Anesth Pain Med 2020;15:478-485 https://doi.org/10.17085/apm.20032 Experimental Research pISSN 1975-5171 • eISSN 2383-7977

Antinociceptive effects of intrathecal cimifugin treatment: a preliminary rat study based on formalin test

Hyun Young Lee1,2, Young Joon Ki2,3, Su Yeong Park2, Soo Yeon Cho2, Jinyoung Seo2, Kyung Joon Lim1,2, and Ki Tae Jung1,2

1Department of Anesthesiology and Pain Medicine, School of Medicine, Chosun Received May 5, 2020 University, 2Department of Anesthesiology and Pain Medicine, Chosun University Revised June 16, 2020 Hospital, 3Department of Medicine, Graduate School, Chosun University, Gwangju, Accepted August 5, 2020

Background: Cimifugin is one of the components of the root of divaricata. The extract derived from S. divaricata is traditionally used as an analgesic. This study was con- ducted to evaluate the analgesic effect of intrathecal cimifugin in the formalin test. Methods: Male Sprague–Dawley rats (n = 20) were randomized into four groups for intra- thecal administration of 70% dimethylsulfoxide and various doses of cimifugin (100 μg, 300 μg, and 1,000 μg). The typical flinch response after the injection of 5% formalin into the hind paw was assessed in two distinct phases: phase 1 until 10 min, and phase 2 from 10

min to 60 min. ED50 values were calculated via linear regression. Results: Intrathecal cimifugin significantly reduced the flinch response in both phases of the formalin test. Significant antinociceptive effects of cimifugin were found with the dose of Corresponding author 300 μg in phase 1 and the dose of 100 μg in phase 2. The ED50 value (95% confidence in- Ki Tae Jung, M.D., Ph.D. tervals) of intrathecal cimifugin was 696.1 (360.8–1,342.8) μg during phase 1 and 1,242.8 Department of Anesthesiology and (42.0–48,292.5) μg during phase 2. Pain Medicine, Chosun University Hospital, School of Medicine, Chosun Conclusions: Intrathecal cimifugin has an antinociceptive effect against formalin-induced University, 365 Pilmun-daero, Dong- pain. Cimifugin has an anti-inflammatory effect at low concentrations, and non-inflammatory gu, Gwangju 61453, Korea analgesic effect at higher concentrations. Tel: 82-62-220-3223 Fax: 82-62-223-2333 E-mail: [email protected] Keywords: Analgesia; ; Chromones; Cimifugin; Nociception; Pain measurement.

INTRODUCTION major targets, including cyclooxygenase (COX), interleuk- ins (IL), and tumor necrosis factor-α [2]. Recent interest in Inflammation is one of the mechanisms of pain. Cyto- chromones as potential candidates for drug development kines and other pro-inflammatory mediators, such as pros- is attributed to their anti-inflammatory effects [2,3]. taglandins, nitric oxide, serotonin, and histamine, are re- Saposhnikovia divaricata and japonicum leased from cells after the tissue damage, resulting in pain belonging to family Umbelliferae, known as ‘Bangpung’ in and hyperalgesia [1]. The inflammatory cascade involves Korea, have been used as traditional herbal medicine to

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Copyright © the Korean Society of Anesthesiologists, 2020

