Pain sensation is a defense mechanism that has evolved to protect tissues against actual or potential damage; obviously, it has played an important role in the survival of animals. Pain is also a symptom in many diseases, which is an important alert to notify that something is going or has already gone wrong in the organism. However, persisting pain may lead to chronic diseases and cause some changes to the central nervous system or the peripheral tissues. Nociceptive signaling dysfunction at different levels of the nervous system can be the major cause of pathological pain (Yam et al., 2018). Pathological pain has a devastating effect on the personal, social, and interpersonal aspects of a human's life. Dealing with pathological pain imposes a huge economic burden on the healthcare system each year worldwide. Globally, it has been estimated that 1 in 5 adults suffers from pain and that another 1 in 10 adults is diagnosed with chronic pain each year (Goldberg and McGee, 2011).
Analgesic agents such as opioid analgesics and nonsteroidal anti-inflammatory drugs have known side effects. This has persuaded researchers to become interested in examining new natural analgesic agents that lack such side effects. Among these novel natural analgesics, herbal medicine has received increasing attention. It is one of the most commonly sought forms of Complementary and alternative medicine, which has attracted growing interest from researchers and has largely been accepted (Jahromi et al., 2021). The herbal market in Persia has a long historical background. Manna is a byproduct of insect activity on host plants. Different types of Manna are available in the Persian herbal market. One of the best-known products in this market with high economic value is the Manna of hedysarum, which is secreted on camel's thorn (Alhagi Maurorum) bushes. Manna of hedysarum has various names such as Taranjebin, Persian Manna, Tar-angabin, Al hagi manna, or camel's thorn manna, to name a few. In English, this Manna is known as Alhagi manna, Caspian manna, Merniabin manna, and Manna of Hedysarum. (Hamedi et al., 2015).
In Persian traditional medicine, various pharmacological properties have been linked with Manna of hedysarum, including antioxidant, anti-inflammatory, and antipyretic ones. It has also been used to treat liver diseases, hyperbilirubinemia states, as a laxative for children, gastrointestinal diseases, joint pains, and blood purifiers in various viral diseases such as chicken-pox measles, and mumps (Ansari, 2019). It has also been used to treat stomach aches, neurological disease, kidney stones (Mardaninejad et al., 2013), rubella, cough, pectoral aches, and vomiting. It has also been used as a thirst quencher (Yazdanparats et al., 2014). Many pharmacological properties of Manna have been attributed to its sacchariferous compound. A 5% humid Manna of hedysarum contains 47.7% melzitoze sugar, 26.44% sucrose, and 11.5% fructose (sharif, 2013). Recently, high selenium accumulating quality of Manna of hedysarum has also been discovered (Ziarati and Hochwimmer, 2018). Despite wide administration of this type of Manna by traditional Persian practitioners or by Iranian folk medicine, little has been done to evaluate its safety or provide evidence for its widely-claimed therapeutic properties. This study aims to evaluate the analgesic effect of Manna of hedysarum. According to the current literature, this will be the first study conducted on this concept.
Animal care and the experimental steps were performed according to the criteria of care and use of institutional Animals Medical Sciences Ethics Committee of Science and Research University, Tehran, Iran (Ethical code: IR.IAU.SRB.REC.1400.029). The protocol and the experiments followed the guidelines on Ethical Standards for investigating experimental pain in animals (Zimmermann, 1983; Kim et al., 2014). In this research, 105 adult male NMRI mice within a weight range of 25−30 grams were used. The animals were housed in a room with a constant temperature (23°C±1) and a 12-hour light/dark cycle in standard cages (8-10 in each cage). Fresh water and chow pellets were allowed with no limitations. The animals were acclimatized to the laboratory environment for one week before performing the experiments, and then the formalin test was performed to determine the antinociceptive effect of Manna of hedysarum. To achieve this goal, five experiments were designed with 4-5 groups in each (n = 6 in each group). The researchers were blinded by the experimental conditions as the requirement of a blind experiment. The experiments were carried out during the light cycle between 8 AM to 2 PM. Thirty minutes before testing, the animals were placed individually in a transparent Plexiglas box (30 × 30 × 25 cm3) equipped with a mirror at a 45° angle below the chamber, which served as an observation chamber.
