Evaluation of Antinociceptive and Anti-Inflammatory Activities of Solvent Fraction of the Roots of Echinops kebericho Mesfin (Asteraceae) In Mice Model

Background Since ancient times, pain and inflammation have been treated using herbal remedies, which are essentially a stockroom of phytochemical components. Due to the numerous adverse effects of the already available anti-pain and anti-inflammatory medications, the search for new potential pharmaceuticals used to relieve pain and inflammation from natural sources is an ongoing process. The present study was therefore, aimed at investigating the antinociceptive and antiinflammatory activities of the solvent fractions of the roots of E. kebericho M. in mice model. Methods Successive maceration was used as a method of extraction using solvents of increasing polarity: methanol and water. The crude extract was then further fractionated using distilled water, ethyl acetate, and chloroform. Each solvent fraction was then evaluated for its peripheral analgesic activities using an acetic acid-induced writing test and central analgesic activities using the hot plate method. The acute and chronic anti-inflammatory activities of the solvent fractions were detected using carrageenan induced paw edema and cotton pellet ear granuloma respectively. The detected doses were 100mg/kg, 200mg/kg, and 400mg/kg. The positive control groups received ASA (150mg/kg) for the writing test, morphine (10mg/kg) for the hot plate method, diclofenac Na for carrageenan induced paw edema and dexamethasone (10mg/kg) for granuloma, while the negative control group received distilled water. Result EA fraction at all test doses employed (100mg/kg, 200mg/kg and 400mg/kg) showed statistical significant (p < 0.05, p < 0.01, p < 0.001 respectively) analgesic effects in both chemical and thermal induced pain stimuli in dose dependant manner. Likewise, EA fraction also exhibited anti-inflammatory activities on carrageenan induced paw edema and cotton pellet-induced granuloma in a dose-dependent manner. The AQ fraction on the other hand produced statistical significant (p < 0.05, p < 0.012) analgesic and anti-inflammatory activities at the doses of 200mg/kg and 400mg/kg, while the CH fraction exhibited statistical significant (p < 0.05) analgesic and anti-inflammatory activity at the dose of 400mg/kg. Conclusion In general, the data obtained from the present study elucidated that the solvent fractions possessed significant analgesic and anti-inflammatory activities and recommended further investigations.


INTRODUCTION
Pain and inflammation are the most common manifestations of variety of conditions that impact many individuals globally, these clinical conditions are the two most important areas of global scientific study (1). Whenever there is actual or potential tissue damage, pain is always a subjective, unpleasant sensory and emotional experience. It is often triggered by painful stimuli, which are subsequently transmitted to the central nervous system (CNS) and recognized as such (1,2). It is an immediate reaction to an undesirable occurrence linked to tissue damage, like an injury, inflammation, cancer, osteo, and rheumatoid arthritis. In addition, severe pain might arise suddenly (trigeminal neuralgia), persist for a long time after the initial injury has healed (phantom limb pain), or be brought on by a harmless stimulus (allodynia) (1, 3, and 4). It serves as the body's means of defense against harm (5). Similar to how it conducts body rescue functions, inflammation also neutralizes, destroys, and dilutes dangerous substances. Even though, the inflammatory processes, that are the body's defense mechanism, aid in the removal of pathogens and other harmful stimuli and start the healing process for the inflammatory reaction, untreated and long term inflammatory processes harm the body organs and resulted organ failures and even death (6,7).
Both pain and inflammations are the most common reasons for patients seeking medical interventions (8).
for the extraction. After all, a total amount of 5kg powdered root was macerated using 99.9% Absolute methanol with distilled water in the ratio of 4:1 for about 72 hrs. The maceration was undertaken with occasional shaking using mini orbital shaker being tuned to 120 rpm for 72 hrs. at room temperature. Then, the extract was filtered first using muslin cloth and then using Whatman filter paper No 1. Filtration and collection of the extract were repeated three times with the whole extraction taking a total of 9 days. After the extraction was completed, methanol and water were evaporated under vacuum using rotary vapor and oven at 40 0 C. The resulting solution was placed in a deep freezer operating at -20 0 C till it formed solid ice and then the remaining solvent (water) will be removed using lyophilizer. After crude extraction was completed, the resulting extract was further subjected to be fractionated using solvents of different polarity i.e. ethyl acetate, distilled water, and chloroform. After all, the resulting fractions were within a deep refrigerator (4 0 c) till the commencement of the main procedure (3,44).