478 KSPS ------479 ]. The inserted]. The 13 [ DMSO at a concentration greater greater DMSO a concentration at ). The rats in the vehicle-treated group group in the vehicle-treated rats ). The Fig. 2 Fig. ( Cimifugin (purity ≥ ChemFac from purchased 98%) was n (each groups four into randomized were rats The = 5) The intrathecal catheter was implanted after the anes the after implanted was catheter intrathecal The using a computerized random number according to the the to according number random computerized a using protocol study as a control 70% DMSO intrathecally administered were test in the different rats The during test. the formalin concen at cimifugin with intrathecal treated were groups during the respectively, of 100, 300, and 1,000 μg, trations formalin test. head and fixed with a 3-O silk. The tip of the catheter was was tip of the catheter with and fixed The head a 3-O silk. inhibit CSF to wire steel stainless plugged with a 30-gauge procedure, catheterization the intrathecal After leakage. in the the anesthesia from recover to allowed were the rats observed was function Ambulatory after cages. individual withoutmotor Only rats the anesthesia. from recovery full with or sensory rats The used in the study. were deficits or sensorymotor immediately euthanized deficits were anesthetics. of volatile with an overdose Drug preparation and grouping dissolved in 70% di Cimifugin powder was es (China). (DMSO). di dissolved solutions The methylsulfoxide were powder Cimifugin levels. μg 1,000 and 300, 100, to luted dissolved fully by was of cimifugin powder concentration maximal 70%. The than in 70% DMSO dissolve to 1,000 μg/10 μl. The was required a pilot study determined cimifugin was of by concentration not shown). (data the cage located in a room maintained at a constant tempera constant a at maintained room a in located cage the food and a under water 23°C), to to (20 access with free ture cycle of 12:12. light/dark Intrathecal catheterization surgical the of sterilea dressing After withthesia isoflurane. of the atlanto-occipital the level at of the rat field, the head was magna incised was cisterna and the membrane inserted was the into catheter opened. A polyethylene-5 for drug space administration intrathecal at catheter end of the intrathecal the placed at was catheter the it into advancing carefully by enlargement the lumbar cath The site. the incision 8.5 cm from about region caudal of cerebrospi the leakage confirmed was by location eter catheter The fluid (CSF)of the catheter. the tip nal through the of area in the frontal skin the through out pulled tip was

. ------) for ) for ]. Bio 4 [ HO ]. These ]. These ] and AR divarica 7 . , S 12 6 [ Antinociceptive effect of cimifugin of effect Antinociceptive [ japonicum Thunb . P O ]. Thus, we hypothesized we hypothesized ]. Thus, 11 ]. We previously demonstrat previously ]. We , 8 [ O 10 [ ]. Recent studies showed anti-in showed studies ]. Recent 9 [ O O ]. Compounds isolated from from isolated ]. Compounds /www.nc3rs.org.uk/arrive-guidelines 2 divaricata . ), which is one of the bioactive substances de substances is one of the bioactive ), which S https:/ ] and anti-inflammatory effects] and anti-inflammatory 5 MATERIALS AND METHODS MATERIALS Fig. 1 Fig. HO Chemical structure of cimifugin. of structure Chemical The animal study was approved by the Institutional Animal Animal the Institutional by approved was study animal The , including sec-O-glucosylhamaudol (SOG), sec-O-glucosylhamaudol hamaudol, , including www.anesth-pain-med.org Fig. 1.Fig. from Damul Science (Korea), and those weighing 225–250 g 225–250 g and those weighing Science (Korea), Damul from used (n in the study were each = housedin were rats The 20). guidelines ( guidelines Specific of experimental in animals. pain the investigation purchased were rats Sprague-Dawley male pathogen-free lowed the guidelines and ethical standards stipulated by the the by stipulated standards ethical and the guidelines lowed of Pain for the Study Association International Experiments) Research:RIVE (Animal Vivo of In Reporting Care and Use Committee (no. CIACUC 2018-S0042). We fol We 2018-S0042). CIACUC (no. Committee Use and Care Animal preparation effective dose (ED) of cimifugin required for the analgesia. doseeffective (ED) of cimifugin required tion-dependent analgesic effect of cimifugin based on a effect of cimifugin based analgesic on a tion-dependent the secondary The test. formalin calculating was outcome cimifugin using a formalin test to confirm the analgesic ef confirm to a formalin test analgesic the cimifugin using fect anti-inflammatory via effect. of cimifugin mediated primary the concentra to The was of the study outcome that the anti-inflammatory effect of cimifugin may result result effect the anti-inflammatory cimifugin may of that we conducted a preliminary Thus, effect. in an analgesic effect determineof intrathecal to the antinociceptive study flammatory effects of cimifugin, a chromone (1,4-benzo chromone a cimifugin, of effects flammatory pyran, rived from via opioid receptors receptors opioid via treat fever, rheumatism, neuralgia, and headache headache and neuralgia, rheumatism, fever, treat which is one of the constituents of is one of the constituents which ledabouriellol, divaricatol, isofraxidin,and cimifugin cimifugin isofraxidin,and divaricatol, ledabouriellol, effect showed an analgesic SOG, effect of intrathecal antinociceptive ed the strong properties of ‘Bangpung’ pharmaceu properties potential suggested [ candidates tical active substances derived from ‘Bangpung’ derived showed anti substances from active [ oxidant ta Anesth Pain Med Vol. 15 No. 4

Intrathecal catheterization (n=20)

Randomization after a week

Vehicle-treated group Cimifugin 100 μg group Cimifugin 300 μg group Cimifugin 1,000 μg group (DMSO 70%) (n = 5) (n = 5) (n = 5) (n = 5)

Intrathecal administration of prepared drugs 10 minutes prior to the formalin injection

Formalin test Dose response calculation

Fig. 2. Flow diagram of the study design. DMSO: dimethylsulfoxide.

