Thymol Nanopolymer Synthesis and Its Effects on Morphine Withdrawal Syndrome in Comparison With Clonidine in Rats

The drug delivery system is valuable in the treatment of the disease. A nanopolymer as a thymol and Thymbra spicata release system was synthesized and its effects on morphine withdrawal syndrome in comparison with clonidine in rats were studied. The nanopolymer was characterized by different methods, namely, IR, HNMR, CNMR, GPC, DLS, and AFM. Thymol in T. spicata extract was assessed. The loading and release rate of thymol and T. spicata extract on the nanopolymer were evaluated by HPLC. The median lethal dose (LD50) of the T. spicata extract, thymol, extract nanopolymer, and thymol nanopolymer was studied. The frequency of jumping, rearing, and teeth chattering in naloxone-induced morphine withdrawal syndrome was studied. Synthesized nanopolymer was desirable as a carrier for the drug. The loaded amount of extract and thymol on nanopolymer was estimated 55 ± 3.2% and 48 ± 2.6% and the drug released was 71 and 68%, respectively. LD50 of the T. spicata extract, thymol, extract nanopolymer, and thymol nanopolymer was 975, 580, 1,250, and 650 mg/kg, respectively. This study showed that thymol nanopolymer was more effective than clonidine to reduce the frequency of morphine withdrawal symptoms. Our results suggest that T. spicata extract, thymol, extract nanopolymer, and thymol nanopolymer are mighty in reducing the narcotic withdrawal signs. The mechanism of action and therapeutic potential is maybe similar to clonidine.


INTRODUCTION
Many benefits, namely, improved drug solubility, stability, reduced clearance rate, increased drug exposure, and fewer side effects, have been reported for nanoscale drug delivery (Moghimi et al., 2005;Wagner et al., 2006;Kim et al., 2010). The size of the nanoparticles carrier plays an important role in the production of nanodrugs to improve biodistribution, tissue penetration, and function (Stano et al., 2012;Tang et al., 2014). The amount of drug-loaded on the nanoparticle depends on factors such as the branches of hydrophobic and hydrophilic polymers (Chu et al., 2013). Citric acid is one of the most inexpensive and compatible substances that is also used in the pharmaceutical and food industries (Naeini et al., 2010). In addition to citric acid, glycerol can be used to synthesize a biocompatible polymer (Lee et al., 2001). Fourier transforms infrared (FTIR), nuclear magnetic resonance (NMR), gel permeation chromatography (GPC), dynamic light scattering (DLS), and atomic force microscopy (AFM) are used to investigate the physicochemical properties of the produced polymeric nanoparticles (Adeli et al., 2013).
Today, dependence on opioids is a world health problem that makes many economic, social, and individual problems. In this regard, the use of nanocarriers loaded with naloxone has been considered in the treatment of opioid overdose (Hasan et al., 2021). Nevertheless, the challenge of using nanoparticles loaded with a bioactive component of medicinal plants remains.
Withdrawal symptoms in morphine addicts are relatively severe and include hypertension, diarrhea and vomiting, and dysphoria (Ballantyne and LaForge, 2007). Mu-, kappa-, and delta-opioid receptor agonists, such as methadone, are effective in helping to quit an addiction with analgesic effects, but there are side effects such as constipation, and respiratory depression, physical dependence, and addiction themselves (Li and Zhang, 2012). However, common pharmacological treatments for withdrawal syndrome include MOR agonists (methadone) or multifunctional opioid/nociception-OFQ agonists (buprenorphine) (Kreek et al., 2002).
Medicinal herbs with a variety of active components have been used potentially in the treatment of withdrawal syndrome (Ward et al., 2011;Tabatabai et al., 2014). Numerous studies have shown that various medicinal plants such as Passiflora incarnate, Hypericum perforatu, Avena sativa, and Valeriana officinalis, with their antianxiety, hypnotic, analgesic, and antispasmodic effects, have improved the symptoms of withdrawal syndrome (Ebrahimie et al., 2015). Although there was no evidence of the morphine withdrawal and anti-anxiety effect of Thymbra spicata extract in the previous studies, T. capitata (L.) extract of the same family had a significant antianxiety effect (Motti and de Falco, 2021). The anti-analgesic and antinociceptive effects of the Thymus vulgaris L. extract have also been documented (Rašković et al., 2020). Many medicinal plant oils such as T. spicata, Thymus vulgaris, Monarda fistulosa, Trachyspermum ammi, Thymus ciliates, and Nigella sativa, contain a monoterpene named thymol (Wattanasatcha et al., 2012), which can be used as a food additive under the Food and Drug Administration regulations (Dhaneshwar et al., 2013). Due to the chemical structure of thymol, it can be used to produce nanoparticles to treat various diseases (Jafari et al., 2018;Robledo et al., 2018). Thymol has previously been shown to attenuate the signs and symptoms of an opiate withdrawal syndrome in humans (Khodayar et al., 2014). Thymol not only has been able to reduce the anxiety associated with diazepam withdrawal but also improves motor and memory impairment in rats (Saleem et al., 2021). In this study, in addition to synthesizing a nanopolymer and binding it with thymol, the effects and possible mechanism on morphine withdrawal syndrome in rats were investigated.

