Evaluation of Aster bakerianus Burtt Davy ex C.A. Sm. Crude Root Extract for Acute Antiinflammatory Activity in Rats

The crude extract of Aster bakerianus roots used for treatment of a variety of ailments in Lesotho was evaluated for anti-inflammatory activity and phytochemical content. Extract was first tested for toxicity at oral dosages of 0 (negative control), 1000, 2000, 3000 and 5000 mg/kg bw with five groups of female mice, each group having four mice. Negative control group received sterile distilled water (1.0 ml/kg bw). Mice were observed for general symptoms of toxicity for 24 hours and left for a further 14 days for any delayed toxicity. None of the extract doses induced toxicity. Anti-inflammatory activity of A. bakerianus was determined using the carrageenan-induced rat paw oedema assay. Five groups of six mice each were orally pre-treated as follows: Negative control group (group 1) received sterile distilled water (1.0 ml/kg bw). Positive control group (group 2) received indomethacin (10mg/kg bw). Three test groups (group 3, 4 and 5) received A. bakerianus extract, 100, 200 and 400 mg/kg bw respectively. After one hour of pre-treatment, all test groups and controls were injected with 0.1 ml carrageenan subcutaneously in the right hind paw and paw thicknesses recorded at the following time intervals: 0, 1, 2, 3, 4, 5, 6, and 24 hours. Statistically (p<0.05) there was no difference observed in the antiinflammatory activity profile of A. bakerianus and that of the drug indomethacin at the different time intervals of the study, implying same efficacy. The anti-inflammatory activity of extract was attributed to presence of terpenoids, saponins, sterols, simple phenols, coumarins, polyphenols, flavonoids, tannins, phlobatannins, anthocyanins, alkaloids, glycosides and amino acids. The results of this study justified the documented use of this plant by Basotho for treatment of inflammatory disorders.