Flinch response peated measures analysis of variance. We decided the total sample size as 20 given the drop-out rate of 20%. The formalin test was conducted from 9:00 a.m. to 12:00 Dose-responsiveness of cimifugin was calculated as the p.m. by two researchers. One investigator administered percentage of control in the two phases as follows. formalin injection and intrathecal drug treatment, and an- other investigator carried out the behavioral test. After ad- aptation in a restrained cylinder for 20 min, different doses of cimifugin or vehicle (DMSO 70%) were administered in a volume of 10 μl solution intrathecally using a gear-oper- The ED50 was defined as a dose of cimifugin that resulted ated Hamilton syringe pump, and 10 μl of saline was in a 50% inhibition of flinch count compared with the con- flushed additionally. After 10 min, 5% formalin (50 μl) was trol. The ED50 was calculated via standard linear regression subcutaneously injected into the center of the hind paw analysis according to the method of Tallarida [15]. ED50 val- with a 30 gauge needle. The flinch responses were assessed ues and confidence intervals (CIs) in each phase were cal- by counting the number of flinches per minute. Phase 1 re- culated. sponse (initial acute phase, 0–9 min) was measured at 1 The time-response or the dose-response data of flinch re- and 5 min after the formalin injection, while phase 2 re- sponses are expressed as the number of flinches or the per- sponse (10–60 min) was measured every 5 min until an centage of control. All data are expressed as mean ± SEM. hour after the formalin injection [14]. The flinch responses in phases 1 and 2 were analyzed sepa- rately because of the distinct biphasic response of the for- Statistical analysis malin test. One-way analysis of variance followed by post- hoc test with Turkey’s test for multiple comparisons was The sample size was calculated using G*Power 3.1 soft- used for the statistical analysis of the flinch responses. P val- ware. The calculated effect size was 1.12 based on the re- ues less than 0.05 were considered statistically significant. sults of a previous study, which showed significant antino- ciceptive effect when the total flinch count was decreased RESULTS to 60.2% compared with the control [9]. The total sample size in the three groups was calculated as 16 with α = 0.05 Typical biphasic flinch responses to the formalin test and 95% power for 13 consecutive flinches based on re- were observed in all rats following intrathecal administra-

480 www.anesth-pain-med.org KSPS

481 , and 5 , * 1,000 μg Figs. 4 60 * 300 μg 55 Phase 2 * Control (DMSO 70%) Cimifugin 100 μg Cimifugin 300 μg Cimifugin 1,000 μg 50 Dose of cimifugin 100 μg 45 40 Control (95% CIs) values of intrathecal cimifugin were cimifugin were of intrathecal values (95% CIs) 50 0

). 50

35 350 300 250 200 150 100 Sum of Flinches of Sum The ED The B statistically significant reduction of flinch response was was response of flinch reduction significant statistically P a dose μg (65.5% of control, of 100 at found = and 0.011), a at occurred response flinch in reduction the maximal P dose (50.6% of control, of 1,000 μg = 0.003, 1 Table μg during696.1 (360.8–1,342.8) (42.0– 1 and 1,242.8 phase 2. 48,292.5) μg during phase 30 Time (min) Time - - 25 * 20 1,000 μg Antinociceptive effect of cimifugin of effect Antinociceptive 15 * ). During phase 2, a a 2, phase ). During 300 μg 10 ). Phase 1 5 Table 1 Table

Fig. 3 Fig. ( Dose of cimifugin 100 μg ). During phase 1, a statistically statistically a 1, phase During ). 1 , and 4 5 ,

