Zinc oxide nanoparticles synthesized from Fraxinus rhynchophylla extract by green route method attenuates the chemical and heat induced neurogenic and inflammatory pain models in mice

Journal Pre-proof Zinc oxide nanoparticles synthesized from Fraxinus rhynchophylla extract by green route method attenuates the chemical and heat induced neurogenic and inflammatory pain models in mice

Zhi Wang, Bin Que, Jianhui Gan, Hao Guo, Qiang Chen, Lina Zheng, Najat Marraiki, Abdullah M. Elgorban, Yi Zhang PII:

S1011-1344(19)31220-5

DOI:

https://doi.org/10.1016/j.jphotobiol.2019.111668

Reference:

JPB 111668

To appear in:

Journal of Photochemistry & Photobiology, B: Biology

Received date:

13 September 2019

Revised date:

16 October 2019

Accepted date:

22 October 2019

Please cite this article as: Z. Wang, B. Que, J. Gan, et al., Zinc oxide nanoparticles synthesized from Fraxinus rhynchophylla extract by green route method attenuates the chemical and heat induced neurogenic and inflammatory pain models in mice, Journal of Photochemistry & Photobiology, B: Biology(2018), https://doi.org/10.1016/ j.jphotobiol.2019.111668

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© 2018 Published by Elsevier.

Journal Pre-proof Zinc oxide nanoparticles synthesized from Fraxinus rhynchophylla extract by green route method attenuates the chemical and heat induced neurogenic and inflammatory pain models in mice

Zhi Wang1, Bin Que2, Jianhui Gan3, Hao Guo1,Qiang Chen1,Lina Zheng1, Najat Marraiki4,

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Abdullah M. Elgorban4,5,Yi Zhang6,* [email protected] Department of Anesthesiology, Shanxi Provincial People's Hospital, Taiyuan, Shanxi Province

Department of anesthesiology, Hangzhou Hospital of Traditional Chinese Medicine, Hangzhou,

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2

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030000, P.R. China

3

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Zhejiang Province, 310007, China

Department of Anesthesiology, the Affiliated Tangshan People Hospital of North China

Department of Botany and Microbiology, College of Science, King Saud University, P.O.Box

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4

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University of Science and Technology, Tangshan ,Heibei Province,063000,China

2455Riyadh 11451, Saudi Arabia

Centre of Excellence in Biotechnology Research, King Saud University, P.O Box 2455, Riyadh,

11451, Saudi Arabia

Department of Anesthesiology, Tongji Hospital Affiliated Tongji Medical College, Huazhong

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6

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5

Science and Technology University，Wuhan, Hubei Province,430030,China *

Corresponding author at: Department of Anesthesiology, Tongji Hospital Affiliated Tongji

Medical College, Huazhong Science and Technology University，Wuhan, Hubei Province,430030,China.

Abstract Fraxinus rhynchophylla belongs to the family of Oleaceae and also called as Chinese ash wood possesses various pharmacological properties such as neuroprotective, antimicrobial, anti-

Journal Pre-proof inflammatory, etc. Therefore we synthesized ZnO nanoparticles using Fraxinus rhynchophylla wood extract as reducing and capping agent. The synthesized nanoparticles were characterized with the aid of UV-Spec, DLS, FT-IR and TEM analysis. Green synthesized ZnO nanoparticles were then assessed for anti-nociceptive property by using various nociception models such as thermal stress-induced, acetic acid, glutamate, capsaicin, and formalin-induced nociception. The sedative effect of synthesized ZnO nanoparticles was evaluated with an open field test. UV-

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Spectroscopic analysis confirms the formation of ZnO nanoparticles and the characterization

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studies DLS, FT-IR, and TEM analysis prove it has ideal nanoparticle can be used as a nano-

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drug. Results of both thermal stress-induced methods hot plate and tail immersion nociception

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test verified the synthesized ZnO nanoparticles are a potent antinociceptive drug. ZnO nanoparticles effectively reduced the abdominal writhes in acetic acid-induced nociception and it

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also significantly decreased the nociception activity in another glutamate, capsaicin, and

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formalin-induced nociception models. Open field experiment proved that synthesized ZnO nanoparticles are less sedative compared to the standard antinociceptive drug morphine. Overall

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our findings authentically confirm ZnO nanoparticles synthesized from Fraxinus rhynchophylla

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wood extract is a novel drug that persuasively reduces nociception in different nociceptive induced mice models and can be the best alternative for allopathic drugs which renders severe side effects. Keywords: Green nanosynthesis, Fraxinus rhynchophylla, Ash wood, ZnONP, antinociception, nociceptive models Introduction Pain is a major physiological response to the mechanical damage of tissues and inflammation which can be stimulated by widespread factors and it is a primary indicator of

Journal Pre-proof tissue injuries [1]. Sensation of pain was distinguished in many categories and each was stimulated by different stimuli, whereas all kinds of pain seriously affected the well beings and result in severe physical uneasiness [2]. The pain is an important indicator of inflammation and it is mediated by various chemical mediators through molecular routes, moreover potassium ions, prostaglandins and bradykinins can unswervingly stimulates the nociceptive receptors [3, 4]. Nanotechnology, a blooming interdisciplinary field evolved from biological, material

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engineering science and technology has wide usage in various applications in agriculture, food,

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drug delivery, bio-imaging, etc [5]. Nanoparticles are the single atoms or molecules which are Nanoparticles are unique in their

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contrived to render various applications independently.