Introduction
Acute inflammation is a rapid, short-lived initial response of tissue to harmful stimuli (such as injury) initiated by cells constituting the relevant tissues (Ambriz-Pérez et al., 2016) and is characterised by accumulation of fluid, plasma proteins and the leukocytes which release a large number of soluble inflammatory mediators which are responsible for the initiation, progression, persistence, modulation (regulation) and eventual resolution of the acute state of inflammation (Oguntibeju, 2018;Nguyen et al., 2020). The classical signs of acute inflammation include oedema, erythema, pain, heat, and primarily loss of function of the affected part of the body (Nathan, 2002;Husein et al., 2012).
The acute inflammatory process involves a cascade of biochemical events comprising the local vascular system, the immune system and different cell types found in the injured tissue (Kulinsky, 2007). Numerous proinflammatory mediators are released during an inflammatory response, including the vasoactive amines; histamine, serotonin, bradykinin (Kulinsky, 2007; El-Shitany et al., 2014) and the cytokines, interleukin 1 β (IL-1 β), IL-6, IL-8, IL-12, tumour necrosis factor-α (TNF-α) and interferon-γ (INF-γ) as well as prostaglandins especially PGE2 through cyclooxygenase-2 (COX-2), leukotrienes through lipooxygenase (LOX), nitric oxide (NO) through the inducible nitric oxide synthase (iNOS) (Husein et al., 2012; Kulinsky, 2007;El-Shitany et al., 2014). Also released is the nuclear factor kappa B (NF-κB), a transcription factor that plays an important role in the transcription of genes, as it induces transcription of its target genes such as COX-2, iNOS, TNF-α, IL-1β, and IL-6, chemokines and adhesion molecules (Karin & Ben-Neriah, 2000) that cause acute inflammation (Nguyen et al., 2020). The cytokines play major roles in the initiation and amplification of inflammatory processes (Calixto et al., 2004). Nitric oxide (NO), a free radical generated by inducible nitric oxide synthase (iNOS), can act as a defence and regulatory molecule with homoeostatic activities; however, it can also be detrimental when produced excessively (Xiong et al., 2000;Husein et al., 2012). Inflammatory reactions are supposed to lead to either the resolution of tissue injury or complete eradication of pathogens in the body in case of infection (Husein et al., 2012). However, when the process of inflammation is not completely resolved, it can be detrimental to tissues and the body as a whole (Ben et al., 2016). The different reactions in the inflammatory response cascade are therapeutic targets, which antiinflammatory agents including medicinal plants interfere with to suppress exacerbated inflammatory responses usually invoked in such disorders as injury, rheumatoid arthritis and infection (Iwueke et al., 2006).
Any interruption of the inflammatory sequence of events results in the reduction of the liberation of the mediators causing the microcirculation to come back to normal hemodynamic state (Danya, 2017).
Conventionally, inhibitors of proinflammatory cytokines and cyclooxygenase (COX) enzymes such as the nonsteroidal antiinflammatory drug (NSAID) indomethacin, is currently the choice of antiinflammatory agents (Lucas, 2016). The NSAIDs block prostaglandin and thromboxane formation by inhibiting cyclooxygenase activity (Danya 2017) and inhibit the NF-κB pathway and various inflammationassociated genes (Yamamoto & Gaynor (2001). However, these drugs, proven to be effective in many cases, can cause undesirable side effects to some degree (Lucas, 2016)  Also, traditional medicines derived from plant extracts are increasingly being used to treat a wide variety of diseases; many being prescribed broadly for the treatment of inflammatory conditions though relatively little knowledge about their mode of action is available (Amala Hazel et al., 2018;Oguntibeju, 2018). While plant derived antiinflammatories are rarely as immediately effective as the steroidal antiinflammatory drugs and NSAIDs, they are very rarely as toxic nore potentially life threatening (Talluri et al., 2016;Oguntibeju, 2018).
Research studies on medicinal plants used in traditional medicine represents a suitable approach for the development of new drugs (Ullah et al., 2014). Herbal medicine has been recognised by the World Health Organisation (WHO) as an important component of primary health care and as such efforts are being made to combine its therapeutic potential with that of orthodox medicine (Kaur and Jaggi, 2010). The WHO considers phytotherapy in its health programs and suggests basic procedures for the validation of drugs from plant origin in developing countries (Ullah et al., 2014) and the carrageenan induced oedema model for acute peripheral inflammation is one of the in vivo models approved. Medicinal plants have therefore become the subject of intense pharmacological studies in the last few decades (Fernandes & Banu, 2012;Oguntibeju, 2018).
Many plant species have shown potential for antiinflammatory activities (Oguntibeju, 2018) due to the presence of a wide variety of phytochemicals that can be a source of anti-inflammatories themselves and could also be used for the discovery of novel antiinflammatory agents with fewer side effects (Talluri et al., 2016;Oguntibeju, 2018). The chemical compounds present in plant products are a part of the physiological functions of living organisms, and hence they are believed to have better compatibility with the human body (Prasad et al., 2012). The therapeutic potential of any herbal drug depends on its form; whether it is part of a plant, or isolated active constituents or crude extracts containing several constituents, which often work together synergistically (Bandaranayake, 2006 (Hutchings and van Staden, 1994). The roots may also be pounded and mixed with water to clean the nostrils. A. bakerianus is also used locally for the treatment of snake-bites, asthma, venereal diseases, syphilis, urinary infections, anthrax, eye infections, stomach aches, colic, psychiatric disorders, shortsightedness and intestinal parasites (van Wyk et al., 1997). The liquid from crushed boiled roots is taken in doses of a teaspoonful once a day for chronic coughs or in larger doses as an emetic and have a purgative action (van Wyk et al., 1997).
According to Moteetee and Seleteng-Kose (2017), A. bakerianus has not yet been evaluated for antiinflammatory activity, and to date no other literature was found on anti-inflammatory activity of A. bakerianus hence this study.
The relationship between traditional use of a plant species and inflammatory processes has been studied using several species (

Plant Material
The whole plant of Aster bakerianus was collected at Ha Nkhema village located north east of the National University of Lesotho Roma Campus with the following co-ordinates: 29 o 26' 56'' S, 27 o 43' 18'' E. Altiude: 1683 m above sea level. The roots were obtained by digging-out the whole plant after which the roots were separated and used in the study. The plant was authenticated by the herbarium curator (Mr. M. Polaki) of the Department of Biology, National University of Lesotho. A voucher specimen of the A. bakerianus plant was deposited in the herbarium in the Department of Biology.