, 5.0 0.0

–5.0 25.0 20.0 15.0 10.0 40.0 35.0 30.0 45.0 Figs. 3 Figs. 4 Flinches/min (

Control

5 0

15 10 35 30 25 20 Sum of Flinches of Sum Antinociceptive effects of intrathecal cimifugin are presented as dose-response data. Intrathecal cimifugin decreased the formalin- the decreased cimifugin Intrathecal data. as dose-response presented are cimifugin intrathecal of effects Antinociceptive Time course data showing the antinociceptive effects of cimifugin after formalin injection. Intrathecal cimifugin significantly decreased decreased significantly cimifugin Intrathecal injection. formalin after cimifugin of effects antinociceptive the showing data course Time A Pre-treatment with intrathecal cimifugin decreased the the cimifugin decreased withintrathecal Pre-treatment www.anesth-pain-med.org induced flinch response significantly during phase 1 (A) and phase 2 (B). In phase 1, a statistically significant reduction in flinch response response in flinch reduction significant a statistically 1, 2 (B). In phase phase 1 (A) and phase during significantly response flinch induced 2, a statistically μg. In phase 1,000 of a dose at occurred response in flinch reduction maximal the 300 μg and of dose with the found was 1,000 of a dose at occurred response in flinch reduction maximal μg. The 100 of dose the at found was response in flinch reduction significant vs. control. ± SEM. *P < 0.001 as mean expressed are μg. Data Fig. 4. Fig. Fig. 3. Fig. 5 rats/group. ± SEM of mean the represents line Each dimethylsulfoxide. DMSO: phases. both during response flinch formalin-induced the sponse was observedsponse was a dose of 1,000 μg (41.0% of con at P trol, < 0.001, in both phases bothin phases using found was response in flinch reduction significant re in flinch reduction and the maximal the dose of 300 μg, tion of vehicle (DMSOtion of vehicle 70%) injection formalin significantly by triggered response flinch Anesth Pain Med Vol. 15 No. 4

A Phase 1 B Phase 2 120.0 120.0

100.0 100.0

80.0 80.0 * * 60.0 60.0 * * 40.0 * 40.0

20.0 20.0 Sum of Flinches (% control) Sum of Flinches (% control) 0.0 0.0 Control 100 μg 300 μg 1,000 μg Control 100 μg 300 μg 1,000 μg Dose of cimifugin Dose of cimifugin

Fig. 5. Dose-response data indicate the antinociceptive effects of intrathecal cimifugin. *P < 0.001 vs. control.

Table 1. Antinociceptive Effects of Intrathecal Cimifugin: Dose-response Data Dose response (% of control) Drugs and dose Phase 1 Phase 2 % of control P value % of control P value Control (DMSO 70%) 100.0 - 100.0 - Cimifugin 100 μg 94.4 0.668 65.5 0.011 Cimifugin 300 μg 70.8 0.045 60.9 0.005 Cimifugin 1,000 μg 41.0 < 0.001 50.6 0.003 DMSO: dimethylsulfoxide. P value, compared to the control.

DISCUSSION pyran-type C-ring. Chromones are an important class of natural products that exhibit antibacterial, anticoagulant, As mentioned above, ‘Bangpung’ was traditionally used anti-inflammatory, antioxidant, and anticancer activities to treat fever and pain. The anti-inflammatory and antioxi- [3]. Several types of compounds in chromones targeting dant activity of the bioactive substances derived from S. di- multiple inflammatory pathways have been reported. The varicata may correspond to those effects [5–7]. According anti-inflammatory effects of chromones are mediated not to the phytochemical studies, Prim-O-glucosylcimifugin is only via inhibition of the COX pathway but also by neutro- the main chromone present in S. divaricata, with antipyret- phil-dependent superoxide anion generation [3]. A recent ic, anti- inflammatory, and analgesic properties [4]. Cimi- study involving RAW264.7 cells showed that cimifugin in- fugin is an aglycone of prim-O-glucosylcimifugin, synthe- hibits the activities of mitogen-activated protein kinase sized during blood absorption [4]. According to Li et al. [16] and NF-κB (nuclear factor kappa-light-chain-enhancer of cimifugin showed the highest concentration in plasma of activated B cells) signaling pathways and suppresses the rats treated with the extracts of S. divaricata orally. They release of inflammatory factors [11]. Another study of re- concluded that cimifugin is the potential pharmacody- ported that cimifugin significantly attenuated allergic in- namic component of S. divaricata formed via biotransfor- flammation by reducing thymic stromal lymphopoietin mation in vivo even though prim-O-glucosylcimifugin is and IL-33 synthesis [10]. the major constituent [4,16]. These studies suggest that cimifugin has an anti-inflam- As far as we know, only one study reported the analgesic matory role resulting in an analgesic effect via inhibition of effect of cimifugin. Okuyama et al. [8] reported that oral the inflammatory process. Neuroinflammation plays an administration of 80 mg/kg of cimifugin showed significant important role in the generation and modulation of pain analgesia in mice during acetic acid-induced writhing test. [17], so the anti-inflammatory effect of cimifugin was The results suggested that the analgesic potency of chro- thought to decrease the induction of inflammatory pain. mones may be attributed to the non-glycosylated dihydro- Thus, we conducted the formalin test as a preliminary