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physical properties, chemical and optical properties which portray them as an ideal material to be used in enormous fields such as environment, medicine, agriculture, cosmetics, communication,

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bio-imaging, drug delivery, etc [6, 7]. Recently synthesis of metal oxide nanoparticles and its

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applications in the field of medicine were focused by the researchers. Metal oxides proved to induce inhibit growth and induce apoptosis in oncogenic cells [8, 9], possess antimicrobial,

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wound healing and anti-inflammatory properties [10, 11].

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Zinc oxide (ZnO) nanoparticles are focused by the researcher to be utilized both in the field of industries and as well in the clinical field. ZnO possesses high transmittance, high electron mobility and it is a versatile semiconductor with increased excitation binding energy at room temperature [12]. Zinc oxide exhibits distinctive physicochemical properties hence it utilized in rubber industries [13], cosmetics [14], food additives, sensors [15], solar cells [16] Zinc is a vital trace element present in brain, bone, skin involves in the neurogenesis, hematopoiesis and various other metabolic activities [17]. Compared to other nanoparticles zinc is highly biocompatible and it is approved by the Food and Drug Administration (FDA) of the

Journal Pre-proof USA. Synthesis of ZnO nanoparticles are inexpensive, they are less toxic and proven to possess anticancer, antidiabetic, anti-inflammatory, antibacterial [18, 19], antifungal, larvicidal, wound healing properties [20, 21]. They are used in the field of biosensing, cell imaging, targeted drug delivery, etc nano-medicine [22]. Zinc oxide nanoparticles are mostly synthesized employing the physical and chemical

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techniques which are often expensive, time-consuming and also the chemical used as a reducing agent are often toxic and non-biocompatible [23]. Hence it is need of today to synthesis zinc

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oxide nanoparticles using an economic, eco-friendly, nontoxic alternative method. Recently it

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has been reported the plant materials serve as a reducing and capping agent that converts

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inorganic metals into metal nanoparticles [24-26]. Fraxinus rhynchophylla commonly known as

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ash tree belongs to the family of Oleaceae [27], possesses various pharmacological properties

and diuretic [28, 29].

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such as anticancer, antioxidant, antiobesity, antimicrobial, hepatoprotective, anti-inflammatory

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Fraxinus rhynchophylla utilized in traditional Chinese medication to cure various

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ailments, therefore, we synthesized biocompatible zinc oxide nanoparticles using Fraxinus rhynchophylla bark extract. Main objective of this current work is to explore the antinociceptive efficiency of FR-ZnONPs in animal model. In this research work, we assessed the antinociceptive property of green synthesized ZnONPs by using glutamate, capsaicin, and formalin-induced nociceptive mice models. Since treating nociception/pain is of global concern of today and the commonly prescribed non-steroidal anti-inflammatory drugs render stern side effects such as renal disorders, edema, hemorrhage, hepatic and cardiovascular disorders [30-32]. Materials & Methods

Journal Pre-proof Chemicals Zinc acetate, Tween 80, morphine, naloxone, diclofenac sodium, capsaicin, glutamate, formalin, and acetic acid were purchased for Sigma Aldrich, USA. All the other chemicals used for the present study were of high-quality molecular grade. Preparation of Fraxinus rhynchophylla

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The stem barks of Fraxinus rhynchophylla were purchased from the local vendors and

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barks were dried nicely in the shade for 48h. The dried barks were then powdered using an

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electric blender and the powder was subjected extraction using a 70% aqueous acetone solution.

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The solution was filtered with Whatman filter paper and evaporated at a vacuum rotor to remove alcohol remnants. The extract was used as a reducing agent for the further synthesis of zinc oxide

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nanoparticles

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Green synthesis of Zinc Oxide nanoparticles

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20ml of bark extract was added drop by drop to 50 ml of 1mM zinc acetate solution placed on the magnetic stirrer at room temperature. The solution was then placed on the water

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bat at 60˚C for 2h for the complete reduction of zinc hydroxide. Zinc oxide nanoparticles precipitates were separated by centrifugation at 10000 rpm for 15min. The centrifugation was repeated thrice by dissolving zinc oxide precipitate in distilled water to obtain pure form of zinc oxide nanoparticles [33]. The precipitate was then vacuum dried and subjected to further characterization. Characterization of FR-ZnONP UV-Visible spectroscopy analysis of FR-ZnONP

Journal Pre-proof The synthesis of FR-ZnONP was confirmed with UV-Vis spectroscopic analysis. FRZnONP were subjected to UV-Vis spectroscopic analysis between the spectrum range of 300700nm, λ25 spectrophotometer, Perkin Elmer. The surface Plasmon resonance peak of FRZnONP was measured by plotting the wavelength on the X axis and the absorbance on the Y axis.

