Journal of Ethnopharmacology 284 (2022) 114779
Available online 27 October 2021
0378-8741/© 2021 Elsevier B.V. All rights reserved.
Antinociceptive effects of flower extracts and the active fraction from Styrax japonicus
Lei He
a,c, Ying Zhou
b,c, Guangjun Wan
d, Wencui Wang
b,c, Nan Zhang
b,c, Lei Yao
b,c,*aDepartment of Resources and Environment, School of Agriculture and Biology, Shanghai Jiao Tong University, 800 Dong Chuan Road, Shanghai, 200240, China
bDepartment of Landscape Architecture, School of Design, Shanghai Jiao Tong University, 800 Dong Chuan Road, Shanghai, 200240, China
cR&D Center for Aromatic Plants, Shanghai Jiao Tong University, 800 Dong Chuan Road, Shanghai, 200240, China
dNanjing Fragrant Jasmine Agricultural Technology Co., Ltd, Liuhe District, Nanjing, 211521, China
A R T I C L E I N F O Keywords:
Styrax japonicus Ethanol extracts Fraction Analgesic Mechanism
A B S T R A C T
Ethnopharmacological relevance: Flowers from Styrax japonicus sieb. et Zucc. have been used as a Chinese folk medicine to alleviate pain such as toothache and sore throat.
Aim of the study: To testify the analgesic effect of flowers from Styrax japonicus, analyze components of the active fraction, and investigate the mechanism of analgesia.
Materials and methods: Flower extracts were obtained by ethanol, petroleum ether and hydrodistillation extraction. Different fractions of ethanol extracts (EE) were isolated by silica gel column chromatography and preparative liquid chromatography. Analgesic effects of EE, petroleum ether extracts (PEE), hydrodistillation extracts (HDE), and fractions of EE were evaluated using hot plate, acetic acid-induced writhing and formalin tests on mice. Components of the active fraction 1 (F1) were determined by the ultrahigh-performance liquid chromatography Q extractive mass spectrometry (UHPLC-QE-MS). Anti-inflammatory and sedative effects involving analgesic mechanisms were evaluated by carrageenan induced hind paw oedema and pentobarbital sodium sleep tests, respectively. In addition, antagonists including naloxone hydrochloride (NXH), flumazenil (FM), SCH23390 (SCH) and WAY100635 (WAY) were used to investigate the possible mechanism of analgesia.
Contents of neurotransmitters and relevant metabolites in different brain regions of mice were also quantified by the ultraperformance liquid chromatography with a fluorescence detector (UPLC-FLD).
Results: EE rather than PEE and HDE at medium and high doses (150 mg/kg and 300 mg/kg) significantly prolonged the latency time of the response of mice to the thermal stimulation in the hot plate test. Moreover, EE significantly decreased number of writhes in the acetic acid-induced writhing test, and reduced licking time in both two phases of the formalin test in a dose-dependent manner. The F1 (50 mg/kg) showed effective anti- nociceptive responses in all mice models. However, fraction 2 (F2) and fraction 3 (F3) at 50 mg/kg performed no analgesic action. Kaempferol-3-O-rutinoside, isorhamnetin-3-O-rutinoside, pinoresinol-4-O-glucoside, forsythin and arctiin were identified from components of the F1. Furthermore, F1 (50 mg/kg) did not significantly affect hind paw oedema of mice induced by carrageenan but significantly shortened sleep latency and increased sleep duration in the pentobarbital sodium sleep test. In addition, the antinociceptive response of F1 was not affected by NXH in two mice models, but significantly blocked by FM and WAY in the hot plate test. In the formalin test, FM avoided the effect of F1 only in the first phase, while the analgesic activity of F1 was totally suppressed by WAY in both two phases. Otherwise, contents of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5- HIAA) increased significantly in hippocampus and striatum of mice in the F1 group.
Conclusion: EE from flowers of Styrax japonicus, and F1, the active part isolated from EE, showed significant antinociceptive activities. The analgesic effect of F1 appeared to be related to the sedative effect, partially mediated by the GABAergic system, and highly involved in the serotonergic system. This was the first study confirming the analgesic effect of Styrax japonicus flower, which provided a candidate for the development of non-opioid analgesics.
* Corresponding author. Department of Landscape Architecture, School of Design, Shanghai Jiao Tong University, 800 Dong Chuan Road, Shanghai, 200240, China.
E-mail addresses: [email protected] (L. He), [email protected] (L. Yao).