482 www.anesth-pain-med.org KSPS

------483 ]. The high concentration of of concentration high ]. The values in phase 2 was large ac large 2 was in phase values 23 50 [ ACKNOWLEDGEMENTS ACKNOWLEDGEMENTS ] suggested that antioxidants such as vitamin E induce vitamin E induce as such antioxidants ] suggested that 22 [ This work was supported by the Korean National Re National the Korean supported was work by This In conclusion, intrathecal cimifugin had antinociceptive antinociceptive cimifugin had intrathecal conclusion, In Although the current study showed an analgesic effect of showed an analgesic study the current Although the actual mechanisms of cimifugin. mechanisms the actual (NRF-2018R1C1B5 grant research Foundation search 085816). higher concentrations of cimifugin (> concentrations higher 1,000 μg/10 μl of 70% we usedas the maximal cimifugin of μg 1,000 DMSO). Thus, cimifugin Moreover, administration. dose for the intrathecal dissolved only in DMSO was powder a concentration at dose of DMSO the control though 70%. Even than greater DMSO effect, showedsome analgesic no reducing had alone effects formalin in the test re affect the study may DMSO used for drug preparation the CI of the ED Third, sults. 48,292.5 μg), which (42.0 to regression the linear to cording the in due to limitation as computational considered was a of cimifugin. Such use to ability higher-concentrations associ of difficulty because CI limits furtherlarge evaluation in studies Further administration. withated the intrathecal with doses large are effects the side of treatment vestigating biological using further investigations needed. Fourth, needed for objective results. are markers that suggesting effects of the formalin in both test, phases concen low effect an anti-inflammatory at cimifugin has higher effects at and non-inflammatory analgesic trations investigate to required are studies Further concentrations. al. sen the central decreasing not only effects by antinociceptive in oxygen the reactive also scavenging but sitization tissue. the peripheral sev were there formalin test, of the in bothcimifugin phases the pharmaco evaluated we merely First, limitations. eral doses of cimifugin in the formalin effect of different logical the mech without elucidating study, in this preliminary test of routes different using studies anisms of action. Further and of specific receptors antagonists using administration as well as other molecular test, formalin methods other than of action of site needed exact determine the to are studies cimifugin. Second, dose of cimifugin (> a higher 1,000 μg) the maximal but effect, analgesic potent yield a more may concentra Higher 1,000 μg. was study dose the current of study this in beadministered not could cimifugin of tions dissolve to and inability difficulties of technical because ------]. Hong et ]. Hong 21 [ Antinociceptive effect of cimifugin of effect Antinociceptive ., which showed a rela a showed ., which ]. The flinch response in in response flinch ]. The , showed that SOG, showed that exhibits 18 [ -adrenergic and opioid recep opioid and -adrenergic 2 α divaricata ]. Therefore, it is suggested that cimi that it is suggested ]. Therefore, . 7 S [ japonicum Thunb . P ]. Our previous study investigating SOG, and other ex investigating study previous ]. Our ]. Thus, other targets may modulate acute peripheral peripheral acute modulate may other targets ]. Thus, 19 20 [ Other hypotheses have been proposed to explain the mech the explain to been proposed have hypotheses Other www.anesth-pain-med.org duced by tissue injury and intrathecal administration of an of an administration injury tissue and intrathecal duced by (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxy) antioxidant in both phases the response reduced effectively formalin-induced pain response. According to the study eval the study to According formalin-induced response. pain test, in the formalin effect of antioxidants the analgesic uating in in the pain role an important play may the pro-oxidant tor tor the mediate effect may nociception. Second, the antioxidant tors [ derived from tracts the μ-opioid recep via pain incisional effect against analgesic nol, the bioactive compound derived from clove, carries not clove, derived from compound nol, the bioactive also but an potential and antioxidant anti-inflammatory only via effects mediated algesic anisms of action. First, derivatives of natural products display display products of natural derivatives anisms of action. First, euge example, For mechanisms. effect multiple via analgesic flammatory effect at low concentrations and via other other and via concentrations low flammatory effect at concentration. high effects at non-inflammatory analgesic tively weak inhibitory activity of cimifugin against both weak inhibitorytively both activity of cimifugin against and COX-2 COX-1 anti-in via effect mediated an antinociceptive fugin has control).These results are consistent with a previous study study with previous a consistent are results control).These effects the anti-inflammatory com of isolated reporting from pounds ceptive effect with the 1,000 μg dose was more effective in effectceptive with the 1,000 μg dose more was 2: 50.6% of phase vs. 1: 41.0% of control 1 (phase phase cant antinociceptive effects were observed upon treatment observed effects were antinociceptive upon treatment cant and 100 μg in with 1 (70.8% of control) 300 μg in phase antinoci its maximal However, 2 (65.5% of control). phase gesic effects in both phases, suggesting effects against effects against suggesting gesic effects in both phases, cimifugin re study, the current non-inflammatory In pain. signifi in both phases;duced response the flinch however, glandins does not play an important role during the early during the early role an important doesglandins not play some nonsteroidal However, phase. the latent but phase anal show drugsanti-inflammatory as paracetamol such horn mediated via inflammatory reaction in the spinal in the spinal inflammatory via horn mediated reaction prosta by mediated the inflammatory pathway Thus, cord. phase 1 is due to a direct effect on the nociceptors, while while effect on the nociceptors, a direct 1 is due to phase both inflammatory in pain represents response 2 phase the in the dorsal changes and functional tissue the peripheral decreased the flinch response to formalin injection to both in response the flinch decreased differ indicate two test in the formalin The phases phases. mechanisms nociceptive ent study to evaluate the analgesic effect of intrathecal cimi effect of intrathecal the analgesic evaluate to study study, current the In inflammation. to its relation fugin and of cimifugin administration we intrathecal confirmed that Anesth Pain Med Vol. 15 No. 4