Preparation of Nanopolymer
By melt polycondensation method, citric acid and glycerol (Carlo Erba) for the synthesis of branched polymer, were combined and dissolved in tetrahydrofuran (THF) (Merck) and filtered. The solution was precipitated in cyclohexane (Merck) and dissolved in THF and then placed in a dialysis bag (Mn cutoff 2000, Sigma-Aldrich) (Adeli et al., 2013). Then, oleic acid was poured into a polymerization capsule and heated at 90, 100, 120, 140, and 160 • C, respectively.

Fourier Transforms Infrared Spectroscopy
The nanopolymer IR spectra were performed with an FT-IR (Nicolet 320 spectrophotometer). The spectra were obtained at a resolution of 4 cm −1 in the range 4,000-500 cm −1 (Dong and Gu, 2002).

Nuclear Magnetic Resonance
Identical NMR spectra were obtained by dissolving samples in D 2 O (Bruker DRX 400 MHz) and the spectra were recorded at 500 MHz. The resulting data were analyzed using ACDLABS/1D NMR software (Neri et al., 1989).

Gel Permeation Chromatography
Using Gel permeation chromatography (Knauer equipped with Smartline Pump 1,000 with an PL Aqua gel-OH mixed-H 8 µm column), molecular weights and distribution of the obtained nanopolymer were determined (Lee, 1978).

Dynamic Light Scattering
Dynamic light scattering data (Malvern Instruments Ltd., United Kingdom were measured in water three times (5 runs to each measurement). The mean size was accounted as the six measurements average (Brown et al., 1992).

Atomic Force Microscopy
A multimode scanning probe microscope (Ara-research Inc., Iran) for atomic force microscopy (AFM) was used and the nanopolymer morphology was determined. The nanopolymer suspension (a droplet) was dried by a freeze dryer (Christ, Germany) onto a clean surface of mica before AFM imaging. The size of the images was 5 × 5 µm. The images were scanned on at least six different areas of the sample (Rezayati Charani et al., 2013).

Thymol Identification by High-Performance Liquid Chromatography
The aerial parts of Thyme spicata were collected from Ilam, Iran, and identity was authenticated by the voucher specimens (NO. 596) deposited in the Department of Horticulture, Faculty of Agriculture, Ilam University. After drying and powdering, the powder was degreased by hexane and 20 g of the plant was used for extraction by a Soxhlet extraction method in a water-ethanol solvent. The plant powder was loaded into the porous cellulose thimble (20 g in a 25-× 80-mm thimble), which was placed inside the Soxhlet extractor. The sidearm was lagged with glass wool. The solvent (250 ml of water-ethanol) was heated and evaporated. By rotary (IKA HB 10, Germany) device, the solvents were removed. The extraction (yield = 5.67%) was lyophilized and kept stored at -20 • C and were dissolved in methanol (Lopez et al., 2013). According to the previous procedure, the highperformance liquid chromatography (HPLC) method was done (Aghamohammadi et al., 2016). A reversed-phase UV-HPLC (Smart line; Knauer, Germany) with a C18 column (Nucleosil H.P.; 25 cm × 0.46 cm internal diameter, particle size 3 µm, 100 Å pore size) was developed for the thymol determination. The T. spicata extract peaks were compared with the thymol standard (Sigma Aldrich). A stock solution (0.1 mg/ml) of thymol standard diluted to obtain 15. 6, 31.25, 62.5, 125, 250, and 500 µM (Moayeri et al., 2018).

