Antinociceptive activity of the Caesalpinia eriostachys Benth. ethanolic extract, fractions, and isolated compounds in mice

Abstract Caesalpinia eriostachys Benth. (CE) is native to the Mexico and multiple effects have been observed from several plants belonging to the same family. CE was subjected to extraction with 95% ethanol, and the components were isolated through column chromatography. The structure of the compound was elucidated based on nuclear magnetic resonance (NMR) spectral data, electron ionization–mass (EI‐MS) spectroscopy, and liquid chromatography–mass (LC‐MS) spectroscopy. In vivo antinociceptive studies were conducted using writhing, 5% formalin, tail‐flick, hot‐plate, and von Frey filament tests. The ethanolic extract showed a significant effect in the acetic acid‐induced pain model and nociceptive behavior in the formalin model (second phase). In hot‐plate test and tail‐flick test, the results showed no difference compared to the control group. The results suggest that the ethanolic extract may act peripherally to reduce pain. In the streptozotocin (STZ)‐induced pain model, the ethanolic extract showed significant effect in the von Frey test model. The n‐Hex (Hexane) and MC (Methylene chloride) fractions and isolated compounds, ellagic acid and agathisflavone, showed increased effect. Based on these results, we confirmed that the CE ethanolic extract and their compounds, ellagic acid and agathisflavone, have antinociceptive effect on diabetes mellitus‐induced pain. Furthermore, the results of this study might be valuable for identifying compounds with antinociceptive activity from natural products.


| INTRODUC TI ON
Pain is a major health problem and one of the most prevalent conditions limiting productivity and diminishing the quality of life (Higgs et al., 2013). Pain has a physiological component called nociception, which is the process by which intense thermal, mechanical, or chemical stimuli are detected by a subpopulation called nociceptors (Regalado et al., 2017). Pain is a symptom associated with several disorders, including inflammation, and it requires analgesic treatment (Fongang et al., 2017). Nonsteroidal anti-inflammatory drugs and opioids are widely used in pain therapy, but prolonged use of these drugs results in side effects, such as gastrointestinal damage, cardiovascular complications, tolerance, and physical and psychological dependence (Saldanha et al., 2017). For these reasons, recently, there is growing interest in the use of herbal medicinal products and the search for sources of new drugs (Fongang et al., 2017). For a long time, medicinal plants have been traditionally used for relief from disease, and certain compounds derived from plants present significant analgesic properties (S. Gorzalczany et al., 2011). Caesalpinia species are distributed throughout the tropic and subtropics, and used folkloric medicine to treat various diseases (Chan et al., 2018). Caesalpinia eriostachys is native species included in the Caesalpinia genus (Fabaceae), commonly used in the west of Mexico as an ornamental tree and forage (Pamatz-Bolaños et al., 2018). Caesalpinia plants belonging to the Caesalpiniaceae family have shown various effects, including hepato-protective effect (Kumar et al., 2010), anti-inflammatory effect (Kale et al., 2010), and antitumor activity (Gupta et al., 2004).
Furthermore, some of the chemical constituents in the plants of the Caesalpinia family have also shown beneficial effects; flavones, isoflavones, and chalcone, isolated from Caesalpinia pulcherrima showed anti-inflammatory activity (Rao et al., 2005), and brazilin isolated from Caesalpinia sappan L. was responsible for antiinflammatory and wound-healing effects (Tewtrakul et al., 2015).
However, no previous studies have evaluated the antinociceptive potential of Caesalpinia eriostachys Benth. (CE). In this study, we confirmed the antinociceptive effects of the crude extract of CE and its individual compounds, indicating the potential for the application as new strategies for pain treatment.

| Extract and isolation
CE was subjected to extraction with 95% ethanol (EtOH) for 3 days at room temperature. The filtrate was concentrated to dryness in vacuo at 37°C. The major components from CE extract were isolated by column chromatography. The CE ethanolic extract was systemically partitioned into five fractions: hexane (n-Hex), methylene chloride (MC), ethyl acetate (EtOAc), butanol (n-BuOH), and water. Among the fractions, the n-Hex fraction was purified using Sephadex LH-20 (100% MeOH) to furnish 22 fractions: Fr.1-Fr.22.

| Experimental animals
Male ICR mice (6 weeks of age), weighing 25-30 g at the beginning of experiments (Koatech, Seoul, Korea), were used. The mice were housed five per one cage in a room that was maintained at 22 ± 1°C with an alternating 12-h light-dark cycles. Food and water were available ad libitum. The animals were allowed to adapt at least 2 h before testing and were only use one time.

