Anti-nociceptive Activity of Quebracho tannin Extract on Pain Induced by Formalin and Writhing Tests in Mice

Background: Based on positive role of the tannins for pain relief, there is no report for possible antinociceptive activity of the Quebracho tannin . Objectives: This study aimed to determine the anti-nociceptive activity of the Quebracho tannin extract (QTE) on pain in mice. Materials and Methods: For this purpose, 340 mice were used for formalin and writhing tests each including 4 experiments with 4 sub-groups. In experiment 1, mice were injected with saline, QTE (100 mg/kg), QTE (200 mg/kg), QTE (400 mg/kg), and morphine (5 mg/kg). In the second experiment, injections included saline, QTE (400 mg/kg), naloxone (2 mg/kg), and QTE + naloxone. Experiments 3 and 4 were similar to experiment 2, except that mice injected were with N G -nitro arginine methyl ester (L-NAME, 10 mg/kg) and cyproheptadine (4 mg/kg) instead of naloxone. Then, formalin (1%) was injected, and time spent for licking the injected paw was recorded until 30 minutes following injection in the first and second phases. Finally, injections in 4 experiment groups were the same, and animals were intraperitoneally injected with acetic acid, and contractions were recorded in the writhing test category. Results: According to the results, QTE (100, 200, and 400 mg/kg) decreased pain in the injected paw ( P = 0.001) and inhibited the pain response by 59.37% ( P = 0.001). Moreover, the injection of naloxone + QTE significantly decreased pain in the injected paw ( P = 0.021). Eventually, the injection of the L-NAME + QTE significantly reduced the anti-nociception effect of the QTE on the formalin test ( P = 0.031) and writhing contractions (55.75%, P = 0.033). Conclusion: These findings suggested anti-nociceptive properties of the QTE mediated by opioidergic and nitrergic systems.

abundant (15%-25% CT), and commercial extracts have approximately 300 mg/g of CT. Extracts are typically taken from the bark and used for leather tanning (12). The Quebracho tannin extracts (QTE) are generally composed of 95% CT and 5% polysaccharides (13). QTE is a commercial source of CT that is widely applied for ruminant feeding to improve digestibility, nitrogen balance, energy partitioning, and milk production (14). It is reported that isolated tannin from Phyllanthus niruri L. has anti-hyperalgesic activity in writhing and formalin test, and this activity is mediated by glutamatergic receptors (15). Additionally, the hydroalcoholic extract of the Satureja khuzistanica Jamzad extract has antinociceptive activity in the formalin test similar to morphine (3 mg/kg), and tannins might be responsible for these anti-inflammatory and anti-nociceptive activities (16). It is suggested that tannic acid is valuable in the treatment of inflammatory pain, osteoarthritis, rheumatic arthritis, and burn pain (17). According to reports, Acacia nilotica (18) and Acacia tortilis (19) have high levels of antioxidants that are useful as anti-inflammatory and anti-nociceptive treatments. Flavones have a beneficial role in numerous physiological for possessing various pharmacological actions. Previous literature revealed the anti-nociceptive and anti-inflammatory activities of many flavone derivatives (20). Considering the lack of any report on the anti-inflammatory and anti-nociceptive effects of the QTE, the current study sought to determine the antinociceptive effects of the QTE on inflammatory pain induced by formalin and writhing tests.

Animals
Overall, 340 adult male NMRI mice (25-30 g) were kept 8-10 mice per cage under standard laboratory conditions (23 ± 1°C ambient temperature, a 12-hour dark/light cycle, and 55%-56% relative humidity) at the Department of Veterinary Medicine, Science and Research Branch, Islamic Azad University, Tehran, Iran. Mice provided chow pellets and water. After 7 days of acclimatization, formalin and writhing tests were employed to determine the antinociceptive effect of the QTE. Accordingly, animals were randomly allocated to 2 classes, including 4 experiments with 4 sub-groups each containing 10 animals (21).