Contents lists available at ScienceDirect
Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm
https://doi.org/10.1016/j.jep.2021.114779
Received 26 July 2021; Received in revised form 16 October 2021; Accepted 22 October 2021
1. Introduction
Styrax japonicus sieb. et Zucc. is an ornamental tree which is widely distributed across East Asian countries. In recent years, medical values of different parts of Styrax japonicus have been investigated. Compounds isolated from the stem bark inhibited the expression of inducible nitric oxide synthase and cyclooxygenase-2 which were induced by lipopoly- saccharide, and showed significant virus-cell fusion inhibitory activities and potent inhibitory activities against matrix metalloproteinases (Kim et al., 2004; Yun et al., 2007; Lee et al., 2010). Four triterpenoid gly- cosides isolated from fresh fruits showed antisweet activities (Yoshi- kawa et al., 2000). To the best of our knowledge, pharmacological research on the flower of Styrax japonicus has not been reported even though it has been used to treat toothache and sore throat in China, and as a folk medicine, the decoction of the dried flowers and citron thorns stewed with duck was administrated orally to pain-suffered patients (G.
Q. Wang, 2014).
Pain is an unpleasant feeling and emotion accompanied by an existing or impending tissue injury according to the International As- sociation for the Study of Pain. This definition focuses on the inner affection of pain. Furthermore, the definition of pain in neurophysiology holds that pain is a kind of sensory information of peripheral tissue injury, which has clear and accurate codes in peripheral nerves, the conduction of the central nerves system and brain (Chapman and Nakamura, 2001). So far, the most effective analgesic drugs are opioids and their derivatives, which exert antinociceptive effects by binding with opioid receptors. However, long-term abuse of opioids usually leads to systemic damage, including drug tolerance, physical depen- dence and addiction (Kosten and George, 2002). Alternatively, tradi- tional Chinese medicines have been a hot spot of the clinical pain treatment due to their significant curative effects, less adverse reactions, and sufficient supplies (Wang et al., 2018). Pharmacological mecha- nisms of traditional Chinese medicines are various, including affecting the cholinergic system or central catecholamine system, initiating endogenous analgesia by activating opioid receptors or increasing opioid peptide levels, regulating 5-HT, γ-aminobutyric acid (GABA) or other neurotransmitter pathways, and inhibiting the process of inflam- mation (Du et al., 2016; Wang et al., 2018).
Several animal models are commonly used for pain assessment and preclinical evaluation of analgesics. Hot plate test is commonly used to evaluate the centrally mediated antinociceptive aspect by investigating the effect of drugs on the latency of thermal stimulation responses of animals (Arrau et al., 2011; Gou et al., 2017). In the writhing test, mice were injected intraperitoneally with 0.6%–1% acetic acid. Acetic acid stimulates the release of inflammatory mediators such as prostaglandin, bradykinin, 5-HT and histamine, resulting in pain and abdominal writhing through peripheral nociceptive sensitization (Hasan et al., 2014; Florentino et al., 2016). Formalin test is divided into two stages:
The first phase is peripheral neuropathic pain caused by the direct stimulation of sensory nerve fibers, and the second phase is a longer lasting pain due to inflammation (Balkrishna et al., 2019). All stimulus from formalin is to make the mice lick their toes.
In this research, since medicinal components and analgesic types of flowers are not clear, antinociceptive effects of flower extracts including volatile, semi-volatile and nonvolatile compounds were evaluated by hot plate, writhing and formalin tests. The active fraction was isolated and its components were identified. In addition, anti-inflammatory and sedative experiments were also carried out to reveal the analgesic mechanisms. And potential mechanisms of opioid, GABAergic, dopa- minergic and serotoninergic systems were investigated.
2. Materials and methods 2.1. Plant materials
Flowers of Styrax japonicus were collected in April from Nanjing
(Jiang Su Province, China) and stored at − 20 ◦C until use. A voucher specimen (No.20210503) was deposited at the herbarium of the R&D Center for Aromatic Plants, Shanghai Jiao Tong University. Accurately weighed flowers (100 g) were immersed in ethanol (300 ml) for 2 h. The slurry was filtered and then evaporated under reduced pressure to obtain 3.6 g non-volatile compounds as EE. Crude extracts of 0.5 g were subjected to isolation through silica gel column chromatography. An eluent system of n-butanol, ethanol and water (8:1:1) was used. The pigment fraction (30 ml) was firstly excluded (122 mg), and the second part (30 ml) was evaporated under reduced pressure and then freeze- dried in vacuum to obtain 138 mg F1. After condensation, 100 μl of the third part (10 ml total) was injected into Waters Prep 150 LC system to acquire 54 mg F2 and 77 mg F3. The detailed separation method of F2 and F3 was recorded in the supplementary data. 300 g flowers were mixed with 1000 ml petroleum ether for 2 h and the extracts were evaporated to obtain 135 mg semi-volatile compounds which were defined as PEE. And 1 kg flowers were loaded into the modified cle- venger type distillation apparatus with 2000 ml distilled water and distilled for approximately 3 h, yielding 125 mg volatile compounds which were signified as HDE.