| Streptozotocin (STZ)-induced diabetic mice
Diabetes was induced in mice via a single intraperitoneal injection of STZ (75 ~ 80 mg/kg in citrate buffer, pH 4.5). STZ was injected for 3 days. Normal groups received the buffer alone.
On the fourth day after STZ administration, glucose levels were measured using a glucometer (ACCU-CHEKPerforma, Roche Diagnostics, Mannheim, Germany). Animals with nonfasting blood glucose concentration above 250 mg/dl were considered diabetic and used in the current study. After one week, five mice each were treated with normal saline (5 ml/kg, b.w., p.o.), ethanolic extract

Acetic acid-induced writhing test
For the visceral pain model, 1% acetic acid was used to the mice model (Koster et al., 1959). Four groups of five mice each were pretreated orally (p.o.) with normal saline (5 ml/kg, b.w., p.o.) and ethanolic extract (5, 10, and 20 mg/kg, b.w., p.o.). About 30 min after the administration, 1% acetic acid was injected intraperitoneally (i.p.) to mice. The number of writhing responses was counted for 30 min and began 5 min after acetic acid injection.

Formalin test
For the formalin pain model (S. Hunskaar et al., 1985;Steinar Hunskaar & Hole, 1987), four groups of five mice each were treated orally with normal saline (5 ml/kg, b.w., p.o.) and ethanolic extract (5, 10, and 20 mg/kg, b.w., p.o.). After 30 min, 10 µl of 5% formalin was injected subcutaneously into the plantar surface of the left hind paw of each animal using a 50-µL Hamilton syringe. The time that the animal spent licking or biting its paws was measured during the first (0-5 min) and the second (20-40 min) phases of the test.

F I G U R E 1 Chemical structure of ellagic acid and agathisflavon
Tail-flick and hot-plate tests Antinociception was determined by the tail-flick (D'Amour & Smith, 1941) and the hot-plate paw-licking tests (Eddy & Leimbach, 1953). For the measurement of the tail-flick latency, mice were held with the tail positioned in the apparatus (EMDIE Instrument Co., Maidens, VA, USA, Model TF6), and the tail-flick response was elicited by applying radiant heat to the dorsal surface of the tail. The test was repeated three times to obtain the mean value of the tail-flick latency. The tail of the mouse not exposed to the heat for more than 20 s to avoid burns. Five mice each were

| Statistical analysis
All values are expressed as the mean ± SD. Comparison between groups was performed using Student's t-test. A p < .05 was considered statistically significant.

| Phytochemical analysis
HPLC analysis of the CE ethanolic extract showed the presence of the compounds ellagic acid (R T : 22.98 min) and agathisflavone (R T : 29.47 min). The fingerprint chromatogram is shown in Figure 2.

| Antinociceptive effect of the CE extract in the tail-flick and hot-plate tests
The result presented in Figure 3 shows that doses of the CE extract at 10, 20, 40, and 80 mg/kg were used for the hot-plate test and tail-flicking test. In the hot-plate test, all CE extract treated group showed similar antinociceptive behavior compared to the control group ( Figure 3a). Compared to the previous experiment, the data of tail-flicking test also showed same results (Figure 3b).

| Antinociceptive effect of CE extract in the acetic acid-induced abdominal writhing test and 5% formalin-induced nociception
The result presented in Figure 4a shows that the CE extract (doses of 5 mg/kg, 10 mg/kg, and 20 mg/kg) significantly inhibited the acetic acid-induced nociception compared to control group at doses of 10 and 20 mg/kg, respectively. As illustrated in Figure 4b, the CE extract at same doses (5 mg/kg, 10 mg/kg, and 20 mg/kg) did not show significant reduction in the nociceptive behavior time in the first phase. But, in the second phase, the CE extract reduced the nociceptive behavior time at same doses. Notably, significant reduction in nociceptive behavior time was observed in the second phase at the dose of 10 mg/kg and 20 mg/kg compared to that in the control group.