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Dynamic Light Scattering measurements of FR-ZnONP

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The average size and distribution pattern of the green synthesized FR-ZnONP was

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FTIR spectroscopic Analysis of FR-ZnONP

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assessed using a Zeta Sizer Dynamic Light Scattering instrument from Malvern, United States.

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FR-ZnONP was subjected to Fourier-transform infrared spectroscopic analysis with a JASCO FTIR instrument-410, United States to assess the functional groups of FR-ZnONP. FR-

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ZnONP were blended with 1% potassium bromide pellets and scanned between the wavelengths

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of 500-4000 cm-1 at room temperature. At a resolution of 4cm/scan, about 50 scans were

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completed and the data were analyzed using the WINFIRST software, Mattson, United States. Transmission electron microscopic analysis of FR-ZnONP FR-ZnONP was assessed using high resolution transmission electron microscope, JEOL, Japan to determine the morphology and size of the synthesized nanoparticles. FR-ZnONP were placed on to a carbon coated 400 mesh copper grid of TEM, dried at room temperature and scanned at 120kV accelerating voltage. The images of FR-ZnONP were captured and 200 particles from several TEM images were analyzed using Image J software to detect the size of the particle.

Journal Pre-proof Antinociceptive property of FR-ZnONP Experimental Animals Healthy young male Swiss Albino mice were employed for the current study to assess the anti-nociceptive property of green synthesized FR-ZnONP. Animals were procured from the institutional animal house, Tongji Hospital Affiliated Tongji Medical College, Huazhong

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Science and Technology University，Wuhan, Hubei Province, China, and the mice were

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acclimatized in the laboratory condition at a temperature of 23ºC±2ºC and relative humidity of

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53±7 %. The mice were bedded with rice husk and 12h light and dark cycle was maintained. The bedding was changed daily and cages were replaced every three days. The mice were

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allowed free to access standard pellet diet and water ad libitum except during behavioral analysis

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the mice fasted overnight on the prior day of the experiment. The behavioral analyses on mice

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were carried out early morning between 5.00am to 8.00am. The mice were treated with the utmost more care and caution, all procedures carried out in the present study were approved by

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the intuitional ethical committee. For experimentation, animals were divided into five groups and

Hot Plate test

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each group contains six animals.

Healthy mice sensitive to thermal stress were only selected for the experiment to avoid false results. The thermal sensitive animals were grouped as Group I mice which were considered to control group which received only the vehicle 1 % tween 80, Group V mice are positive drug control group and Group IV are negative drug control group which were treated with standard antinociceptive drug morphine at dose of 5mg/kg b.wt and opioid antagonist naloxone (2mg/kg b.wt) respectively. Group II, III, and IV were treated with different doses of

Journal Pre-proof green synthesized FR-ZnONP 5, 10 and 15mg/kg b.wt respectively. To confirm the potency of FR-ZnONP antinociceptive property the mice of Group VII, VIII and IX were treated with both 2mg/kg bwt of opioid antagonist naloxone and different dose of green synthesized FR-ZnONP 5, 10 and 15mg/kg b.wt respectively. Group X mice were treated with both 2mg/kg bwt of opioid antagonist naloxone and 5mg/kg bwt of standard drug morphine. Eddy’s hot plate was performed using the protocol of Turner (1965) [34] 30 min after the

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treatment period the mice were placed on to the hot plate maintained at the temperature of 50ºC

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for a period of the 20s, the first behavior changes such as paw licking or hoping were noted. The

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experiment was carried for 2h with an interval of 30min and the readings were recorded.

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The percentage of the maximal possible effect of each mouse was calculated using the equation

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Hot water Tail Immersion test

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%MPE=[{(Postdrug latency)−(Predrug latency)}{(Cut-off time)−(Predrug latency)}]×100.

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The mice which are thermal sensitive whose tail deflection time was 1.5 -2.5 sec were

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only chosen for the tail immersion test. Grouping of hot plate method was followed for the Hot water Tail Immersion test also. Mice were pretreated 30 min before the initiation of the experiment, 5 cm tail of mice were immersed into the hot water maintained at 55ºC, the time taken for the mice to deflect or withdraw the tail was recorded. Each mouse was subjected to an experiment for a period of 20sec and the tail was dried with a clean towel before replacing the mice into the home cage [35]. The experiment was performed until 2h after the treatment period at the interval of 30min and the percentage of MPE was calculated using the formula %MPE=[{(Postdrug latency)−(Predrug latency)}{(Cut-off time)−(Predrug latency)}]×100.