2.2. Chemicals and animals
Silica gel (pore size 20–30A, pore volume 0.35–0.45 ml/g, particle size 20–60 mesh, surface area 650–800 m2/g) was purchased from Sangon Biotech (Shanghai, China). Morphine hydrochloride (MOH) was purchased from the Northeast Pharmaceuticals Group (Shenyang, China). Diazepam (DZP) was obtained from Shanghai Xudong Haipu Pharmaceutical Co., Ltd. (Shanghai, China). 5-HT, 5-HIAA, Glutamate (Glu), GABA, NXH, FM, SCH and WAY were purchased from Sigma- Aldrich (USA). Indomethacin (INDO, Meryer Chemical Technology Co., Ltd., Shanghai). Other reagents were analytical grade and obtained from Sinopharm (Shanghai, China).
Male ICR mice weighing between 25 and 30 g were obtained from the Laboratory Animal Center of Shanghai Jiao Tong University. Mice were kept under a 12 h/12 h light/dark cycle at standard room tem- perature (22 ±2 ◦C) and were supplied freely with enough food and water. All experiments of animal models carried out were in accordance with the rules of Institutional Animal Care and Use Committee at Shanghai Jiao Tong University (Approval No. A2020010).
2.3. Antinociceptive tests of different extracts and fractions in mice models
2.3.1. Hot plate test
Mice were treated with vehicle (physiological saline 10 ml/kg), EE, PEE, HDE (50, 150 and 300 mg/kg), individual fractions (50 mg/kg) and 10 mg/kg reference drug MOH (Padilha et al., 2009), respectively. The administration route was intraperitoneal injection. Then, animals were placed on a hot plate (set at 55 ±0.5 ◦C) 30, 60 and 90 min after drug administration. The latency of reaction to the thermal stimulation (lick hind paw or jump) was recorded and the cut-off time of 30 s was used.
2.3.2. Acetic acid-induced writhing test
Animals were treated with vehicle (10 ml/kg), EE, PEE, HDE (50, 150 and 300 mg/kg), individual fractions (50 mg/kg) and 10 mg/kg reference drug INDO (Lopes et al., 2019), respectively. 30 min after treatment, mice were administered with 0.8% acetic acid at a dose of 10 ml/kg (de Veras et al., 2020). The number of writhes of mice in each group during 30 min was recorded.
2.3.3. Formalin test
Vehicle (10 ml/kg), EE, PEE, HDE (50, 150 and 300 mg/kg), indi- vidual fractions (50 mg/kg), or MOH (10 mg/kg) was administered to animals. 30 min later, each mouse was given an intra-plantar injection of 30 μl of 1% formalin in the sub-planter space of the right-hind paw, the
dose used referred to previous studies (Halder et al., 2012; Hassanpour et al., 2020; Sampaio et al., 2020). The paw licking time was determined at 0–10 min (Phase 1) and 10–30 min (Phase 2) after formalin administration.
2.4. Qualitative analysis of the F1 by UHPLC-QE-MS
UHPLC-QE-MS (Thermofisher, USA) equipped with an ACQUITY UPLC column (BEH C18 100 ×2.1 mm, 1.7 μm, Waters) was applied to analyze the composition of the F1. The sample was diluted with meth- anol, and 5 μl of solution was injected into the system for analysis. The column flow rate was 0.4 ml/min. The mobile phase consisted of (A) water with 0.1% formic acid and (B) acetonitrile with 0.1% formic acid.
A gradient elution program was used: 95% (A)-0 in 10 min, hold 1 min and re-equilibrate in 95% (A) for 2 min. The eluate was analyzed using full MS and product ion scan (1 full scan followed by 6 MS/MS scans) in positive and negative modes. The collision energy to fragment the ions is a normalized collision energy, and nitrogen (99.999%) was used as collision gas. Spray voltage of positive mode was 3.2 kV and negative mode 3.0 kV. The range of full MS scan was between 70 and 1000 m/z.
Data were acquired by software Xcalibur 3.0. and components were identified via searching against both the online mzCloud™ spectral li- brary and in-house Thermo Scientific mzVault spectral libraries as well as literature data.
2.5. Anti-inflammatory and sedative tests of the F1 2.5.1. Carrageenan induced hind paw oedema
In order to evaluate the anti-inflammatory activity of the active F1, a mice model of carrageenan induced hind paw oedema was established as mentioned by previous studies (Gou et al., 2017; Rasheed et al., 2018).
Vehicle (10 ml/kg), F1 (50 mg/kg) and INDO (10 mg/kg) were administrated to mice, respectively. The right hind paw of each mouse was injected subcutaneously with carrageenan (30 μl, 1%) 30 min after the above treatments. Paw oedema was measured using a digital micrometer at 0, 1 2, 3, 4, and 5 h after treatment and the oedema rate was calculated.
