Analgesic potential of dichloromethane leaf extracts of Eucalyptus globulus (Labill) and Senna didymobotrya (Fresenius) in mice models

Introduction Pain is an unpleasant sensory affliction and emotional experience associated with actual or potential tissue damage (1). Pain serves as an instant warning to the nervous system to commence a motor response thus minimizing physical damage (2). Insufficient pain relief is a problem globally (3). In pain management, nonsteroidal anti-inflammatory drugs (NSAIDs) are highly prescribed (4). However, these drugs have many side effects such as gastrointestinal complications namely peptic ulcers, bleeding, and mental dependence. Hence, their clinical uses have been limited in pain management (5). Selective http://www.herbmedpharmacol.com doi: 10.34172/jhp.2020.49


Introduction
Pain is an unpleasant sensory affliction and emotional experience associated with actual or potential tissue damage (1). Pain serves as an instant warning to the nervous system to commence a motor response thus minimizing physical damage (2). Insufficient pain relief is a problem globally (3). In pain management, nonsteroidal anti-inflammatory drugs (NSAIDs) are highly prescribed (4). However, these drugs have many side effects such as gastrointestinal complications namely peptic ulcers, bleeding, and mental dependence. Hence, their clinical uses have been limited in pain management (5). Selective cyclooxygenase-2 inhibitors have some benefits in preventing such side effects, while the risk of cardiovascular adverse effects demands important consideration (6). Narcotics are also used in pain management (7). Narcotics refer to opium, opium derivatives, and their fully synthetic or semi-synthetic substitutes, including leaf of cocoa and cocaine (8).
Senna didymobotrya is a flowering plant that belongs to the legume family. The plant grows to a maximum height of about 30-90 cm. It is found across the country in several types of the habitat. The root extracts of S. didymobotrya are utilized in the management of intestinal worms and malaria (14).
Pain is managed using medicinal plants such as Artemisia dracunculus (15), Acacia mellifera, Carissa spinarum (Linn) (16), Harrisonia abyssinica (17), among others. E. globulus and S. didymobotrya are in traditional use in the management of pain by the people of Embu county. However, there are no scientific data to support this biological activity. Therefore, this study was designed to determine the chemical constituents and in vivo analgesic potential of dichloromethane leaf extracts of E. globulus and S. didymobotrya in mice models.

Materials and Methods
Based on the literature review on ethno-medicinal uses of these plants, leaves from each of these plants were harvested from Embu county, Kenya. The plant samples were put in Khaki bags and then transported to Kenyatta University. Further processing of the plant samples was carried out in the Department of Biochemistry, Microbiology, and Biotechnology. The leaf samples were identified and taxonomically assigned voucher specimen numbers by an acknowledged taxonomist. E. globulus and S. didymobotrya were assigned voucher specimen numbers "JKM001" and "JKM002" respectively. The samples were deposited at Kenyatta University Herbarium for future reference. The plant leaf samples were air-dried at 25°C for two weeks and then ground into a homogenous fine powder. The powder of each sample was kept at room temperature in well-sealed and labeled airtight khaki papers until use in extraction. Five-hundred grams of each plant sample was weighed separately and then put into well-labeled conical flasks, separately. Two liters of dichloromethane (DCM) were put into each conical flask, corked and then let to stand for one day. The mixtures were then filtered separately using Whatman number 1 filter papers. To each of the remnants, 1 L of DCM was added and left to stand for 24 hours. This was followed by a second filtration. This procedure was repeated until the solvent appeared clear. The concentration of each extract was carried out using a rotary evaporator at 40°C. The concentrated extracts were then separately put in clean open beakers to permit the evaporation of the remaining solvents. The extracts obtained were stored at -4°C until use (18).