Experimental Animals
Healthy adult Swiss Albino mice of either sex (25-35g body weight and 6-8 weeks of age) were purchased from Ethiopian Health and Nutrition Research Institute (EHNRI). The animals were kept in cages at room temperature on a 12 hrs. light / dark cycle with access to standard laboratory pellets and water ad libitum. All the experimental animals were allowed to be acclimatized to the laboratory condition for a week before the commencement of the experiment. All animals used in this study were handled in accordance with the internationally accepted standard guidelines for use of laboratory animals

Preliminary Phytochemical Screening
The analgesic and anti-inflammatory activities of the solvent fraction of the roots of E. kebericho were investigated in relation to the presence or lack of secondary metabolites using a conventional phytochemical screening test. Thus, the test for alkaloids, saponins, flavonoids, phenols, steroid, anthraquinone, glycosides, and tannins were performed using standard test procedures (44, 54).

Animal Grouping and Dosing
Swiss albino mice of either sex (25-35g BW) were randomly divided into eleven groups of six mice per group. Group, I was assigned as negative control and received vehicles. Group II served as positive control and was treated with standard drugs; morphine (10mg/kg, s.c) for hot plate test, indomethacin (25 mg/kg, p.o) cotton pellet granuloma, ASA (150 mg/kg, p.o) for writhing. Groups III-XI was used as test groups and was given the solvent fractions at the dose of 100mg/kg, 200mg/kg, and 400mg/kg respectively. Doses were selected based on an acute toxicity study done previously (56,65).

Antinociceptive Activity Test Acetic Acid Induced Writhing Method
This method was conducted to detect the peripheral antinociceptive activities of the solvent fractions of the roots of E. kebericho in mice and was performed by randomly dividing overnight fasted mice of either sex (25-35g) with free water access into twelve groups, consisting of six mice per group. Groups I -IX were assigned as test groups and were given different doses of E.kebericho solvent fraction (100mg/kg, 200mg/kg, and 400mg/kg) which was determined under acute toxicity and dosing done in the previous studies (65,66). Group X was used as negative control and was given a vehicle (distilled water). The eleventh (positive control) group was given 150mg/kg dose of the standard drug ASA. 0.6% v/v of acetic acid (10ml/kg, i.p) was injected into all groups of mice an hour just after the mice were given the extract, vehicle, and the standard drug with their respective doses. Analgesic activity of each fraction was assessed by counting the numbers of writhing which consists of contraction of the abdominal muscle together with stretching of the hind limbs for 30 min after a latency period of 5 min. A reduction in the number of writhes as compared to the control group was considered as evidence for the analgesic potential of each fraction, and was expressed as percent inhibition of writhing, according to the following formula (71). % Analgesic activity = mean writhing count (control groups -treated groups) *100

Hot Plate Method
This method was carried out to evaluate the central antinociceptive potentials of the roots E. kebericho solvent fraction and was performed by introducing the mouse into an open-ended cylindrical space with a floor consisting of a metallic plate that was heated by a thermode. A plate was heated to a constant temperature of 55 0 C ± 1 0 C producing the behavioral components that were measured in terms of their reaction times, namely paw licking, withdrawal of the paw, and jumping. All responses were considered to be supraspinally integrated responses. Each mouse was placed on a hot plate individually with a cut-off time of 15s to avoid lesions to the animals' paws.
The latency to lick the paw or jump from the hot plate was recorded as the reaction time. The reaction times were noted at 0 and 30, 60, 90 and 120 min after the administration of vehicle (distilled water 10ml/kg, standard drug (morphine10mg/kg) and 100mg/kg, 200mg/kg and 400mg/kg of each solvent fraction. Percentage increase in reaction time or pain threshold inhibition was calculated using the formula as follows (72,73).

Carrageenan Induced Paw Edema
This procedure was carried out by giving mice that had been fasting overnight free access to water and inducing an acute inflammation in their paws. Carrageenan (1% w/v in normal saline, 0.05mL) was administered to the mice via injection into the left hind paw's plantar side. To ensure that each mouse's leg could be submerged to the same depth in the plethysmometer's measuring chamber, the skin around the lateral malleolus of each animal was marked just before inflammation was induced.
One hour after giving the appropriate groups of mice the solvent fractions, the vehicle, and the standard medication, carrageenan was administered. Inflammation was expressed in terms of mL i.e., displacement of water by edema using a digital plethysmometer at time 0, 1, 2, 3, 4 after carrageenan injection. The percentage inhibition of edema was calculated in terms of edema volume using the following formula (73). % Edema inhibition = PEC -PET * 100 100 Where; PEC paw edema in control group