Releasing Capacity Assay
Loading capacity was determined by the HPLC method (Lv et al., 2009) and estimate the amount of thymol and extract loaded on the nanopolymer. The mobile phase was 40% methanol and 60% aqueous solution of formic acid (0.1%) (Majumdar et al., 2014). The solution aliquot was injected into the HPLC.
In vitro release of thymol and extract from nanopolymer was done by dissolving of thymol (5 mg) and thymol in extract-loaded nanopolymer in PBS (3 ml, 0.1 M, pH 7.4). At the sampling time, the release medium was replaced by a fresh buffer and injected into the HPLC (Qiao et al., 2005).

Animals
Rats (male albino, 180-260 g, Institute of Medicinal Plants, ACECR, Karaj, Iran) were used. The rats were maintained at standard conditions (22-25 • C, 12 h dark-light cycle, standard rodent feed, and water). In total, eight rats were used for each dose of the thymol, extracts, and drugs (n = 3). The method grouping to study morphine withdrawal syndrome was as Table 1.

Ethical Approval
The study was approved by the Ilam University of Medical Sciences, Ilam, Iran. The ethical approval for this study was obtained from the Animal Care and Ethics Committee (ACEC) of the Ilam University of medical science (IR.MEDILAM.REC.1396.81).

The Median Lethal Dose
To obtain the median lethal dose, different doses of the T. spicata extract, thymol, extract nanopolymer, and thymol nanopolymer were administered (orally gavage, 20 rats in each group). The doses were 50, 200, 400, 600, 800, 1,000, 1,600, and 2,400 mg/kg for T. spicata extract and extract nanopolymer and 50, 100, 200, 300, 400, 500, 600, and 700 mg/kg for thymol and thymol nanopolymer, respectively. Then, 72 h after gavage, the median of dead animals was calculated. Mortality graphs were plotted between log-concentration vs. percent mortality and the LD 50 was determined (Parmar and Prakash, 2006;El Kabbaoui et al., 2017). Therefore, the T. spicata extract and extract nanopolymer with doses of 100, 200, 300 mg/kg and thymol and thymol nanopolymer with doses of 30, 60, and 90 mg/kg was selected to continue the experiments because none of the used doses caused mortality and were considered in the ED 50 range.

Method of Morphine Dependency
Morphine (Darou Pakhsh Pharmaceutical Company, Iran) was injected subcutaneously into the rats two times daily for 7 days. On days 1 and 2, a dose of morphine (2.5 mg/kg) was used two times daily and doubled every day until the day 6, reaching 40 mg/kg. Rats received the last dose (50 mg/kg) on day 7 (Romandini et al., 1984).

Withdrawal Syndrome Method
In all the groups, 4 h after the last dose of morphine, animals received naloxone (3 mg/kg, i.p., Sigma-Aldrich Company, United States). Immediately, the morphine withdrawal symptoms manifestations, namely, jumping, rearing, and teeth chattering, were monitored for half an hour (Feily and Abbasi, 2009). Animals of all groups received intraperitoneally 3 mg/kg naloxone 4 h after the last injection of morphine on day 7 of the morphine treatment. Immediately after the naloxone injection, each animal was placed in a transparent acrylic cylinder to observe the frequency of withdrawal manifestations (jumping, rearing, and teeth chattering) for 30 min.

Method of Coadministration Use of Clonidine Hydrochloride and Morphine on Morphine Dependency and Withdrawal Syndrome
Clonidine hydrochloride (0.2 mg/kg i.p.) was used concomitantly with morphine two times daily (6 days) in group 2. The animals received the last dose on day 7, 4 h before receiving naloxone, to examine jumping, rearing, and teeth chattering.