Preparation of crude extract of A. bakerianus roots
The soil-free roots of Aster bakerianus were gently washed in distilled water and dried in an oven (Labcon) equipped with a fan at 35 o C for two days and then ground to fine powder with a pestle and mortar (Magama et al., 2017). The powdered material (40 g) was extracted with 400 ml methanol (95% v/v in distilled water) for 72 hours at room temperature on an orbital shaker at speed of 120 rotations per minute. The extract was then filtered under suction and the filtrate was concentrated to about a quarter of its original volume under vacuum in a Gallenkamp (Germany) rotary evaporator (Magama et al., 2017). The resultant crude extract was then dried in the oven (Labcon) with fanning at 35 o C until brittle which took 72 hours. The dried extract was stored at 4 o C until use.

2.3
Animals Inbred 20 female nulliparous and non pregnant mice of NIH strain albino mice ranging from 8-12 weeks and weighing between 22-24g were used in the toxicity assay and 36 male Adult Wistar Albino rats between 8-12 weeks old and weighing between 150 and 200 g were used in the carrageenan-induced ratpaw oedema model for acute inflammation. The animals were bred and kept in the animal house of the Department of Biology and allowed free access to food (meadow sheep pellets) and water ad-libitum.

Toxicity assay
The method of the Organization for Economic Cooperation and Development-423 (OECD-423) guidelines (2002) dosing schedule was used to determine the acute oral toxicity of A. bakerianus root extract. Briefly, Twenty (20) healthy female albino mice NIH strain ranging from 8-12 weeks old nulliparous and non pregnant, were obtained from the National University of Lesotho animal house, Department of Biology. The animals were then randomly selected and divided into four groups of four mice each (n =4). Prior to testing, the animals were fed classic horse feed (12% maintenance cubes) and had free access to drinking water but were starved for 12 hours before testing. After oral administration with different fixed single dosages of the A. bakerianus root extract; 1000, 2000, 3000 and 5000 mg/kg body weight and sterile distilled water in the control group using a bulb-ended steel needle, the animals were observed for any toxicological signs and symptoms, and mortality continuously for 1 hour and then hourly for 6 hours and finally after every 24 hours up to 14 days altogether for any delayed toxicity manifestations such as modifications of the skin, the hairs (piloerection), eyes, motor activity and behavior. Emphasis was on the observation of various manifestations of toxicity such as tremors, convulsions, salivation, diarrhoea, lethargy, piloerection, lacrimation, nasal secretion, cyanosis, sleep, coma and mortality (N'Goka et al., 2018; Zahra et al., 2020).

Acute inflammation test
For the determination of the anti-inflammatory activity of the crude extract of A. bakerianus roots, the following safe oral doses were used: 100, 200 and 400 mg/kg bw., which were 1/10 for the first two and 1/12.5 of the 4 th (5000 mg/kg bw) doses used in the acute toxicity tests. In this study, the in vivo acute anti-inflammatory activity of A. bakerianus root extract was evaluated using the inhibition of carrageenan-induced rat hind paw oedema test used in many similar studies ( Anti-inflammatory activity was calculated as a percentage (%) inhibition of paw oedema when the drug indomethacin (or the root extract) was present, relative to the negative control (Group 1) as follows: Vc is the inflammatory increase in paw thickness in control group of animals (Group 1, given only the vehicle of both drug and extract), Vt is the inflammatory increase in paw thickness in drug (or extract) treated animals.  (Thilagavathi et al., 2015). The results were noted as either negative (-) meaning not detected because either absent or below the detection limit) or positive (+) for the particular class of compounds. If positive, the colour intensity was classified as + for low intensity, ++for medium intensity, +++ for high intensity.

