. The UMPS scores for the two animals rescued in the PT were 7 and 8 at the moment of the rescue at T2, and for the animal rescued in the GT the score was 10 at T6.
Regarding the GCMPS, there were no significant differences in the pain scores recorded among any treatments or evaluation times based on this scale (Figure 2).  In the GT, it was also worth noting that ten bitches presented nystagmus and tremors at T1, and six (60%) presented greater bleeding of the surgical wound than others. Development, v. 9, n. 10, e4879108733. 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8733 All phases of the experiments were performed in a fluid and efficient way, without any complications in the pre-, trans-, and postoperative periods, except for the 3 rescues performed.
In terms of the variables of the clinical parameters evaluated all treatments behaved in a similar way.
The increase in HR between T0 and T1 may have been associated with the effects of ketamine because according to Souza et al. (2002) it elevates heart rate and blood pressure as a result of stimulation of the central nervous system. The decrease in RR at T6 may be related to increased manipulation stress by this point. Acepromazine, on the other hand, can cause hypotension and hypothermia (Almeida et al, 2000), which explains the T decrease observed at T1.
The glucose increase observed at T1 was associated with the manipulation of the animals and anaesthetic recovery, as corroborated by observations by Guimarães et al. (2007).
According to Lacerda and Nunes (2008), a neuroendocrine and metabolic response to the painful stimulus during surgery begins immediately and lasts for 24 to 48 hours, which increases the release of cortisol, adrenocorticotropic hormone, and glucagon, in addition to causing an inhibition of insulin secretion that may cause hyperglycemia.
The difference in magnitude in the UMPS score at T6 between GT & PGT, GT & CT and GT & PT indicated that better analgesia was achieved with PGT than in GT. The results also indicates that among the 4 treatments tested, the least effective at this time point was the GT one.
Although none of the 3 rescued animals scored 13, the minimum score that the literature defines are requiring rescue, in this study the surgical team decided to perform rescues if the UMPS score reached levels considered to represent moderate pain by Coutinho (2012), which were those between 6 and 17. Pohl et al. (2011) andComassetto et al. (2017) correlated the UMPS with the Visual Analog Scale in bitches submitted to OH, and agreed that a UMPS score between 6 and 7 requires the administration of analgesic medication, which corroborates what was done in the present study. It should also be noted that the UMPS may have a somewhat subjective score in the period of anesthetic recovery, as seen at T1 (Figure 1), since the parameters evaluated are behavioral and physiological but such parameters can be altered by the use of anesthetics.
The GCMPS considers moderate pain to occur at scores between 6 and 24, but it assesses pain scores based only on behavioral evaluations (Coutinho, 2012); therefore, the analgesic recovery period may mask the actual pain signals from being detected by the observer. All mean values for the animals evaluated in this study were thus very low according to this scale.
However, we agree with this scale for the combined scores of two of the three rescued animals, one of which (in PT) had a score of 6 at T1 and another (in GT) of which had a score of 10 at T6 according to this scale; these rescued animals were thus in moderate pain based on the GCMPS as well as the UMPS.
The presence of nystagmus and tremors at T1 and greater bleeding of the surgical wound are a fact that can be explained by the presence of coumarins in guaco, which inhibit the formation of thromboxane in platelets (Czelusniak et al, 2012). In addition to these signs, the rescued GT animal presented: vocalization, reaction to palpation, and presence of edema in the surgical wound. It is noteworthy that the dose of guaco used was experimental, based only on the dose of propolis that was previously shown to be able to cause analgesia in mice (Batista et al, 2015). Such a dose of guaco appears to have caused adverse effects in the present study, or may even have interacted with another drug used during the anesthetic procedure or for antimicrobial prophylaxis. This demonstrated that further studies are needed to elucidate the function, drug interactions, and optimal dosing of this herbal medicine.
Comparing the analgesic action of the groups tested in relation to CT, and considering that ketoprofen decreases the action of prostaglandins and provides analgesic effects for a period of 24 hours, it was possible to affirm in the present study that PT and GT were effective analgesics in 80% and 90% of the bitches treated, respectively. However, PGT presented similar results to CT, with effectiveness in 100% of the bitches submitted to OH.

Conclusion
It was concluded that the four treatments had similarly satisfactory results in the postoperative analgesia of bitches submitted to OH. However, considering the number of complications and analgesic rescues that occurred when using GT, this was concluded to be the least Satisfactory treatment, while PGT and CT were both found to be more effective and safer.
To former researchers the treatment could be tested in another type of surgeries, using the Guaco and the Red propolis treatment with the same objectives as the pain control. The treatment could be tested in non-invasive clinical trials comparing the effectiveness of the red propolis and the guaco with another AINE's as the control group.