Statistical Analysis
The results were shown as mean ± standard error (SEM). One-way ANOVA followed by Tukey post hoc test was used for data analysis.

Nanopolymer Evaluation
The FT-IR spectrum of the nanopolymer compound was shown (Figure 1). The peak related to the carbonyl ester group, hydroxyl groups, and carbonyl group appears at 1,647, 3,314, and 1,738 cm −1 , respectively.
The nanopolymer 1 HNMR spectrum was shown (Figure 2). Hydrogen A was carbonyl group methane in the oleic acid monomer (3.82 ppm). Hydrogen B was monomer glycerol (2.68 ppm). Hydrogen C was methylated glycerol. Hydrogens D and E were the citric acid hydrocarbon content (3.36 ppm).
The nanopolymer 13 CNMR spectrum was presented (Figure 3). This combination had a 9-peak index in the carbon compounds of this composition (letters A-I). Carbon A the middle carbon of glycerol (74 ppm). Carbons B and C were methyl glycerol carbonates (68 ppm). Carbons D and E were the two Carboniferous carbon (172 ppm). Carbon F was the fourth type of citric acid (44 ppm). Carbons G and H were the methyl group (13 ppm). Carbon G was the carbonyl oleic acid group (183 ppm).
The nanopolymer GPC diagram was shown in Figure 4. This molecular weight was 5,238 g/mol. The size of the nanopolymer was monitored (in water, 25 • C) by DLS (Figure 5). The peak width indicated a particle size sample solution larger than 75 nm, but the diameter of most particles was estimated to be about 100 nm.
The size of the nanopolymer was monitored (in water, 25 • C) by DLS (Figure 5). The peak width indicated a particle size sample solution larger than 75 nm, but the diameter of most particles was estimated to be about 100 nm.   The nanopolymer AFM results allowed morphology at the micro-level (Figures 6A-C). As shown in Figure 6C, its length and width were with a cross-sectional profile in which interchange of heterogeneous wrinkles was visible (Mansouri et al., 2013).

Extraction and Identification
The HPLC chromatogram of thymol was obtained (retention time = 5.400 min and wavelength = 352 nm). The standard chromatogram of T. spicata extract was similar to that of standard thymol in 5.480 min (in the same conditions, Figures 7A,B).  The quantitative analysis revealed that thymol was 3.65 mg/g of T. spicata extract. The method was calibrated with the linear curve (R2 = 0.9989, y = 187,938 × -3,896.03). LOD and LOQ were 52.09 and 173.63, respectively.

Load and Release Evaluation
The loading and release capacity of thymol and thymol in the extract was evaluated by HPLC. The loading rate of thymol and extract was estimated at 55 ± 3.2% and 48 ± 2.6%, respectively, (n = 3).
Thymol and extract were released in two phases (fast and slow) of the nanopolymer. Near to 71% of loaded thymol and 68% of loaded extract in the first 12 h and the remaining 90-92% to 48 h were released at a lower rate (Figure 8).

