Analgesic Effects of the Cressa Cretica Extract on Induced Neuropathic Pain in Rats , and the Potential Role of Opioid Receptors

Article info: Received: 12 Apr 2021 Accepted: 14 Jun 2021 Online Published: 01 Oct 2021 Research Paper: Analgesic Effects of the Cressa Cretica Extract on Induced Neuropathic Pain in Rats, and the Potential Role of Opioid Receptors * Corresponding author: Mohsen Zabihi, PhD. Address: Department of Pharmacology, School of Pharmacy, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. E-mail: mzabihi100@gmail.com


Introduction
amage to the peripheral fibers of sensory nerve cells and central neurons can lead to neuropathic pain. Manifestations of neuropathic pain occur under various circumstances, e.g. diabetes mellitus, chemotherapy, and as the side effects of some medications, such as isoniazid, vincristine, and metronidazole [1][2][3]. Neuropathy is typically characterized by symmetric distal sensory loss, burning sensations, and/ or muscle weakness [4].
Management of the underlying diseases along with symptomatic therapy are the main stay of treating neuropathic pain [4]. Gabapentin, pregabalin, tricyclic antidepressants, and duloxetine are among the drugs that reduce the pain associated with neuropathy and are generally well tolerated [5][6][7][8][9]. Simultaneous treatment with tramadol, Nonsteroidal Anti-Inflammatory Drugs (NSAIDs), or low-dose narcotics may be necessary in some patients for occasional flair-up of pain conditions [10,11].
Cressa cretica (C. cretica) is a blooming plant species of the morning glory family. This plant grows in parts of Africa, Europe, Asia and Australia. It has a long history of application in traditional medicine, with some of its therapeutic effects studied and confirmed. It inhibits bacterial and fungal growth, is used as expectorant and antitussive drug, and improves digestion [12]. It has been effective as an anticancer drug in combination with Tridax procumbens and Euphorbia thymifolia and has been shown to improve testicular function in rats [13,14]. The extract of C. cretica is used in traditional medicine in Iran to treat pain. This medicinal plant is a source of many chemical compounds, including quercetin, which is a flavonoid with analgesic effects [15]. There is little research on the effect of C. cretica on the management of neuropathic pain. This herb is likely to be accepted by patients in Iran as a better treatment option for neuropathic pain than synthetic drugs due to their potential side effects.
Since no research has been done on the mechanism of analgesic action of this plant, and due to the proven role of opioid receptors in pain management, this study was planned to investigate the role this plant extract as a putative analgesic agent for neuropathic pain and an inhibitor of opioid receptors.