Journal Pre-proof Abdominal Writhing test The mice were grouped into five groups consisting of six mice, group I was considered as control mice with 1 % of tween 80, group V are positive drug control mice treated with 10mg/kg b.wt of standard analgesic drug diclofenac sodium. Group II, III and IV were treated with three different concentrations of FR-ZnONP 5, 10 and 15 mg/kg b.wt respectively. One hour after the treatment the mice were treated with 1%v/v acetic acid intraperitoneally and the mice were

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placed into the observation chamber. The numbers of writhing episodes like stretching

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movement, elongation of body and hind limbs were recorded [36]. The number of writes

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performed by mice of each group within 30 min was recorded.

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Excitatory neurotransmitter Glutamate induced nociception test

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Glutamate induced nociception test was performed according to the protocol of Beirith et al. (2002) [37]. 10 μmol/paw of glutamate was injected to the right paw ventral surface of the

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mice which were pretreated with drugs 30 min before initiation of the experiment. The pain

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behavior of mice such as biting or licking of paw performed by each mouse was recorded.

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Neurogenic nociception test

The potency of FR-ZnONP against neurogenic pain was assessed using Capsaicin induced paw licking test model [38]. Mice were pretreated with 1% tween 80 (Control), three different concentrations of FR-ZnONPs (5, 10 and 15 mg/kg b.wt) and standard drug diclofenac sodium (10mg/kg b.wt). After 30 min of treatment, 1.6µg/ paw of capsaicin (20µl) was intraplantar injected into the ventral surface of the right hind paw of the mice. The mice were placed into the observation chamber for 10 min and the number of lickings was recorded for each group of mice.

Journal Pre-proof Formalin induced nociceptive test The antinociceptive effect of FR-ZnONP was confirmed by formalin-induced nociceptive test [39]. 30 min before the initiation of the experiment the mice were grouped into five and treated with 1% tween 80 (Control), three different concentrations of FR-ZnONP (5, 10, 15 mg/kg b.wt) and standard drug morphine (5 mg/ kg b.wt). 2.5% v/v formalin (20µl) solution

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prepared with distilled water injected subcutaneously to the mice right hind paw’s ventral surface. The mice were then placed inside the observation chamber for 35 min and the number of

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lickings performed by the mice was observed in the biphasic, initial phase or nociceptive phase

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(0-5min) and final phase or inflammatory phase (15-35min).

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Open Field test

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The mice were grouped in five and treated with 1% Tween 80 (control), with three varied

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concentrations of FR-ZnONPs (5, 10, 15 mg/kg b.wt) and standard drug morphine (5mg/kg) as positive control. The mice were positioned in the center of open field apparatus which is box

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measuring about 50 cm (length) × 50 cm (width) × 38 cm (height) and divided into 25 squares

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made up of high-density non-porous plastic. The movement of mice was recorded and each mouse was assessed for behavioral changes for 5 min. The number of squares crossed by the mice was counted and the apparatus was wiped with mild ethanol before experimenting with new mice.

Statistical Analysis

Journal Pre-proof All the data recorded during each experiment were evaluated statistically with the help of statistics software Graph pad prism (version 7). The results were evaluated using One-way Analysis of Variance followed by Dunnet’s post hoc test and they were stated as mean ± standard error mean. P values p<0.05, p<0.01 respectively were considered to be statistically significant.

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Results

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Characterization of FR-ZnOPs

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Fig 1a depicts the surface Plasmon resonance peak of green synthesized FR-

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ZnOPs, the absorption observed at 370nm confirms the synthesis of zinc nanoparticles. Fig 1B illustrates the morphological image of FR-ZnONPs observed through high-resolution

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transmission electron microscopy. The nanoparticles were spherical, some agglomeration of

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nanoparticles was observed with an average size of 100-200nm.

Fig 2 signifies the zeta potential of green synthesized Fr-ZnONPs which confirms the

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stability of synthesized nanoparticles. FR-ZnONPs showed a peak at -28.4mV which indicates the increased stability of the nanoparticles. Fig 3 represents the FTIR spectrum peaks of green synthesized FR-ZnONPs. The peaks were seen between 500-4000 cm-1. Sharp peaks were observed at 535, 599, 822, 1058, 1194, 1340, 1567, 1697 cm-1 and few blunt peaks were observed at 3012, 3585 cm-1. The peak observed at 535, 599 cm-1 confirms the formation of zinc oxide and the peak at 1340 cm-1 may due to nitrate bonding. The blunt peak observed at 3010 cm-1 may be due to the C-H stretching and peak 3585 cm-1 contributes to the hydroxyl absorption.