Data analysis
Results for evaluation of A. bakerianus root extract for anti-oedematogenic activity were expressed as the mean value ± standard deviation of the mean paw thickness. Treated groups were compared with the controls for statistically significant differences between the means of each group of 6 rats. (p < 0.05) using paired Student's t-test and the Tukey multiple comparisons analysis of variance. Statistical differences with P < 0.05 were considered significant.

Results
The yield of the crude root extract from the dried powdered roots of A.bakerianus roots after extraction with 95% Methanol (v/v) in distilled water was 8.62%.

Toxicity test for A. bakerianus crude root extract
At the oral dosage range of A. bakerianus crude root extract (1000-5000 mg/kg bw) at which mice were challenged with the extract, no symptoms of toxicity in mice were observed at all dosages of the extract during the 14 days of monitoring, therefore the LD 50 was estimated to be higher than 5000 mg/kg bw.

Weight Progression
The mice of groups 1, 2, 3, and 4 were treated orally with doses of 1000, 2000, 3000 and 5000 mg / kg bw of methanolic root extract of A. bakerianus respectively. The mice of the control group were treated orally with sterile distilled water. The daily weighings of mice during the 14 days of testing for acute toxicity were used to monitor weight progression of the treated and control mice. Figure 1 shows the daily percentage (%) changes in weight for the 14 day study period for acute toxicity. The treated animals gained weight in a dose dependent manner though they were given the same feed. There was no significant (p <0.05) difference in increase in the weight of the mice within groups and between groups compared to the control during the 14 day period of the acute toxicity study in the ANOVA Tukey multiple comparisons.

Antiinflammatory activity of A. bakerianus crude root extract
In Table 1 is presented the results of comparison of mean rat paw thicknesses (mm±SD) of the indomethacin treated group (2) and each extract treated test groups (3)(4)(5) with the negative control (group 2) as well as between test groups themselves, at the seven different time intervals of the experiment and percent inhibition of inflammation in groups 2, 3, 4, 5 and 6 at the time intervals 0, 1, 2, 3, 4, 5, 6 and 24 hours.
Subcutaneous injection of the rat paw with carrageenan without treatment (group 1) as shown in Table 2 induced a time-dependent progressive inflammatory oedema which was recorded as mean paw thickness with a peak during the period from 4 till 6 hours as follows: at 1 hour: 6.31±0.84 mm, at 2 hours: 6.87±0.64 mm, at 3 hours: 6.78±0.67 mm, at 4 hours: 7.50±1.13 mm, at 5 hours : 7.47±1.57mm, and at 6 hours: 7.59±1.15mm post carrageenan injection. After the 6 th hour, a very slight resolution of the inflammation continued till the end of the experiment at 24 hours (7.24±0.67 mm). The increases in paw thickness were less pronounced in the indomethacin and extract treated groups (groups 2-5) than in the negative control (vehicle) treated group (group 1). With reference to the negative control group, it was observed that from the time of subcutaneous injection of the rat paw with carrageenan, up to 2 hours post injection, there was an increase in paw thickness which plateaeud slightly between 2 and 3 hours then began to increase again up till 6 hours and remaining high despite an insignificant deacrese observed at 24 hours (Table 2 and Figure 2). This pattern of the graph was depressed in the treated groups (groups 2-5).
The results ( Table 2) showed that all three test extract doses (100, 200 and 400mg/kg bw) of A. bakerianus root extract protected the rats from carrageenan induced inflammation and the three test extract doses showed a moderate anti-inflammatory activity in a time and dose dependent manner during the period of study. All three doses of the extract and indomethacin induced a significant (p<0.05) timeand dose-dependent inhibitory effect against carrageenan-induced acute peripheral inflammation in the rat paw when compared with the water treated (vehicle) negative control group (group 1) at 1, 2, 3, 4, 6 and 24 hours except at the 5 hour time interval. The inhibitory effects were statistically the the same for both A. bakerianus root and the drug indomethacin treated groups at each of the time intervals and highest during the period of 4-6 hours than during the period 1-3 hours post carrageenan injection. Reduction of paw thickness or oedema (as an indication of reduction in acute inflammation) by the different doses of the crude extract of A. bakerianus roots (100, 200, and 400 mg/kg bw) and the drug indomenthacin (10 mg/kg bw) was observed to be statistically similar (p<0.05) in the Tukey's multiple comparisons analysis using ANOVA at all doses and time intervals as shown in Table 2. There was therefore no statistically significant difference in anti-inflammatory activity between indomethacin and the A. bakerianus extract at all time intervals and doses of the extract used in this study.
In Figure 1 is presented the curves of the changes in paw thickness with time for the three concentrations of A.bakerianus root extract (groups 3-5) and the reference drug indomethacin (group 2). As shown on the graphs, treatment with extracts and indomethacin gave more depressed curves than the negative control group 1 treated with the vehicle, distilled water. Table 2: Group (n = 6) mean of rat paw thickness (mm) ± SD after oral administration with different dosages of A. bakerianus extract (100, 200, 400 mg/kg bw), indomethacin (10 mg/kg bw) and distilled water (1 ml/kg bw), followed one hour later by subcutaneous injection with 0.1 ml carrageenan solution in saline.