Materials and Methods
Extract preparation: The plant, Cressa cretica L., was collected from farms in Yazd, Iran. The scientific name and quality of the plant was confirmed by the Department of Pharmacognosy, Faculty of Pharmacy at Shahid Sadoughi University of Medical Sciences (SSU0043). The aerial parts of the plant were dried at shade and powdered. The hydroalcoholic extract was prepared over four weeks by mixing 100g of the powder in ethanol 80%. The total hydroalcoholic extract was filtered and dried by evaporating the solvent at 40 o C, and the total phenolic and flavonoid concentrations were determined. The desired concentration of the extract was prepared by dilution in distilled water.
Extract standardization: The Folin-Ciocalteu method was used to measure the phenolic compounds [15]. The desired hydroalcoholic extract samples were prepared in distilled water at the concentrations of 0.1, 0.01 or 0.001 μg/mL. The extract samples (200 µL each) were collected into separate test tubes, and distilled water (400 µL) was used as the blank. Then 3 mL and 1.5 mL of Folin-Ciocalteu solution (Merck, Germany) were added to the test tubes, containing the extract and distilled water, respectively, and allowed to incubate at 22°C. After 5 minutes of incubation, 1.5 mL and 3 mL of 0.6% sodium bicarbonate (Merck, Germany) were added to the tubes, containing the extract and distilled water, respectively, and incubated again for 5 minutes at 22°C. The optical absorption of the test tubes was read at the wavelength range of 725 nm on a spectrophotometer (Figure 1). Using the following formula, Y=0.0049 x-0.0016; Y=0.1494, X=3.0163 μg /mL, R ₂ =0.9979, the total phenolic content was determined to be 8.4 μg/mL.
The hydroalcoholic extract samples of the total flavonoid contents were prepared by dilution in distilled water, using quercetin method, At concentrations of 0.1, 0.01 and 0.001μg/mL. Then 300 µL of 5% sodium nitrite (Merck, Germany) was added to the blank and test tubes. After 5 minutes, 300 µL of 10% aluminum chloride (Merck, Germany) and 2 mL of 1M NaOH (Merck, Germany) was added and brought up to a final volume of 100 mL. The absorbances of the samples were read at 510 nm on a spectrophotometer ( Figure 2). Using the below formula: Y=0.0025; X = -0.1495; Y=0.01, X=6.38 μg/mL, R ₂ =0.9975, the total flavonoid content was determined to be 6.38 µ/mL. Chemicals: Ketamine (Rotexmedica, Germany), xylazine (Westberg, Netherland), gabapentin (Dr. Abidi Pharmaceutical Co., Iran), and Naloxone (Kaspian Tamin Co., Iran) were purchased. The hydroalcoholic extract of C. cretica was prepared by dissolving it in distilled water at 100 or 300 mg/mL [16].

Animals:
The study's tests were carried out on adult male Wistar rats, weighing 250±30 g. The animals were housed in cases under a 12-h alternating light-dark cycle at 20-23°C temperature and 50%-60% humidity with free access to standard food and water. All animal experiments were approved by the Institutional Animal Care and Use Committee (Certificate #: IR.SSU.MEDICINE. REC.1397.231) and were conducted according to the institutional guidelines. To minimize animal suffering, additional animal care was provided, and their number was limited to experiments. Efforts were made to minimize animal distress, and the experiments were performed in a quiet room in the morning between 8 to 11 a.m.
Induction of neuropathic pain: Painful peripheral neuropathy was induced by Chronic Constriction Injury (CCI) of the sciatic nerve as described by Bennett and Xie [17] with minimal modifications. General anesthesia was induced by intraperitoneal injection of a mixture of ketamine (50 mg/kg) and xylazine (10 mg/kg). After shaving the area, a 2cm incision was made on the skin of the lateral surface of the left thigh, and another cut was made directly through the biceps femoris muscle to expose the sciatic nerve. Four silk ligatures (4-0) were loosely tied around the nerve, proximal part of the trifurcation with a distance of 1mm between each ligature. Double knots were used to prevent the slippage. The muscular and skin layers were then sutured with silk threads, and the animals were housed in individual cases after their recovery.
To evaluate the effects of treatments, the animals received the medications on days 3, 7, 14, and 21 after surgery, and the behavioral tests were conducted 45 minutes   after taking the medicines in each group. In Naloxone + Cressa group, Naloxone was given to rats 30 minutes after administering the extract. This is because the extract takes time to be absorbed when given orally while the IP given Naloxon is absorbed rapidly. The behavioral tests were subsequently performed after 15 minutes.
Mechanical allodynia test: Mechanical allodynia test was performed, using von Frey test as described earlier by Chacur et al. [19]. The von Frey electronic sensor device (ELVAMED, Iran) was touched sub plantar to examine the rats' withdrawal threshold for a mechanical stimulus. The test was repeated five times at two seconds intervals. The response threshold (g) was recorded.
Thermal hyperalgesia test: A plantar test apparatus (Yo Xun 702, Iran) was used to determine paw withdrawal latency time in response to radiant heat (40 o C, cut-off time: 22 seconds) [20]. The mean latency of the withdrawal response was recorded in seconds.