Journal Pre-proof FR-ZnONP antinociceptive property effect Eddy’s Hot Plate test Table 1 illustrates the potency of mice treated with green synthesized FR-ZnONP to withstand the thermal stress. Compared to the control mice, the FR-ZnONP treated mice shown increased resistance to thermal stress in a dose-dependent manner. 2h after the treatment the

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mice treated with 5, 10, 15 mg/kg b.wt of FR-ZnONPs shown maximal possible effect

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percentage of 36.43%, 42.37%, 54.29% respectively which significantly greater than the MPE

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percentage of control mice. The naloxone opioid antagonist treated mice also responded well to the treatment of 15mg/kg b.wt FR-ZnONP, where the %MPE at 2h was 27.47% which was

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almost equal to the result of mice treated with naloxone and standard drug opioid (33.47%).

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Hot water Tail Immersion test

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Table 2 depicts the mice maximal response time to the thermal stimuli and percentage of maximum possible effect rendered by control and experimental groups. The response time was

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decreased with the duration of time compared to the control mice (6.69±0.13), the FR-ZnONPs

0.82,

9.96

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treated mice shown increased response time in a dose-dependent manner (7.79 ± 0.82, 8.89 ± ±0.74

respectively).

Compared

to

morphine

treatment

(7.79±0.41)

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10 mg/kg/b.wt FR-ZnOPs (9.49±0.47) and 15mg/kg/b.wt FR-ZnOPs (8.89±0.36) treatment shown increased maximal response time in naloxone co-treated mice which depicts the effect of FR-ZnONPs to revert the effect of opioid antagonistic effect of naloxone. Acetic acid induced writhing test Fig 4 depicts the number of abdominal writhings induced by acetic acid in control and FR-ZnONPs treated mice. Compared to control mice (45±3 writhes), the numbers of abdominal

Journal Pre-proof writhed were observed in FR-ZnONPs mice were significantly decreased in a dose-dependent manner (32±4, 26±6, 17±3 writhes respectively). The writhing number was significantly less in standard analgesic drug diclofenac sodium treated mice than the control mice. Glutamate induced nociception test Fig 5 signifies the nociceptive potency of FR-ZnOPs against the excitatory

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neurotransmitter glutamate pain induction. Standard drug diclofenac sodium drastically reduced

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the paw licking to 24±5 licks compared to the control mice 97±4 licks. FR-ZnONPs treatment

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also significantly reduced the licking specifically 15mg/kg b.wt Fr-ZnONPs treated mice shown

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only 37±5 licks which not even 50% of licks observed in control mice.

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Neurogenic nociceptive test

Fig 6 shows the effect of Fr-ZnONPs against the capsaicin-induced neurogenic pain in

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mice model. The number of licks performed by the standard drug diclofenac sodium treated mice

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(30±5 licks) and 15mg/kg bwt FR-ZnONPs treated mice (38±7 licks) were comparatively equal and they are significantly decreased compared to the number of licks performed by the control

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mice (83±4 licks), which confirms the antinociceptive potency of FR-ZnONPs. Formalin induced nociception test Fig 7 depicts the results of FR-ZnONPs treatment against the formalin induced nociception in phase dependent manner. During both the initial phase and the final phase of formalin induced nociception compared to the control mice (95±6, 128±8) the 15 mg/kg bwt FRZnONPs treated mice (35±4, 68±3 respectively) significant reduction in licking numbers and is

Journal Pre-proof comparatively equal to the standard drug diclofenac sodium injected mice (26±5, 52±3 respectively). Open field test Fig 8 signifies the sedative effect induced by the different doses of FR-ZnONPs and the standard drug morphine. Even though the number squares crossed by 15mg/kg bwt FR-ZnONPs (156±8 squares) is significantly reduced compared to the control mice (250±10 squares), it is

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comparatively more than the standard drug morphine treated mice (128±9 squares). Lower dose

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5 mg/kg b.wt FR-ZnONPs treated mice (194±6 squares) significantly increased the number of

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squares compared to the standard drug morphine treated which confirms that FR-ZnONPs

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doesn’t induce sedation in mice.

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Discussion

Zinc oxide naoparticles (ZnONP), once extensively used in the rubber industry, cosmetic

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manufacturing, paint, textile manufacturing, used as anti-reflector, UV light emitters etc

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propagator [40]. Due to the unique physical and chemical properties of ZnONPs it used the clinical diagnostic field and used in the biomedical field to render targeted drug delivery [41,

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42]. Green synthesis of ZnONP which are ecofriendly and biocompatible paved the way for researchers to analyze the potency of ZnONP in treating various diseases. We synthesized the ZnONP using the extract of Fraxinus rhynchophylla and it is confirmed with the UVSpec analysis which had shown surface Plasmon resonance peak at 370nm. The phytochemicals fraxisecoside, esculin, naringenin present in the Fraxinus rhynchophylla extract [43] would have reduced the zinc acetate to zinc oxide nanoparticles [44]. FTIR peaks observed at 535, 599 cm-1 confirms the formation of zinc oxide and the peak at 1340 cm-1 may due to the nitrate bonding. The blunt peak observed at 3010 cm-1 may be due to

Journal Pre-proof the C-H stretching and peak 3585 cm-1 contributes to the hydroxyl absorption which signifies that the phytoconstituents of Fraxinus rhynchophylla acted as capping and stabilizing agent during nanoparticle synthesis. The zinc oxide nanoparticles synthesized were spherical shape and shown -28.4mV zeta potential which indicates that the nanoparticles had minimum surface energy and high thermodynamic stability [45]. Agglomeration of nanoparticles were observed in TEM image which may due to the phytochemicals the results correlates with the previous studies

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synthesized using biological synthesis method [46]. Our characterization analysis confirms the

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green synthesized nanoparticles as an ideal material can be used as drug.