Manna of hedysarum was obtained from a herbal market in Tehran, Iran. The blended Manna was immediately resolved in 80% ethanol (in a ratio of 1:12) and then mixed for 72 hours on a basic shaker (IKA KS 260, Germany). Then, it was filtered and concentrated with a rotary evaporator at a temperature below 50°C (IKA-WERKE, Germany) for 1.5 hours. Finally, the liquid was dried for 24 hours at 50°C, and the resulting powder was mixed with distilled water as a solvent. The mice were injected intraperitoneally with MHEE.
Morphine sulfate, Naloxone, L-NG-Nitro Arginine Methyl Ester (L-NAME), Cimetidine, and Cyproheptadine were purchased from Sigma Chemical Co. (St. Louis, USA), and formalin was purchased from Merck (Darmstadt, Germany). The MHEE and all other drugs were dissolved in distilled water. All drugs were injected intraperitoneally (IP).
In the first experiment, to determine the effective dose of the extract, animals in the control group were treated with normal saline solution (IP); in groups 2-4, animals were injected with MHEE (100 mg/kg, 200 mg/kg, 400 mg/kg), and in group 5 morphine was injected (5 mg/kg); after 30 minutes, in each group, the mice were injected with formalin solution (10 μL, 1%) into the right hind paw. The test was conducted following the guideline delineated by Hunskaar and Hole (Hunskaar and Hole, 1987). The animals were then placed back in the observation chamber (described above), and the nociceptive response was evaluated by measuring the time spent licking the injected paw and expressed as the total licking time in the first phase (phase I; 0–5 min) and the 2nd phase (phase II; 15–30 min) after formalin injection (Azhdari-Zarmehri et al., 2014).
In experiments 2-5, the combination of MHEE and antagonists was injected to assess the neurotransmitter systems. Therefore, the antagonists and MHEE were given 30 minutes and 15 minutes before the formalin challenge, respectively.
In experiment two, animals in the control group were injected (IP) with normal saline, and groups 2-4 were injected with MHEE (400 mg/kg), naloxone (2 mg/kg), and MHEE (400 mg/kg) plus naloxone (2 mg/kg). Then, the nociceptive response was recorded after conducting the formalin test.
In the third experiment, normal saline, MHEE (400 mg/kg), L-NAME (10 mg/kg), and MHEE (400 mg/kg) together with L-NAME (10 mg/kg) were injected. Then, the nociceptive response was evaluated.
The injections of experiment four were as follows: normal saline, MHEE (400 mg/kg), cimetidine (12.5 mg/kg), and MHEE (400 mg/kg) plus cimetidine (12.5 mg/kg). Then, the nociceptive response was recorded.
In experiment five, the mice received IP injections of normal saline, MHEE (400 mg/kg), cyproheptadine (4 mg/kg), and MHEE (400 mg/kg) plus cyproheptadine (4 mg/kg). Then, the nociceptive response was recorded. In this study, the sub-effective doses of antagonist drugs were used, and the applied dose for each antagonist was chosen based on previous studies (Zendehdel et al., 2015; Lin et al., 2016).
The data were analyzed by one-way analysis of variance (ANOVA) using SPSS 16.0 and expressed as mean ± standard error (SE). Data from the first and the 2nd phases were analyzed separately because the formalin test caused a distinct biphasic nociceptive response. For treatments showing the main effect by ANOVA, the analysis was followed by a Tukey post-hoc test. The P-value< 0.05 was considered to be statistically significant.