Mortality Determination
LD 50 values of the T. spicata extract, thymol, extract nanopolymer, and thymol nanopolymer were 975, 580, 1,250, and 650 mg/kg, within 72 h after administration, respectively. No mortality was observed in the treatment range used in this study.
Effect of the Thymbra spicata Extract, Thymol, Extract Nanopolymer, and Thymol Nanopolymer on the Naloxone-Induced Jumping in Morphine-Dependent Rats Administration of 200 and 300 mg/kg of T. spicata extract and extract nanopolymer (p < 0.05 vs. control and p < 0.001 vs. control, respectively) and all doses of thymol and thymol nanopolymer (30, 60, and 90 mg/kg) decreased the jumps number in morphine-dependent rats significantly (p < 0.001 vs. control) precipitated by naloxone administration (5 mg/kg, i.p.) after the morphine last dose. The effect of T. spicata extract and extract nanopolymer was dose-dependent. Clonidine also reduced the jumps number as expected (Figure 9). Effect of the Thymbra spicata Extract, Thymol, Extract Nanopolymer, and Thymol Nanopolymer on the Naloxone-Induced Rearing in Morphine-Dependent Rats Thymbra spicata extracts and extracts nanopolymer at different doses (200 and 300 mg/kg) and thymol and thymol nanopolymer (30, 60, and 90 mg/kg) decreased the rearing in morphinedependent rats precipitated by administration of naloxone (5 mg/kg, i.p.) after the last dose of morphine (p < 0.001 vs. control). The effect of T. spicata extract, thymol, extract nanopolymer, and thymol nanopolymer was dose-dependent. As expected, clonidine reduced the rearing number in animals (Figure 10).
Effect of the Thymbra spicata Extract, Thymol, Extract Nanopolymer, and Thymol Nanopolymer on the Naloxone-Induced Teeth Chattering in Morphine-Dependent Rats Different doses of the T. spicata extract and extract nanopolymer (200 and 300 mg/kg) and thymol and thymol nanopolymer (30, 60, and 90 mg/kg) morphine-dependent rats decreased the teeth chattering in precipitated by administration of naloxone (5 mg/kg, i.p.) after the morphine last dose (p < 0.001 vs. control). The effect of T. spicata extract, thymol, extract nanopolymer, and thymol nanopolymer was dose-dependent. Also, clonidine reduced the teeth chattering number (Figure 11).