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Inflammation sensitizes the nociceptors and somatosensory neurons causing

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hyperalgesia or chronic pain [47]. Most of patients were prescribed with non steroidal anti inflammatory drugs (NSAID) to alleviate pain eventhough it effectively manages pain the long

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term usage leads to various side effects like heartburn, stomachache, dizziness, increased blood

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pressure and allergic reactions [32]. Therefore in the current study we assessed the potency of FR-ZnONP as antinociceptive which may be alternative for NSAID. An organism reflex

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response to an external stimuli termed as nociception which may be triggered chemically,

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mechanically, electrically and thermal induction [48]. The antinociceptive properties of a newly developed drug are usually evaluated with in vivo models. In the present study, we performed thermal induced nociceptive models and chemical induced nociceptive models. The two best models to assess the analgesic effect of drug are the hot plate method and hot water tail immersion test [44,45]. FR-ZnONPs effectively increased the response time to thermal stimulus in comparison to the control. It also rendered a significant analgesic effect when treated with opioid antagonist naloxone. This may be due to

Journal Pre-proof phytoconstituent naringenin present in Fraxinus rhynchophylla [38], which activates the opioid receptors and modulates the spinal reflexes [45,46]. The acetic acid induced writhing model was performed to assess both the antinociceptive and anti-inflammatory property of the FR-ZnONPs. Acetic acid increases the prostaglandins and it also activates the sensory polymodal neurons, causing abdominal writhing in the mice [47-49].

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Previous studies have reported that the exogenous administration of zinc alleviates pain in the aqueous state because ZnO nanoparticles release zinc ions, which alleviates pain and reduces the

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number of writhes in the acetic induced nociceptive model [50,51]. In the aqueous state, ZnONP

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releases zinc ions [52], which may explain the reduction of abdominal writhes in acetic acid

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induced nociceptive mice model treated with FR-ZnONPs.

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Excitatory neurotransmitters play a significant role in the initiation of nociception because they excite the synapses of the sensorial and central nervous system. Glutamate,

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which is a prominent excitatory neurotransmitter, triggers the nociception in peripheral and

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spinal nervous system via N-methyl-Daspartate (NMDA) and non-NMDA receptors [53]. Once

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the noxious stimuli are induced by injecting glutamate into the intraplanar region of the hind paw, the glutamate triggers both peripheral and central neurons via the receptors. It also initiates the synthesis of inflammatory cytokines [54], causing the mice to bite or lick the injected paw. NSAID effectively blocks the prostaglandin release, which reduces the pain induction [55]. Previous reports have suggested that zinc blocks NMDA receptors, thereby preventing glutamate nociceptive signaling [56]. In the present study, comparatively fewer paw licking numbers were observed in FR-ZnONPs treated mice than the control mice, which indicates that the nanoparticles have effectively blocked prostaglandin production and inhibited the excitation glutamate receptors.

Journal Pre-proof The capsaicin induced nociception mice model was performed to assess the analgesic effect of FR-ZnONPs against the neurogenic pain. Capsaicin is an irritant that stimulates specific vanilloid receptors and specifically activates the nociceptor, thereby inducing neurogenic pain [57-59]. Patients with decreased plasma zinc levels complain of neuropathic tongue pain [60]. In the present study, FR-ZnONPs significantly reduced the number of licks performed by FRZnONPs treated mice in the capsaicin induced nociceptive mice model, which may have

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and thus inhibited the capsaicin induced neurogenic pain.

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happened because the zinc ions released from the zinc nanoparticles inhibited vanilloid receptors

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To confirm the analgesic and anti-inflammatory property of FR-ZnONPs, formalin

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induced nociception mice mode was performed. Formalin exerts nociception in biphasic manner in the initial phase, while nociception was triggered by the activation primary afferent sensory

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neurons. During the second phase, nociception was triggered by the release of inflammatory

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cytokines [61,62]. Peripheral acting analgesic drugs only inhibit the second phase by blocking the binding of prostaglandins to the Receptor Potential Vanilloid-1 (TRPA-1) cationic channel,

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which is highly expressed in C-Fiber nociceptors [63]. In the current study, FR-ZnONPs exerted

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an antinociception effect, in both the initial and second phase of formalin induced nociception. The open field test confirms that the low dose of FR-ZnONPs treatment does not induce any sedative effects in mice, whereas a higher dosage produced a minimal sedative effect that is comparatively lesser than the standard analgesic drug morphine.