CONFLICTS OF INTEREST constituents isolated from peucedanum japonicum Thunb. and their cyclooxygenase (COX) inhibitory activity. Korean J No potential conflict of interest relevant to this article Medicinal Crop Sci 2005; 13: 75-9. was reported. 8. Okuyama E, Hasegawa T, Matsushita T, Fujimoto H, Ishibashi M, Yamazaki M. Analgesic components of saposhnikovia root AUTHOR CONTRIBUTIONS (Saposhnikovia divaricata). Chem Pharm Bull (Tokyo) 2001; 49: 154-60. Conceptualization: Ki Tae Jung. Data acquisition: Soo 9. Kim SH, Jong HS, Yoon MH, Oh SH, Jung KT. Antinociceptive Yeon Cho, Jinyoung Seo, Ki Tae Jung. Formal analysis: effect of intrathecal sec-O-glucosylhamaudol on the forma- Hyun Young Lee, Young Joon Ki, Su Yeong Park. Funding: lin-induced pain in rats. Korean J Pain 2017; 30: 98-103. Ki Tae Jung. Supervision: Ki Tae Jung. Writing—original 10. Wang X, Jiang X, Yu X, Liu H, Tao Y, Jiang G, et al. Cimifugin draft: Hyun Young Lee, Ki Tae Jung. Writing—review & ed- suppresses allergic inflammation by reducing epithelial de- iting: Kyung Joon Lim, Ki Tae Jung. rived initiative key factors via regulating tight junctions. J Cell Mol Med 2017; 21: 2926-36. ORCID 11. Han B, Dai Y, Wu H, Zhang Y, Wan L, Zhao J, et al. Cimifugin in- hibits inflammatory responses of RAW264.7 cells induced by Hyun Young Lee, https://orcid.org/0000-0001-5861-3131 lipopolysaccharide. Med Sci Monit 2019; 25: 409-17. Young Joon Ki, https://orcid.org/0000-0001-6203-3690 12. Zimmermann M. Ethical guidelines for investigations of exper- Su Yeong Park, https://orcid.org/0000-0001-7108-4350 imental pain in conscious animals. Pain 1983; 16: 109-10. Soo Yeon Cho, https://orcid.org/0000-0002-7828-7908 13. Yaksh TL, Rudy TA. Chronic catheterization of the spinal sub- Jinyoung Seo, https://orcid.org/0000-0002-5256-6132 arachnoid space. Physiol Behav 1976; 17: 1031-6. Kyung Joon Lim, https://orcid.org/0000-0002-3651-0331 14. Puig S, Sorkin LS. Formalin-evoked activity in identified prima- Ki Tae Jung, https://orcid.org/0000-0002-2486-9961 ry afferent fibers: systemic lidocaine suppresses phase-2 activ- ity. Pain 1996; 64: 345-55. REFERENCES 15. Tallarida RJ, Murray RB. Manual of pharmacologic calculations with computer programs. 2nd ed. New York, Springer. 1987. p. 1. Watkins LR, Milligan ED, Maier SF. Glial activation: a driving 1-95. force for pathological pain. Trends Neurosci 2001; 24: 450-5. 