DISCUSSION
This study showed that the design of drug delivery systems and the production of nanodrugs of T. spicata and its bioactive component thymol can be used more effectively in reducing the symptoms of a morphine withdrawal syndrome in rats. In this regard, to reduce the adverse effects, the researchers are now focusing on drug delivery systems. Thymol, a terpenoids structure, which is a bioactive component of some medicinal plants is used for a variety of disorders and diseases (Alvarez Echazu et al., 2017).
In this study, to get nanopolymer structure, different analytical techniques have been used. The nanopolymer FT-IR spectrum demonstrated carbon derived from the carbonyl group at 1,738 cm −1 and a hydroxyl group at 3,314 cm −1 . The NMR spectrums showed 5 types of hydrogens and 9 types of carbon. The molecular weight of the nanopolymer was obtained by GPC technique around 5,238 g/mol and by DLS tests, the hydrodynamic diameter of the nanopolymer was approximately 75 nm. AFM results showed that nanopolymer was a natural microporous and mesoporous material with polymodal pore size distribution. | The frequency (number of teeth chattering/30 min) of teeth chattering in rats. Group 1; morphine-dependent rats (control). Group 2; morphine-dependent rats and coadministration of clonidine hydrochloride (0.2 mg/kg i.p.) (positive control). Groups 3, 4, and 5; morphine-dependent rats and the coadministration of 100, 200, and 300 mg/kg T. spicata extract, respectively. Groups 6, 7, and 8; morphine-dependent rats and coadministration of 30, 60, and 90 mg/kg thymol, respectively. Groups 9, 10, and 11; morphine-dependent rats and coadministration of 100, 200, and 300 mg/kg extract nanopolymer, respectively. Groups 12, 13, and 14; morphine-dependent rats and coadministration of 30, 60, and 90 mg/kg thymol nanopolymer, respectively. Group 15; non-dependent rats (saline). Data are expressed as mean ± SEM. * P < 0.01 vs. control, * * P < 0.001 vs. control.
Nanopolymer loading capacity was evaluated by HPLC. Based on this analysis, the loading rate of thymol and extract was estimated at 55 ± 3.2% and 48 ± 2.6%, respectively. It has been shown that thymol chitosan nanopolymer had a loading capacity of about 2.5% with antibacterial effects (Medina et al., 2019). But, similar to our study, thymol nanospheres loading was reported to be about 43% (Wattanasatcha et al., 2012).
In this study, it was documented that coadministration of T. spicata extract, thymol, extract nanopolymer, and thymol nanopolymer with morphine reduced the severity of the signs of morphine withdrawal syndrome induced by naloxone. These findings demonstrated that the extract and thymol nanopolymer were able to reduce the symptoms of morphine withdrawal syndrome. The effects observed with extract nanopolymer, thymol nanopolymer, and clonidine in reducing the symptoms of morphine withdrawal syndrome were in one direction. Therefore, one of the mechanisms of action of the nanopolymers may be similar to clonidine. Also, the results showed that increasing the dose of T. spicata extract, thymol, extract nanopolymer, and thymol nanopolymer decreased the frequency of the signs of withdrawal syndrome, namely, jumping, teeth chattering, and rearing. It has been reported that some supplements and drugs such as magnesium, beta-carbolines, and midazolam reduce the symptoms of withdrawal in the rat (Aricioglu-Kartal and Uzbay, 1997). Moreover, for the management of heroin or methadone withdrawal, clonidine and lofexidine are more effective than placebo (Gowing et al., 2014). Recently, the use of medicinal plants to improve the symptoms of morphine withdrawal has been considered.
It has been reported that some medicinal plants, such as Zingiber officinale, Glycyrrhiza glabra, and Ziziphus jujube relieve morphine withdrawal symptoms (Doosti et al., 2013). Thymus species are the medicinal plants whose leaves and flowering parts are widely used as a tonic, antiseptic, antitussive, and carminative (Ghasemi Pirbalouti, 2009;Jahani et al., 2013;Bakhshi et al., 2014) and also used in pharmaceutical, cosmetic, and preservation of several food products (Surburg and Panten, 2006). The medicinal plant Zataria multiflora and Thymus vulgaris, thymolcontaining, reversed the effects of naloxone due to their analgesic effects (Hosseinzadeh et al., 2000;Taherian et al., 2009). Also, Thymus daenensis extract and essential oil attenuate morphine withdrawal behaviors in mice (Khodayar et al., 2014). However, no study has been investigated the effects of the T. spicata extract, extract nanopolymer, and thymol nanopolymer on morphine withdrawal syndrome in rats.
Although the Thymus vulgaris and its main constituents (thymol and carvacrol) have reduced the symptoms of experimental autoimmune encephalomyelitis in an animal model (Mahmoodi et al., 2019), and thymol has anti-Alzheimer's (Azizi et al., 2012), antiseizure (Sancheti et al., 2014), antidepressant (Deng et al., 2015), and nAch receptor activity (Sammi et al., 2017), but the effectiveness of T. spicata on the nervous system was not observed.
It was also found that thymol treatment increased the Aβ protein levels (Azizi et al., 2012) and the prolonged onset of myoclonic jerk and glutathione levels (Sancheti et al., 2014), and decreased oxidative stress (Lee et al., 2015), norepinephrine in the hippocampus (Deng et al., 2015), and synaptic Ach levels (Sammi et al., 2017).
Our results demonstrate that the ameliorative effects of T. spicata extract, thymol, extract nanopolymer, and thymol nanopolymer against withdrawal syndrome by naloxone challenge may be modulated by α2 receptor or PKA pathway in rats. However, extract nanopolymer, and thymol nanopolymer alleviate the morphine withdrawal syndrome more than the total T. spicata extract and thymol.
LD 50 of the T. spicata extract, thymol, extract nanopolymer, and thymol nanopolymer were 975, 580, 1,250, and 650 mg/kg, respectively. It has been documented that agents with LD 50 values in the range of 50-500 and 500-5,000 mg/kg showed moderately and slightly toxic, respectively (Ruiz et al., 2012). By our studies, previous studies have shown that LD 50 of thymol and thymol nanopolymer were 435 and 583 mg/kg, respectively (Karimi et al., 2019), and other studies have shown that LD 50 of thymol was 980 mg/kg (Jamil et al., 2010).

CONCLUSION
Our results suggest that T. spicata extract, thymol, extract nanopolymer, and thymol nanopolymer can reduce the narcotic drugs withdrawal symptoms, and its reducing effect is equivalent to clonidine and may have the potential of narcotic addiction treatment. The efficacy of thymol nanopolymer was greater than the others, and since it affected equivalent to clonidine, its pharmacological mechanism of action is likely to be similar.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ supplementary material.

ETHICS STATEMENT
The animal study was reviewed and approved by the Ilam University of Medical Sciences.