Conclusion

Journal Pre-proof In conclusion green synthesized zinc oxide nanoparticles using Chinese traditional medicinal plant Fraxinus rhynchophylla as a reducing agent was characterized and confirmed the presence of synthesized FR-ZnONPs and fulfills the properties of nanodrug which can be assessed for its pharmacological role. Analgesic effect of FR-ZnONP was analyzed with different nociceptive models, which exhibited persuasive anti nociceptive effect in both thermal induced and chemical induced nociceptive models and also render less sedative effect compared

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to standard analgesic drug. Therefore overall our results confirmed the FR-ZnONPs possessed

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promising antinociceptive activity and also it can be prescribed as analgesic drug with further

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preclinical and clinical investigation.

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Acknowledgment:

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Najat Marraiki and Abdullah M. Elgorban have extended their appreciation to The Researchers

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Conflict of interest

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supporting project number (RSP-2019/56) King Saud University, Riyadh, Saudi Arabia.

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The authors declare no conflict of interest

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Journal Pre-proof Table 1. Antinociceptive effect of zinc oxide nanoparticles synthesised using Chinese ash wood Fraxinus rhynchophylla extract (FR-ZnONP) and reversal effect of naloxone in hot plate induced nociception mice model. pre

Response time(s)(%MPE)

(mg/kg)

treatment

30 min

60 min

90 min

120 min

Control

5.24±0.06

5.39±0.71

5.63±0.93

5.83±0.67

6.29±0.14

ZnONps(5mg)

5.38±0.18

7.99±0.42

9.62±0.47

12.65±0.39

13.19±0.77

(9.63)

(17.98) #

(25.16) *

(31.09 *

7.09±0.19

9.29±0.66

11.96±0.91

13.09±0.73

(11.09)

(24.86) #

(31.43) *

(44.67) *

7.69±0.18

9.33±0.72

11.80±0.53

12.63±1.99

(27.63)

(45.99) #

(53.13) *

(59.92) *

12.42±0.69

15.93±1.22

(44.96)

(51.33) *

(61.44)*

7.37±0.29

7.56±0.66

7.89±0.91

6.81±0.61

7.42±0.68

8.87±0.20

(11.55)*

(24.97) *

Morphine

5.43±0.23

7.63±0.28 (31.29)

NLX(2mg)+

5.69±0.13

6.13±0.17

5.17±0.14

(10mg) NLX(2mg)+

ur

5.29±0.23

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ZnONps

6.25±0.32 (7.43)

ZnONps (5mg) NLX(2mg)+

na

Control NLX(2mg)+

5.49±0.71

ZnONps

ro

9.71±0.33 #

lP

(5mg)

-p

ZnONps(15mg) 5.81±0.29

re

ZnONps(10mg) 5.64±0.29

of

Treatment

(9.41)

#

5.82±0.16

6.14±0.31

7.82±0.30

9.74±0.81

(8.19)

(11.74) #

(21.87) *

(24.59) *

5.96±0.27

7.57±0.54

8.47±0.10

10.23±0.27

(12.47)

(18.41)#

(21.20) *

(25.73) *

7.99±0.41

8.22±0.14

9.27±0.44

11.70±0.22

(3.29)

(9.36) #

(13.77) *

(21.37) *

(15mg) NLX(2mg)+ Morphine (5mg)

5.57±0.11

Journal Pre-proof FR-ZnONP anti nociceptive property was compared with the potency of standard drug morphine. The values depicted in the table are the mean ± SEM of six rats in each group. *p ≤ 0.05 considered to be statistically significant.

Table 2. Antinociceptive effect of zinc oxide nanoparticles synthesised using Chinese ash

of

wood Fraxinus rhynchophylla extract (FR-ZnONP) and reversal effect of naloxone in tail immersion nociception mice model.

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FR-ZnONP anti nociceptive property was compared with the potency of standard drug morphine.

-p

The values depicted in the table are the mean ± SEM of six rats in each group. *p ≤ 0.05

re

considered to be statistically significant. pre

Response time(s)(%MPE)

(mg/kg)

treatment 30 min

60 min

90 min

120 min

Control

7.20

8.59±0.25

8.70 ±0.20

9.09±0.37

9.69±0.14

9.58± 0.40

8.90 ± 0.56 8.04± 0.30 8.80± 0.93

(5.07)

(10.70)

7.25

8.87±0.85

9.50 ± 0.47 9.65± 0.28 9.90±0.93

±0.48

(8.47)#

(9.58)*

7.34

7.49±0.20

9.30 ±0.38 10.40

10.07±0.85

±0.57

(10.58) #

(12.07) *

(15.00)*

lP

Treatment

na

±0.46

7.78±0.30

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ZnONps (5mg)

ZnONps (15mg)