Gas chromatography-mass spectrophotometry (GC-MS) analysis Agilent Gas Chromatograph 7890A/5975C Mass Spectrometer in full scan mode was used to analyze the samples with the following conditions; gas chromatography column "HP-5 MS low bleed capillary column" (0.25 μm, 30 m by 0.25 mm i.d) (J and W, Folsom, California, United States of America), flow rate (Helium) "constant flow mode, 1.25 mL/min", injection split mode, oven temperature of 35 º C for the initial 5 minutes and then raised by 10 º C per minute to 28 º C for 10.5 minutes and run time of 70 minutes.
For analysis, a protocol was used, which was reviewed by the Principal Scientist, and the Head of the Department of Behavioral and Chemical Ecology in the International Centre of Insect Physiology and Ecology (ICIPE), Prof. Baldwyn Torto. A mass of 1.2 mg of DCM leaf extract of E. globulus and 1.1 mg of S. didymobotrya were diluted by partitioning between methanol and hexane. This was followed by vortexes and centrifugation. Bypassing through anhydrous Na 2 SO 4 , the hexane layer was dried and analyzed using GC-MS. Authentic serial dilutions of 1,8-cineole 99% as standard (Gillingham, Dorset, England) (50, 150, 250, 350 and 550 ng/µL) were prepared and analyzed using GC-MS. The peak areas were used for quantification purposes.
Experimental animals Swiss albino mice of both sexes aged between 2-3 months and weighing approximately 20 g were used to assess analgesic activities of the two DCM leaf extracts. The approval for experimentation of animals was obtained from the National Commission for Science, Technology, and Innovation (Reference number NACOSTI/P/16/6765/14525). The animals were taken care of, and handled as per Kenyatta University ethical guidelines and procedures for handling experimentation animals. Mice were selected 24 hours before experimentation based on their normal response to sensorimotor testing. The sensorimotor test was done by holding the mice in a fully extended and inverted position one hour after administration of controls and dosages (19,20).
Experimental design A completely randomized experimental design was adopted in this study. Swiss albino mice were randomly allocated nine groups of 5 mice and treated as follows; group 1 (normal control) comprised mice that received intraperitoneally 3% DMSO. Group 2 (negative control) received 3% DMSO and then pain was induced after 30 minutes by injection of 0.01 mL of 2.5% formalin. Group 3 (positive control), received 0.1 mL of diclofenac sodium at a dose of 15 mg/kg body weight and after 30 minutes were administered with 0.01 mL of 2.5% formalin to induce pain.
The formalin-induced pain was carried out by a previously described method (21), where all the animals received 0.1 mL of treatments intraperitoneally and 30 minutes later with 0.01 mL injection of 2.5% formalin in the left hind paw to induce pain. The time taken on licking, shaking, biting or lifting of the injected paw was scored and recorded (22). The Swiss albino mice were placed inside a transparent plexiglass chamber with a mirror put at the side of the chamber to provide a clear observation of the pain response. Two phases of intensive pain were observed and recorded separately (the early phase of 1-5 minutes and late phase of 15-30 minutes). The percentage of pain inhibition was computed using the following formula. Statistical analysis Data on pain was entered in a Microsoft Excel broadsheet and then transferred to Minitab statistical software (version 17.0) for statistical analysis. Descriptive statistics (mean± SEM) were computed. One-way analysis of variance was employed to analyze for statistical variation among various sets of treatment groups followed by Tukey's post hoc test for mean separations and comparisons. Analgesic activity of the two plant extracts was compared using unpaired t test. The level of significance was set at 99.5% (P ≤ 0.005).

GC-MS results of E. globulus (Labill) and S. didymobotrya (Fresenius)
The GC-MS results revealed that these plants were endowed with several bioactive agents that possessed analgesic activity (Table 2). Representative total ion chromatogram of the DCM leaf extracts of E. globulus and S. didymobotrya with their retention times are respectively presented in Figures 1 and 2.
The molecular and structural formula of compounds identities of DCM leaf extracts of E. globulus and S. didymobotrya are shown in Figure 3.