Discussion
In this study, the crude methanolic extract of A. bakerianus roots used in traditional medicine in the Kingdom of Lesotho for the treatment of various ailments was evaluated for acute toxicity and acute anti-inflammatory activity on the basis of inhibition of carrageenan-induced rat hind paw oedema. The presence of oedema is one of the major signs of inflammation and paw oedema volume has been increasingly used to test new anti-inflammatory drugs (Kumar & Jain, 2014).
In the acute toxicity study, six groups of five mice each were orally administered with sterile distilled water, indomethacin and the following four doses of the crude methanolic extract of A. bakerianus roots (1000, 2000, 3000 and 5000 mg/kg body weight). No sign of toxicity (such as convulsions, ataxy, diarrhoea, increased diuresis) or mortality, in any of the groups therefore the median lethal dose (LD50) of the A. bakerianus roots extract was considered to be higher than the highest dose, 5000 mg/kg bw. The mice gained weight in a dose dependent manner ( Figure 1) even though all groups were fed the same food and water ad libitum. The fact that the LD 50 of the extract was above 5000 mg/kg bw, this was an indication that the extract could be considered as nontoxic, when adimistered orally. In a similar study for acute toxicity, N'Goka et al., 2018 reported that extracts of Vitex madiensis administered orally did not cause any acute toxicity even at 5000 mg/kg bw. All the doses of A. bakerianus root extract caused statistically significant (p<0.05) increases in body mass at the different time intervals compared to the negative control, with the highest increase being induced by 5000 mg / kg during the 14 days of acute toxicity test. The extract was rich in amino acids and phytosterols (Table 3). It has been proven that phytosterols increase body mass and amino acids are required for growth too (Nsonde Ntandou et al., 2015). This effect could therefore be explained by the presence of the phytosterols in the methanolic extracts (N'goka et al., 2018).
The oral doses of A. bakerianus root extract used in the anti-inflammatory tests were chosen as 400 mg/kg bw, 200 mg/kg bw and 100 mg/kg bw, these being much lower than the LD50.
As shown in Table 2 and in Figure 2, group 1 (negative control) rats, it was observed that from the time of sc injection with carrageenan, up to 2 hours post injection, there was an increase in paw thickness which slightly plateaued between 2 and 3 hours. Then, from the 3 rd hour, it began to increase again remaining high till 24hours. The peak period of carrageenan-induced inflammation was from 4 hours.
The changes (increases) in paw thickness in group 1 rats (the negative control group), at the different time intervals as shown in Table 2 were as follows: at 1 hour, 50%; at 2 hours, 54.08%; at 3 hours, 53.46%; at 4 hours, 57.94%; at 5 hours, 57.77%; at 6hours, 58.44% and at 24 hours to 56.45%. The period of maximum paw oedema was from 4 to 24 hours in the untreated group 1. A depressed, similar pattern was observed in the treated groups (groups 2-5). Based on such observations the initiation and progression of oedema after subcutaneous injection of carrageenan into the rat paw has been described as being triphasic and acute (Ishola et al.  Sidhapuriwala et al., 2007) and also the release of protease, lysosomes, and migration of leukocytes into the inflamed site occurs during the late phase of oedema (Talluri et al., 2016). It has been reported that the suppression of carrageenan induced hind paw oedema after the fourth hour correlates reasonably with therapeutic doses of most clinically effective anti-inflammatory drugs, both steroidal and non steroidal such as indomethacin (Panthong et al., 2007;Kumar & Jain, 2014). In our study, this suppression of of carrageenan-induced oedeama by A. bakerianus roots extract and indomethacin was maximal during the period from 4 hours to 6 hours post carrageenan injection corresponding to the late phase of inflammation.