Cotton Wool Induced Granuloma
Cotton wool granuloma which is used to detect anti-proliferative activities of natural products was applied to detect the anti-inflammatory effects of solvent fractions of the roots E. kebericho in chronic inflammation. The procedure was performed by provoked foreign body granuloma in mice by subcutaneous implantation of pellets of compressed cotton. After several days, histologically giant cells and undifferentiated connective tissue was observed besides the fluid infiltration. The amount of newly formed connective tissue was then measured by weighing the dried pellets after removal. The experimental mice were anesthetized with ether and the back skin was shaved and disinfected with 70% ethanol. An incision was made in the lumbar region. By blunted forceps subcutaneous tunnels will be formed and a sterilized cotton pellet will be placed on both sides in the scapular region. The pellets formed from raw cotton which produces a more pronounced inflammation than bleached cotton. The animals were treated for 7 days. Then, the animals were sacrificed; the pellets were prepared and dried until the weight remains constant. The net dry weight, i.e. after subtracting the weight of the cotton pellet was determined (74,75).

Statistical analysis
The data that was obtained from the experiments were expressed as mean ± SEM. The results were statistically analyzed using one-way ANOVA followed by Post Hoc Tukey-tests for multiple group comparison with SPSS version 25 software. The results were considered significantly different at p < 0.05.

Preliminary phytochemical screening
The preliminary phytochemical screening's goal was to investigate the different kinds of secondary metabolites present in each solvent fraction based on qualitative color changes in test reagents, which could provide information on how analgesic and anti-inflammatory effects might relate to the presence of different active phytoconstituents. The solvent fractions from the roots of E.
kebericho showed the presence of numerous secondary metabolites based on preliminary phytochemical screening assays. The only substances found in the chloroform fraction are steroids and alkaloids. Tanning agents, flavonoids, saponins, and cardiac glycosides were present in the aqueous fraction. The richest fraction, however, was the ethyl acetate one since it contained tannins, alkaloids, saponins, terpenoids, and anthraquinones.

Antinociceptive Activity Acetic acid-induced writhing test
This method was conducted to evaluate the peripheral anti-nociceptive effects of the solvent fractions of the roots of E.kebericho to chemical-induced pain stimuli. In this method, the number of writhes (in 15 min) was highest in the negative controlled group (distilled water treated mice) (64.17 ± 0.40) and lowest in AF 400 treated mice (23.2 ± 0.95) ( Table 1). The mean writhing reduction produced by the middle (200mg/kg) and the highest (400mg/kg) doses of the ethyl acetate, aqueous, and chloroform fractions of E. kebericho were statistically significant as compared to the negative control (p < 0.01 and 0.001 respectively) ( Table 1). All the employed test doses of each solvent fraction produced increased inhibition of the numbers of writhing with maximum inhibition observed at the highest doses (400 mg/kg) (p < 0.01 for EA, p < 0.001 for AQ and CH fractions). The dose-dependent writhing reduction was observed in each solvent fraction.
The extent of reduction of writhing in the different doses of each solvent fraction was different, i.e.
(p < 0.001) for 400 mg/kg and (p < 0.01) for 200 mg/kg ethyl acetate fraction. The lowest doses (100mg/kg) of each solvent fraction showed no significant effect on chemical-induced writhing as compared to the negative control groups. On the other hand, the standard drug (ASA 150mg/kg) produced a high percentage of writhing reduction (40.30 %) as compared to other groups. Ethyl acetate fraction produced a 33.08 % writhing reduction while AQ and CH fractions produced a percentage writhing reduction of 29.35% and 25.37% respectively ( Table 1)    Notes: Values are expressed as Mean ± S.E.M (n = 6); analysis was performed with One-Way ANOVA followed by Tukey post hoc test for multiple comparisons. a = as compared to +ve control, b = as compared to -ve control, c = as compared to 100 mg/kg EA, d = as compared to 200 mg/kg EA, e = as compared to 400 mg/kg EA, f = as compared to 100mg/kg AQ, g = as compared to 200mg/kg AQ, h = as compared to 100mg/kg AQ, I = as compared to 100mg/kg CH, j = as compared to 200mg/kg CH, k = as compared to 400mg/kg CH, 1 p < 0.001, 2 p < 0.01, 3 p < 0.05.
Abbreviations: MOR, morphine (10 mg/kg); DW, distilled water (10 mL/kg); AQ aqueous fraction, EA ethyl acetate fraction, CH chloroform fraction.  Analysis was performed with One-Way ANOVA followed by Tukey post hoc multiple comparison test. Data were expressed in mean ± SEM. N = 6.