Cold allodynia test:
Cold allodynia test was performed, using the acetone test (evaporation-evoked cooling) by pouring 1 mL acetone on the hind paws [21]. The test was done five times at 3-min intervals. The paw withdrawal frequency was determined in percentage.
Statistical analyses: Data were expressed as mean±S.E.M, which were analyzed by one-way Analysis of Variance (ANOVA) followed by Tukey's post hoc test. To compare the variables based on repeated observations at different times, repeated measures ANOVA was used. A P-value <0.05 was considered to show significant differences for all comparisons. All statistical analyses were done using SPSS v. 24.

Results
Induction of neuropathic pain: The CCI-induced neuropathic pain did not affect the motor activity of the animals. Closure of the sciatic nerve used in the model reduced the response threshold (g) significantly upon the mechanical allodynia test (Figure 3 Mechanical allodynia response to induced neuropathic pain: The C. cretica extract and gabapentin significantly increased the withdrawal threshold compared to normal saline, and Naloxone mixed with C. cretica extract on all pain evaluations. By adding Naloxone to the C. cretica extract, the withdrawal threshold was reduced Control, CCI rats treated orally with normal saline (4mL/Kg). Sham, rats underwent similar surgery except for sciatic nerves not ligated and were treated orally with normal saline (4ML/Kg). Gabapentin, CCI rats treated orally with gabapentin (70 mg/Kg), Cressa, CCI rats treated orally with C. cretica hydroalcoholic extract (300 mg/Kg), Naloxone, CCI rats treated orally with Naloxone (1 mg/Kg, IP), Naloxone+Cressa, CCI rats treated orally with C. cretica hydroalcoholic extract (300 mg/Kg) and received Naloxone (1mg/Kg, IP) 45 minutes after the extract. C. cretica extract and gabapentin significantly increased the withdrawal threshold compared to Control and Naloxone+Cressa groups on all the evaluation days (P<0.01). ×P<0.001 in comparison with Control group, * P<0.01 in comparison with Control group, #P<0.01 in comparison with Naloxone group. Withdrawal threshold (g)

Control Sham
Gabapentin Cressa Naloxon Naloxon+Cressa Control, CCI rats treated orally with normal saline (4 mL/kg). Sham, rats underwent similar surgery except for sciatic nerves not ligated and were treated orally with normal saline (4 mL/kg). Gabapentin, CCI rats treated orally with gabapentin (70 mg/kg), Cressa, CCI rats treated orally with C. cretica hydroalcoholic extract (300 mg/kg), Naloxone, CCI rats treated orally with Naloxone (1 mg/kg, IP), Naloxone+Cressa, CCI rats treated orally with C. cretica hydroalcoholic extract (300 mg/kg) and received Naloxone ( Thermal hyperalgesia response to induced neuropathic pain: The C. cretica extract and gabapentin significantly increased the withdrawal latency time compared to that of normal saline and Naloxone mixed with C. cretica extract on all pain evaluations. By adding naloxone to the C. cretica extract, the withdrawal latency declined compared to that of the extract alone, while Naloxone alone did not alter this effect (Figure 4, P<0.01). On the thermal hyperalgesia test, there was no difference between the C. cretica extract and gabapentin on days 3, 7, 14, and 21 after surgery.
Cold allodynia response to induced neuropathic pain: The C. cretica extract and gabapentin significantly reduced the paw withdrawal frequency in the rats by acetone test compared to that of the normal saline or Naloxone combined with the C. cretica extract on all pain evaluations. Also, there was no significant difference between the extract and gabapentin for their analgesic effect. By adding Naloxone to the C. cretica extract, the withdrawal frequency was significantly decreased compared to that of the C. cretica extract alone, while Naloxone alone did not alter this parameter ( Figure 5, P<0.01).