Extraction and Drugs
The Quebracho tannin bark was purchased from the local market, and taxonomic identification of plants was conducted at Razi Central Laboratory, Science and Research Branch, Islamic Azad University, Tehran, Iran. The gathered samples were dried at ambient temperature and under sunlight for 5 days. Thirty grams of the plant material was soaked with ethanol (150 mL) for 24 hours at laboratory temperature. Then, the sample was filtered twice using Whatman filter paper until obtaining a clear extract. The obtained filtrates were combined and then evaporated until dryness using a rotary evaporator at 40°C. The extracts were stored in sterile sample tubes at -20°C (22). Morphine, naloxone (nonselective opioid receptor antagonist), nitric oxide inhibitor (L-NAME), and cyproheptadine (serotonergic receptor antagonist) were purchased from Sigma (St. Louis, MO, USA) and, formalin was provided from Merck (Darmstadt, Germany) according to previous research (1).

Writhing Test
In experiment one, mice were intraperitoneally injected with saline (10 mL/kg), QTE (100 mg/kg), QTE (200 mg/kg), QTE (400 mg/kg), and morphine (5 mg/kg). After 30 minutes, acetic acid (10 mL/kg of 0.6%) was intraperitoneally injected, and the writhing test was determined for thirty minutes ( Table 1). The antinociceptive activity was obtained as the percentage of the inhibition of writhing based on the ratio of: (control mean -treatment mean) × 100/control mean (23). Writhing is known as abdominal contractions greater than the abdominal region (25). Then, the QTE at effective dosage was applied for the subsequent experiments. In experiment two, mice were intraperitoneally injected with saline (10 mL/kg), QTE (400 mg/kg), naloxone (2 mg/kg), and QTE + naloxone. In the groups with 2 injections, first, mice were injected with an antagonist, and QTE (10 mg/kg) was injected and the writhing test was conducted ( Table 2) after 15 minutes. In the third experiment, the animals received saline (10 mL/kg), QTE (400 mg/kg), L-NAME (10 mg/ kg), and QTE + L-NAME (Table 3). In experiment four, the mice were intraperitoneally injected with saline (10 mL/kg), QTE (400 mg/kg), cyproheptadine (4 mg/kg), and QTE + cyproheptadine ( Table 4). The dose of the drugs was in accord with the protocols of previous studies (1,23,24). Finally, SPSS 22 was used for data analysis using one-way analysis of variance (ANOVA). The obtained data were presented as the mean ± standard error, and Tukey post-hoc test was applied for the main effect by ANOVA (P < 0.05).

Anti-nociceptive Effects of the QTE
Data on the anti-nociceptive effects of the QTE using formalin and writhing tests are presented in Figure 1 and Table 1. Based on the results, the QTE decreased pain in       the treatment groups compared to control mice (P = 0.001). Further, morphine significantly reduced the licking and biting time of the injected paw (pain response) in phases 1 and 2 and the number of writhing counts, respectively (P = 0.001).

Role of Naloxone on the Anti-nociceptive Activity of the QTE
QTE (400 mg/kg) induced a significant decline in the pain response in phases 1 and 2 compared to the control group (P = 0.001) and inhibition pain response of 59.37% (P = 0.001). Naloxone (2 mg/kg had no significant effect on formalin and writhing tests (P = 0.453). The injection of the naloxone + QTE amplified the pain response in comparison to the QTE group (P = 0.047, Figure 2). Based on the findings, naloxone + QTE lessened pain response in the treatment groups in comparison to the control mice (P = 0.021). These results suggested that the blockade of the opioid receptor with the opioid antagonist has regulated the effects of the QTE. Therefore, the antinociceptive response of the QTE is probably mediated by these receptors. Table 3 and Figure 3, the injection of the QTE (400 mg/kg) significantly reduced the pain response in phases 1 and 2 (P = 0.001) and and writhing movements by 22.17% (P = 0.032). L-NAME (10 mg/kg) had no obvious change, while the co-administration of the L-NAME + QTE suppressed the effect of the QTE (P = 0.031). Additionally, L-NAME + QTE reduced the inhibition number of writhing movements (55.75%, P = 0.033). It seems that the anti-nociceptive response of the QTE is mediated via nitrergic receptors.