Discussion
Despite the availability of standard drugs for the pharmacological management of pain and inflammation, these issues continue to be a global public health concern that affects over 80% of the population. Furthermore, current anti-pain and anti-inflammatory medications have a variety of untoward effects and toxicities. Ethiopian folk medicine practitioners employ traditional herbal treatments to relieve pain and inflammation (5,7,45). The acetic acid-induced writhing test, also commonly known as the abdominal contraction test, is used for a reliable and rapid evaluation of the peripheral analgesic action of natural products. The test has long been used as a screening tool to evaluate the antinociceptive and anti-inflammatory properties of new substances. Pain sensation in this writhing method is elicited by acetic acid is believed to act indirectly by inducing the release of prostaglandins as well as lipooxygenase products into the peritoneum which stimulate the nociceptive neurons on the sensory nerve fibers.
The acetic acid-induced writhing test is a model of visceral pain (1,7,8 Inhibiting the synthesis and release of different endogenous inflammatory mediators, as well as suppressing the sensitivity of peripheral nociceptors in peritoneal free nerve endings to chemicalinduced pain, could be the mechanism by which each solvent fraction elicited peripheral antinociceptive effects in this model. These hypothesized processes are in keeping with the concept that any medication that reduces the amount of whriting will produce analgesia by reducing the creation and release of PGs, as well as inhibiting the transmission of peripheral pain (76).
The second model used was the hot plate method, which is usually used to detect the central analgesic effects of natural products. The detection of opiate (narcotic) analgesics is wellestablished using this model, which depends on nociceptive responses to temperature stimuli.
Analgesics that act on the work by inhibiting NO, which reduces pain and inflammation. Alkaloids, polyphenols, saponins, phytosterols, carotenoids, lignans, sesquiterpene alcohols, acetylenic and thiophene chemicals, terpenoids, and essential oil have all been found in previous phytochemical screenings on EK.
Preliminary phytochemical screening revealed that the root extract of EK included secondary metabolites such as saponin, tannin, alkaloids, phenols, flavonoids, glycosides, and steroids. As a result, the presence of these aforementioned and currently recognized phytoconstituents may be responsible for the extract's analgesic properties (15,16,21,60 Alkaloids, flavonoids, saponin, tannins phenolic substances, glycosides, coumarins, and triterpenoids chemical elements are found in plants that have analgesic and anti-inflammatory effects (45, 57). Tannins, flavonoids, and saponins are well known for their capacity to reduce pain perception and have anti-inflammatory activities by inhibiting enzymes implicated in inflammation, particularly those involved in the arachidonic acid metabolic pathway and prostaglandin formation (3,4). Tannins may influence the inflammatory response by scavenging free radicals and inhibiting iNOS in macrophages. Saponins, on the other hand, work by inhibiting NO, which reduces pain and inflammation (34,34). The presence of active phytoconstituents from each solvent fraction contributes to the anti-inflammatory properties of the plant.

Conclusions and Recommendations
In conclusion, the solvent fractions of the study plant exhibited peripheral analgesic activities and central pain inhibition potentials. The studied plant also showed good anti-inflammatory activity in the chronic inflammatory model. Because of the presence of active secondary metabolites such as alkaloids, flavonoids, saponins, terpenoids, tannins, and essential oils, which have been repeatedly reported to have analgesic and anti-inflammatory properties, these findings could imply the solvent fractions were involved in inhibiting various endogenous inflammatory mediators, pain transmission, and mediators. The research findings provide scientific proof for the traditional claimed usage of Echinops kebericho M. in Ethiopian folk medicine for painful illnesses and inflammation.
Further investigation should be done on constituent isolation, binding studies, and electrophysiological methods to fully elucidate E. Kebericho's anti-pain and anti-inflammatory mechanisms, as well as specific mechanisms associated with these effects.

Data Availability
All data that are analyzed are available on the hand of the corresponding author upon reasonable request

Ethical Approval
Ethical clearance was obtained from the Research and Ethics Committee, College of Health Science, Debre Tabor University to conduct the study in an animal model with the reference number CHS 07-105-21.

Authors' Contributions
All authors made a significant contribution to the work reported; participated in the conception, study design, execution, and acquisition of data, analysis, and interpretation; took part in drafting, revising, or critically reviewing the article; gave final approval of the version to be published; agreed on the journal to which the article has been submitted; and agreed to be accountable for all aspects of the work. TYT conceived the idea and drafted the proposal. SBD, TGY, GTA, and ZDK prepared and critically reviewed the final manuscript for publication. All authors read and approved the final version of the manuscript.