2.5.2. Pentobarbital sodium sleep test
Pentobarbital sodium sleep test is commonly used to evaluate the sedative effect of drugs (dela Pena et al., 2015; Li et al., 2018). Physi- ological saline (10 ml/kg), 1 mg/kg DZP or F1 (50 mg/kg) was injected to mouse of each group. After 15 min, mice were administered with sodium pentobarbital (50 mg/kg) and individually placed in a cage for observation. The interval time between administration and disappear- ance of the righting reflex was recorded as the sleep latency, while the time between disappearance of righting reflex and recovery was recor- ded as the duration of sleep time.
2.6. Analgesic effect of F1 combine with antagonists
To investigate the possible mechanisms of opioid, GABAergic, dopaminergic and serotoninergic systems in the analgesic effect of F1, animals were pretreated with NXH (non-selective antagonist at opioid receptors, 7.5 mg/kg) (Sakurada et al., 2011; Komatsu et al., 2019), FM (a benzodiazepine site antagonist at the GABA-A receptor, 10 mg/kg), SCH (selective antagonist at dopamine D1 receptor, 0.05 mg/kg) and WAY (selective antagonist at 5-HT1A receptor, 0.7 mg/kg), respectively.
The administration route was intraperitoneal injection. Then, 15 min after administration of an antagonist, F1 (50 mg/kg), MOH or vehicle was injected. After 30 min, hot plate test and formalin tests were per- formed successively.
2.7. Quantification of 5-HT and 5-HIAA, Glu and GABA
Mice were decapitated immediately following the formalin test.
Brain tissues (hypothalamus, hippocampus, striatum and cortex) were dissected successively and stored at − 80 ◦C until analysis. The method for extracting 5-HT and 5-HIAA was as the same as literature (N. Zhang et al., 2016). Samples were treated with solution (0.6 mol/l perchloric acid, 0.5 mmol/l EDTA-Na2 and 0.1 g/l L-cysteine) and homogenized by ultrasound, the homogenates were centrifuged at 12,000 rpm for 20 min at 4 ◦C to obtain supernatants. The supernatants were mixed with settling agent (1.2 M dipotassium phosphate and 2 mM EDTA-Na2, 1:1, V/V), centrifuged and filtered through 0.22 μm nylon membrane for analysis. Glu and GABA were extracted and pre-column derivatized as described by a previous report (Zieminska et al., 2018).
UPLC-FLD coupled with a column (BEH C18 100 ×2.1 mm, 1.7 μm, Waters) were applied to analyze neurotransmitters. The column flow rate was 0.3 ml/min. The mobile phase consisted of (A) water with 0.1%
formic acid and (B) acetonitrile with 0.1% formic acid. An isocratic elution (95% A: 5%B) program was used for 5-HT and 5-HIAA tests.
Excitation and emission wavelengths of the fluorescence detector were 280 nm and 330 nm, respectively. Glu and GABA were detected under a gradient elution program: 95% (A)-10% in 8 min and hold 2 min, then re-equilibrate in 95% (A) for 3 min. Excitation and emission wave- lengths were 340 nm and 450 nm, respectively. Neurotransmitters and metabolites were quantified using the external standard method, and standard curves were shown in Fig. S2 (supplementary data).
2.8. Statistical analysis
Data were analyzed by one-way analysis of variance using the RStudio-1.1.456 software, followed by Tukey’s post-hoc test when there were more than two groups. Two-tailed Student’s t-test was used to analyze neurotransmitters data. All data are expressed as a mean ± standard deviation (SD).
3. Results 3.1. Analgesic tests
3.1.1. Effects of different extracts and fractions in the hot plate
In the hot plate test, MOH, and EE in medium or high concentration raised the latency time at 30 min (F(4, 25) =22.02, p <0.001), 60 min (F(4, 25) =7.63, p <0.001), and 90 min (F(4, 25) =10.91, p <0.001) (Fig. 1A). The latency time of mice to thermal stimulation increased considerably (p < 0.01) in the MOH group at each observation time point. EE at 300 mg/kg increased the latency time significantly (p <
0.05) at 30 and 90 min, and groups at doses of 150 and 300 mg/kg showed a remarkable (p < 0.01) increase in latency time at 60 min.
However, mice in PEE and HDE group showed no significant changes in latency time at all doses (Fig. 1B and C). As for fractions, 50 mg/kg F1 from EE raised the latency time significantly (p < 0.05) at 30 (F(4, 25)
=11.77, p < 0.001) and 60 min (F(4, 25) = 8.77, p < 0.001), and extremely significantly (p < 0.01) prolonged the latency time at 90 min (F(4, 25) =13, p <0.001). F2 and F3 at 50 mg/kg showed no significant influences on latency time (Fig. 1D).