Analgesic effects of DCM leaf extracts of E. globulus and S. didymobotrya in mice Two phases were used to assess the antinociceptive activities of DCM leaf extracts of E. globulus and S. didymobotrya on formalin-induced nociception in Swiss albino mice. They included the early phase which lasted between 1-5 minutes and a late phase that lasted between 15-30 minutes after injection of formalin. E. globulus leaf extracts possessed analgesic activity in mice. This was evident by a reduction in paw shaking, licking and lifting time ( Table 3).
The Swiss albino mice that received E. globulus extract at the doses of 25, 50, 100, 150, 200 and 250 mg/kg body weight as well as diclofenac (15 mg/kg), decreased the paw licking time in the early phase by 8.29%, 31.87%, 19.84%, 31.71%, 29.76%, 30.57%, and 31.87%, respectively ( Table  3). The analgesic activity of E. globulus DCM extract at the six dosages exhibited a significant difference in the early phase (P < 0.005, Table 3). On the other hand, the analgesic effect of diclofenac was not statistically significant compared with E. globulus at dosages of 50, 150, 200 and 250 mg/kg body weight in the early phase (P > 0.005, Table 3). The antinociceptive effect of E. globulus extract showed a dose-independent response in the early phase (  Table 3). The analgesic activity of the leaf extract at the six dosages revealed a significant difference in the late phase (P < 0.005; Table 3). In contrast, the analgesic activity of the diclofenac was comparable to that of DCM leaf extract of E. globulus (Labill) at a dose of 250 mg/kg body weight in the late phase (P > 0.005; Table 3). The analgesic effect of the leaf extract of E. globulus showed a dose-dependent response in the late phase ( Table 3). The analgesic effect of DCM extract of E. globulus at all the six dose levels was significantly effective in the late phase compared to the early phase (P < 0.005).
On the other hand, the mice that received DCM leaf extract of S. didymobotrya (Fresenius)had reduced    formalin-induced pain which was evident by reduced paw time licking in the two phases (  (Table 4). However, the plant extract at 25 mg/kg body weight dose level did not show significant analgesic effect in the early phase as shown in Figure 3. In the early phase, the analgesic activity of S. didymobotrya at the six dose levels revealed significant differences (P < 0.005; Table 4). The analgesic effect of the diclofenac was significantly higher compared to that of the DCM extract of S. didymobotrya at all dose levels in the early phase (P < 0.005; Table 4). The antinociceptive effect of S. didymobotrya demonstrated a dose-dependent response in the early phase (  Table 4). The analgesic activity of the S. didymobotrya extract at the six dose levels was statistically significant in the late phase (P < 0.005, Table 4). The analgesic effect of diclofenac was significantly higher compared to those of the six of extracts dose levels in the late phase (P < 0.005, Table 4). The analgesic effect of S. didymobotrya had a dose-dependent response in both phases, except the dose of 250 mg/kg bw dose in the late phase that was not as potent as 200 mg/kg bw (Table  4). In comparison, the analgesic effect of S. didymobotrya DCM leaf extract in the late phase was significantly higher compared to the early phase at the six dose levels tested in mice (P < 0.005, Table 4).
Comparatively, the analgesic effect of E. globulus DCM extract was significantly higher than that of S. didymobotrya DCM extract at all the tested dose levels in the early phase (P < 0.005, Figure 4).
The analgesic effects of S. didymobotrya (Fresenius) and E. globulus DCM leaf extracts demonstrated no significant differences at the doses of 100, 150 and 200 mg/kg body weight in the late phase (P > 0.005, Figure 4). On the other hand, the analgesic effect of the DCM extract of E. globulus was significantly higher compared to that of S. didymobotrya in the late phase the doses of 25, 50 and 250 mg/kg body weight (P < 0.005, Figure 5).