Discussion
Based on the results, the technique used in this study, i.e., Chronic Constriction Injury (CCI) model induced neuropathic pain in experimental rats, as manifested by the behavioral changes in the animals. Neuropathic pain developed three days after the surgery and lasted at least over the course of the study (21 days). Most of the pain occurred on day 14 post surgery and then remained almost constant until the end of the study.
Gabapentin, as an analgesic drug improved the behavioral symptoms resulting from the induced neuropathic pain in rats, confirming the accuracy of neuropathic pain induction [22]. The data analysis shows that the effects of the C. cretica extract (300 mg/kg, orally) in improving the neuropathic pain in animals are very similar to those of gabapentin (70 mg/kg, orally). Further, C. cretica contains edible oils that may be used safely in humans [23]. Numerous studies have shown that it has anti-inflammatory, antipyretic, and analgesic effects [24,25]. Also, the 13 Figure 4: Thermal hyperalgesia test responses in rats in which neuropathic pain was induced by chronic constriction injury (CCI) method.
Control, CCI rats treated orally with normal saline (4mL/Kg). Sham, rats underwent similar surgery except for sciatic nerves not ligated and were treated orally with normal saline (4mL/Kg). Gabapentin, CCI rats treated orally with gabapentin (70 mg/Kg), Cressa, CCI rats treated orally with C. cretica hydroalcoholic extract (300 mg/Kg), Naloxone, CCI rats treated orally with Naloxone (1 mg/Kg, IP), Naloxone+Cressa, CCI rats treated orally with C. cretica hydroalcoholic extract (300 mg/Kg) and received naloxone (1mg/Kg, IP) 45 minutes after the extract. C. cretica extract and gabapentin significantly increased withdrawal latency time compared to Control and Naloxone+Cressa groups on all the evaluation days (P<0.01). ×P<0.001 in comparison with Control group, *P<0.01 in comparison with Control group, #P<0.01 in comparison with Naloxone group.  Control, CCI rats treated orally with normal saline (4 mL/kg). Sham, rats underwent similar surgery except for sciatic nerves not ligated and were treated orally with normal saline (4 mL/kg). Gabapentin, CCI rats treated orally with gabapentin (70 mg/kg), Cressa, CCI rats treated orally with C. cretica hydroalcoholic extract (300 mg/kg), Naloxone, CCI rats treated orally with Naloxone (1 mg/kg, IP), Naloxone+Cressa, CCI rats treated orally with C. cretica hydroalcoholic extract (300 mg/kg) and received naloxone (1mg/kg, IP) 45 minutes after the extract. C. cretica extract and gabapentin significantly increased withdrawal latency time compared to Control and Naloxone+Cressa groups on all the evaluation days (P<0.01). × P<0.001 in comparison with Control group, * P<0.01 in comparison with Control group, # P<0.01 in comparison with Naloxone group.
Quercetin is an anti-inflammatory and analgesic agent. It modulates the inflammatory and immune systems by acting on various cells, including mast cells, dendritic cells, and glial cells. It inhibits Lipopolysaccharide (LPS)-induced Tumor Necrosis Factor α (TNF-α) production in macrophages and LPS-induced IL-8 production in the lung A549 cells. Quercetin inhibits the production of inflammatory enzymes, including cyclooxygenase and lipoxygenase. It significantly inhibits neurological and inflammatory pain by acting on several systems involved, including vanilloid, D2-dopamine, alpha-2-adrenergic, 5-HT3, and glutamate receptors [27]. The antioxidant effects of the flavonoids, rutin and quercetin, inhibit oxaliplatin-induced chronic painful peripheral neuropathy [28,29]. Quercetin has reduced pain in a diabetic neuropathic pain model by modifying opioid mechanisms [30]. In another study, however, Naloxone did not block the analgesic effects of quercetin and the hot-plate test responses, indicating that the opioid system is not involved in its analgesic property [31].