3.1.2. Effects of different extracts and fractions in the writhing test Different extracts and fractions were also assessed by the writhing test, and the results were shown in Fig. 2. EE reduced the number of writhes (F(4, 25) =159.8, p <0.001), INDO avoided mice writhing remarkably (P < 0.001), EE significantly (P < 0.001) decreased the number of writhes in a dose-dependent manner compared to the control group (Fig. 2A). However, PEE and HDE showed no significant inhibi- tive effects on writhing (Fig. 2B and C). F1 rather than F2 or F3 showed a considerable (p < 0.001) decrease (F(4, 25) =47.87, p <0.001) in the number of writhes (Fig. 2D).
3.1.3. Effects of different extracts and fractions in the formalin test In the formalin test, MOH and EE decreased the licking time of mice
at the first phase (F(4, 25) =38.07, p <0.001) and the second phase (F (4, 25) =36.7, p <0.001) (Fig. 3A). Licking time of mice was totally (p
< 0.001) reduced in the MOH group at both two phases. EE showed a dose-related activity, which decreased paw licking time extremely significantly (p < 0.001) at both two phases. PEE and HDE displayed no
significant reductions on licking time at all doses (Fig. 3B and C). In addition, F1 significantly (p < 0.01) decreased licking time at the first phase (F(4, 25) =20.33, p <0.001) and showed a remarkable (p <
0.001) reduction on licking time at the second phase (F(4, 25) =20.98, p <0.001) (Fig. 3D).
Fig. 1. Analgesic effects of flower extracts at different time points in the hot plate test. (A) EE at doses of 50, 150 and 300 mg/kg; (B) PEE at doses of 50, 150 and 300 mg/kg; (C) HDE at doses of 50, 150 and 300 mg/kg; (D) fractions (F1, F2 and F3) from EE at 50 mg/kg. Vehicle (physiological saline, 10 ml/kg), MOH (10 mg/kg).
Data are presented as the mean ±SD (n =6). *p < 0.05, **p <0.01, ***p < 0.001 compared to the vehicle group.
Fig. 2. Analgesic effects of flower extracts in the acetic acid-induced writhing test. (A) EE at doses of 50, 150 and 300 mg/kg; (B) PEE at doses of 50, 150 and 300 mg/kg; (C) HDE at doses of 50, 150 and 300 mg/kg; (D) fractions (F1, F2 and F3) from EE at 50 mg/kg. Vehicle (physiological saline, 10 ml/kg), INDO (10 mg/kg).
Data are presented as the mean ±SD (n =6). *p < 0.05, **p < 0.01, ***p < 0.001 compared to the vehicle group.
Fig. 3. Analgesic effects of flower extracts in the formalin test. (A) EE at doses of 50, 150 and 300 mg/kg (B) PEE at doses of 50, 150 and 300 mg/kg; (C) HDE at doses of 50, 150 and 300 mg/kg; (D) fractions (F1, F2 and F3) from EE at 50 mg/kg. Vehicle (physiological saline, 10 ml/kg), MOH (10 mg/kg). Data are presented as the mean ±SD (n =6). *p < 0.05, **p < 0.01, ***p < 0.001 compared to the vehicle group.
Fig. 4.UHPLC-QE-MS base peak chromatograms of the F1 in the positive (A) and negative (B) modes. 1: kaempferol-3-O-rutinoside, 2: isorhamnetin-3-O-rutinoside, 3: pinoresinol-4-O-glucoside, 4: forsythin, 5: arctiin.
3.2. Identification of compounds
Chromatograms both in positive and negative ion modes were shown in Fig. 4. Five constituents (Table 1) were identified from the F1 based on the exact mass and fragment ions by comparison with available li- braries and literature data. Compound 1: kaempferol-3-O-rutinoside, compound 2: isorhamnetin-3-O-rutinoside, compound 3: pinoresinol- 4-O-glucoside, compound 4: forsythin, compound 5: arctiin. Mass spectrums of compounds were shown in Fig. S (4–8).
3.3. Effects of the F1 in anti-inflammatory and sedative tests
F1 rather than F2 or F3 showed significant analgesic actions in analgesic tests, therefore F1 was determined as the active fraction for further pharmacological evaluation. An anti-inflammatory test was carried out using the mice model of carrageenan induced paw oedema, and the results were shown in Fig. 5A. A significant (P < 0.05) reduction (F(2, 15) =5.83, p <0.05) of paw oedema rate in INDO group was recorded at 1 h after carrageenan injection, and INDO significantly (P <
0.01) inhibited paw oedema after the 1 h. However, compared with the vehicle group, F1 at 50 mg/kg behaved with a lower but not significant oedema rate.