16. Li Y, Wang H, Chen J, Zhao L, Zhang H, Chai Y, et al. RRLC- 2. Gautam R, Jachak SM. Recent developments in anti-inflamma- TOF/MS in identification of constituents and metabolites of tory natural products. Med Res Rev 2009; 29: 767-820. Radix Saposhnikoviae in rat plasma and urine. Acad J Second 3. Vijaya Bhargavi M, Shashikala P, Sumakanth M. Coumarins Mil Med Univ 2010; 30: 760-3. and chromones: a remarkable scaffolds for anti-inflammatory 17. Seol TK, Lee W, Park S, Kim KN, Kim TY, Oh YN, et al. Effect of activity. J Pharm Sci Res 2017; 9: 1483-9. palmitoylethanolamide on inflammatory and neuropathic 4. Li Y, Zhao L, Zhang H, Jia J, Lv L, Zhou G, et al. Comparative pain in rats. Korean J Anesthesiol 2017; 70: 561-6. pharmacokinetics of prim-O-glucosylcimifugin and cimifugin 18. Hunskaar S, Hole K. The formalin test in mice: dissociation be- by liquid chromatography-mass spectrometry after oral ad- tween inflammatory and non-inflammatory pain. Pain 1987; ministration of Radix Saposhnikoviae extract, cimifugin mono- 30: 103-14. mer solution and prim-O-glucosylcimifugin monomer solu- 19. Park SH, Sim YB, Lee JK, Kim SM, Kang YJ, Jung JS, et al. The tion to rats. Biomed Chromatogr 2012; 26: 1234-40. analgesic effects and mechanisms of orally administered euge- 5. Hisamoto M, Kikuzaki H, Ohigashi H, Nakatani N. Antioxidant nol. Arch Pharm Res 2011; 34: 501-7. compounds from the leaves of Peucedanum japonicum thunb. 20. Koh GH, Song H, Kim SH, Yoon MH, Lim KJ, Oh SH, et al. Effect J Agric Food Chem 2003; 51: 5255-61. of sec-O-glucosylhamaudol on mechanical allodynia in a rat 6. Zimecki M, Artym J, Cisowski W, Mazol I, Włodarczyk M, model of postoperative pain. Korean J Pain 2019; 32: 87-96. Gleńsk M. Immunomodulatory and anti-inflammatory activity 21. Hacimuftuoglu A, Handy CR, Goettl VM, Lin CG, Dane S, Ste- of selected osthole derivatives. Z Naturforsch C J Biosci 2009; phens RL Jr. Antioxidants attenuate multiple phases of forma- 64: 361-8. lin-induced nociceptive response in mice. Behav Brain Res 7. Zheng M, Jin W, Son KH, Chang HW, Kim HP, Bae K, et al. The 2006; 173: 211-6.

484 www.anesth-pain-med.org KSPS - 485 . Pieretti S. New insights of dimethyl sulphoxide effects (DMSO) sulphoxide of dimethyl insights New S. Pieretti modelson experimental of nociceptionin vivo and inflamma 2008; 57: 419-25 Res tion. Pharmacol Antinociceptive effect of cimifugin of effect Antinociceptive . effects of vitamin E in formalin-induced nociceptive response effectsin formalin-induced of vitamin E response nociceptive 6: 59-62 2011; Med Pain Anesth in rats. Colucci M, Maione F, Bonito MC, Piscopo A, A, Di Giannuario F, M, Maione Colucci Hong BH, Ko YK, Lee YJ, Han K, Kim Y, Lee Antinociceptive W. K, Y, Han Kim YK, Ko Lee BH, YJ, Hong . . www.anesth-pain-med.org 23 22