Figure 1 demonstrates the antinociceptive effect of MHEE. As illustrated, MHEE significantly decreased the time spent licking and biting in injected paw compared to the control group (P< 0.05) in a dose-dependent manner. The maximum analgesia was observed at a dose of 400 mg/kg of MHEE in the first and the 2nd phases (P<0.05). Pretreatment with naloxone caused a reversal of MHEE-induced analgesic activity, as demonstrated by an increase in the paw licking response compared with the MHEE-treated group (P<0.05), as shown in Figure 2.
Figure 1. Effect of the Manna of hedysarum ethanolic extract on licking and biting the injected paw in male mice (n = 30). Data are expressed as mean ± SE. Different superscripts (a-c) indicate significant differences between groups (P<0.05).
Figure 2. Evaluation of the participation of the opioidergic system on the antinociceptive effect of MHEE in the formalin-induced licking model. The animals were pretreated with naloxone (opioid receptor antagonist) 15 minutes before IP administration of MHEE (400 mg/kg). The results are expressed as mean ± SE of the time the animals spent licking the formalin-injected paw (n=30). Different superscripts (a-c) indicate significant differences between groups (P<0.05).
Cimetidine did not affect the nociceptive behavior (P>0.05). Please see Figure 4. Pretreatment with Cimetidine diminished the analgesic activity of MHEE in both phases (P<0.05), as is shown in Figure 4.
Figure 3. Evaluation of the participation of the nitrergic system on the antinociceptive effect of MHEE in the formalin-induced licking model. The animals were pretreated with L-NAME (antagonist of nitric oxide synthase) 15 minutes before IP administration of MHEE (400 mg/kg). The results are expressed as mean ± SE of the time the animals spent licking the formalin-injected paw (n=30). Different superscripts (a-c) indicate significant differences between the groups (P<0.05).
Figure 4. Evaluation of the participation of the histaminergic system on the antinociceptive effect of MHEE in the formalin-induced licking model. The animals were pretreated with Cimetidine (histamine H2-receptor antagonist) 15 minutes before IP administration of MHEE (400 mg/kg). The results are expressed as mean ± SE of the time the animals spent licking the formalin-injected paw (n=30). Different superscripts (a-c) indicate significant differences between groups (P< 0.05).
Figure 5. Evaluation of the participation of the serotonergic system on the antinociceptive effect of MHEE in the formalin-induced licking model. The animals were pretreated with Cyproheptadine (serotonergic receptor antagonist) 15 minutes before IP administration of MHEE (400 mg/kg). The results are expressed as mean ± SE of the time the animals spent licking the formalin-injected paw (n=30). Different superscripts (a-c) indicate significant differences between groups (P<0.05).
The present study aimed to determine the possible antinociceptive effect of MHEE in vivo since one of the medical properties of Manna of hedysarum, as mentioned in the literature, is its analgesic effect (Mardaninejad et al., 2013; Tavassoli et al., 2020). Based on our knowledge up to the time of preparing this manuscript, no clinical and in vivo studies have investigated this effect. The formalin test was conducted to induce the pain response in this study. In this test, formalin acts as an irritant compound that results in inflammatory pain (Kaliyaperumal et al., 2020). Formalin injection produces a biphasic response. The first phase (0–5 min), which is also known as the neurogenic phase, is due to the activation of C-fibers, and the 2nd phase (inflammatory phase), occurs between 15 and 30 minutes after formalin injection and is mediated by the release of a combination of inflammatory mediators. Attenuation of the nociceptive response is observed during the interphase (5-15 minutes) (Muley et al., 2016). Based on the experiments in our study, MHEE suppressed the nociceptive response, in a dose-dependent manner, in both phases. In this study, morphine (5 mg/kg) was used as a reference drug, and the selected dose was based on previous reports (Mohammadifard and Alimohammadi, 2018).
