Analgesic and anti-inflammatory activities of ethyl acetate and butanol fractions from Vitex polygama hydroalcoholic leaf extract

Abstract This study evaluated the analgesic and anti-inflammatory activities of Vitex polygama. Ethyl acetate and butanol fractions (10-30 mg/kg), obtained from the hydroalcoholic leaf extract, showed an antinociceptive effect in the acetic acid-induced abdominal writhing test, formalin test and modified hot plate test in mice, indicating a peripheral anti-inflammatory action. Ethyl acetate and butanol fractions were effective in inhibiting nitric oxide and TNF-α production, respectively, in RAW 264.7 macrophages. Both fractions (10-30 mg/kg) showed an acute analgesic effect in mice with vincristine-induced neuropathic pain exposed to a thermal stimulus. Through ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-UV-MS/MS) it was possible to identify seven major compounds: isoorientin, orientin, vitexin, isovitexin, O-p-hydroxybenzoyl orientin, O-caffeoyl-orientin, and di-caffeoylquinic acid. Orientin and isoorientin were isolated from ethyl acetate fraction and had their identity confirmed by nuclear magnetic resonance (NMR). Glucosyl flavones appear to be the main metabolites responsible for the anti-inflammatory and analgesic activities observed for V. polygama. Graphical Abstract


Introduction
Pain is considered a health problem worldwide due to its prevalence and impact on quality of life. Plant-derived compounds have greatly contributed to the pharmacological arsenal for the treatment of pain, which is basically composed of non-opioid and opioid analgesics (Walk and Poliak-Tunis 2016). The opioid morphine remains widely used for the treatment of moderate to severe pain (Paul et al. 2021), as well as the non-steroidal anti-inflammatory acetylsalicylic acid for common pain conditions (Hagen and Alchin 2020). In the search for novel analgesic drugs, phytocannabinoids (Vu ckovi c et al. 2018) and polyphenolic compounds (Ferraz et al. 2020;Basu et al. 2021) stand out due to their pharmacological activities.
Vitex polygama Cham. (Lamiaceae) is an endemic species of Brazil that is used in popular medicine to treat kidney diseases and rheumatism (Grandi et al. 1989;Gallo et al. 2008), conditions that are associated with an inflammatory and painful state. We have recently shown that the dichloromethane fraction of V. polygama and isolated compounds have in vitro antimycobacterial and immunomodulatory activities (de Jesus et al. 2022). In this work, we dedicated our efforts to study the antinociceptive and anti-inflammatory effects of the ethyl acetate (EtOAc) and butanol (BuOH) fractions of V. polygama by using experimental models of pain and in vitro assays. Our results contribute to expand current knowledge about the pharmacological potential of this species.