Role of Cyproheptadine on the Anti-nociceptive Activity of the QTE
QTE (400 mg/kg) lessened pain response (P = 0.001) and inhibition in pain response of 69.85 % compared to the control group (P = 0.024). However, cyproheptadine (4 mg/ kg) had no significant anti-nociception effect (P = 0.215, Table 4 and Figure 4). Eventually, the injection of cyproheptadine + QTE exerted had no significant effect on QTE induced nociception and and the inhibition number of writhing movements (68.43%, Table 4, P = 0.754).

Discussion
So far, thousands of studies have focused on finding possible anti-nociceptive and anti-inflammatory activities of medicinal plants. However, there is rising attention in the use of these plants as therapeutic agents (1). Based on the literature, there are novel findings on the interaction of the anti-nociceptive effect of the QTE. The formalin injection in the paw evokes biphasic peripheral pain. The primary phase happens because of the direct stimulation of nociceptors, and the following phase is due to inflammatory pain (26). Based on the obtained data, the QTE (100, 200, and 400 mg/kg) decreased the time spend for licking and biting the injected paw in the formalin test. Furthermore, the QTE (100, 200, and 400 mg/kg) inhibited pain response (37.73, 40.43, and 59.37%, respectively) in the writhing test. A variety of ion channels such as voltage-gated Na + , K + , or Ca 2+ are responsible for pain transmission. The analgesic effect of tannic acid might be related to its modulatory role on ion channel functions (17). For instance, it is assumed that the anti-nociceptive properties of the tannic acid in inflammatory pain are mediated by the activation of the K + and the inhibition of Na + channels and relieving inflammatory mediators such as bradykinin (17). In addition, tannic acid and gallotannins, by inhibiting Cl − secretion, lead to arterial smooth muscle relaxation, which has a potential molecular basis for cardioprotective benefits. However, based on the limitations of the current study, the researchers were unable to determine the effect of the QTE on K + and Na + channels. Perhaps, QTE-induced anti-nociception acts by this mechanism. These therapeutic potentials of the QTE have not been fully elicited and are worthy to be investigated by future studies.
The role of neurotransmitters such as opioidergic, serotonergic, and adrenergic systems in the modulation of nociceptive is well-documented in numerous reports. It is important to determine the possibility of these systems on the nociceptive properties of medical plants as new drugs and medications (27). The results revealed that the injection of naloxone + QTE decreased pain. Naloxone as a nonselective antagonist opioid receptor inhibited the anti-nociceptive effects of the QTE. Perhaps, the antinociceptive response of the QTE is controlled by opioid receptors. All opioid sub-types of receptors are identified in the central nervous system and peripheral tissues are responsible in pain and analgesia (1). However, the researchers of this study could not determine the direct interaction of the QTE with different opioid receptors given the limitations of the present study.
Moreover, the co-administration of L-NAME + QTE decreased the anti-nociception effect of the QTE. However, the co-injection of cyproheptadine + QTE could not significantly affect the anti-nociception effect of the QTE. The inflammatory pain is mediated by synergism in inflammatory mediators such as bradykinin, serotonin, histamine, prostaglandins, and nitric oxide (NO). The antioxidant activity of tannin-rich plants is responsible for the direct inhibition of NO production. The NO pathway has an essential role in the carrageenan-induced inflammatory response and paw edema test (28). Further, NO significantly contributes to the acute and chronic phases of nociception in central and peripheral nervous systems (24). The sub-plantar injection of formalin increased the NO level in the injected site and pretreatment with L-NAME reduced pain in mice (29). Based on the findings of other studies, pretreatment with L-NAME inhibited the anti-nociceptive effect of Melilotus officinalis (Linn.) and Sisyrinchium micranthum extracts in the formalin test (29,30), which is in agreement with previous reports() . Although the direct mechanism for this finding is not determined, NO production in the spinal cord decreases the anti-nociceptive effect of the extract (27). It is assumed that the QTE has anti-nociceptive activity, and this role is mediated via the nitrergic system.