Sedative effect of the F1 was estimated by the pentobarbital sodium sleep test and the results were exhibited in Fig. 5B. DZP and F1 affected the sleep latency (F(2, 15) =30.27, p <0.001) and the sleep time (F(2, 15) = 83.08, p < 0.001) of mice. DZP significantly (p < 0.001) decreased the sleep latency and increased the sleep time of the mice compared to the vehicle group. Similarly, F1 at 50 mg/kg also signifi- cantly (p < 0.001) decreased the sleep latency and increased the sleep time to a certain extent (p < 0.05).
3.4. Effects of antagonists on the analgesic effect of the F1
In the hot plate test (Fig. 6A) and the first (Fig. 6B) and second phases (Fig. 6C) of the formalin test, pretreatment with antagonists only did not show any antinociceptive effect, same as the vehicle group. The anal- gesic activity of F1 was not blocked by SCH almost in two mice models.
NXH significantly (p < 0.001) avoided the analgesic effect of MOH but not influenced the function of F1. However, the analgesic effect of F1 in the hot plate test (F(11, 60) =10.51, p <0.001) and the first phase (F (11, 60) =17.17, p <0.001) of the formalin test were significantly (p <
0.05) inhibited by FM. Furthermore, WAY not only reversed the anal- gesic action of F1 in the same manner (p < 0.05) as FM in the hot plate test, but also totally abolished its response in the first phase (p < 0.001) and the second phase (F(11, 60) =17.29, p <0.001) (p < 0.01) of the formalin test.
3.5. Effects of the F1 on neurotransmitters in brain tissues
Chromatograms of standards and samples were presented in Fig. S3.
Contents of 5-HT and 5-HIAA in the hippocampus and striatum of mice
were affected by F1 (Fig. 7A and B), and both contents of 5-HT and 5- HIAA in the F1 group increased significantly in hippocampus (p <
0.05) and striatum (p < 0.01) compared to the control group. However, no significant changes were observed in hypothalamus and cortex re- gions. Incidentally, the 5-HIAA/5-HT ratio of the F1 group and the control group in four brain regions showed no significant changes. In addition, there were no significant differences in contents of Glu and GABA in the F1 and control groups (Fig. 7D and E).
4. Discussion
Studies on the phytochemicals and pharmacology of flowers from Styrax japonicus have been scarcely reported before. As a Chinese folk medicine, it is often used to relieve pain (G. Q. Wang, 2014). Compo- nents of plant extracts are very complicated, which can be divided into volatile, semi-volatile and non-volatile substances, according to vola- tility (Torri et al., 2010; Balestrini et al., 2021). In this study, in order to comprehensively verify the analgesic effect of the flower extracts, EE, PEE and HDE were obtained and only the chemicals from EE were proved to perform analgesic effect in mice models (Figs. 1–3). It is suggested that non-volatile substances from the flower are the main active chemicals for analgesia. Nevertheless, EE is a crude extract with a lot of impurities. Therefore, after depigmentation, three fractions from the EE were isolated. And only the F1 was proved to be active, simpli- fying the analysis of complex analgesic components.
UHPLC-QE-MS was used to detected components of F1. Two flavo- noids were identified: kaempferol-3-O-rutinoside (Compound 1) and isorhamnetin-3-O-rutinoside (compound 2). Compound1 showed a [M+H]+ion at m/z 595.1653 and a [M-H]- ion at m/z 593.1519, which produced fragments at m/z 449.1071, 287.0544 and 284.0329, 255.0302, respectively. According to a previous report (Yin et al., 2020), kaempferol-3-O-rutinoside displayed a molecular ion [M+H]+at m/z 595.1651 and produced the two same fragments at m/z 449 and 287.
Compound 2 had molecular ions [M+H]+at m/z 625.1758 and [M-H]- at m/z 623.1625, then it was identified as isorhamnetin-3-O-rutinoside considering the loss of rutinoside to produce a major fragment at 315.05 in the negative mode (Tkacz et al., 2020). Compound 3 ([M +NH4]+at m/z 538.2280, [M-H]- at m/z 519.1874) was identified as a lignan. The main fragments (357.1348, 151.0402, 136.0166) corresponded to pinoresinol-4-O-glucoside, which showed a [M-H]+ion at m/z 519.1873 and fragments at 357.1335, 151.0399 and 136.0161 (Zhang et al., 2020). Compound 4 and compound 5 displayed similar molecular ions as well as main fragments (Table 1). A key fragment of compound 4 at m/z 249.1117 in the positive mode corresponded to forsythin (Yang et al., 2013), while compound 5 was tentatively identified as arctiin based on a previous report (Dias et al., 2017).