| Antinociceptive effect of extract, fractions, and compounds isolated from CE on mechanical pain thresholds in STZ-induced pain model
As shown in Figure 5, the extract of CE was potent enough to be highly effective in pain models. Among the concentrations tested, the group that were administered 20-80 mg/kg CE extract showed significantly lower nociception level than the control group at the

| DISCUSS ION
In the present study, we evaluated the antinociceptive effect of the CE ethanolic extract and two isolated compounds, ellagic acid and agathisflavone, for the development of new strategies for the treatment of pain.
Several studies were processed about compounds with antinociceptive effect in natural products, including Urtica circularis (S Gorzalczany et al., 2011), Bryophyllum pinnatum (Crassulaceae) (Ojewole, 2005), and Cenostigma macrophyllum (LEGUMINOSAE) (Alves et al., 2012). In case of mechanism of pain, AMPK activation signaling can effect to several pathologies including chronic neuropathic pain, and metformin directly decreases pain by decreasing the activity of mTORC1 and MAPK signaling in nociceptors (Baeza-Flores et al., 2020). The antinociceptive activities of the extract were evaluated using the acetic acid-induced writhing test, 5% formalininduced pain model, hot-plate test, and tail-flicking test. The hotplate method is suitable for the evaluation for central nociceptive activity (Sharma et al., 2019), and the tail-flicking test measures spinal nociceptive response latencies to a thermal stimulus such as a hot plate (Lima Cavendish et al., 2015). Both tests are simple and commonly used to study analgesic efficiency and less sensitive to non-opioid analgesics (Karna et al., 2019). In the hot-plate test, doses of 40 mg/kg and 80 mg/kg showed slightly increased antinociceptive behavior compared to the control, but not significantly different. The tail-flicking test also showed no significant difference in the treatment groups compared to the control group (p >.05). The acetic acid-induced writhing test is a visceral model for evaluating peripheral antinociceptive activity, and the pain in this model is generated via endogenous mediators (Wang et al., 2014). The CE extract showed a significant decrease in the number of writhing responses in the acetic acid-induced pain model at doses of 10 and 20 mg/kg, suggesting that the CE extract may act to reduce peripheral pain in mice. The formalin-induced pain test was used to evaluate neurogenic pain responses in mice; first phase has been considered to reflect direct chemical stimulation of C-fibers (neurogenic pain) and second phase is dependent on peripheral tissue inflammation (inflammatory pain) (Tjølsen et al., 1992). Additionally, the first phase is induced by the ion channel TRPA 1, and the second phase After evaluating the effect of the CE extract through four pain tests, we confirmed that treatment with the extract, fractions, and compounds of the CE extract reduced the pain threshold, as evaluated by the von Frey filament test in STZ-induced diabetic mice. Diabetes induced by STZ causes glial activation, glutamate toxicity, and neuronal death, which could induce necrotic and apoptotic cell death (Chiu et al., 2016). STZ-induced diabetes is a commonly used experimental model of neuropathic pain (Huang et al., 2016)  antinociceptive effect of ellagic acid, involving mediation by the opioidergic system and l-arginine-NO-cGMP-ATP sensitive K + channels pathway, and ellagic acid showed antinociceptive action in the acetic acid-induced mouse model of pain (Mansouri et al., 2015).
In the case of agathisflavone, a fraction of Cenostigma macrophyllum Tul. Var. acuminate Teles Freire that contained agathisflavone showed antinociceptive action in rodents (Cavalcanti et al., 2017), neuroprotective and anti-inflammatory effects (Dos Santos Souza et al., 2018), and decrease in oxidative stress in the brain (Dumitru et al., 2019). However, so far, there is no available research on the antinociceptive activity of agathisflavone as an isolated compound in various pain models. For this reason, the present study supports the use of CE as a potential analgesic antinociceptive agent. Based on the results, we conclude that CE is a novel antinociceptive agent and can be used as a therapeutic option in the treatment of neuropathic pain associated with diabetes mellitus.

| CON CLUS IONS
In summary, our data demonstrated that administration of the ethanolic extract, fractions, and compounds of CE exhibits antinociceptive activity according to different pain tests induced by acetic acid, a hot plate, 5% formalin, and STZ. The CE extract showed a significant decrease in the number of writhing responses in the acetic acidinduced pain model and in nociceptive behavior in the second phase of the formalin model. In the hot-plate test, the CE groups showed increased antinociceptive behavior compared to the control group, but this effect was not significant. The results of the tail-flicking test also showed no significant difference between the treatment groups and the control group. In the STZ-induced pain model, the n-Hex and MC fractions of the CE extract showed an effect at the 2-to 4-h time point, and two compounds from the n-Hex fraction, ellagic acid and agathisflavone, showed increased effect with the passage of time. Based on these results, the antinociceptive effect of the CE extract, ellagic acid, and agathisflavone was confirmed for peripheral and neurogenic pain. The results of this study might be valuable for identifying compounds with antinociceptive activity from natural products.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

E TH I C A L S TATEM ENT
This study was approved by the Institutional Review Board of Hallym University.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.