The efficacy of A. bakerianus root extract and that of indomethacin were statistically (p<0.05) in this study. The trend observed in our study was in agreement with observations by other authors using the carrageenan test for acute inflammation. Sarada et al., 2012 studied the leaf and bark of extract of Naringi crenulata at oral doses of 250 and 500 mg/kg bw and reported that the pattern of anti-inflammatory activity and percent inhibition in paw volume induced by these extracts was similar to that of indomethacin (10mg/kg bw p.o.) which, to these authors suggested that, the activity of the leaf and bark extracts of Naringi crenulata used in the study could be mediated by COX-1 and COX-2 inhibition since indomethacin is a COX-1 and COX-2 inhibitor. In another study the oral doses 150 mg/kg and 300 mg/kg of ethanol extracts of Barleria courtallica prepared from stem, root and leaf produced significant inhibition of carrageenan induced paw oedema at 3rd hour comparable to that brought about by the reference drug indomethacin (10 mg/kg bw) (Ponmathi et al., 2017). Rodrigues et al., 2016 observed that indomethacin was effective at reducing oedema at all time intervals of a 5 hour study with maximum activity at 5 hours in the same way as oral doses of Ocimum basilicum oil (100 and 50 mg/kg bw p). In a similar study, Ishola et al., 2014 observed that oral administration of methanol root extracts of Alafia barteri, Combretum mucronatum, and Capparis thonningii (100-200mg/kgbw) produced dose-related and time dependent significant suppression of carrageenan-induced inflammation in the middle and late phases when compared with the vehicle-treated negative control group with peak inhibition from the 3rd to the 24th hour at 200mg/kg p.o. treatment. Other reported studies of depression of carrageenan-induced rat paw acute inflammation include studies with methanolic extracts of C. rottleri root, leaf and stem at 125 mg/kg, 250 mg/kg and 500mg/kg bw by Talluri et al., 2016. These doses significantly inhibited the maximal paw edema response during the 6 hours study of the carrageenaninduced rat paw acute inflammation together with the standard inflammatory drug, indomethacin the authors were still to isolate the active componds. Popoola et al., 2016 reported that the ethanolic extracts of Garcinia kola stem bark, Uvaria chamae roots and Olax subscorpioidea roots produced significant time and dose dependent reduction in carrageenan-induced inflammation during the 6 hour study. For G. kola maximum inhibition of 73% was observed at 200 mg/kg, for U. chamae 86.7% produced by 100 mg/kg and for O. subscorpioidea at 92.31% produced by 400 mg/kg bw 6 hours post carrageenan injection.
In Table 3 is presented the results of the qualitative phytochemical analysis of A. bakerianus root extract. The following relevant classes of phytochemicals were detected in the root extract: alkaloids, glycosides, reducing sugars, terpenoids, saponins, phytosterols, simple phenols, polyphenols, flavonoids, tannins, phlobatannins, anthocyanins, betacyanins, coumarins and amino acids. All these compounds have been reported to exhibit antiinflammatory activity (Kosala et  Antioxidant phytochemicals in plant extracts are repored to reduce oxidative stress resulting from neutrophil activity during inflammation by possibly increasing the activity of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and glutathione (GSH) and by reducing ROS generation by neutrophils (Akamatsu et al., 1991; Barragán-Zarate et al., 2020). Alkaloids are said to reduce the intensity of oedema caused by carrageenan by inhibiting vascular permeability induced by histamine (Perez, 2001;Owolabi et al 2018). Some alkaloids may also prevent inflammation through blocking the metabolic pathway of arachidonic acid (Barik et al., 1992;Ullah et al., 2014) and also through inhibition of production of nitric oxide, IL-6 and through down-regulating messenger ribonucleic acid expression of proinflammatory key players such as IL-6, IL-1β, inducible nitric oxide synthase, TNF-α, and cyclooxygenase-2 (Bribi et al., 2015). Compounds with aromatic rings and alcohol groups as seen in phenols and polyphenols (such as coumarins, flavonoids, tannins, phlobatannins, anthocyanins, betacyanins) have been known to modulate inflammation at different levels by decreasing the production of reactive nitrogen and oxygen species . Terpenoids in general have a high tendency to inhibit inflammatory processes by inhibiting the production of pro-inflammatory cytokines and iNOS production (Kim et al., 2009;Owolabi et al., 2018). Phytosterols are known to reduce inflammation through the inhibition of Phospholipase A2, which hydrolyses arachidonic acid from membrane phospholipids. This prevents the formation of prostaglandins and leukotrienes via the cyclooxygenase and lipooxygenase pathways respectively, and this could result in reduction of inflammation (Yuan et al., 2019). Phytosterols, just like polyphenols, have also been reported to possess anti-inflammatory activity by decreasing various mediators of inflammation such as prostaglandins, NO, cytokines TNF-α IL-6, and IL-1 (Rizvi et al., 2014).
The observed anti-inflammatory activity of the A. bakerianus root extract in this study could therefore be ascribed to terpenoids, alkaloids, simple phenols including coumarins and polyphenols, including flavonoids and tannins as well as glycosides among other bioactive compounds acting either as distinct entities or a combination of these phytochemicals acting synergistically (Barragán-Zarate et al., 2020; Popoola et al., 2016). Kosala et al., 2018 attributed the antiiflammatory activity of the methanolic extract of Coptosapelta flavescens roots that they observed to the presence of polyphenols, terpenoids, steroids, anthraquinones and saponins. In a study of the antiinflammatorry activity of ethanolic extracts roots of Cichorium intybus (chicory roots) by Rizvi et al., 2014, the observed anti-inflammatory activity was attributed to the presence of polyphenols, flavonoids, sterols, glycosides, tannins, and terpenoids reported in chicory roots (Street et al., 2013).
Th findings of this study on A. bakerianus extract suggest that the bioactive phytochemical constituents in the crude extract of A. bakerianus roots suppressed all the phases of acute inflammation, with the inhibition being more effective in the late phase, probably by interfering with the release and/or activity of the chemical mediators, such as histamine, serotonin, bradykinin, prostaglandins. Similar to the mechanisms of action of other NSAIDs, both the therapeutic and adverse event profiles of indomethacin are caused by decreased production of prostaglandins (Lucas 2016). For this purpose, ethnobotanical studies represent an increasingly attractive approach for applying indigenous knowledge of plant use to modern societies, with the final aim of developing new remedies.

Conclusion
The anti-inflammatory activity of A. bakerianus root extract in this study was statistically similar in efficacy to that of the reference drug indomethacin, a COX-1 and COX-2 inhibitor. All doses of A. bakerianus root extract used in this study statistically had the same efficacy as the reference drug indomethacin at all the time intervals of the study. Administration of A. bakerianus root extract inhibited the carrageenan-induced inflammatory oedema starting from the first hour after aseptic subcutaneous injection of carrageenan into the rat hind paw and during all phases of inflammation. The inhibition of inflammation was attributed to the presence of various phytochemical classes in the extract, most of which are known to exhibit antioxidant activity. These detected phytochemicals might have reduced the plasma concentrations of pro-inflammatory mediators of the early, intermediate and late phase mediators as well as oxidant species in all phases. The present study confirms the efficacy of the root extract of A. bakerianus as an efficient acute phase anti-inflammatory medication, traditionally used by Basotho in the treatment of various types of inflammation.