Based on the compounds found in the C. cretica extract as mentioned earlier, quercetin and rutin are considered as known compounds in the extract, justifying the effect of the extract on neuropathic pain. Further, there is evidence to suggest that opioid receptors are involved in the analgesic effects of rutin and quercetin [32,33]. In this study, Naloxone alone, as an opioid receptor antagonist, did not affect the behavioral tests in the rats with induced neuropathic pain, whereas the administration of Naloxone (1 mg/kg, IP) 15 minutes after giving the C. cretica extract (300 mg/kg) to the rats orally, significantly reduced the neuropathic analgesic effects of the extract. Considering that opioid receptors are present in peripheral, spinal, and supraspinal areas, and the fact that Naloxone inhibits all types of opioid receptors (µ, δ, and κ), it may be suggested that opioid receptors may be involved in the neuropathic analgesic effects of the C. cretica extract [34][35][36][37][38].
Due to the lack of sufficient information about the effects of other compounds in the C. cretica hydroalcoholic extract, further studies are warranted to fully understand other compounds present in the C. cretica extracts, able to relieve neuropathic pain. Also, it is important to study the Control, CCI rats treated orally with normal saline (4mL/Kg). Sham, rats underwent similar surgery except for sciatic nerves not ligated and were treated orally with normal saline (4mL/Kg). Gabapentin, CCI rats treated orally with gabapentin (70 mg/Kg), Cressa, CCI rats treated orally with C. cretica hydroalcoholic extract (300 mg/Kg), Naloxone, CCI rats treated orally with Naloxone (1 mg/Kg, IP), Naloxone+Cressa, CCI rats treated orally with C. cretica hydroalcoholic extract (300 mg/Kg) and received Naloxone (1mg/Kg, IP) 45 minutes after the extract. C. cretica extract, and gabapentin significantly decreased paw withdrawal frequency by acetone test compared to Control and Naloxone+Cressa groups on all the evaluation days (P<0.01). ×P<0.001 in comparison with Control group, *P<0.01 in comparison with Control group, #P<0.01 in comparison with Naloxone group. Control, CCI rats treated orally with normal saline (4 mL/kg). Sham, rats underwent similar surgery except for sciatic nerves not ligated and were treated orally with normal saline (4 mL/kg). Gabapentin, CCI rats treated orally with gabapentin (70 mg/Kg), Cressa, CCI rats treated orally with C. cretica hydroalcoholic extract (300 mg/Kg), Naloxone, CCI rats treated orally with Naloxone (1 mg/Kg, IP), Naloxone+Cressa, CCI rats treated orally with C. cretica hydroalcoholic extract (300 mg/Kg) and received Naloxone (1 mg/Kg, IP) 45 minutes after the extract. C. cretica extract, and gabapentin significantly decreased paw withdrawal frequency by acetone test compared to Control and Naloxone+Cressa groups on all the evaluation days (P<0.01). × P<0.001 in comparison with Control group, * P<0.01 in comparison with Control group, # P<0.01 in comparison with Naloxone group.
primary mechanism of action of the C. cretica extract and its molecular interaction with opioid receptors.

Conclusions
The hydroalcoholic extract of C. cretica reduced CCIinduced neuropathic pain in experimental rats, and Naloxone, as an opioid receptor antagonist, counteracted the analgesic effects of the C. cretica extract in induced neuropathic pain.

Compliance with ethical guidelines
All animal experiments were approved by the Institutional Animal Care and Use Committee (Registered #: IR.SSU.MEDICINE.REC.1397.231) and were conducted according to the institutional guidelines. To minimize animal suffering, additional animal care was provided, and their number was limited to experiments. Efforts were made to restrict animal distress, and the experiments were performed in a quiet room in the morning, 8-11 a.m.

Funding
This paper was extracted from a PhD. dissertation conducted by Nasrin Zare at the Department of Pharmacology, School of Pharmacy, Shahid Sadoughi University of Medical Sciences, Yazd. Also, the study was supported by the School of Pharmacy, Shahid Sadoughi University of Medical Sciences, Yazd.