Among these five compounds, kaempferol-3-O-rutinoside isolated from Ouratea fieldingiana has a potential pharmacological effect for the treatment of acute orofacial pain, and extracts from Carthamus tinctorius L. possess remarkable antinociceptive effect possibly due to kaempferol- 3-O-rutinoside (Y. Wang et al., 2014; do Nascimento et al., 2018).
Table1
Analysis of the F1 by UHPLC-QE-MS.
RT (min)
Compound Formula m/z Adducts MS/MS
+ – + – + –
4.14 Kaempferol-3-O-
rutinoside C27 H30
O15
595.1653 593.1519 M +H M-H 449.1071, 287.0544 284.0329, 255.0302
4.19 Isorhamnetin-3-O-
rutinoside C28 H32
O16
625.1758 623.1625 M +H M-H 317.0651 315.0512, 314.0435,
299.0199 4.32 Pinoresinol-4-O-
glucoside C26 H32
O11
538.2280 519.1874 M +
NH4 M-H 235.0962, 175.0753, 137.0596 357.1348, 151.0402, 136.0166
4.89 Forsythin C27 H34
O11
552.2435 579.2087 M +
NH4 M +CH2O2-
H 355.1534, 337.1429, 249.1117,
189.0908 371.1502, 356.1269
4.99 Arctiin C27 H34
O11
552.2439 579.2090 M +
NH4 M +CH2O2-
H 373.1638, 355.1534, 337.1428,
151.0752 371.1503, 356.1270
Moreover, Glycosides of isorhamnetin and pinoresinol have the anti- nociceptive activities (Kawano et al., 2009; Kucukboyaci et al., 2016). It could be inferred that kaempferol-3-O-rutinoside is at least an analgesic substance in the active fraction. However, there were still some
substances that are difficult to be identified. Further separation and more accurate identification of the F1 components are needed to find out the potential analgesic compounds.
In analgesic assays, the hot plate test is helpful to estimate central Fig. 5.(A) Effect of the F1 at 50 mg/kg in the carrageenan induced hind paw oedema test; (B) Effect of the active F1 at 50 mg/kg in the pentobarbital sodium sleep test. Vehicle (physiological saline, 10 ml/kg), DZP (1 mg/kg). Data are presented as the mean ±SD (n =6). *p < 0.05, **p < 0.01, ***p < 0.001 compared to the vehicle group.
Fig. 6.Analgesic effect of the F1 at 50 mg/kg in combination with antagonists in the hot plate test (A); the first phase (B) and the second phase (C) of formalin test.
Vehicle (physiological saline, 10 ml/kg), MOH (10 mg/kg), NXH (7.5 mg/kg), FM (10 mg/kg), SCH (0.05 mg/kg) and WAY (0.7 mg/kg). Data are presented as the mean ±SD (n =6). *p < 0.05, **p <0.01, ***p < 0.001 compared to the vehicle group. #p < 0.05, ##p < 0.01, ###p < 0.001 compared to MOH or F1 group.
Fig. 7. Changes in contents of neurotransmitters in four brain regions of the mice after treatment with F1 at 50 mg/kg. Contents of 5-HT (A), 5-HIAA (B) and the ratio of 5-HIAA/5-HT (C) in four brain regions; Contents of Glu (D) and GABA (E) in four brain regions. Control (physiological saline, 10 ml/kg). Data are presented as the mean ±SD (n =6). *p < 0.05, **p < 0.01 compared to the control group.
nociception, the writhing test to assess peripheral nociception, and the formalin test to evaluate peripheral neurogenic nociception in the first phase and inflammatory nociception in the second phase as described in previous researches (Tlili et al., 2018; Lopes et al., 2019; Tesfaye et al., 2020). Similar to MOH, EE and the active F1 relieved pain in all test models. They prolonged the latency time of mice to thermal stimulation at each time point in the hot plate test (Fig. 1A, D), reduced the number of writhes of mice in writhing test (Fig. 2A, D), and decreased the licking time of mice in both phases of formalin test (Fig. 3A, D). However, it is doubtful whether the analgesic mechanism of EE is the same as that of MOH, which perform analgesia or antinociception via binding to opioid receptors (Ahmad et al., 2019). Then the active F1 at an effective dose of 50 mg/kg was adopted to implement experiments to disclose the anal- gesic mechanisms.