Results and discussion
The acetic acid-induced writhing test was used for investigating the antinociceptive effect of the hydroalcoholic leaf extract of V. polygama (EVP) and its fractions. Although this test lacks specificity, it is considered sensitive even to weak analgesics (Le Bars et al. 2001;Patil et al. 2019). EVP, ethyl acetate (EtOAc), and butanol (BuOH) fractions, at 10 mg/kg, caused inhibition of 61.3, 35.0, and 49.2% (P < 0.05), respectively, of the writhing response induced by acetic acid ( Figure S1a). EtOAc ( Figure S1b) and BuOH ( Figure S1c) fractions (10-30 mg/kg) produced dose-dependent antinociception when assessed in the acetic acid-induced writhing test in mice, which was similar to the effect of the positive control indomethacin. In contrast, hexane (HEX), dichloromethane (DCM), and aqueous (AQU) fractions did not show a significant antinociceptive effect ( Figure S1a). Therefore, EtOAc and BuOH fractions were selected to further studies.
EtOAc and BuOH fractions were effective in the formalin test, where the early phase results from the activation of nociceptors and the late phase occurs due to the local release of inflammatory mediators (Patil et al. 2019). At 1 st phase, EtOAc fraction reduced licking time by 30.6, 33.8 and 51.1% (P < 0.05) at 10, 20 and 30 mg/kg, respectively ( Figure S2a). At 2 nd phase, licking time was reduced by 41.1, 39.1, and 41.0% (P < 0.05), respectively ( Figure S2b). Similar results were observed with the BuOH fraction ( Figure S2c and S2d). The effect of both fractions was similar to that of morphine in the 1 st phase (P > 0.05), but not in the 2 nd phase of formalin test (P < 0.05).
Then, EtOAc and BuOH fractions were evaluated at the hot plate test, which is a specific model for the detection of analgesic compounds of central effects, such as opioids (Le Bars et al. 2001). Although morphine significantly increased the latency time throughout the experimental period, both fractions did not alter pain tolerance in the hot plate test (Table S1). With the goal of investigating the anti-inflammatory activity of EtOAc and BuOH fractions, they were evaluated at the modified hot plate test. This test is a variant of the classic hot plate method and aims to detect inflammatory nociception and peripheral hyperalgesia induced by carrageenan (Lavich et al. 2005). EtOAc and BuOH fractions were able to significantly reduce the D latency between the paws at all evaluation times, indicating analgesic activity (Table S2). At the dose of 30 mg/kg, both fractions were as effective as indomethacin.
Taken together, these results indicate that the antinociceptive activity of EtOAc and BuOH fractions is due, at least in part, to a peripheral anti-inflammatory action, which could be related to the inhibition of the synthesis and/or release of inflammatory mediators. Corroborating these results, in experiments using RAW 264.7 macrophages, the EtOAc fraction was able to inhibit the production of nitric oxide (NO) (IC 50 : 30.6 ± 1.1 lg/mL), while the BuOH fraction, 500 lg/mL, inhibited tumour necrosis factor-alpha (TNF-a) production in 55.83 ± 0.39% ( Figure S3). EtOAc fraction was not tested at the concentration of 500 lg/mL because it was cytotoxic ( Figure S4).
Chemotherapy-induced peripheral neuropathy (CIPN) is a painful side-effect of frequently used chemotherapeutic agents, but no effective treatment currently exists for preventing or limiting the occurrence and severity of CIPN (Burgess et al. 2021;Finnerup et al. 2021). Therefore, we investigated the acute effects of EtOAc and BuOH fractions on vincristine-induced neuropathic pain in mice. This experimental model involves an inflammatory response, with activation of NO synthase and increased expression of TNF-a (Kiguchi et al. 2008;Burgess et al. 2021). Hind paw withdraw latency in the hot plate test was significantly reduced in vincristine-treated mice when compared to the control group, a sign of thermal hyperalgesia ( Figure S5). EtOAc and BuOH fractions (10 and 30 mg/kg) increased the hind paw withdrawal latency 1 and 3 hours after i.p. administration, indicating an acute analgesic effect in mice exposed to thermal stimulus ( Figure S5). Although more studies are necessary to characterize the chronic effects of V. polygama on neuropathic pain, our results highlight the pharmacological potential of this species. Other medicinal plants of the Lamiaceae family have shown anti-neuropathic effects. Ocimum sanctum attenuated vincristine-induced neuropathic pain in rats (Kaur et al. 2010), while Sideritis bilgerian was effective in the partial sciatic nerve ligation model of neuropathic pain in mice (Cavalcanti et al. 2021).
According to the pharmacological results, the phytochemical analysis of EtOAc and BuOH fractions was performed by HPLC-DAD and UPLC-UV-MS/MS. The retention time (t R ), MS spectral data and wavelength of UV maximal absorption (k max ) for the major constituents of EtOAc and BuOH fractions are listed in Table S3. EVP and fractions showed a similar profile in the chromatogram ( Figure S6). In addition, peaks with coincident retention times and UV k max typical of flavonoids (240, 269 and 349 nm) suggested the presence of the same major flavonoids in the EVP and fractions. After comparing the retention time, UV absorption and MS/MS fragmentation pattern with the literature data (Leitão and Delle Monache 1998;Pereira et al. 2005;de Jesus et al. 2022), seven major peaks were identified: isoorientin, orientin, vitexin, isovitexin, O-phydroxybenzoyl orientin, O-caffeoyl-orientin, and di-caffeoylquinic acid ( Figure S7). Peak 8 was partially identified as a caffeic acid derivative. The MS spectrum of major compounds (peaks 1 and 2) showed a protonated molecule with m/z 449 [M þ H] þ. , compatible with the molecular formula C 21 H 20 O 11 . The fragmentation pattern in MS/ MS showed losses of 120 and 150 u, characteristic of mono-C-hexosyl moieties, resulting in fragment ions at m/z 329 and 299, respectively (Figures S8 and S9). This information demonstrated that these flavonoids are isomers and evidenced that luteolin is the aglycone. These isomers were isolated from the EtOAc fraction (as described in the supplementary material experimental section) and differentiated according to their NMR spectrum by the H-6 and H-8 chemical shifts, characteristic due to the different A-ring glycosylation position (de Oliveira et al. 2013). Isoorientin (luteolin-6-C-glucoside peak 1t R 9.26 min) showed a singlet at 6.51 ppm (H-8) with the absence of H-6, and, orientin (luteolin-8-C-glucosidepeak 2t R 9.62 min) showed a singlet at 6.24 ppm (H-6) with the absence of H-8 (Figures S10 and S11).
Isoorientin and orientin are probably the main responsible for the analgesic effects of EtOAc and BuOH fractions of V. polygama, as we have observed for the antimycobacterial and anti-inflammatory effects of DCM fraction (de Jesus et al. 2022). Our findings are consistent with previous studies that showed that orientin and isoorientin present analgesic activity (K€ upeli et al. 2004;da Silva et al. 2010;Wedler et al. 2014). In the same way, another C-glycosylflavone, vitexin, was shown to have analgesic activity through anti-inflammatory mechanisms (Borghi et al. 2013), as well as the aglycone luteolin (Fan et al. 2018).

Conclusions
This study contributed to improve the knowledge about the pharmacological and phytochemical properties of V. polygama and highlights its potential for the treatment of inflammatory pain. Further studies are needed to deepen the effects of this species on neuropathic pain.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This study was supported by CNPq and FAPERJ. In addition, this work was supported, in part, by FAPERJ fellowship to I.F.O.R. (E-26/201.132/2020).