Discussion
This study aimed at determining the analgesic activity of DCM leaf extracts of E. globulus and S. didymobotrya on formalin-induced nociception in mice. The E. globulus and S. didymobotrya leaf extracts showed significant analgesic effects by reducing pain in mice in the early and late phases. The demonstrated effects were both peripheral and central (23). The central analgesic activity could have been due to inhibition of the nociceptive effects of noradrenaline, bradykinin, prostaglandins, adrenaline, adenosine, serotonin, and acetylcholine. On the other hand, the peripheral analgesic effect could be attributed to inhibition of the discharge of endogenous pain mediators like prostaglandin-2 (PGE 2) and PGE 2 -α in peritoneal fluids including lipoxygenase which triggers the nociceptive neurons (24).
The significant antinociceptive effects of DCM leaf extracts of these plants could be attributed to the existence of analgesic components that acted by blocking the prostaglandin pathways (25). The DCM leaf extracts mechanisms of action can be postulated to be similar to those of NSAIDs like diclofenac and ibuprofen. These Values are expressed as mean ± SEM for 5 mice in each group. Values with the different superscript letters are statistically significant along the same column using one-way ANOVA accompanied by Tukey's post hoc test (P ≤ 0.005). The values in brackets represent % pain inhibition. drugs block the synthesis of prostaglandins by truncating the cyclooxygenase-1 pathway (26). This inhibition lowers the peripheral nervous tissue sensitization leading to less nerve stimulation and pain reduction (27). The DCM leaf extracts of the two plants exhibited analgesic effects by decreasing paw licking time in early and late phases on formalin-induced pain in mice (28). GC-MS results revealed the presence of bioactive phytochemicals that possessed analgesic activity. According to previous study, Aniba canelilla was found to have analgesic effects on the acetic acid-induced writhing, hot plate test and formalin-induced pain at doses of 50, 100 and 200 mg/kg bw. The study also found that the plant contains essential oils possessed significant analgesic activity (29). Studies by (30), on analgesic activity of p-cymene in glutamate, formalin and capsaicin tests in mice models, showed that this compound possessed analgesic potential. p-Cymene also possesses antinociceptive activity and has been shown to reduce the acetic acid-induced writhing in rodents. Alpha-terpineol is a monoterpenoid alcohol found in the essential oils of  several species of Eucalyptus. α-Terpineol on acetic acid, formalin, glutamate-induced pain, and hot plate induced pain (32). The GC-MS analysis of DCM leaf extracts of E. globulus revealed the presence of α-eudesmol, a component of essential oil. A study by (33), on seasonal variation, chemical composition, and analgesic and antimicrobial activities of the essential oil from leaves of Tetradenia riparia in Southern Brazil reported that α-eudesmol reduced acetic acid-induced abdominal writhing in mice.
A study carried out by Radulović et al (34), on the analgesic activity of F. ovina using acetic acid abdominal constrictions, hot plate, tail immersion and dynamic hot plate tests in mice at doses 50, 100 and 200 mg/kg revealed that essential oils of which camphor was a component possessed antinociceptive activity. A review by Nuutinen (35), reported that endo-fenchol is able to induce hyperalgesia in mice. Limonene also a component of essential oils belongs to monoterpenoids. It has been reported by Erasto and Viljoen in their review on biosynthetic, ecological and pharmacological relevance that limonene possessed analgesic activity (36). Studies reported by Paula-Freire et al (37), on antihypernociceptive activity of O. gratissimum essential oil, reported that myrcene at doses of 10, 20 and 40 mg/kg body weight showed significant antinociceptive activity against von Frey and hot plate tests models in mice. Similarly, a study by Shah et al (38), reported that myrcene had antinociceptive potential, by reducing latency time in tail-flick and hot plate test models in rodents.

Conclusion
In conclusion, the DCM leaf extracts of E. globulus and S. didymobotrya were found to contain bioactive compounds that were able to significantly reduce formalin-induced analgesia in mice. This study validates the traditional use of these plants in the management of pain.