An inflammatory model of carrageenan induced mice paw oedema was established, and revealed that F1 at dose of 50 mg/kg was enough to incur analgesia (Figs. 1–3) but showed no significant anti-inflammatory response (Fig. 5A). Admittedly, this was not sufficed to indicate that F1 could not exhibit an anti-inflammatory action, since the dose may be insufficient, but it can be inferred that the analgesic effect of F1 was not achieved by inhibiting the process of inflammation, especially for the pain caused by inflammation. Interestingly, the mice developed drowsiness when treated with EE and F1, then the pentobarbital sodium sleep test was performed which proved that F1 had a sedative effect, which shortened the sleep latency and prolonged the sleep time of mice (Fig. 5B). Most of the sedative principles of sedative drugs involve neurotransmitter systems, especially the serotonergic and GABAergic systems (F. Y. Zhang et al., 2016; Lee et al., 2017; Oh et al., 2019; Shi et al., 2019). Some plant extracts such as glucuronoxylan from chamo- mile tea and phenolic compounds from Vernonia patula have both sedative and analgesic effects (Chaves et al., 2020; Siraj et al., 2021), and peshawaraquinone isolated from Fernandoa adenophylla presents both effects which are mediated by the same pathway (Alhumaydhi et al., 2021). Those results suggested that the analgesic activity of EE or F1 was related to sedative effect, which was probably mediated by neurotransmitter rather than the anti-inflammatory pathway.
The hippocampus plays an important role in brain function which includes the response to pain, and striatum can receive the processing information of pain signals (Yang et al., 2008; Ahmadi et al., 2016).
These seem to be verified in this study as significant increases of contents of 5-HT and 5-HIAA in F1 group were observed in the hippocampus and striatum of mice (Fig. 7A and B). Meanwhile, a stable ratio of 5-HIA- A/5-HT in this study indicated that F1 could not regulate the meta- bolic rate of 5-HT.
The transmission of neurotransmitters requires the cooperation of corresponding receptors. Among varieties of receptors of 5-HT, the 5- HT1A receptor occupies an important position as a target for treatment of pain (Newman-Tancredia et al., 2018). Unlike MOH, the analgesic effect of F1 cannot be reversed by NXH, but abolished by WAY (a selective antagonist for 5-HT1A receptor) in the hot plate test (Fig. 6A) and almost completely suppressed by WAY in the formalin test (Fig. 6B and C), which indicated that the mechanism of F1 is closely dependent on the 5-HT/5-HT1A pathway but not the opioid system. As for GABA/GABA-A
pathway, no obvious changes were observed in the contents of Glu and GABA in different brain regions of mice (Fig. 7D and E), but the anal- gesic effect of the F1 was partially inhibited by FM (a benzodiazepine site antagonist at the GABA-A receptor), which prefigured that the further research should be focused on receptors. As a consequence, the analgesic effect of the F1 was not mediated by the opioid system, but partially involved in the GABAergic system and highly related to the serotonergic system.
5. Conclusion
This is the first time to report that EE from flowers of Styrax japonicus and F1, the active fraction from EE, performed antinociceptive effects at
central and peripheral levels. Kaempferol-3-O-rutinoside, isorhamnetin- 3-O-rutinoside, pinoresinol-4-O-glucoside, forsythin and arctiin were identified from components of F1. And kaempferol-3-O-rutinoside is likely to be one of the analgesic compounds. The analgesic effect of F1 appeared to be related to the sedative effect, partially mediated by the GABAergic system, and highly involved in the serotonergic system, but stayed away from the opioid system and the anti-inflammatory pathway.
Our study confirmed the analgesic effect of the flower used as a folk medicine and preliminarily explored its analgesic mechanism, which proposed EE as a good candidate for non-opioid analgesics.
Funding
This work was supported by the National Key Research and Devel- opment Program of China (grant number 2019YFA0706200), and Nanjing Fragrant Jasmine Agricultural Technology Co., Ltd [grant number 19H2H22000212].
CRediT authorship contribution statement
Lei He: Investigation, Visualization, Writing – original draft, prep- aration, Software. Ying Zhou: Investigation, Visualization, Writing – review & editing. Guangjun Wan: Conceptualization, Methodology.
Wencui Wang: Investigation, Visualization. Nan Zhang: Investigation, Visualization, Writing – review & editing. Lei Yao: Conceptualization, Methodology, Writing – review & editing, Supervision.
Declaration of competing interest
The authors declared that there are no conflicts of interest.
Acknowledgments
We are grateful to Nanjing Fragrant Jasmine Agricultural Technol- ogy Co., Ltd for supplying flowers of Styrax japonicus for this project.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jep.2021.114779.
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Glossary EE: ethanol extracts
PEE: petroleum ether extracts HDE: hydrodistillation extracts
UHPLC-QE-MS: ultrahigh-performance liquid chromatography Q extractive mass spectrometry
UPLC-FLD: ultraperformance liquid chromatography with a fluorescence detector 5-HT: 5-hydroxytryptamine
GABA: γ-aminobutyric acid MOH: Morphine hydrochloride DZP: Diazepam
NXH: naloxone hydrochloride FM: flumazenil
SCH: SCH23390 WAY: WAY100635 INDO: Indomethacin