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ZnONps(10mg)

(10.35)

(11.74) *

±0.97

(9.47)

(15.20)*

(14.23)* Morphine

7.48±0.20

#

(12.85)

(5mg) NLX(2mg)+ Control

9.69±0.58

7.59

9.90 ± 0.70 10.87±0.20 10.98±0.39 (18.40) *

(20.47)*

±0 7.79 ± 0.58 7.98 ± 0.85 8.69± 0.21

(22.86) * 8.94 ±0.98

.20 NLX(2mg)+ ZnONps (5mg) 7.70±0.20

8.48 ±0.58

8.69±0.40

8.84±0.20

8.03±0.58

(6.36) #

(6.89) *

(9.70) *

(10.25) *

Journal Pre-proof NLX(2mg)+

ZnONps 7.61±0.24

10.39±0.21 10.89±0.57 10.04±0.50 10.57±0.55

(10mg)

(6.77) #

(7.63) *

(8.07) *

(9.07) **

NLX(2mg)+ZnONps(15mg) 7.07±0.42

8.52±0.96

8.79±0.48

8.99±0.64

9.91±0.42

(9.65) #

(10.31) *

(10.25) *

(13.09) *

7.30± 0.38

7.27 ± 0.69 7.86±0.71

8.81±0.52

(6.14) #

(7.69)*

(12.07) *

7.56±0.29

NLX(2mg)+ Morphine (5mg)

of

Figure Legends

(6.58) *

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Fig 1A: The UV–visible spectrum absorption pattern of zinc oxide nanoparticles synthesised

-p

using Chinese ash wood Fraxinus rhynchophylla extract (FR-ZnONP)

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Fig 1B: High Resolution Transmission electron microscopy of zinc oxide nanoparticles

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synthesised using Chinese ash wood Fraxinus rhynchophylla extract (FR-ZnONP)

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Fig 2. Zeta potential of zinc oxide nanoparticles synthesised using Chinese ash wood Fraxinus

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rhynchophylla extract (FR-ZnONP)

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Fig 3. Fourier-transform infrared spectroscopy analysis of zinc oxide nanoparticles synthesised using Chinese ash wood Fraxinus rhynchophylla extract (FR-ZnONP)

Fig 4. Antinociceptive effect of zinc oxide nanoparticles synthesised using Chinese ash wood Fraxinus rhynchophylla extract (FR-ZnONP) in acetic acid induced nociception mice model. FR-ZnONP anti nociceptive property was compared with the potency of standard drug diclofenac sodium. The values depicted in the table are the mean ± SEM of six rats in each group. #P < 0.05, *p < 0.01 were considered to be statistically significant.

Journal Pre-proof

Fig 5. Antinociceptive effect of zinc oxide nanoparticles synthesised using Chinese ash wood Fraxinus rhynchophylla extract (FR-ZnONP) in glutamate induced nociception mice model. FR-ZnONP anti nociceptive property was compared with the potency of standard drug diclofenac sodium. The values depicted in the table are the mean ± SEM of six rats in each

of

group. #P < 0.05, *p < 0.01 were considered to be statistically significant.

ro

Fig 6. Antinociceptive effect of zinc oxide nanoparticles synthesised using Chinese ash wood

-p

Fraxinus rhynchophylla extract (FR-ZnONP) in neurogenic nociception mice model.

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FR-ZnONP anti nociceptive property was compared with the potency of standard drug diclofenac sodium. The values depicted in the table are the mean ± SEM of six rats in each

na

lP

group. #P < 0.05, *p < 0.01 were considered to be statistically significant.

Fig 7. Antinociceptive effect of zinc oxide nanoparticles synthesised using Chinese ash wood

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Fraxinus rhynchophylla extract (FR-ZnONP) in formalin induced nociception mice model.

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Initial phase of nociception induction (A) and the inflammatory phase of glutamate (B). FRZnONP anti nociceptive property was compared with the potency of standard drug morphine. The values depicted in the table are the mean ± SEM of six rats in each group. #P < 0.05, *p < 0.01 were considered to be statistically significant.

Fig 8. Sedative effect of zinc oxide nanoparticles synthesised using Chinese ash wood Fraxinus rhynchophylla extract (FR-ZnONP) in formalin induced nociception mice model.

Journal Pre-proof FR-ZnONP anti nociceptive property was compared with the potency of standard drug morphine. The values depicted in the table are the mean ± SEM of six rats in each group. #P < 0.05, *p <

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na

lP

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-p

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of

0.01 were considered to be statistically significant.

Journal Pre-proof

Highlights 

Zinc oxide nanoparticles are focused by the researcher to be utilized both in clinical field



Synthesized ZnO nanoparticles using Fraxinus rhynchophylla wood extract as reducing and capping agent Synthesized ZnO nanoparticles are less sedative compared to the standard antinociceptive

ur

na

lP

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-p

ro

of

drug morphine

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

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8