Manna of hedysarum contains high levels of selenium compounds (Ziarati and Hochwimmer, 2018). Selenium (Se) is an essential trace element that plays a pivotal role in the cell's antioxidant defense system. Antioxidants reduce inflammation by neutralizing excessive free radicals (Arulselvan et al., 2016). The analgesic effect of MHEE seen in the second phase of the formalin test may be due to the amount of Se content. However, antinociceptive activity was seen in both phases. Therefore, regarding the present study results, it might be suggested that besides Se, there are also other mechanisms involved in MHEE-induced antinociception activity.
To investigate the neurotransmitter systems involved in Manna of hedysarum antinociceptive activity, the MHEE reaction was examined in the formalin test with opioidergic, nitrergic, histaminergic, and serotonergic receptors.
As observed, injection of naloxone did not demonstrate any analgesic activity in the first and the 2nd phases (Figure 2); this is consistent with previous studies (Azhdari-Zarmehri et al., 2014). Naloxone is a competitive antagonist at mu, kappa, and delta receptors (Whalen et al., 2019). Opioid receptors are contributed to antinociception. Pretreatment with naloxone decreased the analgesic effect of MHEE (Figure 2). Therefore, we hypothesized that the analgesic activity of MHEE is related to the opioid system.
The other drug used in this research, L-NG-Nitro Arginine Methyl Ester (L-NAME), is a non-specific nitric oxide synthase (NOS) inhibitor (Liu et al., 2019). Nitric oxide (NO) is an important neurotransmitter with an essential role in acute and chronic pain states at central and peripheral levels. NO plays a complex and diverse role in modulating the nociceptive process. In the dorsal horn of the spinal cord, NO contributes to the development of central sensitization. Moreover, the Subplantar injection of irritant compounds increases the NO level in the injected site, and pretreatment with L-NAME prevented the increase in the pro-inflammatory cytokines.
Additionally, evidence demonstrates that NO induces analgesia and is involved in the peripheral antinociceptive effect of analgesic drugs (Cury et al., 2011). As can be seen, co-administration of L-NAME and MHEE decreased the antinociceptive effect of MHEE (Figure 3). This information may suggest that the antinociceptive activity of the Manna of hedysarum is mediated through the nitrergic system.
Cimetidine, a specific competitive histamine H2-receptor antagonist, was injected to observe the histaminergic system's role in the antinociceptive activity of Manna of hedysarum. Histamine plays a dual role in the processing of nociceptive information, that is, antinociceptive activity in the CNS versus nociceptive activity in the PNS. In the PNS, tissue injuries lead to histamine release and result in pain hypersensitivity through the sensitization of polymodal nociceptors (Obara et al., 2020). Co-administration of Cimetidine and MHEE also decreased the antinociceptive effect of MHEE. Accordingly, the Manna of hedysarum antinociceptive activity might be associated with the histaminergic system (Hasanein 2011).
Cyproheptadine is a potent competitive antagonist of serotonin and histamine H1 receptors (Maddison et al., 2008). Serotonin plays a complex role in the perception of pain. It is assumed that serotonin can both inhibit and promote pain perception by different physiological mechanisms (Marks et al., 2009). Co-administration of Cyproheptadine and MHEE also decreased the antinociceptive effect of MHEE. Therefore, the analgesic activity of MHEE can be attributed to the serotonergic system.
In the current study, no procedure was applied to identify the ingredients of MHEE and the results of the previous analysis of Manna of hedysarum were used (Sharif, 2013; Ziarati and Hochwimmer, 2018). This can be considered a limitation in this study.
In summary, MHEE has antinociceptive activity in mice, mediated via opioidergic, nitrergic, histaminergic, and serotonergic systems; however, the precise mechanism of the activities mentioned above has to be clarified in the future studies. In future studies, these results can be used as a guideline to determine the antinociceptive mechanism of action of Manna of hedysarum.
The authors extend their gratitude to the Faculty of Veterinary Medicine, Science and Research Branch, Islamic Azad University, Tehran, Iran. This paper was carried out from the D.V.M thesis of the first author.
The authors declared that there is no conflict of interest.